CD73-IN-1-HNMR-28411-MedChemExpress

Hirsch Teil1. What are chemical sensors?- Definition !!!2. Selectivity- Definition- Equilibrium based selectivity: free energy, dielectric constant and distance,- Kinetic based selectivity: steady-state regime3. Recognition Methods- Ion recognition: recognition-electric charge, selectivity-size,transduction-potentiometric, optical methodse.g. PH electrode ----> part 3- Recognition by affinity interactions: reversible, non-covalent bonds-ionic bonds, hydrogen bonds, van der Waals interaction => result in a molecular assiciation complex; also respect to shape and chemical reactivity; indicated by stability constant (very stable)- antibody - antigen interaction => immunochemical reactionantibody: glycoprotein produced by immune system to identify and neutralizepathogen microorganisms.antigen: the part of the pathogen that reactions with the antibody.use specific antibody receptor => identify pathogenuse antigen receptor => identify antibody (the detection of infection byparticular pathogen)- lectin proteins recognize caborhydrates (agglutinins, hemagglutinin)carbohydrate-binding modules link to the catalytic part of glycosidehydrolases => result in degradation of cell wall, storage of polysaccharide- A Molecularly Imprinted Polymer (MIP) is a polymer that has been processed usingthe molecular imprinting technique which leaves cavities in polymer matrix withaffinity to a chosen "template" molecule.In chemistry, molecular imprinting is a technique to create template-shaped cavities in polymer matrices with memory of the template molecules to be used in molecular recognition.-Nucleic acid aptamers are nucleic acid species that have been engineered throughrepeated rounds of in vitro selection to bind to various molecular targets such assmall molecules, proteins, nucleic acids, and even cells, tissues and organisms.Aptamers are useful in biotechnological and therapeutic a pplications as they offer molecular recognition properties that rival that of the commonly used bimolecular antibodies.- Recognition by nucleic acids: hydrogen bonds between two distinct pairs of nucleobases => two complementary nucleic acids form a double strand association complex => called hybridizationnucleic acid sensors: short single strand NA as receptor to recognize a particular NA sequence in the analyte NA => detection of genetic anomalies and pathogen mircoorganism- Recognition by enzyme: dynamic processEnzyme: protein compound that function as catalysts in chemical reaction occurring in living system.- Recognition by cells and tissues: advantages of enzyme incorporated in biological materials => in their natural environmentsee part 3, Wegener - Recognition by gases and vapors: based on sorption at solid material => surface-adsorption, inner-absorption; purely physical phenomenon or chemical reaction.4. Transduction MetohdosChemical transduction: monitoring the change of chemical composition of the sensing element in response to the recognition process. => change in concentration/amount is measured => detect primary product -> secondary product or coreagent -> labeling productLABEL can be a simple molecular species or nanoparticals that can be detected by available physiochemical methods => enzyme, fluorescent dyes, luminescent dyes, electroactive compoundsPhysical transduction: a specific physical property of the sensing element that is affected by its interaction with the analyte is monitored. => mass, reflective index, dielectric properties, electrical resistivity => LABEL-FREE- Thermometric transductionRecognition of the analyte leads to change in temperature => only catalytical processes generate sufficient heat to the measurement => application: combustible gases react with O2 at the surface of a catalyst.- Transduction based on mechanical effectsRecognition leads to change in mass of the sensing element => monitored by mass tranducer based on quartz crystal microbalance (QCM)----------------------------------------------------------------------------------------------------------------- QCM, correct name: Thickness shear modePiezoelectric effect:generation of electrical charges on the surface of a solid by strain, pressure or torsion (mechanical deformation of solid) =>electricity resulting from pressureI nverse piezoelectric effect:application of charges to surfaces of piezoelectricsolid generates mechanical deformation (elongation, contraction, torsion)QCM is based on Inverse piezoelectric effect!# AT cut => 35`15`=> minimum temperature coefficient at 50~70 CIt makes the AT-cut well suited to applications requiring high degree of frequency stability over wide temperature ranges.## Electrodes are applied on both sides, and AC voltage applied.DC cannot flow across the crystal because it consists of an insulator material;however the crystal somewhat behaves as capacitor and allow an AC current to f low along the left-hand loop.AC voltage applied => leads to shear oscillation of crystal => when the voltage frequency matches the intrinsic vibration frequency of the crystal => the vibration amplitude is at maximal => the resonant => resonant frequency (f0) => depend on crystal thickness (e.g. d q= 330 um, f0= 5MHZ), density and elasticity of piezoelectric material### AT-cut resonator: thickness: ~0.2 mm, diameter of the active area: 5~20 mm #### Deposition of a homogenous mass film (a rigid overlay)Sauerbrey equation:Cf indicate sensitivity of QCMcondition of this equation: rigid deposited mass; △m<2% of crystal mass;operated in vacuum or in gaseous atmopphereIn liquid: the liquid breaks the vibration by friction => lessen f0Thickness of the layer must be greater than the wave decay lengththat is of 250 nm of 5 MHz resonator at water. ----> part 2!!!##### QCM in practice => see p.41----------------------------------------------------------------------------------------------------------------- - Resistive and capacitive transductionRecognition leads to changes in the electrical property of this materialResistive transduction: gases interact with MOS => change in electrical resistivity Capactive transduction => dielectric constant- Electrochemical transductionsee part 2, Matysik - Optical transductionOptical transduction can be based on light emission or light absorption, also by physical quantity (reflective index) and light scattering.5. Sensor Configuration and Fabrication- Lateral flow assayA typical test strip consists of the following components:1. Sample pad – an absorbent pad onto which the test sample is applied2. Conjugate pad –this contains antibodies specific to the target analyte;conjugated to coloured particles (e.g. gold nanoparticles)3. Reaction membrane –typically a hydrophobic nitrocellulose or celluloseacetate membrane onto which anti-target analyte antibodies are immobilized in a line across the membrane as a capture zone or test line, and a control zonecontaining antibodies specific for the conjugate antibodies.4. Wicking pad –a further absorbent pad designed to draw the sample acrossthe reaction membrane by capillary action and collect it.Double antibody sandwich assays: the sample migrates from the sample pad through the conjugate pad where any target analyte present will bind to the c onjugate.=> The sample then continues to migrate across the membrane until it reaches the test line where the target or conjugate complex will bind to the immobilized antibodies producing a visible line on the membrane. => The sample then migrates further along the strip until it reaches the control line, where excess conjugate will bind and producea second visible line on the membrane.This control line indicates that the sample has migrated across the membrane as intended. Two clear lines on the membrane is a positive result. A single line in the control zone is a negative result. Double antibody sandwich assays are most suitable for larger analytes, such as bacterial pathogens and viruses, with multiple antigenic sites. 6. Methods and Material in Sensor Preparation- Immobilization at solid surface => integration of a transducer with the receptor Physical adsorption at a solid supportNon covalent immobilization at solid surface => hydrophobic interaction, hydrogen bonding, electrostatic attraction; monolayer; no restrict access; not stable; Langmuir isotherm -> equilibrium interactionSupport material: silica, cellulose acetate, PVCCovalent bonding to the solid supportCovalent conjugation => stable, covalent bond, time consuming, expensiveCommon reactive group: -OH, -NH2, -C=O, -SH- Carboxylic acid with DCC- Glutaraldehyde reacts with the a.a. of lysine in protein => widely used Support: porous material => high specific area, high density of immobilized compounds => hydrogel: immobilized by entrapment/covalent corsslink - Natural polymers: Cellulose, Dextran- Synthetic polymers: Polystyrene- Active polymers: Epoxide (without preliminary activation) -->DNA array !!!- Inactive Polymers: Vicinal hydroxyls actived by CNBr- Inorganic support: Silica, AL2O3, TiO2 => stable at extreme PH- Metal support: noble metals, thiols on golds --> self assembled monolayers!Affinity reaction: avidin-biotin !!!Thin molecular layers: one or several molecular layers in solid support - Self-assembly of amphiphilic compounds: preparation of liposome andmicelles; liposome can be used of entrapment of molecular- Bilipid layer membranes: Langmuir-Blodgett technique- Layer by Layer assembly- Sol-Gel chemistry methods: silica gel => -O-Si-O-- Hydrogels: Xerogel, aerogel- Conducting polymers: Polyacetylene, polyaniline --> gas senor based on CP (----> part 3 !!!); also as entrapment matrix for biological receptors- Mesoporous materials: porous materials with pore (diameter: 2-50 nm,close to protein) => enzyme immobilization by entrapment (crosslinking withglutaraldehyde)- Deposition of polymers onto solid surfaces: dip coating, drop coating, spin coating ----> part 2 !!!Perm-selective memberanes: Nafin ----> Clark oxygen electrode Support-free crosslinkingEntrapment in a polymer networkEncapsulation7. Microfabrication Methodes- Spot Arraying: Contact-based & Noncontact-based; DNA microarray !!!!!Pros & Cons- Thick-film Technology: screen-printing technique (5-50 um thick layer)- Thin-film Technology: Photolithography (2 um)- Softlithography ----> experiment !!!!- Microcontact printing ----> experiment !!!!8. Optical Sensors- Electromagnetic RadiationOptical sensor => interaction of electromagnetic radiation with sensor layer - frequency; wavelength; photon energy (definition)- Structure: integration with wavelength-selection (optical filters) device and light sources (lasers), light detectors (phototransistors)- Optical Waveguides- Optical FibersOptical fibers' structuretotal internal reflection => evanescent wave- Spectrochemical Transduction MethodsSpectrochemical method analysis => light absorption or emission by sample => optical label performs absorption or emission (organic dye or metal complexes) - Light absorption: absorbance => concentration; sensitivity => thickness, absorpyivity, absorptivity => wavelength- Diffuse reflectance spectrometry: refelctance => concentration; suitable forsolid in near IR- Luminescence: Fluorescence spectromerty => fluorophore (label, organic dye or metal complexes, luminescent nanparticle ); steady-statefluorescence measurement, Time-resolved fluormetry; fluorescencequenching; resonance energy transfer (FRET); chemical- andbioluminescence => luminol; electrochemicaluminescence; Ramanspetrometry- Surface Plasmon Resonance Spectroscopy (SPR)。

