doi10.1093comjnlbxl053 Describing Use-Case Relationships with Sequence Diagrams

CHEMICAL INDUSTRY AND ENGINEERING PROGRESS 2017年第36卷第2期·548·化 工 进 展离子液体及负载型钯催化剂在加氢反应中的研究进展张文林，张佳莉，靳斐，高展艳，陈瑶，李春利（河北工业大学化工学院，化工节能过程集成与资源利用国家地方联合工程实验室，天津 300130） 摘要：离子液体因其独特的溶剂性能、催化性能及结构可设计性，在催化体系中可作溶剂、催化活性中心、稳定剂、分散剂等。

负载型钯催化剂具有比表面积大、金属分散性和热稳定性好等优点，但存在着催化剂对目标产物选择性差、成本高、反应机理尚不明确等问题。

本文以苯酚选择性加氢为探针反应，综述了该反应对催化剂的要求及贵金属钯的优势。

将苯酚加氢催化剂分为无机负载钯催化剂、聚合物负载钯催化剂和离子液体-聚合物负载钯催化剂三类，并分析了载体性质、助剂、离子液体、钯盐等对催化性能的影响。

分析表明：具有一定规则微观形貌、含P 、N 等元素的非多孔性多官能团碱性载体催化效果较好，且载体中含有较多碱性中心，有利于催化剂活性和选择性的提高；助剂Na 、K 、Al 、Ni 、Ca 、Cs 等的加入可在降低成本的同时提高催化性能；钯盐Pd(OAc)2加氢性能优于Pd(acac)2、PdCl 2、Pd(NO 3)2；离子液体的引入不仅使反应体系易分离、反应条件降低，而且提高了催化剂活性和选择性。

今后的主要发展方向是深入研究离子液体在催化加氢过程中的作用、催化加氢机理、催化剂稳定性等。

关键词：离子液体；钯基催化剂；载体；加氢；选择性中图分类号：TQ032.4 文献标志码：A 文章编号：1000–6613（2017）02–0548–07 DOI ：10.16085/j.issn.1000-6613.2017.02.021Progress of ionic liquids and supported palladium catalysts in thehydrogenation reactionsZHANG Wenlin ，ZHANG Jiali ，JIN Fei ，GAO Zhanyan ，CHEN Yao ，LI Chunli（National-Local Joint Engineering Laboratory for Energy Conservation of Chemical Process Integration and ResourcesUtilization ，School of Chemical Engineering ，Hebei University of Technology ，Tianjin 300130，China ）Abstract ：Ionic liquids can be used as solvents ，catalytic active sites ，stabilizers ，dispersants et al owing to their unique solvent performances ，catalytic performances and designable structures. Supported palladium catalysts have many advantages such as high specific surface area ，metal dispersion and good thermal stability ，as well as problems of low selectivity of target selectivity ，high cost and unclear reaction mechanism. Taking selective hydrogenation of phenol as the probe reaction ，this paper summarizes the requirements for the catalysts and the advantages of palladium. Hydrogenation catalysts of phenol are divided into inorganic supported Pd catalysts ，polymer supported Pd catalysts and ionic liquid-polymer supported Pd catalysts. The influences of carriers ，ionic liquids ，palladium salts ，additives on the catalytic performance are analyzed. The results show that non-porous ，polyfunctional alkaline carriers with certain microstructures ，and certain elements such as P ，N have better catalytic performance. And carriers with more basic sites are more likely to have higher activity and selectivity. Additives such as Na 、K 、Al 、Ni 、Ca 、Cs et al can improve the catalytic performance while reducing costs. The hydrogenation performance of Pd(OAc)2 is better than that of the other three. The application of ILs makes it easier to separate the catalysts from the reaction system ，relieves the第一作者及联系人：张文林（1968—），男，教授，主要从事分离与纯化技术以及绿色化工方面的研究。

激素依赖性皮炎的研究进展沈旭成1,2张怡1谢志敏1王艳芳1叶兴东11广州医科大学皮肤病研究所广州市皮肤病防治所皮肤科510095；2佛山市第二人民医院皮肤科528000通信作者：叶兴东，Email：【摘要】激素依赖性皮炎概念尚未统一，通常指不恰当的外用糖皮质激素制剂治疗皮肤疾病后原有皮损消退，但停药后复发或加重，患者不得不继续使用激素的一种亚急性皮肤炎症，临床典型表现为灼热、刺痒、局部干燥紧缩感，该病发病率逐渐增高，发病机制尚未十分明确，需进一步研究探讨；本文就激素依赖性皮炎概念、动物模型研究、发病机制、病理生理、临床特点及治疗进行综述。

