tosoh protein a 5

Have you heard of 5’-Deoxy-5’-methylthioadenosine (5’-dA)? It’s like the superhero of nucleosides, involved in the methionine salvage pathway and also a key player in the world of epigenetics! Picture this: it’s like adenosine went to a makeover and came back with a cool methylthio group attached to its 5’ carbon, giving birth to the awesome 5’-dA. This modified nucleoside doesn’t just stop there – it can jump into RNA or take a detour into the methionine salvage pathway. But wait, there’s more! 5’-dA is not just some ordinary molecule – it has a knack for gene regulation and might even be involved in causing or preventing diseases. Who knew a tiny nucleoside could have such a big impact? Stay tuned for more exciting updates on the adventures of 5’-dA!你听说过5 ' 脱氧核糖核酸5 ' 甲基乙二醇（5 ' dA）吗？就像核苷酸的超级英雄，想象一下：这就像阿迪诺辛去改头换面，带着一个冷却的甲基硫酸组附着在它的5 ' 碳上，产下了令人赞叹的5 ' dA。

P0303903Packed Columnsfor TOYOPEARL® ScreeningToyoScreen® Series INSTRUCTION MANUALTOSOH CORPORATIONKeep this manual with the product for future reference.To help protect you and/or your property from potential damage and ensure personal safety, please read this manual thoroughly before using the product.Indicates a hazard with a medium level of risk which, if not avoided, couldresult in death or serious injury.Indicates a hazard with a low level of risk which, if not avoided, could result in minor or moderate injury.Not taking proper precautions when using flammable solvents could result in fire, explosion, or poisoning. Keep away from fireSafety Precautions[Notational Conventions ]Notation ExplanationWARNING WARNINGCAUTIONNOTECAUTIONUse only in well-ventilated areasIn case of insufficient ventilation, flammable and toxic solvents can cause fire, explosion, or poisoning.Do not spill solventsSpillage and leakage can cause fire, electric shock, poisoning, injury, or corrosion.Wear appropriate protective gear when cleaning up a spill.Wear protective eye gear and glovesOrganic solvents and acids should not come into direct contact with the skin.Handle the package with careInappropriate handling may cause rupturing and/or splattering of the product.Only use this product for its intended useThis product is intended for the separation and purification of small molecules and proteins. Do not use it for any other purpose.Monitor the backpressureThe maximum pressure of the column hardware is 1 MPa. Make sure the backpressure does not exceed the maximum.Make sure compounds are safeCheck that the target compounds and solutions after separation and purification are safe.Proper disposalDispose in accordance with local laws and regulations.Precautions: Shipping Solvents Note a) Shipping solvent; aqueous solution containing 20 % ethanolToyoScreen DEAE-650M, ToyoScreen SuperQ-650M, ToyoScreen QAE-550C,ToyoScreen GigaCap Q-650M, ToyoScreen Q-600C AR, ToyoScreen GigaCapDEAE-650M, ToyoScreen CM-650M, ToyoScreen GigaCap CM-650M,ToyoScreen SP-650M, ToyoScreen SP-550C, ToyoScreen MegaCap II SP-550EC,ToyoScreen GigaCap S-650M, ToyoScreen AF-Chelate-650M,ToyoScreen AF-Blue HC-650M, ToyoScreen AF-Red-650M,ToyoScreen AF-Heparin HC-650M, ToyoScreen AF-rProtein A-650F,ToyoScreen AF-rProtein A HC-650F, ToyoScreen MX-Trp-650Mb) Shipping solvent; 1.8 mol/L ammonium sulfateToyoScreen Ether-650M, ToyoScreen Phenyl-650M, ToyoScreen Butyl-650M,ToyoScreen Hexyl-650C, ToyoScreen PPG-600M, ToyoScreen Butyl-600M,ToyoScreen SuperButyl-550Cc) Shipping solvent; 3.6 mol/L ammonium sulfateToyoScreen Phenyl-600MFirst Aid Inhalation Move the person to an area with fresh air and rinse the mouthwith plenty of water. a) b) c)Call immediately for medical attention. a)Skin exposure Wash the exposed area with plenty of soap and water.Eye exposure Open the eyes as wide as possible and rinse with clean water for atleast 15 minutes. Call immediately for medical attention.Ingestion Rinse the mouth with plenty of water. Call immediately for medical attention.Ventilation Handling and Storage Provide adequate air ventilation to keep organic vaporconcentrations below approved level. a)Disposal methods Waste Disposal Dispose in accordance with local laws and regulations. a)General considerationsPlease pay attention to all safety precautions with respect tothe handling and storage of this product.Container handling Container may break if not handled with care. a)Wear appropriate protective equipment Use solvent-resistant gloves and protective eye gear when usingthis product. Use of a gas mask, additional protective clothing orrubber boots could be appropriate when handling this product. a)Use rubber gloves and protective eye gear when using thisproduct. b) c)Hazardous substance storage If any flammable solvents are used for shipping or storage of thisproduct, keep away from fire and open heat sources. a)Storage temperature Avoid storing this product at very low temperatures ( 0 ) toprevent product from freezing.Precautions: ToyoScreen Chromatographic Column Note ToyoScreen products contain combustible packings based on a methacrylate polymer.First Aid Inhalation Move the person to an area with fresh air and rinse the mouthwith plenty of water. Call immediately for medical attention.Skin exposure Wash the exposed area with plenty of soap and water.Eye exposure Open the eyes as wide as possible and rinse with clean water forat least 15 minutes. Call immediately for medical attention.Ingestion Rinse the mouth with plenty of water. Call immediately for medical attention.Fire precautions Handling and Storage Do not expose this chromatographic resin to fire or open heatsources.Disposal methods Waste Disposal Dispose in accordance with local laws and regulations. Seebelow for additional precautions.General considerations Please pay attention to all safety precautions with respect to thehandling and storage of this product.Disposal precautionThis product can be safely incinerated. Appropriate nitrogen oxides exhaust emission precautions shouldbe taken specifically for ToyoScreen DEAE-650M,ToyoScreen SuperQ-650M, ToyoScreen QAE-550C,ToyoScreen GigaCap Q-650M, ToyoScreen Q-600C AR,ToyoScreen GigaCap DEAE-650M,ToyoScreen AF-Chelate-650M,ToyoScreen AF-Heparin HC-650Mand ToyoScreen MX-Trp-650M. Appropriate sulfur exhaust emission precautions should be takenspecifically for ToyoScreen SP-650M, ToyoScreen SP-550C,ToyoScreen GigaCap S-650M andToyoScreen MegaCap II SP-550EC. Appropriate sulfur and nitrogen oxides exhaust emissionprecautions should be taken specifically forToyoScreen AF-Red-650M, ToyoScreen AF-Blue HC-650M,ToyoScreen AF-rProtein A-650F andToyoScreen AF-rProtein A HC-650F.Ventilation Provide adequate air ventilation to keep organic vaporconcentrations below approved level.Wear appropriate protective equipment Use solvent-resistant gloves and protective eye gear when usingthis product. Use of a dust respirator could be appropriate whenhandling dried chromatographic media.Hazardous substance storage If any flammable solvents are used for shipping or storage of thisproduct, keep away from fire or open heat sources.Table of Contents1. Introduction (1)2. Column Dimensions and Basic Properties of Packing Material (1)3. Column Components (2)4. Column Attachment (2)5. Connecting ToyoScreen Columns to an Instrument (4)6. Mobile Phases (5)7. Analytical Conditions (6)8. Storage (6)9. Remarks (8)10. Warranty (8)5. Connecting ToyoScreen Columns to an Instrument5−1 FittingsT he column should be attached to the injector and detector using 1/16”tubing. Narrow internal diameter PEEK (polyetheretherketone) or plastic tubing is recommended to prevent sample dilution. Metal fittings may damage the column, therefore plastic or PEEK fittings should be used to attach tubing to the ToyoScreen columns. The PEEK fittings are sold separately (Part No. 0016566).W hen using an FPLC system, a T-F union should be used (sold separately, Part No. 0020028).Note FPLC is a registered trademark of GE Healthcare.5−2 Flow DirectionT he mobile phase should only flow through the column in the direction indicated on the label.5−3 Prevent Air from Entering the ColumnW hen attaching or detaching the column from the instrument, take great care to prevent air from entering the column. Flush all air bubbles from the tubing using mobile phase before attaching the column to the instrument. Air bubbles introduced onto the column may result in uneven flow (channeling) and lower performance.5−4 Connection to InstrumentP rior to connecting the column, connect two pieces of tubing (one piece to connect the column to the injector, the other connecting the column to the detector) with a 1/16”union, and pump mobile phase through the tubing in order to expel the air from the column inlet tubing.T he next step is to remove any air, if present, from the column. Connect the outlet end of the column to the injector or pump and slowly pump in the reverse direction, mobile phase through the column to expel any remaining air bubbles. It is important to prevent a sudden surge of mobile phase or pressure as this may lower the performance of the column. Therefore, slowly step-up the mobile phase flow rate until it reaches the desired value (we recommend 1 mL/min for the 1 mL columns and 5 mL/min for 5 mL columns). After confirming that there are no bubbles eluting from the inlet (top) of the column, stop the flow and disconnect the column. Reposition the column in the direction of the flow arrow and carefully reconnect the top of the column to the injector/ pump. Please note that if a detector is connected, the column backpressure may increase significantly due to resistance of tubing inside the detector. In this case, either replace the tubing causing the higher pressure or lower the flow rate.5−5 Prior to AnalysisA fter the column is properly installed between the injector and detector, avoid sudden pressurization by slowly stepping-up the flow rate of the mobile phase until the desired flow rate is obtained (see section 7; for flow rate guidelines).5−6 Post AnalysisA fter operation, do not detach the column from the instrument until the pump stops and the flow of the mobile phase has stopped. If the column is detached before the flow has stopped, the column will be subjected to a sudden pressure drop that may cause the performance to deteriorate.6. Mobile Phases6−1 Mobile Phase ViscosityW hen using a highly viscous mobile phase, such as mixtures of organics with aqueous buffer, use a lower flow rate because the backpressure increases with viscosity of the mobile phase. High backpressure may damage the column.6−2 Impurities in Mobile PhaseIn order to prevent ghost peaks, analytical grade solvents are recommended.6−3 Dissolution of SampleS elect a solvent that completely dissolves the sample. If there is precipitate in the injected sample, the inlet tubing and/or inlet frit may become plugged resulting in lower performance.6−4 DegassingI f the mobile phase is not adequately degassed, bubbles may enter the column and decrease column performance. To prevent this problem, degas the mobile phase thoroughly. If only a few bubbles are present in the column, performance may be recovered by running a well-degassed mobile phase (such as distilled water) through the column.1) Remove the screw plug located at the top of the column, leaving the bottom screw plug in place. Fill the inlet of the Inlet Cap with distilled water (G) and then slowly remove the Inlet Cap (H) from the holder.2) Loosen the Outlet Cap of the holder, and detach the column from the Column Holder Body.3) Fill the column top with water. Confirm that liquid drops exit from the bottom of the column through gravity flow and that no air bubbles are present in the column outlet (I).4) Fill Protective End Cap (J) with water using a wash bottle or other suitable device and attach the cap to the column outlet side.5) Attach the custom snap-on Seal Cap (K) to top of the column.K eep the packing material from drying out according to the procedure described above. Drying the packing material can cause significant deterioration of columnperformance.1)4)(K)2)3)9. RemarksToyoScreen MX-Trp-650M should be kept away from oxidizing agent, protected from light and stored in the dark.10. Warranty1) Inspect the following immediately after receiving the product:a. product package for any signs of damage.b. each of the columns if it has not been dried.2) If you should find any of the above defects, contact the local Tosoh salesrepresentative within 2 weeks after receiving the product. After two weeks, we will assume that you have received a satisfactory product.Part No. 0021360 0021361 0021362 0021363 0021364 0021365 0021859 0021860 0021992 0021993 0021366 0021367 0021951 0021952 0021368 0021369 0021370 0021371 0021870 0021871 0021868 0021869 0021372 0021373 0021374 0021375 0021376 0021377 0021378 0021379 0021380 0021381 0021494 0021495 0021892 0021893 0021382 0021383 0021384 0021385 0021386 0021387 0021388 0021389 0021390 0021391 0022809 0022810 0022811 0022824 0022825 0022872 0022873Product DescriptionToyoScreen DEAE-650MToyoScreen DEAE-650MToyoScreen SuperQ-650MToyoScreen SuperQ-650MToyoScreen QAE-550CToyoScreen QAE-550CToyoScreen GigaCap Q-650MToyoScreen GigaCap Q-650MToyoScreen Q-600C ARToyoScreen Q-600C ARToyoScreen CM-650MToyoScreen CM-650MToyoScreen GigaCap CM-650MToyoScreen GigaCap CM-650MToyoScreen SP-650MToyoScreen SP-650MToyoScreen SP-550CToyoScreen SP-550CToyoScreen MegaCap II SP-550ECToyoScreen MegaCap II SP-550ECToyoScreen GigaCap S-650MToyoScreen GigaCap S-650MToyoScreen Ether-650MToyoScreen Ether-650MToyoScreen Phenyl-650MToyoScreen Phenyl-650MToyoScreen Butyl-650MToyoScreen Butyl-650MToyoScreen Hexyl-650CToyoScreen Hexyl-650CToyoScreen PPG-600MToyoScreen PPG-600MToyoScreen Butyl-600MToyoScreen Butyl-600MToyoScreen Phenyl-600MToyoScreen Phenyl-600MToyoScreen SuperButyl-550CToyoScreen SuperButyl-550CToyoScreen AF-Chelate-650MToyoScreen AF-Chelate-650MToyoScreen AF-Blue HC-650MToyoScreen AF-Blue HC-650MToyoScreen AF-Red-650MToyoScreen AF-Red-650MToyoScreen AF-Heparin HC-650MToyoScreen AF-Heparin HC-650MToyoScreen AF-rProtein A-650FToyoScreen AF-rProtein A-650FToyoScreen AF-rProtein A-650FToyoScreen MX-Trp-650MToyoScreen MX-Trp-650MToyoScreen GigaCap DEAE-650MToyoScreen GigaCap DEAE-650M1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 each1 mL × 5 each5 mL × 1 each5 mL × 5 each1 mL × 6 each5 mL ×6 each1 mL × 6 each5 mL ×6 eachPackageProduct List of ToyoScreen Series1 mL × 5 Grades × 1 each5 mL × 5 Grades × 1 each1 mL × 5 Grades × 1 each5 mL × 5 Grades × 1 each1 mL × 6 Grades × 1 each5 mL ×6 Grades × 1 each1 mL × 6 Grades × 1 each5 mL ×6 Grades × 1 eachToyoScreen A-IEC(DEAE-650M, SuperQ-650M, QAE-550C,GigaCap Q-650M, Q-600C AR)ToyoScreen A-IEC(DEAE-650M, SuperQ-650M, QAE-550C,GigaCap Q-650M, Q-600C AR)ToyoScreen C-IEC(CM-650M, SP-650M, SP-550C,GigaCap CM-650M, GigaCap S-650M)ToyoScreen C-IEC(CM-650M, SP-650M, SP-550C,GigaCap CM-650M, GigaCap S-650M)ToyoScreen IEC(GigaCap Q-650M, SuperQ-650M, Q-600C AR,GigaCap CM-650M,GigaCap S-650M, SP-550C)ToyoScreen IEC(GigaCap Q-650M, SuperQ-650M, Q-600C AR,GigaCap CM-650M,GigaCap S-650M, SP-550C)ToyoScreen HIC(PPG-600M, Butyl-600M, Phenyl-650M,Butyl-650M, Phenyl-600M, Hexyl-650C)ToyoScreen HIC(PPG-600M, Butyl-600M, Phenyl-650M,Butyl-650M, Phenyl-600M, Hexyl-650C)Column holder body × 1 each,Inlet cap (male end) × 1 each (for 1 mL column),Inlet cap (male end) × 1 each (for 5 mL column),Outlet cap (female end) × 1 each (for 1 mL and 5 mL column),Screws plug × 2 eachToyoScreen Holder002139200213930021394002139500213960021397002139800213990021400Part No.0023436002343000234310023432Product DescriptionToyoScreen AF-rProtein A HC-650F ToyoScreen AF-rProtein A HC-650F ToyoScreen AF-rProtein A HC-650F ToyoScreen AF-rProtein A HC-650F 1 mL × 1 each 1 mL × 5 each 5 mL × 1 each 5 mL × 5 each PackageBIOSCIENCE DIVISIONShiba-Koen First Bldg.3-8-2 Shiba, Minato-ku, Tokyo 105-8623, JapanPhone: +81-3-5427-5180 Fax: +81-3-5427-5220Web site: /HPLC database: www2.tosoh.co.jp/hlc/hlcdb.nsf/StartE?OpenFormTOSOH BIOSCIENCE GmbHZettachring 6, 70567 Stuttgart, GermanyPhone: +49-711-132570 Fax: +49-711-1325789E-mail:******************Web site: /TOSOH BIOSCIENCESHANGHAI CO., LTD.Room 301, Plaza B, No.1289 Yi Shan Road,Xu Hui District, Shanghai 200233, China Phone: +86-21-3461-0856 Fax: +86-21-3461-0858E-mail:**************.cnWeb site: /TOSOH ASIA PTE. LTD.63 Market Street #10-03 Singapore 048942Phone: +65-6226-5106 Fax: +65-6226-5215E-mail:*******************Web site: /TOSOH BIOSCIENCE LLCTOSOH CORPORATION3604 Horizon Drive Suite 100,King of Prussia, PA 19406, USAPhone: +1-800-366-4875 Fax: +1-610-272-3028E-mail:******************Web site: /TSKgel, TSKgel SuperMultipore, TSKgel STAT, BioAssist, Lipopropak, TOYOPEARL, ToyoScreen, TOYOPEARL GigaCap, TOYOPEARL MegaCap, TOYOPAK and EcoSEC are registered trademarks of Tosoh Corporation in Japan, United States, European Union, etc.HLC is the registered trademark of Tosoh Corporation in Japan and China.This manual may not be reprinted or copied in whole or in part without written consent of Tosoh Corporation.The contents of the manual are subject to change without notice.Printed in Japan。