华蟾毒素(CBG)酶联免疫吸附检测试剂盒货号：ELK8271规格：96T（本试剂盒仅供体外研究使用，不用于临床诊断）灵敏度：91.3ng/mL检测范围：312.5-20000ng/mL特异性：可检测重组或天然的CBG，且不与其它相关蛋白交叉反应。

试剂盒组成中文名称英文名称规格保存条件48T96T酶标板（可拆）Pre-coated Microplate 6条x8孔12条x8孔4°C/-20°C（6个月）冻干标准品Standard(lyophilized)124°C/-20°C（6个月）标准品&样品稀释液Standard/Sample Diluent Buffer10mL20mL4°C生物素结合物（100×）Biotinylated-Conjugate(100x)30μL60μL4°C/-20°C（6个月）生物素结合物稀释液Biotinylated Conjugate Diluent5mL10mL4°C浓缩HRP酶结合物（100×）Streptavidin-HRP(100×)60μL120μL4°C/-20°C（6个月）酶结合物稀释液HRP Diluent6mL12mL4°C浓缩洗涤液（25×）Wash Buffer(25×)10mL20mL4°C显色底物溶液（TMB）TMB Substrate Solution6mL10mL4°C（避光）反应终止液Stop reagent3mL6mL4°C封板覆膜Plate Covers12常温产品说明书Instruction manual1份1份常温特别说明1.打开包装后请及时检查所有物品是否齐全完整。

所有试剂的批号见标签。

2.试剂盒保存：-20°C（长时间存放，试剂按照标签提示温度保存）；2-8°C（一周内使用）；避免反复冻融，过期后请勿使用。

实验研究依达拉奉右莰醇通过抑制TLR4/NF-κB信号通路减轻实验性自身免疫性脑脊髓炎小鼠炎症反应晚丽，李作孝△摘要：目的探讨依达拉奉右莰醇对实验性自身免疫性脑脊髓炎（EAE）小鼠炎症反应的影响及其机制。

方法30只雌性C57BL/6小鼠随机分为空白组、模型组、依达拉奉右莰醇干预组各10只。

除空白组外，其余2组小鼠均采用髓鞘少突胶质细胞糖蛋白35-55（MOG35-55）多肽诱导EAE模型。

从造模次日开始，依达拉奉右莰醇干预组腹腔注射依达拉奉右莰醇12.5mg/kg，空白组及模型组腹腔注射等量生理盐水，1次/d，连续14d。

观察小鼠发病情况，并行神经功能障碍评分；HE和LFB染色观察脊髓组织病理改变；实时荧光定量PCR检测脑组织匀浆中白细胞介素（IL）-1β、IL-6及肿瘤坏死因子（TNF）-αmRNA表达水平；蛋白免疫印迹法检测脊髓组织中Toll样受体4（TLR4）、核因子κB p65（NF-κB p65）蛋白表达水平。

结果空白组小鼠均未发病，其余2组小鼠不同程度发病。

与模型组相比，依达拉奉右莰醇干预组小鼠的发病潜伏期、高峰期延迟，高峰期神经功能障碍评分降低（P＜0.01）。

空白组小鼠脊髓组织未见异常；模型组脊髓组织大量炎性细胞浸润、髓鞘结构紊乱；依达拉奉右莰醇干预组较模型组的炎性细胞浸润减少、髓鞘结构紊乱情况改善。

与空白组相比，其余2组小鼠脑组织匀浆中IL-1β、IL-6、TNF-αmRNA表达水平以及脊髓组织中TLR4、NF-κB p65蛋白表达水平显著升高，以依达拉奉右莰醇干预可逆转建模引起的上述改变（P＜0.05）。

结论依达拉奉右莰醇可减轻EAE小鼠炎症反应，其机制可能与抑制TLR4/NF-κB信号通路活化有关。

关键词：脑脊髓炎，自身免疫性，实验性；Toll样受体4；NF-κB；炎症；白细胞介素类；肿瘤坏死因子α；依达拉奉右莰醇；TLR4/NF-κB信号通路中图分类号：R744.51文献标志码：A DOI：10.11958/20212362Edaravone dexborneol reduces inflammation in mice with experimental autoimmuneencephalomyelitis by inhibiting TLR4/NF-κB signaling pathwayWAN Li,LI Zuoxiao△Department of Neurology,the Affiliated Hospital of Southwest Medical University,Luzhou646000,China△Corresponding Author E-mail:****************Abstract:Objective To investigate the effect and mechanism of edaravone dexborneol on the inflammatory response in mice with experimental autoimmune encephalomyelitis(EAE).Methods Thirty female C57BL/6mice were randomly divided into the blank group,the model group and the edaravone dexborneol intervention group,with10mice in each group. Except for the blank group,EAE model was induced by myelin oligodendrocyte glycoprotein35-55(MOG35-55) polypeptide in the other two groups.From the day after modeling,mice in the edaravone dexborneol intervention group were intraperitoneally injected with edaravone dexborneol12.5mg/kg,while the mice in the blank group and the model group were intraperitoneally injected with the equal amount normal saline,once a day for consecutive14days.The behavioral changes of mice were observed,and neurological dysfunction scores were performed.HE and LFB staining were used to detect spinal cord pathological changes.The mRNA expression levels of interleukin(IL)-1β,IL-6and tumor necrosis factor-α(TNF-α)in brain homogenate were detected by real-time fluorescence quantitative PCR.The protein expression levels of Toll-like receptor4(TLR4)and nuclear factorκB p65(NF-κB p65)in spinal cord tissue were detected by Western blot assay.Results None of the mice in the blank group had the disease,and the other two groups of mice had different degrees of pared with the model group,the incubation period and peak period were delayed in the edaravone dexborneol intervention group,and neurological deficit scores in peak period decreased(P＜0.01).No abnormality was found in spinal cord tissue structure in mice of the blank group,and a large number of inflammatory cell infiltration,myelin structure 基金项目：泸州市人民政府-西南医科大学科技战略合作基金项目（2018LZXNYD-ZK17）作者单位：西南医科大学附属医院神经内科（邮编646000）作者简介：晚丽（1994），女，硕士在读，主要从事神经免疫方面研究。

Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSDXing-Yun Chai1, Song-Lin Li2, Ping Li1*1Key Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China2Institute of Nanjing Military Command for Drug Control, Nanjing, 210002, People’s Republic of China*Corresponding author: Ping LiKey Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China.E-mail address: lipingli@Tel.: +86-25-8324-2299; 8539-1244; 135********Fax: +86-25-8532-2747AbstractA new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidinB (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb.Simultaneous separation of these seven saponins was achieved on a C18 analytical column with a mixed mobile phase consisting of acetonitrile(A)-water(B)(29:71 v/v) acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min on linear gradient, and then keep isocratic elution with 54%B from 25 to 30min.The drift tube temperature of ELSD was set at 106℃, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2 0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0% respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.Keywords: HPLC-ELSD; Flos Lonicerae; Saponins; Quantification1. IntroductionFlos Lonicerae (Jinyinhua in Chinese), the dried buds of several species of the genus Lonicera (Caprifoliaceae), is a commonly used traditional Chinese medicine (TCM) herb. It has been used for centuries in TCM practice for the treatment of sores, carbuncles, furuncles, swelling and affections caused by exopathogenic wind-heat or epidemic febrile diseases at the early stage [1]. Though four species of Lonicera are documented as the sources of Flos Lonicerae in China Pharmacopeia (2000 edition), i.e. L. japonica, L. hypoglauca,L. daystyla and L. confusa, other species such as L. similes and L. macranthoides have also been used on the same purpose in some local areas in China [2]. So it is an important issue to comprehensively evaluate the different sources of Flos Lonicerae, so as to ensure the clinical efficacy of this Chinese herbal drug.Chemical and pharmacological investigations on Flos Lonicerae resulted in discovering several kinds of bioactive components, i.e. chlorogenic acid and its analogues, flavonoids, iridoid glucosides and triterpenoid saponins [3]. Previously, chlorogenic acid has been used as the chemical marker for the quality evaluation of Flos Lonicerae,owing to its antipyretic and antibiotic property as well as its high content in the herb. But this compound is not a characteristic component of Flos Lonicerae, as it has also been used as the chemical marker for other Chinese herbal drugs such as Flos Chrysanthemi and so on[4-5]. Moreover, chlorogenic acid alone could not be responsible for the overall pharmacological activities of Flos Lonicerae[6].On the other hand, many studies revealed that triterpenoidal saponins of Flos Lonicerae possess protection effects on hepatic injury caused by Acetaminophen, Cd, and CCl4, and conspicuous depressant effects on swelling of ear croton oil [7-11]. Therefore, saponins should also be considered as one of the markers for quality control of Flos Lonicerae. Consequently, determinations of all types of components such as chlorogenic acid, flavonoids, iridoid glucosides and triterpenoidal saponins in Flos Lonicerae could be a better strategy for the comprehensive quality evaluation of Flos Lonicerae.Recently an HPLC-ELSD method has been established in our laboratory for qualitative and quantitative determination of iridoid glucosides in Flos Lonicerae [12]. But no method was reported for the determination of triterpenoidal saponins in Flos Lonicera. As a series studies on the comprehensive evaluation of Flos Lonicera, we report here, for the first time, the development of an HPLC-ELSD method for simultaneous determination of seven triterpenoidal saponins in the Chinese herbal drug Flos Lonicerae, i.e.macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) (Fig. 1).2. Experimental2.1. Samples, chemicals and reagentsFive samples of Lonicera species,L. japonica from Mi county, HeNan province (LJ1999-07), L. hypoglauca from Jiujang county, JiangXi province (LH2001-06), L. similes from Fei county, ShanDong province (LS2001-07), L. confuse from Xupu county, HuNan province (LC2001-07), and L. macranthoides from Longhu county, HuNan province (LM2000-06) respectively, were collected in China. All samples were authenticated by Dr. Ping Li, professor of department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. The voucher specimens were deposited in the department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. Seven saponin reference compounds: macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) were isolated previously from the dried buds of L. confusa by repeated silica gel, sephadex LH-20 and Rp-18 silica gel column chromatography, their structures were elucidated by comparison of their spectral data (UV, IR, MS, 1H- NMR and 13C-NMR) with references [13-15]. The purity of these saponins were determined to be more than 98% by normalization of the peak areas detected by HPLC with ELSD, and showed very stable in methanol solution.HPLC-grade acetonitrile from Merck (Darmstadt, Germany), the deionized water from Robust (Guangzhou, China), were purchased. The other solvents, purchased from Nanjing Chemical Factory (Nanjing, China) were of analytical grade.2.2. Apparatus and chromatographic conditionsAglient1100 series HPLC apparatus was used. Chromatography was carried out on an Aglient Zorbax SB-C18 column（250 4.6mm, 5.0µm）at a column temperature of 25℃.A Rheodyne 7125i sampling valve (Cotati, USA) equipped with a sample loop of 20µl was used for sample injection. The analog signal from Alltech ELSD 2000 (Alltech, Deerfield, IL, USA)was transmitted to a HP Chemstation for processing through an Agilent 35900E (Agilent Technologies, USA).The optimum resolution was obtained by using a linear gradient elution. The mobile phase was composed of acetonitrile(A) and water(B) which acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min in linear gradient, and back to the isocratic elution of 54%B from 25 to 30 min.The drift tube temperature for ELSD was set at 106℃and the nitrogen flow-rate was of 2.6 l/min. The chromatographic peaks were identified by comparing their retention time with that of each reference compound tried under the same chromatographic conditions with a series of mobile phases. In addition, spiking samples with the reference compounds further confirmed the identities of the peaks.2.3. Calibration curvesMethanol stock solutions containing seven analytes were prepared and diluted to appropriate concentration for the construction of calibration curves. Six concentrationof the seven analytes’ solution were injected in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The results were demonstrated in Table1.2.4. Limits of detection and quantificationMethanol stock solution containing seven reference compounds were diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions were injected into HPLC for analysis.The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ for each compound were shown in Table1.2.5. Precision and accuracyIntra- and inter-day variations were chosen to determine the precision of the developed assay. Approximate 2.0g of the pulverized samples of L. macranthoides were weighted, extracted and analyzed as described in 2.6 Sample preparation section. For intra-day variability test, the samples were analyzed in triplicate for three times within one day, while for inter-day variability test, the samples were examined in triplicate for consecutive three days. Variations were expressed by the relative standard deviations. The results were given in Table 2.Recovery test was used to evaluate the accuracy of this method. Accurate amounts of seven saponins were added to approximate 1.0g of L. macranthoides,and then extracted and analyzed as described in 2.6 Sample preparation section. The average recoveries were counted by the formula: recovery (%) = (amount found –original amount)/ amount spiked ×100%, and RSD (%) = (SD/mean) ×100%. The results were given in Table 3.2.6. Sample preparationSamples of Flos Lonicerae were dried at 50℃until constant weight. Approximate 2.0g of the pulverized samples, accurately weighed, was extracted with 60% ethanol in a flask for 4h. The ethanol was evaporated to dryness with a rotary evaporator. Residue was dissolved in water, followed by defatting with 60ml of petroleum ether for 2 times, and then the water solution was evaporated, residue was dissolved with methanol into a 25ml