【关键词】激素依赖性皮炎；动物模型；研究进展Research progress of corticosteroid dependent dermatitisShen Xucheng1,2,Zhang Yi1,Xie Zhimin1,Wang Yanfang1,Ye Xingdong11Department of Dermatology,Guangzhou Institute of Dermatology,Dermatology Institute of Guangzhou Medical University,Guangzhou510095,China;2Department of Dermatology,The Second People's Hospital of Foshan,Foshan528000,ChinaCorresponding author:Ye Xingdong,Email:【Abstract】The concept of corticosteroid dependent dermatitis has not been unified.The disease is subacute skin inflammation caused by the abuse of corticosteroids,usually refers to inappropriate topical glucocorticoid preparations for the treatment of skin diseases,which makes the original skin lesions temporarily subsided,but relapses or worsens after withdrawal,so that patients have to continue to use glucocorticoids.Its clinical manifestations are burning,itching,local dryness and tightness,the incidence of the disease is gradually increasing,its pathogenesis is not yet clear,which needs further research.This article reviews the concept of disease,animal model research,pathogenesis,pathophysiology,clinical features,and treatment.【Key words】Corticosteroid dependent dermatitis;Animal model;Research progress激素依赖性皮炎（corticosteroid dependent dermatitis，CDD），顾名思义，是指治疗炎症性皮肤病的过程中，为控制临床症状而不得不继续外用激素制剂的一种临床现象。

doi: 10.11978/2022266 南海热带岛礁生物土壤结皮中细菌的分离及其固砂特性初步研究黄谕1, 2, 王琳1, 麦志茂1, 李洁1, 张偲1, 31. 中国科学院热带海洋生物资源与生态重点实验室(中国科学院南海海洋研究所), 广东广州 510301;2. 中国科学院大学, 北京 100049;3. 南方海洋科学与工程广东省实验室(广州), 广东广州 511458摘要: 南海热带岛礁生物土壤结皮中有大量可分泌胞外多糖的微生物, 这一特性对固砂具有重要的促进作用。

本研究利用TSA、改良TSA、MA三种培养基对采自南海永暑礁及三亚鹿回头海岸带的生物土壤结皮进行可培养细菌的分离纯化。

本研究共分离纯化70株细菌, 并进行基于16S rRNA基因序列的鉴定, 发现所分离的菌株属于3门5纲12目19科25属, 厚壁菌门(Firmicutes)为优势门, 芽孢杆菌属(Bacillus)为优势属。

此外, 有22株菌与已知物种16S rRNA基因相似性低于98.65%, 为潜在的新物种。

纯化菌株的胞外多糖采用乙醇沉淀法提取、苯酚硫酸法检测含量, 发现19株菌胞外多糖含量高于0.013mg·mL−1。

选取其中胞外多糖含量最高的9株菌进行土壤团聚能力的测定, 发现将菌株SCSIO 17111(Lysobacter sp.)菌液喷洒于珊瑚砂表面能够使土壤团聚体保持稳定。

针对粒径小于0.2mm的细砂, 菌株SCSIO 17111固结的砂土在经过干筛处理后, 保留量可达92.7%。

本研究可为岛礁珊瑚砂的固定、减少土壤流失提供高产胞外多糖菌株资源, 还可以为促进岛礁生物土壤结皮的形成提供新的研究思路和方法。

关键词: 南海岛礁; 可培养细菌多样性; 胞外多糖; 固砂中图分类号: S154.3 文献标识码: A 文章编号: 1009-5470(2023)06-0101-10Isolation and characterization of sand fixation ability of bacteria in biological soil crusts of the tropical islands, South China SeaHUANG Yu1, 2, WANG Lin1, MAI Zhimao1, LI Jie1, ZHANG Si1, 31. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, ChinaAbstract: There are many microbial resources present in biological soil crusts of tropical reefs in the South China Sea, and the extracellular polysaccharides secreted by these microorganisms play an important role in promoting sand consolidation. In this study, three media, TSA, modified TSA and MA, were used to isolate and purify culturable bacteria from biological soil crusts in the Yongshu Reef and Luhuitou, Sanya, South China Sea. A total of 70 bacterial strains were isolated and purified in this study. And based on 16S rRNA gene identification, these isolated strains belonged to 3 phyla, 5 classes, 12 orders and 19 families, 25 genera. The dominant phylum was Firmicutes and the dominant genus was Bacillus. In addition, 22 strains had less than 98.65%16S rRNA gene similarities with known species, which were potential novel species. The extracellular polysaccharide content of purified strains was measured by ethanol precipitation method and phenol sulfate method, and the contents of 19 strains were more than 0.013收稿日期：2022-12-29; 修订日期：2023-02-27。