[收稿日期]2021-08-25　[修回日期]2022-09-23[基金项目]湖北省卫生健康委2021-2022年度青年人才项目(WJ2021F032)[作者简介]冷冬月(1985-),女,硕士,主治医师.[文章编号]1000⁃2200(2024)02⁃0146⁃06㊃基础医学㊃SOX5对大鼠成骨细胞增殖及核因子⁃κB 信号通路的影响冷冬月1,李旭峰2,方兴刚1(1.湖北省十堰市太和医院中西医结合科,442000;2.湖北省十堰市人民医院中医科,442000)[摘要]目的:探究Y⁃box 蛋白5(SOX5)对大鼠成骨细胞(OB)增殖及核因子⁃κB(NF⁃κB)信号通路的影响㊂方法:取新生24h SPF 级SD 乳鼠,应用酶消化法分离大鼠颅骨OB㊂形态学观察和ALP 染色鉴定成骨细胞;取生长良好的第4代OB,分为空白对照组㊁pcDNA3.1组㊁pcDNA⁃SOX5组㊁si⁃NC 组㊁siRNA⁃SOX5组㊂转染48h 后qRT⁃PCR 检测细胞中SOX5mRNA 的表达;MTT 法检测细胞增殖活力;ALP 活性检测试剂盒测量ALP 活性;茜素红染色观察钙结节的形成情况;Western blotting 检测OB 分化及NF⁃κB 信号通路相关蛋白Runt 相关转录因子2(Runx2)㊁Ⅰ型胶原蛋白(Collagen Ⅰ)㊁NF⁃κB p65及其磷酸化蛋白㊁KB 抑制蛋白激酶α(IκBα)㊁肿瘤坏死因子α(TNF⁃α)的表达㊂结果:与空白对照组相比,pcDNA⁃SOX5组大鼠OB 中SOX5mRNA 水平㊁p⁃NF⁃κB p65/NF⁃κB p65㊁TNF⁃α蛋白表达显著升高(P <0.05),OB 增殖活力㊁ALP 活性㊁钙化结节数量和面积㊁Runx2㊁Collagen Ⅰ㊁IκBα表达显著降低(P <0.05);siRNA⁃SOX5组大鼠OB 中SOX5mRNA 水平㊁p⁃NF⁃κB p65/NF⁃κB p65㊁TNF⁃α蛋白表达明显降低(P <0.05),OB 增殖活力㊁ALP 活性㊁钙化结节数量和面积㊁Runx2㊁Collagen Ⅰ㊁IκBα表达明显升高(P <0.05)㊂结论:SOX5具有调控OB 增殖㊁分化的作用,其作用机制可能与调控NF⁃κB 信号通路有关㊂[关键词]骨质疏松;Y⁃box 蛋白5;成骨细胞;核因子⁃κB[中图法分类号]R 681 [文献标志码]A DOI :10.13898/ki.issn.1000⁃2200.2024.02.002Effects of SOX5on rat osteoblast proliferation and NF⁃κB signaling pathwayLENG Dongyue 1,LI Xufeng 2,FANG Xinggang 1(1.Department of Integrated Traditional Chinese and Western Medicine ,Taihe Hospital ,Shiyan Hubei 442000;2.Department of Traditional Chinese Medicine ,Shiyan People′s Hospital ,Shiyan Hubei 442000,China )[Abstract ]Objective :To explore the effects of Y⁃box protein 5(SOX5)on rat osteoblasts (OB)proliferation and nuclear factor κB (NF⁃κB)signaling pathway.Methods :The newborn 24h SPF grade SD suckling rats were taken,and the OB of the rat skull was separated by enzyme digestion method.Morphological observation and ALP staining were used to identify osteoblasts.The fourth generation OBs with good growth were divided into blank control group,pcDNA3.1group,pcDNA⁃SOX5group,si⁃NC group,and siRNA⁃SOX5group.After 48h of transfection,qRT⁃PCR was used to detect the expression of SOX5mRNA in the cells;MTT method was used to detect cell proliferation viability;ALP activity detection kit was used to measure ALP activity;alizarin red staining was used to observe the formation of calcium nodules;Western blotting was used to detect OB differentiation and the expression of NF⁃κB signaling pathway⁃related proteins [Runt⁃related transcription factor 2(Runx2),type Ⅰcollagen (collagen Ⅰ),nuclear factor κB p65(NF⁃κB p65)and its phosphorylated protein,KB inhibits protein kinase α(IκBα),tumor necrosis factor α(TNF⁃α)].Results :Compared with the blank control group,the SOX5mRNA level,p⁃NF⁃κB p65/NF⁃κB p65,and TNF⁃αproteins expression in the OB of rats in the pcDNA⁃SOX5group were significantly increased (P <0.05),the OB proliferation activity,ALP activity,the number and area of calcified nodules,Runx2,collagen Ⅰ,and IκBαexpression were significantly reduced (P <0.05);the SOX5mRNA level,p⁃NF⁃κBp65/NF⁃κB p65,and TNF⁃αproteins expression in the OB of rats in the siRNA⁃SOX5group were significantly reduced (P <0.05),the OB proliferation activity,ALP activity,the number and area of calcified nodules,Runx2,collagen Ⅰ,and IκBαexpression were significantly increased (P <0.05).Conclusions :SOX5can regulate the proliferation and differentiation of OB,and its mechanism may be related to the regulation of NF⁃κB signaling pathway.[Key words ]osteoporosis;Y⁃box protein 5;osteoblasts;nuclear factor⁃κB 骨质疏松症(OP)是一种骨代谢疾病,其特征在于骨量下降,伴随着骨组织和微结构的恶化以及骨矿物质密度的下降[1]㊂在OP 中,骨形成与吸收之间的不平衡最终导致了骨的变性和易骨折[2]㊂成骨细胞(osteoblasts,OB)是一类特殊的具有成骨潜能的细胞,在骨重建及骨稳态维持中都发挥着重要的作用[3];OB活性下降是OP的主要原因[4]㊂Y⁃box蛋白5(SOX5)是SOX家族SoxD组的成员,编码控制细胞命运的各种转录因子并在许多谱系中进行分化,包括神经元㊁软骨细胞和B细胞[5-7]㊂研究[8-9]表明SOX5与类风湿关节炎和软骨形成密切相关,是治疗绝经后OP的有希望的分子靶标㊂已经在卵巢切除小鼠的骨髓细胞中发现了差异表达的SOX5;且与绝经前健康女性的骨髓样本相比,绝经后OP病人骨髓中SOX5的mRNA和蛋白表达水平显著上调;SOX5过表达可抑制人间充质干细胞的成骨分化[10]㊂而SOX5对OB增殖的分子功能及作用机制尚不明确;因此,本研究以大鼠OB为对象,探讨SOX5对OB增殖的影响及其潜在的作用机制,为OP的治疗及药物开发提供参考㊂1　材料与方法1.1　实验材料　出生24h的SPF级SD乳鼠10只,雌雄不限,质量(10±3)g,由北京维通利华实验动物技术有限公司提供,许可证为SCXK(京)2019⁃0009㊂饲养温度(20±2)℃,相对湿度为45%~60%㊂胎牛血清㊁胰蛋白酶㊁RPMI1640培养基均购自美国GIBCO公司;TRIzol RNA分离试剂及SYBR®Premix Ex Taq TM 试剂盒均购买于日本TaKaRa公司;Lipofectamine TM 2000转染试剂盒购自美国Thermo公司;pcDNA⁃SOX5和pcDNA3.1载体㊁siRNA⁃SOX5和其阴性对照(si⁃NC)以及PCR引物序列由上海GenePharma 公司设计合成;氯化十六烷基吡啶鎓(美国Sigma⁃Aldrich,货号:C9002);碱性磷酸酶(Alkaline phosphatase,ALP)检测试剂盒(P0321S)㊁MTT试剂盒(C0009S)购自碧云天生物科技公司;茜素红染色试剂(北京索莱宝科技有限公司,货号:G8550);兔抗大鼠Runt相关转录因子2(runt⁃related transcription factor2,Runx2)(ab236639)㊁Ⅰ型胶原蛋白(CollagenⅠ)(ab270993)㊁核因子⁃κB p65 (nuclear factor kappa⁃B p65,NF⁃κB p65) (ab16502)㊁p⁃NF⁃κB p65(ab76302)㊁肿瘤坏死因子α(TNF⁃α)(ab205587)㊁KB抑制蛋白激酶α(KB inhibits protein kinaseα,IκBα)(ab109300)㊁山羊抗兔IgG H&L(HRP)(ab205718)㊁β⁃actin(ab8227)均购自英国abcam公司㊂细胞培养箱(美国Thermo Fisher Scientific公司);ABI Prism®7300型荧光定量PCR系统(美国);倒置荧光显微镜(IX73,购自日本Olympus公司);iMark680多功能酶标仪㊁蛋白转膜装置购自美国Bio⁃Rad公司㊂1.2　成骨细胞的分离、鉴定　应用酶消化法分离大鼠颅骨成骨细胞[11]㊂取新生24h内SD乳鼠,处死后,乙醇浸泡5min,在无菌条件下,取出头盖骨,PBS冲洗后切成1mm3的骨颗粒,37℃水浴中用0.1%的胶原酶以1∶10的比例将骨颗粒消化分离20min,然后再次用胶原酶分离约1h㊂将分离获得的悬浮液以1200r/min离心,去上清液,用PBS漂洗3次,并加入含有15%胎牛血清㊁青霉素(100U/mL)和链霉素(100U/mL)的培养液㊂在37℃㊁5%CO2下孵育,1周换液2次,待细胞融合约80%时传代,换用成骨诱导培养基(基础培养基+50μg/mL维生素C+10mmol/Lβ⁃甘油磷酸钠+10nmol/L地塞米松)㊂取第3代细胞通过ALP染色和形态观察行成骨细胞鉴定,取生长良好的第4代细胞用于实验㊂鉴定成骨细胞:(1)形态学观察:在倒置相差显微镜下观察原代和继代培养成骨细胞的生长和形态变化,并在随机选择的视野中拍照㊂(2)ALP染色:将第3代的成骨细胞培养7d,并参照ALP染色试剂盒的说明进行ALP染色㊂去除成骨细胞的培养基,PBS清洗细胞2~3次,并用4%多聚甲醛固定10min㊂清洗后加入300μL显色液,避光于37℃的培养箱中孵育2h,显微镜下观察㊂1.3　分组及转染　取生长良好的第4代成骨细胞,按每孔2×106个接种于24孔板,分成5个处理组:(1)空白对照组,不进行任何转染;(2)pcDNA3.1组,用Lipofectamine2000按照说明书将100nmol/L pcDNA3.1转染至成骨细胞;(3)pcDNA⁃SOX5组,用Lipofectamine2000按照说明书将100nmol/L pcDNA⁃SOX5转染至成骨细胞;(4)si⁃NC组,将si⁃NC转染至成骨细胞;(5)siRNA⁃SOX5组,将siRNA⁃SOX5转染至成骨细胞㊂转染48h后收获细胞以进行后续实验㊂1.4　qRT⁃PCR检测细胞中SOX5mRNA的表达　使用TRIzol试剂提取细胞总RNA,使用分光光度计测量总RNA的浓度㊂按照试剂盒说明书将RNA反转录为cDNA,进行PCR扩增(见表1)㊂反应体系(20μL):cDNA(200ng/μL)2μL,SYBR®Premix Ex Taq TM(2×)10μL,上下游引物各0.4μL,ddH2O7.2μL㊂循环条件:94℃持续10min,然后在94℃持续10s,60℃持续20s和72℃持续1min进行40个循环㊂用β⁃actin作为对照,相对SOX5mRNA的表达量采用2-ΔΔCt方法计算㊂表1　RT⁃PCR引物序列基因引物序列SOX5F:5′⁃CAG CCA GAG TTA GCA CAA TAG G⁃3′R:5′⁃CTG TTG TTC CCG TCG GAG TT⁃3′β⁃actinF:5′⁃TTG CGT TAC ACC CTT TCT TG⁃3′R:5′⁃TGT CAC CTT CAC CGT TCC A⁃3′1.5　MTT法检测细胞增殖活力　转染48h后将成骨细胞以2×103细胞/孔的浓度接种到96孔板中,继续培养24h,然后每孔中加入20μL的MTT溶液(5mg/mL),于培养箱中孵育4h,吸去上层清液后加入150μL的DMSO, 490nm波长处测定各孔吸光度值(OD),计算细胞增殖率㊂细胞增殖率=(实验组OD值-空白孔OD 值)/(对照组OD值-空白孔OD值)×100%㊂1.6　ALP活性测定　ALP是成骨细胞分化的早期标志㊂将转染的成骨细胞用成骨培养基培养7d㊂然后,除去OM培养基,并使用0.1%TritonX⁃100裂解细胞获得细胞裂解液,然后12000g离心10min,取上清液为样品㊂使用ALP活性检测试剂盒测量上清液中ALP活性㊂于405nm处测定各组OD值,使用对硝基苯酚绘制标准曲线;另用BCA法测定每个样品的总蛋白浓度㊂并将每个样品的ALP活性标准化为相应的总蛋白含量㊂1.7　茜素红染色　采用茜素红染色观察钙结节的形成数量,评估成骨细胞的矿化能力㊂成骨细胞按1.3方法进行分组处理,转染后将细胞培养2周,弃去培养液,细胞用PBS洗涤2次,并在室温下用4%多聚甲醛固定30min㊂弃去多聚甲醛,PBS洗涤后在37℃下用0.1%茜素红染液染色1h㊂倒置光学显微镜下观察图像并拍照,并使用Image⁃Pro Plus6.0软件统计钙化结节染色阳性区域的面积和数量㊂1.8　Western blotting检测成骨细胞NF⁃κB信号通路相关蛋白的表达　使用RIPA裂解液提取细胞中蛋白质㊂使用BCA试剂盒对蛋白质浓度进行定量㊂取等量蛋白质样品上样(每泳道30μg),10%SDS⁃PAGE分离蛋白质,并通过半干转移将其转移到PVDF膜上㊂将膜用5%脱脂牛奶封闭,并在4℃下与一抗(Runx2㊁CollagenⅠ㊁NF⁃κB p65㊁p⁃NF⁃κB p65㊁TNF⁃α㊁IκBα㊁β⁃actin,1∶1000)孵育过夜㊂第2天,将膜与偶联辣根过氧化物酶的二抗孵育(1∶2000)2h,然后使用化学发光试剂盒(ECL)进行显色㊂以β⁃actin为内参,分析结果㊂1.9　统计学方法　以上所有实验均重复3次㊂采用单因素方差分析(One⁃way ANOVA),Tukey的事后检验用于单因素方差分析后的成对比较㊂2　结果2.1　成骨细胞原代培养和鉴定　原代培养第3天,倒置相差显微镜下见成骨细胞从碎骨块周围爬出,呈放射状贴壁生长,形态不规则,呈多边形或三角形,有较多突起,单核卵圆形, 1~2个核仁,胞质丰富清晰,ALP染色呈阳性,蓝黑色颗粒沉积在细胞质及细胞核ALP活性部位(见图1)㊂2.2　各组成骨细胞中SOX5mRNA的表达　与空白对照组相比,pcDNA⁃SOX5组大鼠OB 中SOX5mRNA水平显著升高(P<0.05),siRNA⁃SOX5组大鼠OB中SOX5mRNA水平明显降低(P <0.05)(见表2)㊂表2　各组大鼠OB中SOX5mRNA水平比较(x±s;n i=3)分组SOX5mRNA空白对照组 1.00±0.00 pcDNA3.1组 1.02±0.01 pcDNA⁃SOX5组 4.65±0.37*si⁃NC组 1.01±0.01 siRNA⁃SOX5组0.41±0.05* F315.94 P<0.01 MS组内0.028 q检验:与空白对照组比较*P<0.052.3　各组成骨细胞增殖活力　与空白对照组相比,pcDNA⁃SOX5组大鼠OB增殖活力显著降低(P <0.05),siRNA⁃SOX5组大鼠OB 增殖活力明显升高(P <0.05)(见表3)㊂表3　各组大鼠OB 增殖活力比较(x ±s ;n i =3)分组增殖率/%空白对照组100.00±0.00pcDNA3.1组102.13±10.01pcDNA⁃SOX5组64.75±6.16*si⁃NC 组104.06±9.31siRNA⁃SOX5组137.31±7.42* F144.83 P<0.01 MS 组内55.976 q 检验:与空白对照组比较*P <0.052.4　各组成骨细胞ALP 活性　与空白对照组相比,pcDNA⁃SOX5组大鼠OB中ALP 活性显著降低(P <0.05),siRNA⁃SOX5组大鼠OB 中ALP 活性明显升高(P <0.05)(见表4)㊂表4　各组大鼠OB 中ALP 活性比较(x ±s ;n i =3)分组ALP 活性空白对照组 1.00±0.00pcDNA3.1组 1.03±0.08pcDNA⁃SOX5组0.75±0.05*si⁃NC 组 1.02±0.06siRNA⁃SOX5组1.54±0.09* F 60.49 P<0.01 MS 组内0.004 q 检验:与空白对照组比较*P <0.052.5　各组成骨细胞钙结节形成情况　倒置显微镜下观察可见细胞呈多层重叠生长,各组细胞间均可见橘红色钙化结节;与空白对照组相比,pcDNA⁃SOX5组大鼠OB 钙化结节数量和面积显著降低(P <0.05),siRNA⁃SOX5组大鼠OB 钙化结节数量和面积明显增加(P <0.05)(见图2㊁表5)㊂2.6　各组成骨细胞分化及NF⁃κB 信号通路相关蛋白的表达　与空白对照组相比,pcDNA⁃SOX5组大鼠OB 中p⁃NF⁃κB p65/NF⁃κB p65㊁TNF⁃α蛋白表达显著升高,Runx2㊁Collagen Ⅰ㊁IκBα表达显著降低(P <0.05),siRNA⁃SOX5组大鼠OB 中p⁃NF⁃κB p65/NF⁃κB p65㊁TNF⁃α蛋白表达明显降低,Runx2㊁Collagen Ⅰ㊁IκBα表达明显增加(P <0.05)(见图3㊁表6)㊂3　讨论 OB 是骨形成的主要功能单位,负责骨骼重塑过程中骨骼基质的合成㊁分泌和矿化㊂OB 在维持骨量和减少骨质流失中起关键作用㊂刺激OB 增殖并促进其分化成熟是调节骨代谢㊁促进新骨形成㊁修复骨缺损的重要方法[12]㊂MTT 法是检测细胞增殖的指标,可以反映生活细胞的代谢水平㊂本研究采用MTT 法检测SOX5对OB 增殖活性的影响,结果显示SOX5mRNA 过表达后,对体外培养的OB 增殖有明显的抑制作用,而采用siRNA 技术干扰SOX5的表达后,OB 增殖率明显增加;表明沉默SOX5具有促进OB 增殖的作用㊂ OB 的增殖通常通过测量细胞总蛋白㊁ALP 活性和Collagen