flask. One ml of the methanol solution was drawn and transferred to a 5ml flask, diluted to the mark with methanol. The resultant solution was at last filtrated through a 0.45µm syringe filter (Type Millex-HA, Millipore, USA) and 20µl of the filtrate was injected to HPLC system. The contents of the analytes were determined from the corresponding calibration curves.3. Results and discussionsThe temperature of drift tube and the gas flow-rate are two most important adjustable parameters for ELSD, they play a prominent role to an analyte response. In ourprevious work [12], the temperature of drift tube was optimized at 90°C for the determination of iridoids. As the polarity of saponins are higher than that of iridoids, more water was used in the mobile phase for the separation of saponins, therefore the temperature for saponins determination was optimized systematically from 95°C to 110°C, the flow-rate from 2.2 to 3.0 l/min. Dipsacoside B was selected as the testing saponin for optimizing ELSD conditions, as it was contained in all samples. Eventually, the drift tube temperature of 106℃and a gas flow of 2.6 l/min were optimized to detect the analytes. And these two exact experimental parameters should be strictly controlled in the analytical procedure [16].All calibration curves showed good linear regression (r2 0.9922) within test ranges. Validation studies of this method proved that this assay has good reproducibility. As shown in Table 2, the overall intra- and inter-day variations are less than 6% for all seven analytes. As demonstrated in Table 3, the developed analytical method has good accuracy with the overall recovery of high than 96% for the analytes concerned. The limit of detection (S/N=3) and the limit of quantification (S/N=10) are less than 0.26μg and 0.88μg respectively (Table1), indicating that this HPLC-ELSD method is precise, accurate and se nsitive enough for the quantitative evaluation of major non- chromaphoric saponins in Flos Lonicerae.It has been reported that there are two major types of saponins in Flos Lonicerae, i.e. saponins with hederagenin as aglycone and saponins with oleanolic acid as the aglycone [17]. But hederagenin type saponins of the herb were reported to have distinct activities of liver protection and anti-inflammatory [7-11]. So we adoptedseven hederagenin type saponins as representative markers to establish a quality control method.The newly established HPLC-ELSD method was applied to analyze seven analytes in five plant sources of Flos Lonicerae, i.e. L. japonica,L. hypoglauca,L. confusa,L. similes and L. macranthoides(Table 4). It was found that there were remarkable differences of seven saponins contents between different plant sources of Flos Lonicerae. All seven saponins analyzed could be detected in L. confusa and L. hypoglauca, while only dipsacoside B was detected in L. japonica. Among all seven saponins interested, only dipsacoside B was found in all five plant species of Flos Lonicerae analyzed, and this compound was determined as the major saponin with content of 53.7 mg/g in L. hypoglauca. On the other hand, macranthoidin B was found to be the major saponin with the content higher than 41.0mg/g in L. macranthoides,L. confusa, and L. similis, while the contents of other analytes were much lower.In our previous study [12], overall HPLC profiles of iridoid glucosides was used to qualitatively and quantitatively distinguish different origins of Flos Lonicerae. As shown in Fig.2, the chromatogram profiles of L. confusa, L. japonica and L. similes seem to be similar, resulting in the difficulty of clarifying the origins of Flos Lonicerae solely by HPLC profiles of saponins, in addition to the clear difference of the HPLC profiles of saponins from L. macranthoides and L. hypoglauca.Therefore, in addition to the conventional morphological and histological identification methods, the contents and the HPLC profiles of saponins and iridoids could also be used as accessory chemical evidence toclarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.4. ConclusionsThis is the first report on validation of an analytical method for qualification and quantification of saponins in Flos Lonicerae. This newly established HPLC-ELSD method can be used to simultaneously quantify seven saponins, i.e. macranthoidin B, macranthoidin A, dipsacoside B, hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester, macranthoside B, macranthoside A, and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside in Flos Lonicerae. Together with the HPLC profiles of iridoids, the HPLC-ELSD profiles of saponins could also be used as an accessory chemical evidence to clarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.AcknowledgementsThis project is financially supported by Fund for Distinguished Chinese Young Scholars of the National Science Foundation of China (30325046) and the National High Tech Program（2003AA2Z2010）.[1]Ministry of Public Health of the People’s Republic of China, Pharmacopoeia ofthe People’s Republic of China, V ol.1, 2000, p. 177.[2]W. Shi, R.B. Shi, Y.R. Lu, Chin. Pharm. J., 34(1999) 724.[3]J.B. Xing, P. Li, D.L. Wen, Chin. Med. Mater., 26(2001) 457.[4]Y.Q. Zhang, L.C. Xu, L.P. Wang, J. Chin. Med. Mater., 21(1996) 204.[5] D. Zhang, Z.W. Li, Y. Jiang, J. Pharm. Anal., 16(1996) 83.[6]T.Z. Wang, Y.M. Li, Huaxiyaoxue Zazhi, 15(2000) 292.[7]J.ZH. Shi, G.T. Liu. Acta Pharm. Sin., 30(1995) 311.[8]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 209.[9]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 213.[10]J.ZH. Shi, L. Wan, X.F. Chen.ZhongYao YaoLi Yu LinChuang, 6 (1990) 33.[11]J. Liu, L. Xia, X.F. Chen. Acta Pharmacol. Sin., 9 (1988) 395[12]H.J. Li, P. Li, W.C. Ye, J. Chromatogr. A 1008(2003) 167-72.[13]Q. Mao, D. Cao, X.SH. Jia. Acta Pharm. Sin., 28(1993) 273.[14]H. Kizu, S. Hirabayashi, M. Suzuki, et al. Chem. Pharm. Bull., 33(1985) 3473.[15]S. Saito, S. Sumita, N. Tamura, et al. Chem Pharm Bull., 38(1990) 411.[16]Alltech ELSD 2000 Operating Manual, Alltech, 2001, p. 16. In Chinese.[17]J.B. Xing, P. Li, Chin. Med. Mater., 22(1999) 366.Fig. 1 Chemical structures of seven saponins from Lonicera confusa macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)Fig. 2Representative HPLC chromatograms of mixed standards and methanol extracts of Flos Lonicerae.Column: Agilent Zorbax SB-C18 column（250 4.6mm, 5.0µm）, temperature of 25℃; Detector: ELSD, drift tube temperature 106℃, nitrogen flow-rate 2.6 l/min.A: Mixed standards, B: L. confusa, C: L. japonica, D: L. macranthoides, E: L. hypoglauca, F: L. similes.Table 1 Calibration curves for seven saponinsAnalytes Calibration curve ar2Test range(μg)LOD(μg)LOQ(μg)1 y=6711.9x－377.6 0.9940 0.56–22.01 0.26 0.882 y=7812.6x－411.9 0.9922 0.54–21.63 0.26 0.843 y=6798.5x－299.0 0.9958 0.46–18.42 0.22 0.724 y=12805x－487.9 0.9961 0.38–15.66 0.10 0.345 y=4143.8x－88.62 0.9989 0.42–16.82 0.18 0.246 y=3946.8x－94.4 0.9977 0.40–16.02 0.16 0.207 y=4287.8x－95.2 0.9982 0.42–16.46 0.12 0.22a y: Peak area; x: concentration (mg/ml)Table 2 Reproducibility of the assayAnalyteIntra-day variability Inter-day variability Content (mg/g) Mean RSD (%) Content (mg/g) Mean RSD (%)1 46.1646.2846.2246.22 0.1346.2245.3647.4226.33 2.232 5.385.385.165.31 2.405.285.345.045.22 3.043 4.374.304.184.28 2.244.284.464.024.255.204 nd1)-- -- nd -- --5 1.761.801.821.79 1.701.801.681.841.77 4.706 1.281.241.221.252.451.241.341.201.26 5.727 tr2)-- -- tr -- -- 1): not detected; 2): trace. RSD (%) = (SD/Mean) ×100%Table 3 Recovery of the seven analytesAnalyteOriginal(mg) Spiked(mg)Found(mg)Recovery(%)Mean(%)RSD(%)1 23.0823.1423.1119.7122.8628.1042.7346.1351.0199.7100.699.399.8 0.722.692.672.582.082.913.164.735.515.7698.197.6100.698.8 1.632.172.152.091.732.182.623.884.404.6598.8103.297.799.9 2.94nd1)1.011.050.980.981.101.0297.0104.8104.1102.0 4.250.880.900.910.700.871.081.561.752.0197.197.7101.898.9 2.660.640.620.610.450.610.751.081.211.3397.796.796.096.8 0.97tr2)1.021.101.081.031.111.07100.9102.799.1100.9 1.81): not detected; 2): trace.a Recovery (%) = (Amount found –Original amount)/ Amount spiked ×100%, RSD (%) = (SD/Mean) ×100%Table 4 Contents of seven saponins in Lonicera spp.Content (mg/g)1 2 3 4 5 6 7 L. confusa45.65±0.32 5.13±0.08 4.45±0.11tr1) 2.04±0.04tr 1.81±0.03 L. japonica nd2)nd 3.44±0.09nd nd nd nd L. macranthoides46.22±0.06 5.31±0.13 4.28±0.10 tr 1.79±0.03 1.25±0.03 tr L. hypoglauca11.17±0.07 nq3)53.78±1.18nd 1.72±0.02 2.23±0.06 2.52±0.04 L. similes41.22±0.25 4.57±0.07 3.79±0.09nd 1.75±0.02tr nd 1): trace; 2): not detected.. 3) not quantified owing to the suspicious purity of the peak.。