Compare drug release profiles of water poor soluble drugs from a novel chitosan and polycarbophil interpolyelectrolytes complexation (PCC) and hydroxylpropyl - methylcellulose (HPMC) based matrix tabletsZhilei Lu*, Weiyang Chen, Eugene Olivier, Josias H., HammanDepartment of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, P retoria, 0001, South Africa资料个人收集整理，勿做商业用途*Corresponding author:Zhilei Lu (Dr.)Department of Pharmaceutical Sciences,Tshwane University of Technology,Private Bag X680,Pretoria, 0001,South Africa (e-mail: luzj@tut.ac.za)AbstractThe aim of this study was to compare the drug release behaviours of water poor soluble drugs from an interpolyelectrolyte complex (IPEC) of chitosan with polycarbophil (PCC) and hydroxylpropylmethylcellulose (HPMC) based matrix tablets. A novel interpoly - electrolyte complex (IPEC) of chitosan with polycarbophil (PCC) was synthesized and characterized. Water poor soluble drugsHydrochlorothiazide and Ketoprofen were used in this study as model drugs.Polymers (including PCC, HPMC K100M and HPMC K100LV) based matrix tablets drug controlled release system were prepared using direct compression method.The results illustrate PCC based-matrix tablets offer a swelling controlled release system for water poor soluble drug and drug release mechanism from this matrix drug delivery system can be improved by addition of microcrystalline cellulose (Avicel).Analysis of the in vitro release kinetic parametersof the matrix tablets, PCC based matrix tablets exhibited similar or higher drug release exponent (n) and mean dissolution time (MDT) values compared to the HPMC based matrix tablets. It demonstrated that PCC polymer can be successfully used as a matrixcontrolled release system for the water poor soluble model drugs such as hydroxylpropylmethylcellulose (HPMC). 资料个人收集整理，勿做商业用途1 IntroductionOver the last three decades years, as the expense and complications involved in marketing new drug entities have increased, with concomitant recognition of the therapeutic advantages of controlled drug delivery, greater attention has been focused on the development of novel and controlled release drug delivery systemsto provide a long-term therapeutic of drugs at the site of action following a single dose (Mandal, 2000; Jantzen and Robinson, 2002). Many formulation techniques have been used tobuild t”he barrier into the peroral dosage form to provide slow release of the maintenance dose. These techniques include the use of coatings, embedding of the drug in a wax, polymeric or plastic matrix, microencapsulation, chemical bindingto ion-exchange resins and incorporation into an osmotic pump (Collett and Moreton, 2002:293). Among different technologies used in controlled drug delivery, polymeric matrix systems are the most majority because of the simplicity of formulation, ease of manufacturing, low cost and applicability to drugs with wide range of solubility (Colombo, et al., 2000; Jamzad and Fassihi, 2006). 资料个人收集整理，勿做商业用途Drugs release profiles from polymeric matrix system can influence by different factors, but the type, amount, and physicochemical properties of the polymers used play a primary role (Jamzad and Fassihi, 2006). Hydroxylpropyl-methylcellulose (HPMC) is the most important hydrophilic carrier material used for oral drug sustained delivery systems (Colombo, 1993; Siepmann and Peppas, 2001). BecauseHPMC is water soluble polymer, it is generally recognized that drug release fromHPMC matrices follows two mechanisms, drug diffusion through the swelling gellayer and release by matrix erosion of the swollen layer (Ford et al., 1987; Raoet al., 1990; Colombo, 1993; Tahara et al., 1995; Reynolds, Gehrke et al., 1998; Siepmann et al., 1999; Siepmann and Peppas, 2001). However, diffusion, swelling and erosion are most important rate-controlling mechanisms of commercial available controlled release products (Langer and Peppas, 1983), the major advantages of swelling/erosionHPMC based matrix drug delivery system are: (i) minimum the drug burst release; (ii) the different physicochemical drugs release rate approach a constant; (iii) the possibility to predict the effect of the device design parameters (e.g. shape, size and composition of HPMC-based matrix tablets) on the resulting drug release rate, thus facilitating the development of new pharmaceutical products (Colombo, 1993;Siepmann and Peppas, 2001资).料个人收集整理，勿做商业用途Interpolyelectrolyte complexes (IPEC) are formed as precipitates by two oppositely charged polyelectrolytes in an aqueous solution, have been reported as a new class of polymer carriers, which play an important role in creating new oral drug delivery systems (Peppas and Khare, 1993; Berger et al., 2004). A variety chemical structure and stoichiometry of both components in interpolyelectrolyte complexes depends onthe pH values of the media, ionic strength, concentration, mixing ratio, and temperature (Peppas, 1986; Dumitriu and Chornet, 1998; Berger et al., 2004;Moustafine et al., 2005a). Chitosan is a positively charged (amino groups) deacetylated derivative of the natural polysaccharide, chitin (Paul and Sharma, 2000).Chitosan has already been successfully used to form complexes with natural anionic polymers such as carboxymethylcellulose, alginic acid, dextran sulfate,carboxymethyl dextran, heparin, carrageenan,pectin methacrylic acid copolymers ? (Eudragit polymers) and xanthan (Dumitriu and Chornet, 1998, Berger et al., 2004,Sankalia et al., 2007, Margulis and Moustafine, 2006)资. 