Ⅰ分泌来评估㊂ALP 是OB 分泌的一组膜结合糖蛋白,是OB 早期分化的特异性标志,ALP 活性的高低,能较客观地反映OB 分化成熟的程度[13]㊂Runx2㊁Collagen Ⅰ是OB 相关的蛋白,在成骨分化活动中起重要的促进作用[4],在增殖阶段,OB 分泌Collagen I 以帮助矿化并减少骨质流失㊂OB 分化成熟后多层重叠生长形成结节样结构,并不断分泌基质和矿物质,形成矿化结节㊂茜素红染色可以评估成骨分化晚期细胞外基质矿化情况钙化结节的形成情况[14]㊂本实验中,将转染的OB 用成骨培养基培养7d 后,通过ALP 活性检测了SOX5对OB 分化的影响,结果显示SOX5过表达后,ALP 活性值较低,OB 钙化结节数量和面积降低,且Runx2㊁Collagen Ⅰ表达减少,分析可能与细胞增殖㊁分化受到抑制有关;而SOX5mRNA 表达下调时,ALP 活性㊁细胞钙化程度及Runx2㊁CollagenⅠ表达增加㊂分析沉默SOX5促进成骨细胞ALP活性的原因可能是:(1)与促进细胞增殖有关,OB数量的增加,分泌的ALP也随之增加;(2)上调ALP mRNA表达水平,促进ALP的分泌,调节OB的分化㊂表5　各组大鼠OB钙结节形成情况(x±s;n i=3)分组钙化结节数量钙化结节面积/mm2空白对照组898.62±51.4360.54±5.71 pcDNA3.1组901.05±72.6161.12±5.63 pcDNA⁃SOX5组616.47±60.54*33.28±4.17* si⁃NC组895.09±58.3062.35±4.24 siRNA⁃SOX5组1174.81±73.42*89.76±6.79* F28.7041.08 P<0.01<0.01 MS组内4074.34729.154 q检验:与空白对照组比较*P<0.05表6　各组大鼠OB分化及NF⁃κB信号通路相关蛋白的表达(x±s;n i=3)分组Runx2CollagenⅠ　p⁃NF⁃κB p65/NF⁃κB p65　IκBαTNF⁃α空白对照组0.98±0.10 1.12±0.11　0.49±0.04　0.89±0.080.32±0.02 pcDNA3.1组 1.01±0.09 1.10±0.10*0.51±0.050.91±0.070.31±0.03 pcDNA⁃SOX5组0.66±0.07*0.74±0.07* 1.28±0.11*0.40±0.05*0.87±0.06* si⁃NC组0.99±0.06 1.08±0.090.52±0.070.88±0.040.34±0.04 siRNA⁃SOX5组 1.25±0.08* 1.31±0.12*0.33±0.04* 1.22±0.06*0.16±0.03* F20.0312.8892.2768.09150.30 P<0.01<0.01<0.01<0.01<0.01 MS组内0.0070.0100.0050.0040.001 q检验:与空白对照组比较*P<0.05 NF⁃κB控制主要骨骼细胞类型(破骨细胞[15]㊁成骨细胞[16]和软骨细胞[17])的分化或活性㊂生理和病理性骨重塑的刺激都会影响NF⁃κB信号转导[18];NF⁃κB的启动子活性主要是由核移位和NF⁃κB磷酸化引起[19]㊂在静息细胞中,NF⁃κB以NF?κB/IκBα复合物的形式存在于细胞质;在病理条件下,刺激激活核因子抑制剂IκB激酶(IκB kinases, IKKs),该激酶通过靶向将IκBα蛋白降解,释放NF⁃κB并允许其核移位和DNA结合,促进TNF⁃α㊁IL⁃1β等炎性因子的表达,减少骨细胞形成,使OB增殖㊁分化能力降低㊂研究发现NF⁃κB的激活下调OB分化[20];抑制IκBα的降解,稳定NF⁃κB/IκBα复合物,可抑制NF⁃κB的活化,减少炎性因子的释放[21-22]㊂HUANG等[23]发现在人牙槽骨OB分化中,三七皂苷R1可通过抑制NF⁃κB通路,逆转TNF⁃α诱导的钙结节和ALP活性的降低㊂杨青坡等[24]发现姜黄素能降低骨组织中NF⁃κB表达,明显改善OP大鼠骨密度㊂在本研究中,我们通过pcDNA3.1质粒转染构建SOX5过表达OB,发现大鼠OB中磷酸化NF⁃κB p65㊁TNF⁃α蛋白表达升高, IκBα表达降低,说明NF⁃κB信号通路被激活;而沉默SOX5的表达后,IκBα表达增加,磷酸化NF⁃κB p65㊁TNF⁃α表达降低,提示NF⁃κB信号通路被抑制㊂综上所述,沉默SOX5具有促进OB增殖的作用,并可调节OB的分化,其作用机制可能与抑制NF⁃κB信号通路的激活有关,过表达SOX5则发挥相反作用㊂但本研究尚存在一定不足,未对NF⁃κB 信号通路进行干扰从反面进行验证;此外,由于体内外环境的巨大差异,在体内沉默SOX5是否发挥相同的作用,有待进一步研究㊂[参考文献][1]　MENG YC,LIN T,JIANG H,et al.miR⁃122exerts inhibitoryeffects on osteoblast proliferation/differentiation in osteoporosis byactivating the PCP4⁃Mediated JNK pathway[J].Mol Ther NucleicAcids,2020,20:345.[2]　杨菁,聂子淮,滕斌,等.基于FRAX风险评估的分层管理在社区老年骨质疏松症病人中的应用研究[J].蚌埠医学院学报,2022,47(2):254.[3]　CHOTIYARNWONG P,MCCLOSKEY EV.Pathogenesis ofglucocorticoid⁃induced osteoporosis and options for treatment[J].Nat Rev 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Free Sulfhydryl in Recombinant Monoclonal AntibodiesWei Zhang*and Marta J.CzuprynWyeth BioPharma,Genetics Institute Campus,One Burtt Road,Andover,Massachusetts01810 Monoclonal antibody(mAb)therapy applications have been growing rapidly in recentyears.Like other recombinant protein drugs,therapeutic mAb’s need to be wellcharacterized to ensure their structural and functional integrity.IgG mAb’s arecomposed of two heavy and two light chains covalently linked by interchain disulfidebonds.Each domain of the heavy or light chain contains one additional disulfide bond.Native IgG mAb’s,with completely formed disulfide bonds,should not bear any freesulfhydryl.This report describes detection and quantification of free sulfhydryl inrecombinant mAb’s produced in Chinese hamster ovary(CHO)cells using a fluorescenttechnique.The method utilizes the fluorescent probe N-(1-pyrenyl)maleimide(NPM).The purified mAb’s appear to be homogeneous under native conditions with ap-proximately0.02mol of free sulfhydryl per mole of protein.Upon denaturation,minorspecies related to the mAb’s are observed on sodium dodecyl sulfate polyacrylamidegel electrophoresis(SDS-PAGE),and the free sulfhydryl level is determined to beapproximately0.1mol/mol of protein.These results suggest that a small portion ofthese recombinant mAb’s lack in intermolecular disulfide bonds but remain nonco-valently associated under native conditions.The formation of the free sulfhydrylcontaining mAb species is likely to occur during the culture process and/or proteinfolding process in the endoplasmic reticulum(ER).IntroductionThe rapid advancement of recombinant monoclonal antibody(mAb)therapy has necessitated detailed char-acterization and analysis of this protein family.Thera-peutic mAb’s derived by recombinant DNA technology, like other genetically engineered protein pharmaceuti-cals,require extensive and stringent characterization of purity,structural integrity,and stability,as molecular modifications may result in undesirable adverse biologi-cal effects,such as increased immunogenicity.Though the general structural features of antibodies have been well documented in literature(1,2),a number of studies have revealed structural variations of recombinant mAb therapeutics.These include isoaspartate formation re-sulting from asparagine deamidation and/or aspartate isomerization(3-5),processing of C-terminal lysine resides(6),methionine and cysteine(Cys)oxidation(7), variability in glycosylation(8-10),mutation introduced during transfection(11),and crossover event between variable genes of heavy and light chains during tran-scription(12).These modifications,due to host-dependent and/or in vitro process-dependent factors,are epigenetic and thus not predicable from the genetic structure of construct alone.They may alter the biological activities or pharmacokinetic properties of therapeutic mAb’s and consequently require substantive characterization to ensure product quality and to provide understanding of structure/function relationships.IgG antibodies have a four-chain structure composed of two heavy chains(HCs)and two light chains(LCs) covalently linked by interchain disulfide bridges(Figure 1).This four-chain structure is also maintained by strong,noncovalent interactions between the N-terminal half of HC-LC pairs and between the C-terminal regions of HC. In addition to interchain disulfide bonds,one intrachain disulfide bond is present and is shielded within each -barrel domain of HC and LC(1,2).While the disulfide linkage between HC and LC always involves the last Cys residue,Cys213according to the Kabat numbering system(13)of the LC,the Cys residue from the HC varies,depending on the IgG subtype(13).In IgG1mAb, the fifth Cys residue(Cys217)of HC forms an inter HC and LC disulfide bond.In IgG2and IgG4mAb’s,it is the third Cys amino acid of HC(Cys123)that links HC and LC.In the hinge region,different IgG subtypes have different numbers of disulfide bond connecting the HCs. In IgG1and IgG4,the two HCs are covalently linked by two disulfide bonds,while in IgG2,there are four disulfide bonds in its hinge region(1,2).In a correctly folded IgG antibody,regardless of the subtype,all Cys residues are involved in disulfide bond formation,there-fore,no free sulfhydryl should be present.During our work on recombinant mAb’s,we detected the presence of some free sulfhydryl as well as nonco-valently associated mAb fragments.We investigated the noncovalently associated species in mAb produced in Chinese hamster ovary(CHO)cells.To this end,we analyzed the structural integrity of these molecules under native and denaturing conditions.Free sulfhydryl content in the mAb’s was determined following deriva-tization with a fluorescent probe.Our results suggest that these noncovalently associated mAb fragments may result from incomplete formation of interchain disulfide bonds within the mAb molecules.