离子色谱法测定甲磺酸伊马替尼中残留的二甲胺嵇海澄;李孝壁;李琴;姜涛;胡春勇【摘要】Objective To establish a method the determination of dimethylamine in imatinib mesylate by ion chromatography. Methods The determination was carried out on a Dionex IonPacCS12A(250mm×4.6mm)column with 20mmol/L methanesulfonic acid solution as eluent by electrical conductivity detector.The flow rate was1.0mL/min,The column temperature was 30℃,and temperature of conductivity detection pool was 35℃. Results There was a good linearity over the range of 0.10-9.84μg/mL with a the correlation coefficient r higher than 1.0000(n=6).The RSD for repeatition was 0.11%(n=6),The average recovery was 97.5%with RSD of accuracy at 1.8%. Conclusion The Methods was simple,accurate and good reproducibility,which can be used for the determination of dimethylamine for imatinib mesylate.%目的：建立了一种离子色谱法测定伊马替尼中二甲胺的方法。

DOI：10.16658/ki.1672-4062.2024.01.174硫辛酸注射液联合胰激肽原酶肠溶片对DPN的临床疗效及生存质量的影响王莉，朱海峰濉溪县中医医院内分泌科，安徽淮北235100[摘要]目的探讨硫辛酸注射液联合胰激肽原酶肠溶片对2型糖尿病周围神经病变（Diabetic Peripheral Neu⁃ropathy， DPN）患者的临床疗效、生存质量及安全性的影响。

方法选取2021年2月—2022年4月濉溪县中医医院60名DPN患者作为研究对象。

通过随机数表法分为两组，每组30例。

对照组采用常规治疗，观察组在对照组基础上加用硫辛酸注射液和胰激肽原酶肠溶片治疗。

比较两组患者的神经病变评分、神经电生理指标、生存质量评分、安全性指标和不良反应发生率。

结果治疗后，观察组神经病变评分（6.2±0.9）分低于对照组（7.6±1.1）分，差异有统计学意义（t=5.438，P<0.05）；观察组神经电生理指标、生存质量评分、安全性指标均优于对照组，差异有统计学意义（P均<0.05）；两组患者不良反应发生率比较，差异无统计学意义（P>0.05）。

结论硫辛酸注射液联合胰激肽原酶肠溶片对DPN患者有良好的临床疗效，能够改善神经功能、改善神经电生理指标、提高生存质量，且安全性高。

[关键词] 硫辛酸注射液；胰激肽原酶肠溶片；2型糖尿病周围神经病变；临床疗效[中图分类号] R587.2 [文献标识码] A [文章编号] 1672-4062（2024）01（a）-0174-05Effect of Lipoic Acid Injection Combined with Pancreatic Kininogenase Enteric-coated Tablets on Clinical Efficacy and Quality of Survival in DPN WANG Li, ZHU HaifengDepartment of Endocrinology, Suixi County Hospital of Traditional Chinese Medicine, Huaibei, Anhui Province, 235100 China[Abstract] Objective To investigate the effects of lipoic acid injection combined with pancreatic kininogenase enteric-coated tablets on the clinical efficacy, quality of survival and safety of patients with type 2 diabetic peripheral neuropathy (DPN). Methods 60 DPN patients admitted to Suixi County Hospital of Traditional Chinese Medicine from February 2021 to April 2022 were selected as the study objects. They were divided into two groups with 30 cases in each group by random number table method. The control group received conventional treatment, and the observation group was treated with lipoic acid injection and pancreatic kininogenase enteric-coated tablets on the basis of control group. Neuropathy score, neuroelectrophysiological index, quality of life score, safety index and incidence of adverse reactions were compared between the two groups. Results After treatment, the neuropathy score of observation group (6.2±0.9) points was lower than that of control group (7.6±1.1) points, and the difference was statistically significant (t= 5.438, P<0.05). Neuroelectrophysiological indexes, quality of survival scores and safety indexes of the observation group were better than those of the control group, and the differences were statistically significant (all P<0.05). There was no significant difference in the incidence of adverse reactions between the two groups (P>0.05). Conclusion Li⁃[作者简介]王莉（1982-），女，本科，主治医生，研究方向为糖尿病周围神经病变。

生物技术进展 2023 年 第 13 卷 第 4 期 596 ~ 603Current Biotechnology ISSN 2095‑2341研究论文Articles重组贻贝粘蛋白的表征及功效评价李敏 ， 魏文培 ， 乔莎 ， 郝东 ， 周浩 ， 赵硕文 ， 张立峰 ， 侯增淼 *西安德诺海思医疗科技有限公司，西安 710000摘要：为了推进重组贻贝粘蛋白在医疗、化妆品领域的应用，对大肠杆菌规模化发酵及纯化生产获得的重组贻贝粘蛋白进行了表征及功效评价。

经Edman 降解法、基质辅助激光解吸电离飞行时间质谱、PITC 法、非还原型SDS -聚丙烯酰胺凝胶电泳法、凝胶法、改良的Arnow 法对重组贻贝粘蛋白进行氨基酸N 端测序、相对分子量分析、氨基酸组成分析、蛋白纯度分析、内毒素含量测定、多巴含量测定；通过细胞迁移、斑马鱼尾鳍修复效果对重组贻贝粘蛋白进行功效评价。

结果显示，获得的重组贻贝粘蛋白与理论的一级结构一致，蛋白纯度达95%以上，内毒素<10 EU ·mg -1,多巴含量大于5%；重组贻贝粘蛋白浓度为60 μg ·mL -1时能够显著促进细胞增殖的活性（P <0.01）；斑马鱼尾鳍面积样品组与模型对照组相比极显著增加（P <0.001）。