料个人收集整理，勿做商业用途In this study, a novel polymer - IPEC between chitosan and polycarbophil (PCC) was synthesized, characterized and used as direct compressedexcipients in the matrix tablet. Although it have been well known that various IPEC have been used as a polymer carriers in drug controlled release system (Peppas and Khare, 1993, Garcia and Ghaly, 1996, Lorenzo-Lamoza et al., 1998, Soppirnath and Aminabhavi, 2002,Chen et al., 2004, Nam et al., 2004, Moustafine et al., 2005b), IPEC chitosan and polycarbophil was used as a polymer carriers have been investigated by Lu et al., (2006, 2007a, 2007 b, 2008a; 2008b资料个人收集整理，勿做商业用途The aim of this study was to comparein vitro water poor soluble drugs release profile of HPMC based matrices system to PCC based matrices system at same formulation.Water poor soluble model drugs Hydrochlorothiazide and ketoprofen were used in thisstudy. Two types HPMC (K100M and K100LV) and PCC polymers were used indirect compressedpolymers based matrix drug release system. The results of the hydration and erosion studies showed PCC based matrix systems have superior swelling properties. Drug release exponent (n) of each formulation PCC based matrices tablets are higher than HPMC based matrices tablets at pH 7.4 buffer solutions. It demonstrated that PCC has high potential to use in polymer based matrix drug con trolled released delivery for water poor soluble drugs资料个人收集整理，勿做商业用途2. Materials and methods2.1 MaterialsChitosan (Warren Chem Specialities, South Africa, Deacetylation Degree =91.25%),Polycarbophil (Noveon, Cleveland, USA), Hydroxylpropylmethylcellulose (MethocelK100M, K100LV Premium, Colorcon Limited, Kent, England), Ketoprofen (Changzhou Siyao Pharma. China), Hydrochlorothiazide (Huzhou Konch Pharmaceutical Co., Ltd. China), Microcrystalline cellulose (Avicel, pH101, FMC corporation NV, Brussels, Belgium), Sodium carboxymethyl starch (Explotab, Edward Mendell Co., Inc New York, USA). All other chemicals were of analytical grade and used as receive资料个人收集整理，勿做商业用途2.2 Preparation of interpolyelectrolytes complexation between chitosan and P olycarb op hil (PCC)资料个人收集整理，勿做商业用途Chitosan (30 g) was dissolved in 1000 ml of a 2% v/v acetic acid solution andpolycarbophil (30 g) was dissolved in 1000 ml of a 2% v/v acetic acid solution. Thechitosan solution was slowly added to the polycarbophil solution underhomogenisation (5200 rpm, ZKA , Germany) over a period of 20 minutes. Themixture was then mechanically stirred for a period of 1 hour at a speed of 1200 rpm(Heidolph RZR2021, Germany). The gel formed was separated by centrifuging for 5 min at 3000 rpm and then washed several times with a 2% v/v acetic acidsolution toremove any unreacted polymeric material. The gel was freeze dried for a period of 48 hours (Jouan LP3, France) and the lyophilised powder was screened through a 300prn sieve资料个人收集整理，勿做商业用途2.3Differential scanning calorimetry (DSC)DSC thernograns of the PCC were recorded with a Shinadzu DSC50 (Kyoto, Japan) instrument. The thermal behaviour was studied by sealing 2 mg samples of the material in aluminium crimp cells and heating it at a heating rate of 10o C per min under the flow of nitrogen at a flow rate of 20 ml/min. The calorimeter was calibrated with 2 mg of indium (Kyoto, Japan, melting point 156.4o C) at a heating rate of 10o C per min.资料个人收集整理，勿做商业用途2.4Fourier transforn infrared (FT-IR)Fourier transforn infrared (FT-IR) spectral data of the PCC polyner was obtained on a FTS-175C spectrophotoneter (BIO-RAD, USA) using the KBr disk nethod. 资料个人收集整理，勿做商业用途2.5Preparation of the natrix tabletsIn order to conpare the release profiles of water poor soluble drugs fron polyner based natrix tablets, nonolithic natrix type tablets containing hydrochlorothiazide or ketoprofen were prepared by conpressing a nixture of the ingredients with varying concentrations of the PCC, HPMC K100M and HPMC K100LV as indicated in Table 1. The ingredients of the different fornulationswere nanually pre-nixed by stirring in a 1000 nl glass beaker for 30 ninutes with a spatula. After the addition of 0.05 g of nagnesiun stearate (0.5% w/w), the powder nass was nixed for 10 nin. The powdernixture was conpressed using a rotating tablet press (Cadnach, India) fitted with round, shallow pun ches to p roduce matrix type tablets with a 6 mm diameter资料个人收集整理，勿做商业用途26 Weight, hard ness, thick ness and friability of tablets资料个人收集整理，勿做商业用途Weight variation was tested by weighing 20 randomly selected tablets individually, the n calculati ng the average weight and comparing the in dividual tablet weights to the average. The specification for weight variation is 10% from the average weight if the average weight < 0.08 g (USP 2006资料个人收集整理，勿做商业用途The hardnessof ten randomly selected matrix type tablets of each formulation was determined using a hardness tester (TBH 220 ERWE K A, Germany). The force (N) n eeded to break each tablet was recorded料个人收集整理，勿做商业用途The thick ness of each of 10 ran domly selected matrix type tablets were measured witha vernier calliper (accuracy = 0.02 mm). The thickness of the tablet should be within 5% variation of the average value资料个人收集整理，勿做商业用途A friability test was con ducted on the tablets using an Erweka Friabilator (TA3R,Germany). Twenty matrices were randomly selected from each formulation and any loose dust was removed with the aid of a soft brush. The selected tablets were weighed accurately and p laced in the drum of the friabilator. The drum was rotated at 25 rpm for 4 minu tes after which the matrices were removed. Any loose dust was removed from the matrices before they were weighed again. The friability maximal limit is 1% (USP 2006) was calculated using the following equation资料个人收集整理，勿做商业用途F (%) = W before (g)「W曲(g)X 100%(1)W after (g)Where F is the friability, W before is the initial weight of the matrices and W after is the weight of the matrices after test ing资料个人收集整理，勿做商业用途2.7 Swelli ng and erosi on studiesSwelling and erosion studies were carried out for all formulations matrix tablets. The matrices were weighed in dividually before they were pl aced in 900 ml p hos phate buffer (pH 7.4) at 37.0 0.