*To whom correspondence should be addressed.Email: wxzhang@.Tel:(978)247-2997.Fax:(978)247-2834.509Biotechnol.Prog.2002,18,509−51310.1021/bp025511z CCC:$22.00©2002American Chemical Society and American Institute of Chemical EngineersPublished on Web04/11/2002Materials and MethodsMaterial.The recombinant mAb’s analyzed in the present work were produced in CHO cells and purified at Wyeth BioPharma,Genetics Institute Campus in Andover,MA.Samples included one IgG1,two IgG2(IgG2-I and IgG2-II herein),and one IgG4mAb.N -Acetyl-L -cysteine (Ac-Cys),N -(1-pyrenyl)maleimide (NPM),and N ,N -dimethylformamide (DMF)were obtained from Sigma.Size Exclusion Chromatography (SEC -HPLC).SEC -HPLC analysis of was performed on a Waters Alliance system with a Waters model 2487dual wave-length absorbance detector and a refrigerated autosam-pler controlled at 4°C.A TosoHaas G3000SW XL (7.8mm ×300mm,5µm,250Å)column was equilibrated with a mobile phase consisting of 10mM sodium phosphate,250mM NaCl,pH 7.2at a flow rate of 0.75mL/min.A 20-µl sample of mAb solution at a concentration of approxi-mately 1mg/mL was injected.The elution was monitored at 214nm.The analysis time was 20min.Sodium Dodecyl Sulfate Polyacrylamide Gel Elec-trophoresis (SDS -PAGE).The mAb samples were analyzed using precast Novex 4-20%Tris-Glycine gels.Nonreducing sample buffer consisted of 125mM Tris-HCl,pH 6.8,30%(v/v)glycerol,4%(w/v)SDS,and 0.05%(w/v)bromphenol blue.Reducing sample buffer was made by mixing 5vol of 1M dithiothreitol and 95vol of nonreducing buffer.Next,10µL of the mAb solutions at a concentration of 1mg/mL was mixed with equal volume of sample buffer.The mixture was heated at 100°C for 5min for nonreducing SDS -PAGE and 1min for the reducing gel.The gels were run at 150V (constant voltage)for 1.5h.After electrophoresis,the gels were stained in 0.05%(w/v)Coomassie blue and destained using 10%(v/v)acetic acid,15%(v/v)methanol.Measurement of Sulfhydryl.Sulfhydryl content of the mAb’s was measured by a fluorescent assay.Free sulfhydryl was derivatized by adding 10µL of 2mM NPM in DMF to the following three mAb sample prepara-tions:100µL of mAb solution diluted with 500µL of phosphate-buffered saline (PBS),pH 7.3;100µL of mAb solution diluted with 500µL of 6M guanidine hydro-chloride (GuHCl)in PBS buffer,pH 7.3;100µL of mAb solution diluted with 500µL of 6M GuHCl in PBS,pH 7.3,incubated under argon at 37°C for 2h.After theNPM solution was added,the mixtures were incubated at room temperature for 5min and quenched with 10µL of 50%acetic acid.A set of Ac-Cys standards in PBS,pH 7.3with the concentration ranging from 0.25to 25µM,and a PBS,pH 7.3blank were also prepared by the above procedure.Fluorescence measurement was performed on a Waters model 474scanning fluorescence detector equipped with a cuvette holder.This setting allows measurement of the fluorescence of a sample without using a chromatographic system and therefore simplifies sample handling.A quartz cuvette with 1.0-cm path length was used for fluorescence measurement.The excitation wavelength was set at 330nm,and the emission wavelength was set at 380nm.All samples were analyzed in triplicate.The measured sulfhydryl content was expressed as mole of sulfhydryl per mole of mAb.ResultsSEC -HPLC.Under the native chromatographic con-ditions in this work,all four mAb’s eluted between 10and 11min as a single peak (Figure 2),consistent with a molecular weight of ca.150kDa.The observed level of high molecular weight species (earlier eluting material)was below 1%for these mAb’s.No low molecular weight protein species were detected (the peak at ∼15min was related to salts in the buffer).These results confirmed the identities of these recombinant mAb’s,and they appeared to be homogeneous under the native SEC chromatographic conditions.SDS -PAGE.The four mAb’s had similar banding patterns in both reducing and nonreducing SDS -PAGE (Figure 3).Under reducing conditions,predominantly a single HC band migrating at approximately 50kDa and a LC band migrating at approximately 25kDa were observed (Figure 3A).The band below the LC of the IgG4mAb (Figure 3A,lane 6)was the result of deamidation-related peptide bond cleavage,as identified by electro-blotting and Edman sequencing (data not shown).Under nonreducing conditions,all four mAb’s migrated pre-dominantly as a single band of approximately 150kDa,corresponding to a full mAb molecule consisted of 2HC and 2LC (Figure 3B).However,an additional array of minor bands was also present with apparent molecular weights lower than 150kDa (Figure 3B).Peptidemap-Figure 1.Schematic drawing of IgG antibody.Each mAb consists of two HCs and two LCs covalently linked by interchain disulfide bonds.In IgG1and IgG4antibodies,two disulfide bonds are in the hinge region connecting the two HCs,whereas in IgG2antibody four disulfide bonds are in the hinge region.In addition,Cys213of the LC forms inter-HC and LC disulfide bond with Cys217in IgG1,or Cys123in IgG2and IgG4.In all IgG antibodies,each -barrel domain of the HC and LC contains one intrachain disulfide linkage.ping of these mAb’s detected very little peptide bond cleavage (data not shown).Electroblotting and subse-quent Edman sequencing of these lower bands suggested that they may be mAb missing one LC,HC dimer,a half molecule (one HC and one LC),single HC and LC (data not shown).The bands migrating around 25kDa were all identified to be LC-related (Figure 3B).It has been reported that proteins containing disulfide bonds bind less SDS than their reduced forms (14).Hence,the formation of the multiple LC bands may be due to incomplete reduction of the -barrel disulfide bonds in the LC (15).Under the denaturing SDS -PAGE condi-tions,mAb minor species were resolved,while under the native SEC -HPLC conditions,the mAb’s eluted as one peak.Therefore,these minor species are most likely present in solution as noncovalently associated mAb molecules,or partially linked via disulfide bond.Measurement of Sulfhydryl.To elucidate the origin of these mAb minor species under denaturing conditions,free sulfhydryl content was measured.Though Ellman’s assay has been used for quantification of protein sulfhy-dryl,it lacks adequate sensitivity for sulfhydryl content below the low micromolar range (16).Therefore,the sulfhydryl level in the mAb’s was assessed by a fluores-cent assay.NPM was used as the fluorescent probe because of its specificity,sensitivity,and ease of use (17).In particular,NPM is essentially nonfluorescent until sulfhydryl conjugated,which allows measurement of the fluorescence of the sample without prior reagent separa-tion (17).The fluorescence emission intensities of a series of Ac-Cys standards derivatized by NPM were used to establish a standard curve of fluorescence vs sulfhydryl concentration.The standard curve was linear from 0.25to 25µM with a R 2value of 0.9999(Figure 4)and was then used to determine the sulfhydryl content in the mAb samples.The results were summarized in Figure 5.When measured under nondenaturing conditions in PBS,less than 0.02mol of free sulfhydryl was detected per mole of the IgG2or IgG4mAb,while the IgG1mAb was found to contain approximately 0.03mol of sulfhydryl.To access the potentially buried sulfhydryl,the mAb’s were dena-tured with 5M GuHCl and then modified with NPM.In all cases,sulfhydryl level increased to nearly 0.08mol/mol of protein,3-4times higher than that in the native state.In a third set of experiments,the mAb samples in 5M GuHCl were incubated at 37°C for 2h.The two IgG2mAb’s were found to contain approximately 0.1mol of sulfhydryl per mol of protein,while the IgG1and IgG4mAb’s contained approximately 0.09sulfhydryl per pro-tein molecule.These results indicated that all mAb’s analyzed have similar levels of sulhydryl with the major-ity not accessible in the native state of protein.The increased sulfhydryl levels under denaturingconditionsFigure 2.SEC -HPLC profiles of recombinant mAb’s.(A)IgG1mAb.(B)IgG2-I mAb.(C)IgG2-II mAb.(D)IgG4mAb.Figure 3.SDS -PAGE profiles of recombinant mAb’s under (A)reducing and (B)nonreducing ne 1,molecular weight marker;lane 2,blank;lane 3,IgG1mAb;lane 4,IgG2-I mAb;lane 5,IgG2-II mAb;lane 6,IgG4mAb.also implied the presence of noncovalently associated mAb molecules in solution.DiscussionThe sulfhydryl group of Cys is potentially the most reactive nucleophile of all the functional side chains in proteins.It can exist in the reduced free state or the oxidized state to form disulfide bond.The state of sulfhydryl is often important to the structure and func-tion of a protein (18).Therefore,an assessment of the presence and state of sulfhydryl groups in recombinant protein pharmaceuticals allows an evaluation of their structural and functional integrity.In IgG antibodies,all Cys residues form inter-or intramolecular disulfide bonds,and therefore no free sulfhydryl should exist (Figure 1).However,in the present study,all four recombinant IgG mAb’s produced in CHO cells were found to contain free sulfhydryl groups,and the measured level was elevated upon denaturation.This implied that some Cys residues in these mAb’s stay in the reduced state and do not form either inter-or intrachain disulfide bond.The elevated accessibility of free sulfhydryl under denaturing condi-tions suggested that the majority of these sulfhydryls may reside within a solvent-inaccessible region.This result,combined with those from SEC -HPLC and SDS -PAGE,indicated the presence of noncovalently associated mAb molecules under native conditions.The noncovalent interchain interactions are known to be strong enough to maintain the four-chain structure of IgG antibodies,as reduced and alkylated IgG1did not measurably dissociate under native conditions (1,2).It has been shown that a chimeric IgG1mAb in which the hinge Cys residues were mutated to