研究结果表明，重组贻贝粘蛋白具有显著的促细胞迁移和修复愈合的功效，具备作为生物医学材料的潜质。

关键词：贻贝粘蛋白；基因重组；生物材料；表征；功效评价DOI ：10.19586/j.2095­2341.2023.0021 中图分类号：S985.3+1 文献标志码：ACharacterization and Efficacy Evaluation of Recombinant Mussel Adhesive ProteinLI Min ， WEI Wenpei ， QIAO Sha ， HAO Dong ， ZHOU Hao ， ZHAO Shuowen ， ZHANG Lifeng ，HOU Zengmiao *Xi'an DeNovo Hith Medical Technology Co.， Ltd ， Xi'an 710000， ChinaAbstract ：In order to promote the application of recombinant mussel adhesive protein in the medical and cosmetics field ， the recombi⁃nant mussel adhesive protein obtained from scale fermentation and purification of Escherichia coli was characterized and its efficacy was evaluated. Amino acid N -terminal sequencing ， relative molecular weight analysis ， amino acid composition analysis ， protein purityanalysis ， endotoxin content ， dihydroxyphenylalanine （DOPA ） content of recombinant mussel adhesive protein were determined by the following methods ： Edman degradation ， matrix -assisted laser desorption ionization time -of -flight mass spectrometry （MALDI -TOF -MS ）， phenyl -isothiocyanate （PITC ）， nonreductive SDS -polyacrylamide gel electrophoresis （SDS -PAGE ）， gel method ， modified Ar⁃now. The efficacy of recombinant mussel adhesive protein was evaluated by cell migration and repairing effect of zebrafish tail fin. Re⁃sults showed that the obtained recombinant mussel adhesive protein was confirmed to be consistent with the theoretical primary structure ， protein purity of more than 95%， endotoxin <10 EU ·mg -1， DOPA content above 5%. When the recombinant mussel adhesive protein concentration was 60 μg ·mL -1， the effect of promoting cell proliferation was the most obvious ， and it had very significant activity （P <0.01）. The caudal fin area of zebrafish in sample group was significantly increased compared with model control group （P <0.001）. The results indicated that recombinant mussel adhesive protein can promote cell migration and repair healing and has the potential to be used as biomedical materials.Key words ：mussel adhesive protein ； gene recombination ； biological materials ； representation ； efficacy evaluation贻贝粘蛋白（mussel adhesive protein ， MAP ）也称作贻贝足丝蛋白（mussel foot protein ，Mfps ），收稿日期：2023⁃02⁃24； 接受日期：2023⁃03⁃31联系方式：李敏 E -mail:*******************;*通信作者 侯增淼 E -mail:***********************.cn李敏，等：重组贻贝粘蛋白的表征及功效评价是海洋贝类——紫贻贝（Mytilus galloprovincalis）、厚壳贻贝（Mytilus coruscus）、翡翠贻贝（Perna viri⁃dis）等分泌的一种特殊的蛋白质，贻贝中含有多种贻贝粘蛋白，包括贻贝粘蛋白（Mfp 1～6）、前胶原蛋白（precollagens）和基质蛋白（matrix proteins）等［1］。