寸C.± The medium was stirred with a paddle at a rotation speed of 50 rpm in a USP dissolution flask. At each time point, three tablets of each formulatio n were removed from the dissoluti on flask and gen tly wiped with a tissue toremove surface water, weighed and then placed into a plastic bowel. The matrix tablets were dried at 60°C until constant weight was achieved. The mean weights were determ ined for the three tablets at each time in terval. The data obta ined from this exp erime nt was used to calculate the swelli ng in dex and p erce ntage mass loss.料个人收集整理，勿做商业用途2.7.1Swelli ng indexThe swelli ng in dex (or degree of swelli ng) was calculated accord ing to the followi ngequati on资料个人收集整理，勿做商业用途s,=WJ—00W dWhere SI is the swelling index, W s and W d are the swollen and dry matrix weights, resp ectively, at immersio n timet in the buffer soluti on.资料个人收集整理，勿做商业用途2.7.2P erce ntage of matrix erosi onThe p erce ntage of matrix erosi on is calculated in relatio n to the in itial dry weight of the matrices, accord ing to the followi ng equation 资料个人收集整理，勿做商业用途Erow 册件"00%Where: dry weight (t) is the weight at time t.28 Assay of hydrochlorothiazide and ket oprofen in matrix tablets.料个人收集整理，勿做商业用途The drug content of the matrix type tablets was determ ined by crush ing 10 ran domly selected tablets from each formulatio n in a mortar and p estle. App roximately 80 mg po wder from the hydrochlorothiazide or ket oprofen containing matrices were weighed accurately and individually transferred into a 200 ml volumetric flasks, which were then made up to volume with p hos phate buffer soluti on (pH 7.4). This mixture was stirred for 30 minutes to allow compiete release of the drug. After filtration through a 0.45 阿filter membrane, the solution was assayed using ultraviolet (UV) spectrophotometry (Helios a Thermo , England) at a wavelength of 271 nm for hydrochlorothiazide and 261 nm for ketoprofen. The assay for drug content wasperformed in triplicate for each formulation. The percentage drug content of the tablets was calculated by mea ns of the followi ng equation:料个人收集整理，勿做商业用途DC (% w/w^W dru^x100%WmtWhere DC is the drug content, W drug is the weight of the drug and W mt is the weight of the matrix tablet .资料个人收集整理，勿做商业用途2.9 Release an alysisThe USP (2006) dissoluti on app aratus 2 (i.e. p addle) was used to determ ine the in vitro drug release from the different polymers based matrix tablets. The dissolution medium (900 ml) consisted of phosphate buffer solution (pH 7.4) at 37 0.5 o C and a ± rotation speed of 50 rpm was used. Three hydrochlorothiazide or ketoprofen matrix tablets of each formulatio n were in troduced into each of three dissoluti on vessels (i.e.?in triplicate) in a six station dissolution apparatus (TDT-08L, Electrolab , India).Samp les (5 ml) were withdraw n at sp ecially in tervals, and 5 ml of p reheated dissolution medium was replaced immediately. Sink conditions were maintained throughout the study. The samp les were filtered through a 0.45 阿membra ne, hydrochlorothiazide or ketoprofen content in the solution was determined using ultraviolet (UV) spectrop hotometry at a wavele ngth of 271 or 261 nm, res pectively.An alyses were p erformed in tripli cate资料个人收集整理，勿做商业用途2.9.1 Kin eticCon trolled release drug delivery systems may be classified accord ing to their mecha ni sms of drug release, which in cludes diffusi on-con trolled, dissoluti on con trolled, swelli ng con trolled and chemically con trolled systems (La nger et al., 1983). Drug release from sim pie swellable and erosi on systems may be described by the well-known power law expression and is defined by the following equation(Ritger and Pepp as, 1987; P illay and Fassihi, 1999资料个人收集整理，勿做商业用途Where M t is the amount of drug released at time t, M is the overall amount of drug released, K is the release con sta nt; n is the release or diffusi onal exponent; M/M is the cumulative drug concen trati on released at time t (or fractio nal drug release)料个人收集整理，勿做商业用途The release exponent (n) is used for the in terpretati on of the release mecha nism from poly meric matrix con trolled drug release systems (Peppas 1985). For the case of < 0.45 corrosFickdantdiffusi on release (Case I<an89homalous (non-Fickia n) transport, n = 0.89 toa zero-order (Case II) release kin etics, and n > 0.89to a super Case II transport (Ritger and Pepp as, 1987资料个人收集整理，勿做商业用途 The dissoluti on data were modelled by using the Po wer law equati on (Eq 7) withgraphs analysis software (Origin Scientific Graphing and Analysis software, Version 7, Origi nLab Corpo rati on, USA) using the Gaussia n-Newt on (Leve nberg-Hartely) app roach 资料个人收集整理，勿做商业用途2.9.2 Mea n dissolutio n time (MDT)MDT is a statistical moment that describes the cumulative dissolution process and provides a quantitative estimation of the drug release rate. It is defined by the following equation (Reppas and Nicolaides, 2000; Sousa^t al., 2002):资料个人收集整理，勿 做商业用途nMDTt i M t/M^ i =±Where MDT is the mean dissolution time, M t is the amount of the drug released at time t; t i is the time (min) at the midpoint between i and i-1 and M 乂 is the overall amount of the drug released.