Ser was able to maintain thefour-chain structure in solution but was found to be predominantly of the half molecule form on nonreducing SDS -PAGE gels (19).Therefore,in the present study,the noncovalently associated species were undetectable under native SEC -HPLC conditions and were only revealed under denaturing conditions,such as SDS -PAGE and in GuHCl solution.The presence of free sulfhydryl groups in these mAb’s implied incomplete formation of disulfide bonds among the detected mAb fragments,which is required for covalent assembly of the four-chain folding of IgG anti-bodies.In CHO and other eukaryotic cells,disulfide bond formation and protein folding proceed within the lumen of the endoplasmic reticulum (ER),where protein oxida-tion takes place upon translocation of nascent peptides into the ER lumen (20).For antibodies,disulfide bond formation is additionally complicated with the balanced transcription of two different genes and the assembly of four polypeptide chains.It has been found that assembly of LC and HC to a complete antibody molecule is the primary rate-limiting step in antibody production and can be speeded up by a high expression rate of LC (21).Therefore,it is likely that the incomplete disulfide bond formation of these mAb’s occurs in vivo in the ER.It is worth pointing out that during the production of the mAb’s analyzed in the present study,low concentrations of both cystine and cysteine were used in the cell culture media,so it is also possible that formation of these hypodisulfide-bonded species occurs in vitro after the mAb’s are secreted into the culture media.However,during the production of recombinant murine interleukin 12in CHO cells using culture media containing cystine and cysteine,a glutathione adduct was found,indicating the incomplete disulfide bond formation in vivo (22).Though the origin of the incomplete disulfide bond formation of these mAb’s is unknown at this time,the presence of free sulfhydryl and noncovalently associated species may be undesirable.Also,accumulation of mis/unfolded proteins has been reported to cause ER stress (23,24)and,subsequently,apoptosis and loss of cell viability (25-27).Moreover,the long-term effect of these undisulfide-bonded mAb species on the stability and biological activity remains to be determined.ConclusionsWe have measured the free sulfhydryl content in recombinant mAb’s by a fluorescent assay.The detected sulfhydryl level was approximately 0.02and 0.1mol/mol of protein under native and denaturing conditions,respectively.The mAb’s appeared to be homogeneous as assessed by SEC -HPLC and reducing SDS -PAGE.However,in addition to the bands corresponding to the intact mAb’s,an array of minor binds was observed on nonreducing SDS -PAGE gels.These results suggest that noncovalently associated species are present in these mAb’s and that these mAb fragments may result from incomplete disulfide bond formation between the HC and LC.The formation of these undisulfide-bonded mAb fragments may occur during the protein folding process in the ER and/or the cell culture process.The long-term effects of these free sulfhydryl-containing minor species on the stability and potency of these mAb’s require further investigation.The fluorescent assay for assess-ment of free sulfhydryl is sensitive and quantitative and is applicable to production monitoring and structural analysis of other recombinant protein and mAb pharma-ceuticals.Figure 4.Ac-Cys fluorescence standard curve.The fluorescence was measured after derivatization with NPM.The excitation and emission wavelengths were set at 330and 380nm,respectively.The curve was linear in the range of 0.25-25µM.Figure 5.Free sulfhydryl content in recombinant mAb’s.Experimental conditions are described in Materials and Meth-ods.AcknowledgmentWe would like to thank Dr.Hubert Scoble for his support on this project and Drs.Mark Hardy,Yin Luo, and Lisa Marzilli for their critical review of the manu-script.We are grateful to Philip Boyle and Jeff Banas for their technical assistance.References and Notes(1)Dorrington,K.J.The structural basis for the functionalversativility of immunoglobulin G.Can.J.Biochem.1974, 56,1087-1101.(2)Burton,D.R.Immunoglobulin G:functional sites.Mol.Immunol.1985,22,161-206.(3)Cacia,J.;Keck,R.;Presta,L.G.;Frenz,J.Isomerization ofan aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE:identifica-tion and effect on binding affinity.Biochemistry1996,35, 1897-1903.(4)Perkins,M.;Theiler,R.;Lunte,S.;Jeschke,M.Determina-tion of the origin of charge heterogeneity in a murine monoclonal antibody.Pharm.Res.2000,17,1110-1117. (5)Harris,R.J.;Kabakoff,B.;Macchi,F.D.;Shen,F.J.;Kwong,M.J.;Andya,D.S.;Shire,J.;Bjork,N.;Totpal,K.;Chen,A.B.Identification of multiple sources of charge heterogeneityin a recombinant antibody.J.Chromatogr.B2001,752,233-245.(6)Harris,R.J.Processing of C-terminal lysine and arginineresidues of proteins isolated from mammalian cell culture.J.Chromatogr.A1995,705,233-245.(7)Kroon,D.J.;Baldwin-Ferro,A.;Lalan,P.Identification ofsites of degradation in a therapeutic monoclonal antibody by peptide mapping.Pharm.Res.1992,9,1386-1393.(8)Weitzhandler,M.;Hardy,M.;Co,M.S.;Avdalovic,N.Analysis of carbohydrates on IgG preparations.J.Pharm.Sci.1994,83,1670-1675.(9)Sheeley,D.M.;Merrill,B.M.;Taylor,L.C.Characterizationof monoclonal antibody glycosylation:comparison of expres-sion systems and identification of terminal R-linked galactose.Anal.Biochem.1997,247,102-110.(10)Bihoreau,N.;Ramon,C.;Lazard,M.;Schmitter,J.M.Combination of capillary electrophoresis and matrix-assisted laser desorption ionization mass spectrometry for glycosyl-ation analysis of a human monoclonal anti-Rhesus(D)anti-body.J.Chromatogr.B1997,697,123-33.(11)Harris,R.J.;Murnane,A.A.;Utter,S.L.;Wagner,K.L.;Cox,E.T.;Polastri,G.D.;Helder,J.C.;Sliwkowski,M.B.Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody.Bio/Technology 1993,11,1293-1297.(12)Wan,M.;Shiau,F.Y.;Gordon,W.;Wang,G.Y.Variantantibody identification by peptide mapping.Biotechnol.Bioeng.1999,62,485-488.(13)Kabat,D.J.;Wu,T.T.;Reid-Miller,M.;Perry,H.M.;Gottesman,K.S.Sequences of Proteins of ImmunologicalInterest,4th ed;U.S.Department of Health and Human Services:Washington,DC,1987.(14)Pitt-Rivers,R.;Impiombato,F.S.A.The binding of sodiumdodecyl sulphate to various proteins.Biochem.J.1968,109, 825-830.(15)Kikuchi,H.;Goto,Y.;Hamguchi,K.Reduction of the buriedintrachain disulfide bond of the constant fragment of the immunoglobulin light chain:global unfolding under physi-ological conditions.Biochemistry1986,25,2009-2013. (16)Wright,S.K.;Viola,R.E.Evaluation of methods for thequantitation of cysteines in proteins.Anal.Biochem.1998, 265,8-14.(17)Winters,R.A.;Zukowski,J.;Ercal,N.;Matthews,R.H.;Spitz,D.R.Analysis of glutathione,glutathione disulfide, cysteine,homocysteine,and other biological thiols by high-performance liquid chromatography following derivatization by n-(1-pyrenyl)maleimide.Anal.Biochem.1995,227,14-21.(18)Liu,T.-Y.The role of sulfur in proteins.In The Proteins,3rd ed;Neuvath,H.,Hill,R.L.,Eds.;Academic Press: London,1977;pp329-402.(19)Gillies,S. D.;Wesolowski,J.S.Antigen binding andbiological activities of engineered mutant chimeric antibodies with human tumor specificities.Hum.Antibod.Hybridomas 1991,1,47-54.(20)Frand,A.R.;Cuozzo,J.W.;Kaiser,C.A.Pathways forprotein disulfide bond formation.Trends Cell Biol.2000,10, 203-210.(21)Strutzenberger,K.;Borth,N.;Kunert,R.;Steinfellner,W.;Katinger,H.Changes during subclone development and aging of human antibody-producing recombinant CHO cells.J.Biotechnol.1999,69,215-226.(22)Nickbarg,E.B.;Vath,J.;Pittman,D.D.;Leonard,J.E.;Waldburger,K.E.;Bond,M.Structural characterization of the recombinant P40heavy chain subunit monomer and homodimer of murine IL-12.Bioorg.Chem.1995,23,380-396.(23)Dorner,A.J.;Wasley,l.C.;Raney,P.;Haugejorden,S.;Green,M.;Kaufman,R.J.The stress response in Chinese hamster ovary cells.J.Biol.Chem.1990,265,22029-22034.(24)Kaufman,R.J.Stress singaling from the lumen of theendoplasmic reticulum:coordination of gene transscriptional and translational controls.Genes Dev.1999,13,1211-1233.(25)Al-Rubeai,M.;Singh,R.P.Apoptosis in cell culture.Curr.Opin.Biotechnol.1998,9,152-156.(26)Mastrangelo,A.J.;Betenbaugh,M.J.Overcoming apop-tosis:new methods for improving protein-expression systems.Trends Biotechnol.1998,16,88-95.(27)Murphy,T.C.;Woods,N.R.;Dickson,A.J.Expression ofthe transcription factor GADD153is an indicator of apoptosis for recombinant Chinese hamster ovary(CHO)cells.Biotech-nol.Bioeng.2001,75,621-629.Accepted for publication March5,2002.BP025511Z。