血管外膜细胞钙化及其钙化机制研究谭小青,张旭升,樊小容,黄战军摘要 目的:研究经体外诱导钙化建立大鼠血管外膜细胞钙化模型,检测钙化过程中成骨相关指标及凋亡㊁自噬相关蛋白的表达变化,旨在为心血管疾病模型提供更精确的细胞模型,并初步探讨其钙化机制㊂方法:原代提取大鼠胸主动脉外膜纤维细胞,取3~6代细胞使用诱导培养基(高糖DMEM +10%胎牛血清+10mmol/L β-甘油磷酸+0.05mmol/L 抗坏血酸+100mmol/L 地塞米松)诱导钙化,诱导时间为3d ㊁6d ㊁9d ㊁12d ㊁15d ,筛选出诱导细胞钙化的最佳时间㊂对细胞采用茜素红S 染色㊁细胞内钙含量测定和碱性磷酸酶(ALP )活性检测,鉴定是否成功构建钙化模型㊂采用实时定量聚合酶链式反应(PT -PCR )检测成骨相关因子骨形态生成蛋白2(BMP2)和核心结合因子α1(Runx2)的mRNA 含量,蛋白免疫印迹法(Western Blot )检测凋亡蛋白Bax ㊁Bcl -2和自噬相关蛋白微血管相关蛋白(LC3)㊁Beclin -1的表达水平,找出血管外膜细胞钙化的潜在机制㊂结果:当诱导钙化时间为15d 时,血管外膜细胞中主要钙化指标胞内钙含量及ALP 活性上调(P <0.05),茜素红S 染色显示钙化组有明显钙盐沉积㊂血管外膜细胞经钙化诱导后,BMP2和Runx2的mRNA 水平上调,Bax 蛋白水平上调,Bcl -2和Beclin -1蛋白水平下调,LC3-Ⅱ/LC3-Ⅰ比值上调(P <0.05)㊂结论:钙化诱导培养基培养血管外膜细胞15d 可成功构建钙化细胞模型,血管外膜细胞钙化可能与细胞向成骨样表型转化有关,血管外膜细胞钙化过程涉及细胞自噬及凋亡调控㊂关键词 血管外膜细胞;钙化;成骨样表型转化;自噬与凋亡;实验研究d o i :10.12102/j.i s s n .1672-1349.2023.18.010 Calcification of Vascular Adventitial Cells and Its MechanismTAN Xiaoqing,ZHANG Xusheng,FAN Xiaorong,HUANG Zhanjun Longgang District People 's Hospital of Shenzhen,Shenzhen 518172,Guangdong,China Corresponding Author ZHANG Xusheng,E -mail:*****************Abstract Objective:To investigate the mananism of calcification of rat vascular adventitial cells,establish the calcification model of rat vascular adventitial cells,and detect the expression changes of osteogenesis -related indicators,apoptosis,and autophagy -related proteins during the calcification process.It aimed to provide more accurate cell models for cardiovascular disease and initially explore the mechanism of calcification.Methods:Rat thoracic aortic adventitial fibroblasts were extracted from the primary generation,and the 3rd to 6th generation cells were used for induction medium(high glucose DMEM +10%fetal bovine serum +10mmol/L β-glycerophosphate +0.05mmol/L ascorbic acid +100mmol/L dexamethasone)to induce calcification,the induction time was 3,6,9,12,and 15d,and the optimal time for inducing cell calcification was selected.The cells were stained with alizarin red S,detected by intracellular calcium content and alkaline phosphatase(ALP)to identify whether the calcification model was successfully constructed.Real -time quantitative reverse transcription polymerase chain reaction(RT -PCR)was used to detect the mRNA levels of osteogenesis -related factors bone morphogenetic protein(BMP2)and runt -related transcription factor 2(Runx2);Western Blot was used to detect the apoptosis proteins Bax,Bcl -2,the autophagy -related proteins LC -3,and Beclin -1expression level;then the potential mechanism of vascular adventitial cell calcification would be revealed.Results:When calcification was induced for 15days,the intracellular calcium content in the adventitial cells of the main calcification indicators and ALP activity were up -regulated(P <0.05).Alizarin red S staining showed obvious calcium deposits in the calcification group.After calcification was induced in adventitial cells,the mRNA levels of BMP2and Runx2up -regulated,the protein levels of Bax up -regulated,the protein levels of Bcl -2and Beclin -1down -regulated,and the ratio of LC3-Ⅱ/LC3-Ⅰdown -regulated(P <0.05).Conclusion:Adventitial cells cultured in the calcification -inducing medium for 15days could successfully construct a calcified cell model.calcification of adventitial cells might be related to the transformation of cells to an osteoblast -like phenotype.The Calcification process of adventitial cells involved autophagy and apoptosis regulation.Keywords adventitial cells;calcification;osteogenic phenotype transformation;autophagy and apoptosis;experimental study血管钙化常见于动脉粥样硬化㊁血脂异常㊁高血压㊁糖尿病㊁慢性肾病及衰老等人群[1],血管钙化引起血管硬度增加㊁顺应性降低,导致心肌缺血㊁心力衰竭㊁血栓形成等,增加脑卒中㊁心脏病㊁动脉粥样硬化斑块破裂等的风险,被认为是影响心血管疾病的重要因素之一[2-4]㊂目前关于血管内膜㊁中膜和心脏瓣膜钙化的关注和研究相对较多㊂临床工作中发现,血管外膜也可发生钙化,然而调查发现,现阶段对血管外膜钙化的作者单位 深圳市龙岗区人民医院(广东深圳518172)通讯作者 张旭升,E -mail :*****************引用信息 谭小青,张旭升,樊小容,等.血管外膜细胞钙化及其钙化机制研究[J ].中西医结合心脑血管病杂志,2023,21(18):3347-3350.关注较少,因此,需要更多的研究来阐明血管钙化的致病机制㊂最初血管钙化被认为是被动和退行性病变,标志着血管老化,但是越来越多研究表明血管钙化是类似于胚胎骨形成的病理生物学过程[5-6]㊂Bostr öm 等[7-8]研究发现,钙化过程中大鼠血管中膜细胞由原有收缩表型转变成为成骨样细胞表型,原有的收缩标志物如平滑肌肌动蛋白α(α-SMA )等表达减少,并表达核心结合因子α1(Runx2)㊁骨形态生成蛋白2(BMP2)等多种成骨样标志物,从而介导骨基质在血管中沉积㊂细胞凋亡与自噬为2种细胞死亡的方式,与血管钙化息息相关,研究表明,血管中膜细胞在细胞凋亡过程中释放凋亡小体,促进细胞钙化,而细胞自噬通过多种机制调控细胞钙化[9-10]㊂本研究对大鼠血管外膜细胞进行体外诱导钙化,建立大鼠血管外膜细胞钙化模型,并检测钙化过程中成骨相关指标及凋亡㊁自噬相关蛋白的表达变化,旨在为心血管疾病模型提供更精确的细胞模型,并初步探讨其钙化机制㊂1材料与方法1.1试剂胎牛血清(FBS,Gibco),青霉素,链霉素(Gibco,美国),茜素红S溶液,β-甘油磷酸,抗坏血酸,地塞米松(Sigma,美国),抗GAPDH抗体(Bioworld),抗Bcl-2, Bax,Bcelin1和微血管相关蛋白(LC3)抗体(CST),碱性磷酸酶检测试剂盒㊁钙(Ca)检测试剂盒(南京建城生物工程研究所)㊂1.2大鼠血管外膜细胞分离与培养取10只4~6周龄雄性Wistar-Kyoto大鼠(体质量120~180g)胸主动脉分离血管外膜,采用组织黏附法培养㊂使用添加10%胎牛血清的高糖DMEM培养基(Gibco dmem)在37ħ㊁5%二氧化碳条件下培养细胞㊂当细胞增殖至80%~90%融合时,用0.25%胰酶消化传代㊂使用第3代至第6代的细胞进行后续实验㊂1.3体外钙化模型的建立钙化诱导培养基为含10%胎牛血清,10mmol/L β-甘油磷酸钠,0.05mmol/L抗坏血酸和100mmol/L 地塞米松的高糖DMEM培养液㊂将第3代至第6代细胞分为对照组和钙化组,待细胞长至50%融合时,使用钙化诱导培养基培养,每3d更换1次培养基,连续培养15d㊂1.4碱性磷酸酶(ALP)酶活测定细胞钙化诱导后,弃去培养基,1ˑ磷酸缓冲盐溶液(PBS)洗细胞3次,加入裂解液500μL(1%T ritonX-100),冰上裂解40min后,离心,取上清液㊂使用上清液根据试剂盒说明书检测ALP活性及总蛋白含量㊂1.5细胞内钙含量检测细胞钙化诱导后,弃去培养基,1ˑPBS洗细胞3次,每孔加入500μL0.6mol/L的盐酸4ħ脱钙过夜,取上清,根据钙测试试剂盒说明书检测钙含量㊂将脱钙后的细胞用4ħPBS洗3次,每孔加入500μL NaOH/0.1%SDS裂解细胞,取上清,用二喹啉甲酸法(BCA)测定细胞蛋白含量㊂1.6茜素红S染色细胞钙化诱导15d,弃去培养基,1ˑPBS洗细胞3次,加入0.5mL4%多聚甲醛室温固定15min,用双蒸水洗3次,加入1mL0.1%茜素红室温孵育15min,吸去染液,双蒸水洗3次,在倒置显微镜下观察㊂1.7实时定量聚合酶链式反应(RT-PCR)检测细胞钙化诱导后,弃去培养基,1ˑPBS洗细胞3次,使用TaKaRa MiniBEST Universal RNA Extraction Kit提取总RNA,使用PrimeScrip TM RT reagent Kit将所提取的RNA逆转录合成cDNA,以cDNA为模板,通过SYBR Green I嵌合荧光定量RT-PCR检测BMP-2㊁Runx2和GAPDH的表达量㊂引物序列见表1㊂表1引物序列基因方向序列Runx2正向5'-TGGCTTTGGTTTCAGGTTAGG-3'反向5'-TGGAGATGTTGCTCTGTTCG-3' BMP-2正向5'-TGAGGATTAGCAGGTCTTTGC-3'反向5'-TCTCGTTTGTGGAGTGGATG-3' GAPDH正向5'-GGCTGCCCAGAACATCAT-3'反向5'-CGGACACATTGGGGGTAG-3'1.8蛋白免疫印迹法(Western