料个人收集整理，勿做商业用途cyli ndrical, i n sp ecially, n diffusi on al), 0.45 < n2.9.3 Differe nt factor f i and Similarityfactor f 2 The different factor f i is a measure of the relative error between two dissoluti on curves and the similarity factor f 2 is a measure of similarity in the p erce ntage dissoluti on betwee n two dissoluti on curves (Moore and Fla nn er, 1996). Assu ming that the p erce ntage dissolved values for two p rofiles cannot be higher tha n 100, the differe nt factor f 1 can have values from 0 (whe n no differe nee the two curves exists) to 100 (when maximum differenee exists). With the same assumption holding, the similarity factor f 2 can have values from 100 (when no differenee between the two curves exists) to 0 (when maximum differenee exists) (Pillay and Fassihi, 1999;Moore and Fla nn er, 1996; Re ppas and Nicolaides, 2000). In this study, these factors were used to confirm the relative of release p rofiles of water poor soluble model drugs from poly mers based matrix tablets of the same formulati ons. They are defi ned bythe followi ng equati ons:资料个人收集整理，勿做商业用途 f^100xn Z |Rt —Tt t 吕 n z R f2"0^「100hXG (Rt -T , I V ny 丿］Where n is the number of sample withdrawal points, R t is the percentage of the refere nee dissolved at time t, T is the p erce ntage of the test dissolved at time 资料个人 收集整理，勿做商业用途 3 Results and discussion 3.1 Prep arati on and characterisati on ofPCC The ion ic bond of the interpo "electrolyte comp lex (IP EC) betwee n chitosa n andpo lycarb op hil was con firmed by means p reviously p ublished differe ntial sea nning calorimetry (DSC) (Lu et al., 2007b) and Fourier tran sform in frared (FT-IR). Fig.1shows the FT-IR sp ectra of chitosa n, po lycarb op hil and the PCC poly mer.资料个人收集整理，勿做商业用途-1A peak that appears at 1561 cm on the IR spectrum of the PCC, which might be assigned to the carboxylate groups that formed ionic bonds with the protonated amino groups of chitosan as previously illustrated for the interaction between Eudragit E andEudragit L (Moustafine at al., 2005). This ionic bond seems to be the primary bin di ng force for the formatio n of a comp lex betwee n chitosa n and po lycarb op hil.资料个人收集整理，勿做商业用途Chitosan is a cationic polymer of natural origin with excellent gel and film forming properties. Polycarbophil can also be considered as polyanions with negatively charged carboxylate groups. Mixing chitosan and polycarbophil in acidic solution (2% acetic acid solution was used in the study), ionic bonds should form between the protonated free amino groups of chitosan and carboxylate groups of polycarbophil.According to the results obtained from DSC and FT-IR, the possible process of formatio n of interpo "electrolyte comp lexes may be described as illustrated in Fig.2资料个人收集整理，勿做商业用途3.2Physical characteristics and drug content of poly mers based matrix tablets^ 料个人收集整理，勿做商业用途As summarised in Table 2, the physical characteristics of matrix tablets showed the good thickness uniformity, as ranged from 3.40 0.04 to 4.12± 0.0±4mm, a variationof matrix tablets weight from 73.3 2.4 mg to±87.9 4.0±mg, furthermore the weight variation of all formulation tablets is very low (< 10% from the average weight) (USP 2006). Hardness of the matrix tablets shows a range from 68 ±14 to 94 ±12N.The tablets also pasted the friability test (<1%), confirm that all formulations tablets are within USP (2006) limits. Drugs content of all formulations ranged from 4.60 0.65 to 5.01 0.1±1%.资料个人收集整理，勿做商业用途3.3Swelli ng and erosi on prop erties of the poly mers based matrices tablets资料个人收集整理，勿做商业用途Investigation of matrix hydration and erosion by gravimetrical analysis is a valuable exercise to better understand the mechanism of release and the relative importance of participating parameters (Jamzad and Fassihi, 2006). Fig.3 and Fig.4 illustrate the water uptake profiles and Fig.5， Fig.6 illustrate percentage of matrix erosion of all formulation tablets, respectively. Swelling properties of the all formulation matrix tablets based on the content of PCC, HPMC K100M and HPMC K100LV in the matrices tablets. Water uptake and percentage of matrix erosion values of these matrix tablets show superior swelling characteristics either HPMC K100LV based matrix tablets, or PCC based matrix tablets. 资料个人收集整理，勿做商业用途IPEC betwee n chitosa n and po lycarb op hil is a three -dime nsional n etworks water insoluble poly (acrylic acid) polymer with free hydroxy groups. Hydroxy groups ofPCC contribute hydrophilic capacity significantly and polymer erosion characteristics depend on the reaction ratio of chitosan and polycarbophil while polymer synthesis.While the PCC based matrixes were put into the buffer solution, the electrostatic repulsion between fixed charges (hydroxy groups) uncoiled the polymers chains.The counterion diffusion inside the PCC gel creates an additional osmotic pressure difference across the gel, consequently lead to higher water uptake (Peppas and Khare, 1993; Lu, et al., 2007b). During the matrix erosion, the ionic bonds between chitosan and polycarbophil were not broken by the matrix swelling. PCC based matrix