蛋白A(Protein A)酶联检测试剂盒(可拆卸)说明书产品目录编号# AB000109C此试剂盒包含检测生物药物内蛋白A (Protein A) 杂质所需要的全部组分。

请在操作之前仔细阅读此说明书。

本试剂盒只能用于科学研究，不能用于医学诊断。

背景资料在单克隆抗体药物的纯化过程中，以蛋白A（Protein A）为基础的亲合层析是广泛应用的纯化方法之一，单克隆抗体可以有效的在此步骤中得到纯化。

蛋白A 的最初来源是细菌（Staphylococcus aureus）细胞壁中的一个蛋白质，研究表明蛋白A 可能对人体有潜在的危害，它的生物学作用包括刺激人体多种细胞素（IFNγ, TNFα, IL-1α,IL-1β, IL-2, IL-4）的释放。

由于在蛋白A 亲合层析柱使用过程中会有微量的蛋白A 从层析柱上脱落下来，因此用精确灵敏的方法来确证单克隆抗体药物中蛋白A 的残留处于一个较低的和稳定的水平是单克隆抗体药物的质量标准之一。

蛋白A (Protein A) 酶联检测试剂盒是用来定量分析生物药物中蛋白A杂质的试剂盒。

首先，用不同浓度的蛋白A标准蛋白制作一条标准曲线，然后再根据标准曲线来分析生物药物中残留蛋白A的含量。

具体方法为：先将蛋白A标准蛋白或者待测样品适当稀释后加入已包被好抗蛋白A抗体的96孔平板中。

经过1.5小时的保温，蛋白A与已经固定在平板上的抗体结合形成抗原－抗体复合物。

此复合物被随后加入的生物素标记的抗蛋白A的抗体结合并进一步形成更高级的复合物，反应45分钟后洗去未结合的物质。

再加入链霉亲合素-辣根过氧化物酶复合物（Streptavidin-HRP Conjugate），静置30分钟。

因链霉亲合素可以与任何带生物素标记的抗体相结合，从而使链霉亲合素-辣根过氧化物酶复合物连接到平板上。

接下来冲洗掉未结合的物质，加入TMB底物。

固相上的酶催化底物产生颜色反应，待颜色反应一段时间后，终止反应。

用酶标仪在450 nm波长下测定其结果，此结果能间接地反应结合到平板上的蛋白A数量的多少。

腺苷酸激酶5(AK5)ELISA试剂盒说明书本试剂仅供研究使用标本：体液腺苷酸激酶5(AK5)ELISA试剂盒说明书试验原理：BFGF试剂盒是固相夹心法酶联免疫吸附实验（ELISA）.已知BFGF浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。

先将BFGF和生物素标记的抗体同时温育。

人碱性成纤维细胞生长因子ELISA 检测试剂盒洗涤后，加入亲和素标记过的HRP。

再经过温育和洗涤，去除未结合的酶结合物，然后加入底物A、B，和酶结合物同时作用。

产生颜色。

颜色的深浅和样品中BFGF的浓度呈比例关系。

腺苷酸激酶5(AK5)ELISA试剂盒说明书自备材料1．蒸馏水。

2．加样器：5ul、10ul、50ul、100ul、200、500ul、1000ul。

3．振荡器及磁力搅拌器等。

安全性1．避免直接接触终止液和底物A、B。

一旦接触到这些液体，请尽快用水冲洗。

2．实验中不要吃喝、抽烟或使用化妆品。

3．不要用嘴吸取试剂盒里的任何成份。

腺苷酸激酶5(AK5)ELISA试剂盒说明书操作注意事项1．试剂应按标签储存，使用前恢复到室温。

稀稀过后的标准品应丢弃，不可保存。

2．实验中不用的板条应立即放回包装袋中，密封保存，以免变质。

3．不用的其它试剂应包装好或盖好。

不同批号的试剂不要混用。

保质前使用。

4．使用一次性的吸头以免交叉污染，吸取终止液和底物A、B液时，避免使用带金属部分的加样器。

5．使用干净的塑料容器配置洗涤液。

使用前充分混匀试剂盒里的各种成份及样品。

6．洗涤酶标板时应充分拍干，不要将吸水纸直接放入酶标反应孔中吸水。

7．底物A应挥发，避免长时间打开盖子。

底物B对光敏感，避免长时间暴露于光下。

避免用手接触，有毒。

实验完成后应立即读取OD值。

8．加入试剂的顺序应一致，以保证所有反应板孔温育的时间一样。

9．按照中标明的时间、加液的量及顺序进行温育操作。

腺苷酸激酶5(AK5)ELISA试剂盒说明书样品收集、处理及保存方法1、血清-----操作过程中避免任何细胞刺激。

Solutions for Monoclonal Antibody Analysis and CharacterizationSolutions for Your Separation Tasks from Lab to Production Tosoh Bioscience is a leading supplier of chromatographic columns, media and gel permeation chromatography (GPC) instruments with over 500 specialty products to meet your analysis and purification needs.TOYOPEARL® TSKgel & Ca++Pure-HA® ResinsTOYOPEARL and TSKgel chromatography resins are specifically designed for the purification of biomolecules. Ca++Pure-HA is a hydroxyapatite resin and has unique separation properties for biomolecules. These resins show excellent physical strength and ideal flow characteristics for industrial downstream processing.EcoSEC® GPC SystemsFor more info visit: Solutions for Monoclonal Antibody Analysis and CharacterizationTSKgel U/HPLC Columns for BioseparationsWebVisit for videos, product information and ordering.Our technical service staff is ready to answer questions:*************************In PersonA technical seminar can bearranged on-site or via the web. ****************************Protein, monoclonal antibody (mAb) and antibody drug conjugate (ADC) biopharmaceuticals form a major part of thegrowing biologics drug market. During development and production of these products, it is essential to detect, characterize and quantify impurities as well as structural variants and modifications, and to monitor product stability. Figure 1 highlights several approaches to mAb and ADC characterization including size exclusion chromatography (SEC) for aggregate andfragment analysis, SEC/mass spectrometry (MS) for accurate mass determination, hydrophobic interaction chromatography (HIC) for ADC drug-to-antibody ratio (DAR) analysis, cation exchange (CEX) for charge variant analysis, and affinity chromatography (Protein A) for titer analysis.and consistent results.1LC and LC/MS protein analytical chemistry methods for testing biological productsCall customer service: 866-527-3587, technical service: 800-366-4875, option #3harvested cell culture media can be captured and accuratelyFigure 3 demonstrates the high durability and wide dynamic load range of the TSKgel Protein A-5PW column prior to and after 2,009 injections without being cleaned.TSKgel Protein A-5PW column has code-modified hexamer of the C domain which enables (Figure 4).20.20.7 1.2 1.7 2.2Human IgG 10.20.7 1.2 1.7 2.2Human IgG 20.20.7 1.2Retention time (minutes)Retention time (minutes)Retention time (minutes)Retention time (minutes)1.72.2Human IgG 40.20.71.2 1.72.2Mouse IgGmAUmAUmAUmAUFast capture of various lgG subclasses using 05001,0001,5002,0002,500050100150200250P e a k a r e aSample load (µg)Durability and dynamic range of TSKgel Protein A-5PW columnFor more info visit: 3maintaining resolution in the range acceptable by USP guidelines.Comparison of mAb aggregate analysis between TSKgelCall customer service: 866-527-3587, technical service: 800-366-4875, option #3of mAb A on both columns at pH 7. For this IgG, theAs shown in Figure 8, this is not the case for mAb B,where the strong cation exchange column shows a better separation. Retention and resolution of the charged isoforms are dependent on the buffer pH, as can be seen in Figure 9 for the analysis of mAb B on TSKgel SP-STAT .Weak and strong cation exchange columns provide differentselectivities for the analysis of charge heterogeneity of proteins. In order to reach the best separation of acidic and basic isoforms from the main peak, both types should be evaluated at various pH values of the mobile phase during method development. TSKgel STAT series columns provide a high resolution of isoforms in a short analysis time and are ideally suited for the QC of biotherapeutics by UHPLC or HPLC.Columns: TSKgel SP-STAT, 7 µm, 4.6 mm ID × 10 cmTSKgel CM-STAT, 7 µm, 4.6 mm ID × 10 cm Mobile phase: A: 10 mmol/L sodium phosphate buffer, pH 7.0 (Figures 7, 8, 9)10 mmol/L sodium phosphate buffer, pH 6.0 (Figure 9) 10 mmol/L sodium acetate buffer, pH 5.0 (Figure 9) B: 100 mmol/L phosphate, pH 7.0 + 500 mmol/L NaCl (Figures 7, 8, 9)100 mmol/L phosphate, pH 6.0 + 500 mmol/L NaCl (Figure 9)100 mmol/L acetate, pH 5.0 + 500 mmol/L NaCl(Figure 9)Gradient: 0 - 100% B in 30 min Flow rate: 1 mL/min Detection: UV @ 280 nm Injection vol.: 10 µLSamples: mAb A (2 g/L), mAb B (2 g/L)Analysis of mAb B at various pH values using TSKgel SP-STAT5as a major single peak at approximately 9.5 minutes (Figure 10, upper panel). This single peak indicated that the unconjugated Trastuzumab consisted of mostly homogeneous molecules. The profile of the drugconjugated Trastuzumab exhibited well resolved peakswith different retention times than that of the unconjugated drug and with baseline separation (Figure 10, upper panel). As more drug is conjugated to the mAb vehicle, the ADC becomes more hydrophobic and is retained longer by the HIC stationary phase, allowing resolution of the different drug loaded species. The chromatogram shows well resolved peaks ranging from a DAR of 0 to 8 based upon peak mobility (Figure 10, lower panel).Column: TSKgel Butyl-NPR, 4.6 mm ID × 10 cm Mobile phase: A: 25 mmol/L phosphate buffer, pH 7.0 including 1.5 mol/L ammonium sulfate B: 25 mmol/L phosphate buffer, pH 7.0 / 2-propanol = 8 / 2Gradient: 0 100% B (20 minutes)Flow rate: 0.5 mL/min Detection: UV @ 280 nm Injection vol.: 10 µLSample Conc.: Trastuzumab; 0.24 g/L, ADC (Trastuzumab-vcMMAE); 2.2 g/LRetention time (minutes)Call customer service: 866-527-3587, technical service: 800-366-4875, option #3addition, the analysis was completed in half the run time.column is capable of separating Columns: A. TSKgel UP-SW3000, 2 µm, 4.6 mm ID × 30 cmB. SEC column, 5 µm, 7.8 mm ID × 30 cm Instruments: A. UltiMate 3000RS UHPLC System B. Agilent 1260Mobile phase: 0.2 mol/L potassium phosphate/0.25 mol/L KCl, pH 6.2 Flow rate: A. 0.35 mL/min B. 0.5 mL/min D e t e c t o r r e s p o n s e (m A U )Retention time (minutes)D e t e c t o r r e s p o n s e (m A U )Retention time (minutes)mAb analysis using a 5 μm SEC column versus Retention time (minutes)D e t e c t o r r e s p o n s e (m A U )Retention time (minutes)Columns: A. SEC column, 5 µm, 7.8 mm ID × 30 cm × 2 B. TSKgel UP-SW3000, 2 µm, 4.6 mm ID × 30 cm Mobile phase: 100 mmol/L phosphate buffer + 100 mmol/L sodiumsulfate + 0.05% sodium azide, pH 6.7Flow rate: A. 1.0 mL/min B. 0.35 mL/min Detection: UV @ 280 nm Temperature: 25 °C Injection vol.: 10 µL Samples: mouse-human chimeric IgG, monoclonal 1. trimer 2. dimer 3. monomer 4. fragmentglycan structure is demonstrated in Figure 13 using the example of N-glyco mapping of the ZP-domain of murine transforming growth factor beta type3 receptor (TGFR-3). Recombinant proteins were purified and submitted to exoglycosidasecleavage. Glycans were fluorescently labeled with 2aminobenzamid (2-AB), compared to the dextran ladder, and separated by HILIC..CABDEFGradient: 0 - 35 min: 75 - 35% B Flow rate: 0.22 mL/min(A) Dextran ladder, (B) PNGase digest, (C-F) sequential Sialidase A (Abs), α-Fucosidase (Bkf), ß-Galactosidase (Btg),ß-N-Acetylhexoamidase (Guh).ABC(A) Mass spectrum of 2-AB-labeled glycans released fromof m/z 2243, (C) MS2 mass spectrum of M/Z 1930The TSKgel UP-SW3000, 2 μm SEC column can be effectivelyused for accurate mass determination of monoclonal antibodiesand related products using SEC/MS.A bispecific T cell engager (BiTE®) consisting of two single-chain variable fragments (scFvs) recombinantly linked by anonimmunogenic five-amino-acid chain was analyzed by SEC/MS using a TSKgel UP-SW3000, 2 μm column.The ~55 kDa BiTE and parent mAbs (data not shown) weresubsequently injected onto a TSKgel UP-SW3000 columncoupled to a mass spectrometer for molar mass determination.Figure 15 shows the (a) total ion chromatogram, (b) massspectrum and (c) deconvoluted mass spectrum of the BiTE.A main peak can be seen at m/z 54,143; adjacent peaks atm/z 54,181, 54,219 and 54,086 correspond to different saltadducts.Prior to analysis, a blank injection was run in order to assesscolumn particle shedding. Figure 16a shows the total ionchromatogram of a blank injection. MS data indicates thatthere is no shedding from the TSKgel UP-SW3000 columnprior to sample injection. Additionally, a blank injectionwas run between each of the sample injections in order tomonitor sample carryover. Figure 16b shows the total ionchromatogram of a blank injection run between the BiTE andparent mAb showing no evidence of carryover. The lack ofshedding and carryover indicate that the TSKgel UP-SW3000column is suitable for use with MS.Column: TSKgel UP-SW3000, 2 µm, 4.6 mm ID × 30 cmFlow rate: 0.35 mL/minparent mAb shown, 0.5 mg/mL (Creative Biolabs)Ionization mode: Electrospray ionization, positive modeSEC/MS analysis of the BiTETSKgel, TOYOPEARL, Ca ++Pure-HA, and Tosoh Bioscience are registered trademarks of Tosoh Corporation BiTE is a registered trademark of Amgen Inc. Corporation Prominence and Nexera are registered trademarks of Shimadzu Corporation Q Exactive is a trademark of Thermo Fisher Scientific Inc.UltiMate is a registered trademark of the Dionex Corporation*The guard column can be directly connected to the analytical column without tubing between the two columns.A male-type outlet endfitting on the guard column enables the direct connection to the screw-type endfitting of the analytical column.**Columns are available with 2, 3, 5 and 10 μm particles and a wide range of column geometriesAggregate Analysis, Intact mAb and Fragment Analysis, Accurate Mass Determination。