Blot)检测细胞钙化诱导15d,弃去培养基,1ˑPBS洗细胞3次,提取细胞总蛋白㊂使用12%SDS-PAGE胶电泳分离,并转移到聚偏二氟乙烯膜(PVDF)上,封闭后,加入一抗(Bax1ʒ1000,Bcl-21ʒ1000,Beclin11ʒ1000, LC31ʒ1000,GAPDH1:1000)稀释液,4ħ孵育过夜;加入二抗稀释液(1ʒ10000)室温孵育1h后,使用ECL发光试剂盒显影并计算灰度值㊂1.9统计学处理应用SPSS19.0软件进行统计处理,符合正态分布的定量资料以均数ʃ标准差(xʃs)表示,比较采用t检验,以P<0.05为差异有统计学意义㊂2结果2.1大鼠血管外膜细胞可在体外被诱导钙化为验证高磷是否能诱导大鼠血管外膜细胞钙化,使用钙化诱导培养基培养细胞,在不同时间点检测ALP活性和胞内钙含量㊂随着培养时间延长,ALP活性逐渐上升,在培养第12天达到峰值,与对照组比较差异有统计学意义(P<0.05);诱导第3天开始所测得的胞内钙含量与对照组比较升高(P<0.05),ALP 活性和钙含量升高具有时间依赖性㊂详见图1㊁图2㊂诱导15d所测得钙含量最高,因此,后续实验选择的诱导时间为15d㊂对钙化诱导15d的细胞进行茜素红S染色,结果显示,对照组细胞呈长梭形,而钙化组细胞变成菱形㊂茜素红S染色后,钙化组可观察到大量的橘红色钙结节(见图3),而对照组完全没有㊂这也证明大鼠血管外膜细胞可在体外被钙化培养基诱导钙化㊂图1钙化诱导培养基诱导外膜细胞后ALP含量(与0d时比较,*P<0.05)图2钙化诱导培养基诱导外膜细胞后胞内钙含量(与0d时比较,*P<0.05)图3培养15d时细胞经茜素S红染色切片图(ˑ100)2.2血管外膜细胞钙化与细胞向成骨样表型转化有关血管钙化的增加与成骨细胞特异性标志物如BMP2㊁和Runx2的增加有关[11]㊂RT-PCR结果显示,与对照组比较,钙化组的成骨细胞特异性标志物BMP2和Runx2mRNA表达量增加,与对照组比较差异有统计学意义(P<0.05)㊂详见图4㊂图4外膜细胞钙化过程中BMP2和Runx2mRNA表达量(与对照组比较,*P<0.05)2.3血管外膜细胞钙化过程涉及细胞自噬及凋亡调控通过Western Blot检测凋亡和自噬相关蛋白的表达量变化㊂与对照组比较,钙化组促凋亡蛋白Bax表达上调,抑凋亡蛋白Bcl-2表达下调(P<0.05)㊂详见图5㊂钙化组自噬相关蛋白Beclin1表达上调,LC3-Ⅱ/ LC3-Ⅰ比例上调(P<0.05),说明钙化诱导培养后细胞内凋亡水平上调㊁自噬水平升高㊂详见图6㊂图5诱导钙化后促凋亡蛋白及抑凋亡蛋白表达变化图6诱导钙化后凋亡及自噬蛋白Beclin1等表达变化3讨论血管钙化作为心血管疾病病人的并发症之一,其发病率与严重程度逐年增高及加重,是导致心血管疾病病人高死亡率的重要因素㊂血管钙化缺乏有效的治疗药物㊂因此,探究血管钙化发病机制,在分子水平寻找有效的诊断和防治靶点是急需开展的基础研究工作㊂本研究证明,使用10mmol/Lβ-甘油磷酸+0.05 mmol/L抗坏血酸+100mmol/L地塞米松培养外膜细胞即可诱导大鼠血管外膜细胞在体外发生钙化,这是通过茜素红S染色㊁ALP活性检测及胞内钙含量检测结果得以确定的㊂血管钙化过程中,血管中膜细胞向成骨样细胞表型转变并表达相关成骨标志物,从而引起骨基质的沉积,是血管钙化的重要特点及机制[5]㊂本实验所用的血管外膜细胞钙化条件与血管中膜细胞钙化条件一致,说明血管外膜细胞钙化的机制可能与中膜细胞钙化的机制部分一致㊂血管中膜细胞钙化过程中,细胞表达成骨相关的转录因子如Runx2等,进而促进下游表达骨相关蛋白如骨形态发生蛋白BMP2等的表达,从而促使细胞向成骨样细胞主动分化[12-13],本研究也观察到类似的机制㊂通过PT-PCR检测,发现钙化培养基培养大鼠血管外膜细胞15d后,BMP2和Runx2的mRNA表达水平升高㊂本研究通过对钙盐沉积与成骨样细胞表型转变2个维度的探讨,证明血管外膜细胞可在体外被诱导钙化,丰富了血管钙化的分型㊂血管钙化的发生机制复杂,涉及多种信号通路,如细胞自噬和凋亡㊁Wnt/β-catenin信号通路激活㊁内质网应激等均参与调控血管钙化的过程㊂自噬作为一种细胞应激的适应性反应,在维持血管结构与功能中十分关键㊂研究表明,血管钙化过程中自噬水平增高[14-15]㊂在体外实验中,高磷可提高大鼠血管中膜细胞的自噬水平,增加细胞内自噬体数量,从而抑制凋亡与钙化[16]㊂还有研究表明,自噬可通过抑制大鼠血管中膜细胞氧化应激,抑制血管内皮细胞的炎症反应,对三酰甘油等脂代谢进行调控,从而减轻血管钙化[17-18]㊂LC3和Beclin1是2种典型的自噬标志物,Western Blot实验结果表明,用钙化培养基诱导大鼠血管外膜细胞15d,LC3-Ⅱ/LC3-Ⅰ比率升高,Beclin1蛋白水平表达升高,说明细胞内自噬水平升高㊂多项研究表明,细胞凋亡参与促进血管钙化的发生,抑制细胞凋亡和抑制钙化[16-17]㊂在对大鼠的体内研究发现,成纤维细胞生长因子21通过内质网应激调控Caspase-12信号通路来减少血管内中膜细胞凋亡,从而抑制血管钙化[18]㊂另外,提高培养基中的Pi 或Ca2+浓度,可诱导细胞质膜形成并释放基质囊泡(如凋亡小体),从而导致细胞外基质钙化,这种基质钙化可能成为血管钙化的成核位点[19]㊂Bax和Bcl-2是2种典型的凋亡和抑制凋亡蛋白,本实验结果证明,利用钙化培养基对血管外膜细胞诱导钙化过程中,细胞内凋亡水平升高㊂同时细胞内自噬水平也升高,这可能是细胞自我调控以对抗钙化的结果㊂本研究证实血管外膜细胞可在体外被诱导钙化,且外膜钙化过程与骨组织钙化过程类似,为主动可调控的过程㊂血管钙化是一个复杂的过程,涉及细胞凋亡和自噬等调控通路,仍需进一步研究㊂参考文献:[1]梁英权,段亚君,韩际宏.血管钙化分子机制研究进展[J].中国动脉硬化杂志,2020,28(11):921-929.[2]NICOLL R,HENEIN M Y.The predictive value of arterial andvalvular calcification for mortality and cardiovascular events[J].Int J Cardiol Heart Vessel,2014,3:1-5.[3]JOHNSON R C,LEOPOLD J A,LOSCALZO J.Vascularcalcification:pathobiological mechanisms and clinical implications[J].Circulation Research,2006,99(10):1044-1059.[4]YAMADA S,GIACHELLI C M.Vascular calcification in CKD-MBD:roles for phosphate,FGF23,and Klotho[J].Bone,2017,100:87-93.[5]LIN M E,CHEN T M,WALLINGFORD M C,et al.Runx2deletion insmooth muscle cells inhibits vascular osteochondrogenesis andcalcification but not atherosclerotic lesion formation[J].Cardiovascular Research,2016,112(2):606-616.[6]DURHAM A L,SPEER M Y,SCATENA M,et al.Role of smoothmuscle cells in vascular calcification:implications in atherosclerosis andarterial stiffness[J].Cardiovascular Research,2018,114(4):590-600.[7]BOSTRÖM K I,RAJAMANNAN N M,TOWLER D A.The regulationof valvular and vascular sclerosis by osteogenic morphogens[J].Circulation Research,2011,109(5):564-577.[8]SPEER M Y,YANG H Y,BRABB T,et al.Smooth muscle cells giverise to osteochondrogenic precursors and chondrocytes incalcifying arteries[J].Circulation Research,2009,104(6):733-741.[9]PROUDFOOT D,SKEPPER J N,HEGYI L,et al.Apoptosisregulates human vascular calcification in vitro:evidence forinitiation of vascular calcification by apoptotic bodies[J].Circulation Research,2000,87(11):1055-1062.[10]AN S J,BOYD R,ZHU M,et al.NADPH oxidase mediatesangiotensin II-induced endothelin-1expression in vascularadventitial fibroblasts[J].Cardiovascular Research,2007,75(4):702-709.[11]ZEADIN M,BUTCHER M,WERSTUCK G,et al.Effect of leptin onvascular calcification in apolipoprotein E-deficient mice[J].Arterioscler Thromb Vasc Biol,2009,29(12):2069-2075. [12]LEOPOLD J A.Vascular calcification:mechanisms of vascularsmooth muscle cell calcification[J].Trends in CardiovascularMedicine,2015,25(4):267-274.[13]刘聿秀.高尿酸诱导血管钙化的机制研究[D].青岛:青岛大学,2015.[14]LIU Q,LUO Y,ZHAO Y,et al.Nano-hydroxyapatite acceleratesvascular calcification via lysosome impairment and autophagydysfunction in smooth muscle cells[J].Bioact Mater,2022,8:478-493.[15]LIANG J,HUANG J,HE W,et al.β-Hydroxybutyric Inhibits vascularcalcification via autophagy enhancement in models induced byhigh phosphate[J].Front Cardiovasc Med,2021,8:685748. [16]CICERI P,ELLI F,CAPPELLETTI L,et al.A new in vitro model todelay high phosphate-induced vascular calcification progression[J].Mol Cell Biochem,2015,410(1/2):197-206.[17]BYON C H,JAVED A,DAI Q,et al.Oxidative stress inducesvascular calcification through modulation of the osteogenictranscription factor Runx2by AKT signaling[J].The Journal ofBiological Chemistry,2008,283(22):15319-15327.[18]OUIMET M,FRANKLIN V,MAK E,et al.Autophagy regulatescholesterol efflux from macrophage foam cells via lysosomal acidlipase[J].Cell Metabolism,2011,13(6):655-667.[19]REYNOLDS J L,JOANNIDES A J,SKEPPER J N,et al.Humanvascular smooth muscle cells undergo vesicle-mediatedcalcification in response to changes in extracellular calcium andphosphate concentrations:a potential mechanism for acceleratedvascular calcification in ESRD[J].Journal of the AmericanSociety of Nephrology,2004,15(11):2857-2867.(收稿日期:2022-03-30)(本文编辑王雅洁)。