tablets (F1 and F7 formulation) have superior swelling behaviors compare to the HPMC based matrices. Swelling index values of F1 and F7 formulation matrix tablets are 1599.62±216.68 % and 1579.82 ±118.05 % at 12 hours, respectively.Furthermore, addition of microcrystalline cellulose (Avicel) can increase matrices erosion significantly. Compare the erosion behaviors of F1, F7 and F2, F8formulation (containing 20% Avicel), F1 and F7 matrix tablets erode 5.74 1.62 % and 6.59 1±.18 % on 12 hours only, cont rary F2 and F8 matrix tablets erode 55.59 1.43 and 100 % respectively. Microcrystalline cellulose (Avicel) is widely used in pharmaceutical, primarily as a binder/diluent, also has some disintegrant properties on oral tablet and capsule formulations where it is used in both wet granulation and direct-compression process (Wheatley, 2000). In this study, matrix erosion behaviours were act by microcrystalline cellulose facilitating the transport of liquid into the pore of matrix tablets. It demonstrates that PCC polymer have capacity to form swelli ng only or swelli ng-erosi on matrix drug delivery system.资料个人收集整理，勿做商业用途It also was confirmed that PCC based matrix tablets have much better swelling behaviors than HPMC based matrix tablets by comparing swelling curves in Fig.3 andFig.4. Swelling index values of F1 and F7 formulation matrix tablets are 1599.62216.68 % and 1579.82 11±8.05 % at 12 hours, contrary F3 and F9 matrix tablets are545.96 ±4.32% and 547.72 2±6.27%. HPMC K100LV based matrix tablets have excellence erosion curves in this study, F5, F6, F11 and F12 formulation matrixtablets eroded 100% on 12 hours, but F2 and F8 (PCC based tablets) formulation matrix tablets can eroded 55.59 ±1.43 and 100 % with microcrystalline cellulosefacilitating. 资料个人收集整理，勿做商业用途3.4Drug releaseIn vitro drug release was performed in pH 7.4 phosphate buffer solution for 12 hours.Results of percentage drug release versus time for hydrochlorothiazide and ketoprofen in different formulations matrices tablets are presented in Fig.7 and Fig. 8, while theMDT and drug release kinetics values were present in Table 3资.料个人收集整理，勿做商业用In this study, water poor soluble model drugs hydrochlorothiazide and ketoprofen release from polymers based matrix tablets was controlled by the polymer matrices swelling or swelling combination with erosion. Percentage of drug release, matrix swelling and erosion of F7 were summarised in Fig 9. The percentageketoprofen release curve follows the percentagematrix tablets swelling curve, it demonstrates that PCC based matrix drug delivery system is the swelling dependent drug release system for water poor soluble model drugs. Same as F7 matrix tablets, F1 matrix tablets is also a swelling only drug delivery system, in these matrix systems drugs release behaviour primarily depend on the matrix swelling characteristics. Because as the superior swelling capacity of PCC based matrix tablets, liquid environments inside of the matrix provide that the model drugs release are zero order drug release.As described in Table 3, release exponentsn)( of F1 and F7 are 0.83 0.03 a±nd 0.99 ± 0.02 during the experimental time, respectively. 资料个人收集整理，勿做商业用途Addition of microcrystalline cellulose (Avicel) influence the model drugs release profiles from PCC based matrix tablets significantly. Cumulative drug release of F2 and F8 formulation tablets is 93.7 4.13 % a±nd 99.6 4.2±5% at 12 hours, relativelyF1 and F7 formulation tablets is 73.8 1.13 % an±d 47.2 4.5±3 % only. This can be explained by drugs release mechanism were swelling and erosion instead of swelling only, consequently accelerate the drugs release. The adjustable capacity of PCC based matrix drug delivery system by addition of microcrystalline cellulose (Avicel) dem on strates the poten tial useful of PCC poly mer in drug con trolled release field 资料个人收集整理，勿做商业用途Compare to the PCC based matrix tablets, the drugs release profiles of HPMC based matrix tablets were adjusted difficultly. The relatives f1 and f2 values of difference polymers including PCC, HPMC 100M, HPMC 100LV based matrix tablets containing hydrochlorothiazide under same formulation were show in Table 4. As describedf1 and f2 values in Table 4, F3 and F4, F5 and F6 formulation tablets have similar drug release behaviours, but F1 and F2 formulation tablets illustrate different drug release behaviours. This phenomenacan be explained by the superior water uptake capacity of PCC polymer, more water containing can easier broken the physical tensility between the polymer particles. 资料个人收集整理，勿做商业用途However, HPMC 100LV polymer has excellence erosion characteristics, in this study model drugs release from HPMC 100LV based matrix tablets illustrate matrix erosion dependent properties. In generally, drug release from swelling and erosion matrix system shows zero order release pare the drug release exponentsn(), release constant (k1), and mean dissolution time (MDT) of F2 to F5, F6, they have not significantly different as described in Tablet 3, furthermore the relatives f1 and f2 values between F2 and F5, F6 in Table 4 show they are similar release profiles. It imply PCC based matrix tablets can become a swelling and erosion drug delivery system by the addition of microcrystalline cellulose (Avicel), this drug delivery system illustrate similar drug release p rofiles as HPMC 100LV based matrix tablets资料个人收集整理，勿做商业用途Although it is very complex process that the model drugs release from swelling and。