胃蛋白酶水解大豆分离蛋白的研究庞美蓉;丁秀臻;孔祥珍;华欲飞【摘要】采用胃蛋白酶对大豆分离蛋白进行酶法水解，随着酶解时间的延长，蛋白质的水解度逐渐增大，水解6h，DH为7．90％。

采用分子筛凝胶过滤色谱（SE—HPLC）和十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）对不同的酶解时间下大豆分离蛋白酶解物的分子结构进行表征，结果表明，胃蛋白酶选择性地酶解大豆11s球蛋白。

大豆分离蛋白经胃蛋白酶水解6h后，分子量大于10ku的部分占21．34％，其中主要是7S球蛋白；分子量小于5ku的部分所占比例为68．30％。

对酶解物中的游离巯基和二硫键含量测定结果表明，随着酶解的进行，游离巯基的含量呈现先增大后减小的趋势，二硫键的含量总体变化不大。

该研究旨在更好地了解胃蛋白酶水解大豆分离蛋白的机理，并为开发大豆分离蛋白酶解物产品提供理论依据。

%Soy protein extract was enzymatically hydrolyzed with Pepsin. With the increase of soy protein hydrol- ysis, the degree of hydrolysis values varied from 0% to 7.90% after 360 min of incubation. The molecular weight dis- tribution of soy protein hydrolysates prepared with Pepsin was determined by SE-HPLC and SDS-PAGE. The results showed that soy glyeinin （ 11 S） was selectively hydrolyzed by Pepsin. The molecular weight distribution of the hydrol- ysates obtained after 6 h of incubation were as follows ： 21.34 % were above 10 ku and 68.30 % were below 5 kDa. Soy conglycinin played a big role on the MW above 10 kDa. With the hydrolysis, free sulfhydryl content first in- creased and then decreased, while the content of disulfide bonds had no obvious change. The results herein were help- ful for understanding themechanism of soy protein hydrolysis with Pepsin, and could serve as a guide for the develop- ment of soy protein hydrolysate products.【期刊名称】《食品与发酵工业》【年(卷),期】2012(038)007【总页数】5页(P103-107)【关键词】大豆蛋白;胃蛋白酶;水解;分子量【作者】庞美蓉;丁秀臻;孔祥珍;华欲飞【作者单位】江南大学食品科学与技术国家重点实验室,食品学院,江苏无锡214122;江南大学食品科学与技术国家重点实验室,食品学院,江苏无锡214122;江南大学食品科学与技术国家重点实验室,食品学院,江苏无锡214122;江南大学食品科学与技术国家重点实验室,食品学院,江苏无锡214122【正文语种】中文【中图分类】TS214.2大豆蛋白中90%的蛋白质以储藏蛋白的形式存在，主要分为大豆球蛋白(glycinin)和β-伴大豆球蛋白(β-conglycinin)[1］。

protein a磁珠结合抗体的洗脱方式全文共四篇示例，供读者参考第一篇示例：蛋白A磁珠（Protein A Magnetic Beads）是一种常用的蛋白结合亲和层析实验工具，能够快速、高效地富集含有His标签的蛋白。

在分子生物学研究中，蛋白A磁珠通常与抗体一起使用，用于特异性地富集目标蛋白。

蛋白A磁珠结合抗体的洗脱过程在实验中起着至关重要的作用，直接影响到后续蛋白的纯化和分析结果。

正确的洗脱方式可以保证蛋白的完整性和活性，提高实验的准确性和灵敏度。

本文将介绍一些常用的蛋白A磁珠结合抗体的洗脱方式，希望能为相关实验提供参考。

一、低pH洗脱法低pH洗脱法是最常用的蛋白A磁珠结合抗体的洗脱方式之一。

在此方法中，可以使用含有低pH值的缓冲液（如乙酸、乙酸钠等）将蛋白A磁珠上的抗体洗脱下来。

一般来说，将蛋白A磁珠悬浮于适当的低pH缓冲液中，并进行轻轻的振荡混匀，使抗体与磁珠分离。

需要注意的是，在低pH条件下，蛋白的稳定性可能会受到影响，部分蛋白可能会失去活性或变性。

在选择低pH洗脱法时，需要针对不同的蛋白和抗体进行优化，并在实验过程中及时监测洗脱效果，以确保不影响实验结果。

二、高盐洗脱法高盐洗脱法的优点在于操作简单、温和，对大部分蛋白的活性影响较小。

但需要注意的是，过高浓度的盐类可能会影响实验后续的应用，因此在选择高盐洗脱法时，需要针对具体实验要求进行优化。

三、竞争性洗脱法竞争性洗脱法是一种新兴的蛋白A磁珠结合抗体的洗脱方式，相对于传统的洗脱方法，竞争性洗脱法更具选择性和特异性。

在此方法中，可以添加特定的抗体蛋白，使其与蛋白A磁珠上的抗体发生竞争结合，并最终将抗体洗脱下来。

竞争性洗脱法的优势在于能够实现对目标抗体的特异性洗脱，减少非特异性结合和杂质的干扰。

但也需要注意，选择合适的竞争性抗体和条件是关键，需要在实验中进行多次优化以获得最佳的洗脱效果。

蛋白A磁珠结合抗体的洗脱方式对实验结果具有重要影响，选择合适的洗脱方法能够有效保证蛋白的完整性和活性。

美国药典（USP）中规定的色谱柱类型Chromatographic ReagentsThe following list of packings (L), phases (G), and supports (S) is intended to be a convenient reference for the chromatographer. [note—Particle sizes given in this listing are those generally provided. Where other, usually finer, sizes are required, the individual monograph specifies the desired particle size. Within any category of packings or phases listed below, there may be a wide range of columns available. Where it is necessary to define more specifically the chromatographic conditions, the individual monograph so indicates.]Change to read:PackingsL1—Octadecyl silane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 &micro;m in diameter, or a monolithic silica rod.L2—Octadecyl silane chemically bonded to silica gel of a controlled surface porosity that has been bonded to a solid spherical core, 30 to 50 &micro;m in diameter.L3—Porous silica particles, 3 USP31 to 10 &micro;m in diameter, or a monolithic silica rod. USP31L4—Silica gel of controlled surface porosity bonded to a solid spherical core, 30 to 50&micro;m in diameter.L5—Alumina of controlled surface porosity bonded to a solid spherical core, 30 to 50 &micro;m in diameter.L6—Strong cation-exchange packing–sulfonated fluorocarbon polymer coated on a solid spherical core, 30 to 50 &micro;m in diameter.L7—Octylsilane chemically bonded to totally porous silica particles, 1.5 to 10 &micro;m in diameter, or a monolithic silica rod. USP31L8—An essentially monomolecular layer of aminopropylsilane chemically bonded to totally porous silica gel support, 3 to 10 &micro;m in diameter.L9—Irregular or spherical, totally porous silica gel having a chemically bonded, strongly acidic cation-exchange coating, 3 to 10 &micro;m in diameter.L10—Nitrile groups chemically bonded to porous silica particles, 3 to 10 &micro;m in diameter.L11—Phenyl groups chemically bonded to porous silica particles, 1.5 to 10 &micro;m in diameter.L12—A strong anion-exchange packing made by chemically bonding a quaternary amine to a solid silica spherical core, 30 to 50 &micro;m in diameter.L13—Trimethylsilane chemically bonded to porous silica particles, 3 to 10 &micro;m in diameter.L14—Silica gel having a chemically bonded, strongly basic quaternary ammoniumanion-exchange coating, 5 to 10 &micro;m in diameter.L15—Hexylsilane chemically bonded to totally porous silica particles, 3 to 10 &micro;m in diameter.L16—Dimethylsilane chemically bonded to porous silica particles, 5 to 10 &micro;m in diameter.L17—Strong cation-exchange resin consisting of sulfonated cross-linkedstyrene-divinylbenzene copolymer in the hydrogen form, 7 to 11 &micro;m in diameter.L18—Amino and cyano groups chemically bonded to porous silica particles, 3 to 10 &micro;m in diameter.L19—Strong cation-exchange resin consisting of sulfonated cross-linkedstyrene-divinylbenzene copolymer in the calcium form, about 9 &micro;m in diameter.L20—Dihydroxypropane groups chemically bonded to porous silica particles, 5 to 10 &micro;m in diameter.L21—A rigid, spherical styrene-divinylbenzene copolymer, 5 to 10 &micro;m in diameter.L22—A cation-exchange resin made of porous polystyrene gel with sulfonic acid groups, about 10 &micro;m in size.L23—An anion-exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary ammonium groups, about 10 &micro;m in size.L24—A semi-rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the matrix surface, 32 to 63 &micro;m in diameter.[note—Available as YMC-Pack PVA-SIL manufactured by YMC Co., Ltd. and distributed by Waters Corp. ().]L25—Packing having the capacity to separate compounds with a molecular weight range from 100–5000 (as determined by polyethylene oxide), applied to neutral, anionic, and cationicwater-soluble polymers. A polymethacrylate resin base, cross-linked with polyhydroxylated ether (surface contained some residual carboxyl functional groups) was found suitable.L26—Butyl silane chemically bonded to totally porous silica particles, 3 to 10 &micro;m in diameter.L27—Porous silica particles, 30 to 50 &micro;m in diameter.L28—A multifunctional support, which consists of a high purity, 100 , spherical silica substrate that has been bonded with anionic exchanger, amine functionality in addition to a conventional reversed phase C8 functionality.L29—Gamma alumina, reverse-phase, low carbon percentage by weight, alumina-based polybutadiene spherical particles, 5 &micro;m in diameter with a pore volume of 80 .L30—Ethyl silane chemically bonded to totally porous silica particles, 3 to 10 &micro;m in diameter.L31—A hydroxide-selective, strong anion-exchange resin-quaternary amine bonded on latex particles attached to a core of 8.5-&micro;m macroporous particles having a pore size of 2000 and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene.L32—A chiral ligand-exchange packing–l-proline copper complex covalently bonded to irregularly shaped silica particles, 5 to 10 &micro;m in diameter.L33—Packing having the capacity to separate dextrans by molecular size over a range of4,000 to 500,000 Da. It is spherical, silica-based, and processed to provide pH stability.[note—Available as TSKgel G4000 SWXL from Tosoh Biosep ().]L34—Strong cation-exchange resin consisting of sulfonated cross-linkedstyrene-divinylbenzene copolymer in the lead form, about 9 &micro;m in diameter.L35—A zirconium-stabilized spherical silica packing with a hydrophilic (diol-type) molecular monolayer bonded phase having a pore size of 150 .L36—A 3,5-dinitrobenzoyl derivative of l-phenylglycine covalently bonded to 5-&micro;m aminopropyl silica.L37—Packing having the capacity to separate proteins by molecular size over a range of2,000 to 40,000 Da. It is a polymethacrylate gel.L38—A methacrylate-based size-exclusion packing for water-soluble samples.L39—A hydrophilic polyhydroxymethacrylate gel of totally porous spherical resin.L40—Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica particles, 5 to 20&micro;m in diameter.L41—Immobilized 1-acid glycoprotein on spherical silica particles, 5 &micro;m in diameter.L42—Octylsilane and octadecylsilane groups chemically bonded to porous silica particles, 5 &micro;m in diameter.L43—Pentafluorophenyl groups chemically bonded to silica particles by a propyl spacer, 5 to 10 &micro;m in diameter.L44—A multifunctional support, which consists of a high purity, 60 , spherical silica substrate that has been bonded with a cationic exchanger, sulfonic acid functionality in addition to a conventional reversed phase C8 functionality.L45—Beta cyclodextrin bonded to porous silica particles, 5 to 10 &micro;m in diameter.L46—Polystyrene/divinylbenzene substrate agglomerated with quaternary amine functionalized latex beads, about 10 &micro;m in diameter.L47—High-capacity anion-exchange microporous substrate, fully functionalized with trimethlyamine groups, 8 &micro;m in diameter.[note—Available as CarboPac MA1 and distributed by Dionex Corp. ().]L48—Sulfonated, cross-linked polystyrene with an outer layer of submicron, porous,anion-exchange microbeads, 15 &micro;m in diameter.L49—A reversed-phase packing made by coating a thin layer of polybutadiene onto spherical porous zirconia particles, 3 to 10 &micro;m in diameter.[note—Available as Zirchrom PBD, manufactured by ZirChrom Separations, Inc., distributed by Alltech, .]L50—Multifunction resin with reversed-phase retention and strong anion-exchange functionalities. The resin consists of ethylvinylbenzene, 55% cross-linked with divinylbenzene copolymer, 3 to 15 &micro;m in diameter, and a surface area not less than 350 m2 per g. Substrate is coated with quaternary ammonium functionalized latex particles consisting of styrene cross-linked with divinylbenzene.[note—Available as OmniPac PAX-500 and distributed by Dionex Corp. ().]L51—Amylose tris-3,5-dimethylphenylcarbamate-coated, porous, spherical, silica particles, 5 to 10 &micro;m in diameter.[note—Available as Chiralpak AD from Chiral Technologies, Inc., ().]L52—A strong cation-exchange resin made of porous silica with sulfopropyl groups, 5 to 10&micro;m in diameter.[note—Available as TSK IC SW Cation from Tosoh Biosep ().]L53—Weak cation-exchange resin consisting of ethylvinylbenzene, 55% cross-linked with divinylbenzene copolymer, 3 to 15 &micro;m diameter. Substrate is surface grafted with carboxylic acid and/or phosphoric acid functionalized monomers. Capacity not less than 500 &micro;Eq/column.[note—Available as IonPac CS14 distributed by Dionex Corp. ().]L54—A size exclusion medium made of covalent bonding of dextran to highly cross-linked porous agarose beads, about 13 &micro;m in diameter.[note—Available as Superdex Peptide HR 10/30 from Amersham Pharmacia Biotech ().]L55—A strong cation-exchange resin made of porous silica coated with polybutadiene–maleic acid copolymer, about 5 &micro;m in diameter.[note—Available as IC-Pak C M/D from Waters Corp. ().]L56—Propyl silane chemically bonded to totally porous silica particles, 3 to 10 &micro;m in diameter.[note—Available as Zorbax SB-C3 from Agilent Technologies (/chem).]L57—A chiral-recognition protein, ovomucoid, chemically bonded to silica particles, about 5&micro;m in diameter, with a pore size of 120 .[note—Available as Ultron ES-OVM from Agilent Technologies (/chem).]L58—Strong cation-exchange resin consisting of sulfonated cross-linkedstyrene-divinylbenzene copolymer in the sodium form, about 6 to 30 &micro;m 1S (USP31) in diameter.[note—Available as Aminex HPX-87N from Bio-Rad Laboratories, (2000/01 catalog,#125-0143) .]L59—Packing having the capacity to separate proteins by molecular weight over the range of 10 to 500 kDa. It is spherical (10 &micro;m), silica-based, and processed to provide hydrophilic characteristics and pH stability.[note—Available as TSKgel G3000SW Column (analytical column) and TSKgel Guard (guard column) from Tosoh Biosep (part numbers 05789 and 05371, respectively)().]L60—Spherical, porous silica gel, 10 &micro;m or less in diameter, the surface of which has been covalently modified with alkyl amide groups and endcapped.[note—Available as Supelcosil ABZ from Supelco (/supelco).]L61—A hydroxide selective strong anion-exchange resin consisting of a highly cross-linked core of 13 &micro;m microporous particles having a pore size less than 10 units and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene with a latex coating composed of 85 nm diameter microbeads bonded with alkanol quaternary ammonium ions (6%).[note—Available as Ion Pac AS-11 and AG-11 from Dionex ().]L62—C30 silane bonded phase on a fully porous spherical silica, 3 to 15 &micro;m in diameter.L63— Glycopeptide teicoplanin linked through multiple covalent bonds to a 100- units spherical silica.[Note—Available as Chirobiotic T from Astec ().] 1S (USP31)美国药典(USP)规定的色谱柱编号见下面，是对应的色谱柱类型。

Chin J Hemorh.2020;230(3)326收稿日期：2020-07-14 作者简介：韩俊霞(1987—),女,山东菏泽人,硕士,主治医师,研究方向:脂肪细胞因子与2型糖尿病及其并发症的相关性。

通讯作者：王秋萍，E-mail ：****************doi:10.3969/j.issn.1009-881X.2020.03.015血清CTRP12水平与2型糖尿病和肥胖的相关性研究韩俊霞1，王秋萍2，成兴波1（1.苏州大学附属第一医院内分泌科，江苏 苏州 215006；2.苏州大学附属第一医院内分泌科实验室，江苏 苏州 215006）摘要：目的 探讨血清补体C1肿瘤坏死因子相关蛋白12（CTRP12）水平与2型糖尿病（T2DM ）和肥胖的相关性及临床意义。

方法 将56 例初诊T2DM 患者分为肥胖组（A 组）和非肥胖组（B 组），52 名正常对照组分为肥胖组（C 组）和非肥胖组（D 组），测量身高、体重、血压、腰围、臀围，测定空腹血糖（FBG ）、糖化血红蛋白（HbA1c ）、总胆固醇（TC ）、甘油三酯（TG ）、高密度脂蛋白胆固醇（HDL-C ）及低密度脂蛋白胆固醇（LDL-C ），计算体重指数（BMI ）、腰臀比（WHR ）。

结果 糖尿病组血清CTRP12水平显著低于正常对照组，正常肥胖组显著低于正常非肥胖组（P ＜0.01）。

相关分析显示：CTRP12与腰围、WHR 、FBG 和HbA1c 呈负相关（P ＜0.05），与INS 呈正相关（P ＜0.05），TG 、BMI 、HbA1c 为血清CTRP12的独立危险因素（P ＜0.05）。

结论 血清CTRP12为T2DM 的保护因素，血清CTRP12水平的降低可能预测T2DM 的发生和发展。

关键词：CTRP12；肥胖；2型糖尿病中图分类号：R587.1；R589.2 文献标识码：A 文章编号：1009-881X(2020)03-0326-03Association Between Serum CTRP12 and Type 2 Diabetes Mellitus with ObesityHAN Jun-xia, WANG Qiu-ping, CHENG Xing-bo(Department of Endocrinology, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006,China)Abstract ：Objective To evaluate the association between serum CTRP12 and type 2 diabetes mellitus (T2DM) with obesity. Methods 56 newly diagnosed patients with T2DM were divided into obese group (group A) and lean group (group B). 52 subjects with normal glucose tolerance were divided into obese group (group C) and lean group (group D). Height, weight, blood pressure, waist circumference (WC), hip circumference (HP), fasting blood glucose (FBG), HbA1c, total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were determined in all subjects. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated. Results Compared with group C + D, the levels of CTRP12 in group A + B were lower. Compared with group D, the levels of CTRP12 in group C were lower (P ＜0.05). Simple correlation analysis showed that CTRP12 was positively correlated with INS (P ＜0.05), but negative with WC, WHR, FBG and HbA1c (P ＜0.05). Furthermore, multiple regression analysis showed that TG, BMI and HbA1c were the independent factors of serum CTRP12 level (P ＜0.05). Conclusion serum CTRP12 is a protective factor for T2DM. The decrease of serum CTRP12 level may predict the occurrence and development of T2DM.Key words ：CTRP12; obesity; T2DM近年来我国2型糖尿病（T2DM ）发病率逐年增加，发病年龄呈年轻化趋势，其发病机制为胰岛素抵抗及分泌缺陷，病因极其复杂。

ATG5分子量1. 介绍ATG5（Autophagy-related protein 5）是一种参与自噬过程的关键蛋白质。

自噬是一种细胞内的降解过程，通过将细胞内的有害物质、损坏的细胞器和蛋白质降解并回收，维持细胞的稳态和生存能力。

ATG5作为自噬过程中的重要分子，发挥着调控自噬的重要作用。

2. ATG5的结构和功能2.1 结构ATG5是一种约275个氨基酸残基的蛋白质，它由多个结构域组成。

其中，ATG5蛋白质的N端包含ATG12结合位点，C端含有ATG7结合位点。

ATG5还包含一个共轭位点，用于与ATG12蛋白质发生共轭反应。

2.2 功能ATG5主要通过与ATG12蛋白质发生共轭反应，形成ATG5-ATG12复合物，参与自噬的调控。

ATG5-ATG12复合物与另一个复合物ATG16形成ATG5-ATG12-ATG16L1三聚体，进一步参与自噬小体的形成和自噬过程的进行。

3. ATG5在自噬过程中的作用自噬是一个高度调控的过程，ATG5在其中扮演着重要的角色。

以下是ATG5在自噬过程中的几个关键作用：3.1 自噬小体的形成ATG5-ATG12-ATG16L1复合物是自噬小体的关键组分之一。

它能够通过与其他自噬相关蛋白相互作用，促进自噬小体的形成。

自噬小体是自噬过程中的主要结构，它能够包裹并降解细胞内的有害物质和损坏的细胞器。

3.2 自噬的调控ATG5-ATG12复合物能够与其他ATG家族蛋白相互作用，调控自噬的进行。

例如，ATG5-ATG12复合物可以与ATG16L1相互作用，促进自噬小体的形成。

它还能够与ATG3、ATG7等蛋白相互作用，参与自噬相关蛋白的修饰和调控。

3.3 自噬的选择性ATG5还参与了自噬的选择性过程，即通过特定的信号途径选择性地降解细胞内的特定蛋白质或细胞器。

ATG5能够与其他选择性自噬相关蛋白相互作用，调控特定物质的降解。

4. ATG5分子量的测定方法测定ATG5的分子量是研究其结构和功能的重要一步。