最新：溶血磷脂在慢性阻塞性肺疾病中的研究进展摘要慢性阻塞性肺疾病(COPD)是呼吸系统中的常见病，其临床表现为呼吸困难、慢性咳嗽、咳痰，以持续气流受限为特征。

因肺功能进行性减退，严重影响患者的劳动力和生活质量，造成巨大的社会和经济负担。

因此COPD 的早诊断早治疗以及开发新的治疗靶点意义重大。

溶血磷脂作为重要的信号传递介质，与气道慢性炎症、氧化应激、细胞凋亡、肺组织纤维化等COPD 病生理过程密切相关，有望成为COPD临床标志物及治疗靶点。

COPD是呼吸系统中的常见病和多发病，是以持续气流受限为特征的可预防和治疗的疾病。

其气流受限呈进行性发展，与气道和肺组织的异常慢性炎症反应有关。

因肺功能进行性减退，严重影响患者的劳动力和生活质量。

COPD发病率和患病率居高不下，造成巨大的社会和经济负担[1]。

预计到2020年，COPD将成为世界第三大死亡原因[2]。

肺功能检查对确定气流受限有重要意义。

根据慢性阻塞性肺疾病全球倡议[3]，在吸入支气管扩张剂后，第1秒用力呼气容积(forced expiratory volume in one second，FEV1)/FVC<0.7表明存在持续气流受限。

尽管COPD传统的定义建立在肺功能检查上，人们发现随着年龄的增长，肺功能逐渐下降[4]，因此将FEV1/FVC<0.7作为诊断标准有可能导致健康的中年人和老年人的COPD 误诊。

另一方面，因为肺功能明显下降之前就启动COPD前期病理过程，有可能导致早期的轻度COPD患者的漏诊[5]。

最新研究表明不同表型的COPD肺功能有所差异，例如肺气肿型[6]、慢性支管炎型[7]或频繁急性加重型[8]。

这些表型的具体分子机制仍不清楚，但是有些研究通过使用若干组生物标志物的组合，发现炎症在鉴别这些表型中起到重要作用[9,10]。

在此背景下，有学者把研究方向转向了COPD发病机制以及相关分子代谢产物，试图从中寻找新的生物标记物以及治疗靶点。

Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryfindings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantification of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsflanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-specific primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-specific primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Purification 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-specific primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into afilter cartridgewithin a collection bel thefilter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the samefilter.17.Centrifuge at10,000rpm for15s at RT.18.Collect thefiltrate(theflow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theflow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newfilterbel thefilter as small RNA.When you have >700μL of thefiltrate mixture,apply it in successive appli-cation to the samefilter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theflow-through and repeat until all of thefiltratemixture is passed through thefilter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to thefilter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theflow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to thefilter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theflow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer thefilter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of thefilter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave thefilter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube briefly.3.Incubate for1h at37◦C.3.3.Purification 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a purificationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard thefiltrate(theflow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of thefilter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let thefilter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube briefly.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-specific primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube briefly.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-specific primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which thefluorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method amplifies over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-specific primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefficiency.Good efficiencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially amplifies maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). 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专题论述MEAT RESEARCHCHINA MEAT RESEARCH CENTER肉汤中特征风味体系的形成机理及分析方法研究进展关海宁，徐筱君，孙薇婷，刘登勇*，刁小琴*（渤海大学食品科学与工程学院，辽宁省食品安全重点实验室，生鲜农产品贮藏加工及安全控制技术国家地方联合工程研究中心，辽宁 锦州 121013）摘 要：肉汤风味良好，营养丰富，而且具有一定的保健功效。

本文从肉汤的营养价值、基本分类入手，重点阐述肉汤风味体系的形成机理、影响因素、肉汤在贮藏、加工过程中风味的变化规律以及用于鉴定肉汤风味的典型分析方法，简要阐述关键性风味的成分属性，为后期传统肉汤制品的加工、品质升级及风味研究提供一定的理论依据。

关键词：肉汤；特征风味；形成机理；影响机制；分析方法Recent Progress in the Formation Mechanism and Analytical Methods for Characteristic Flavor Compounds in BrothGUAN Haining, XU Xiaojun, SUN Weiting, LIU Dengyong *, DIAO Xiaoqin *(National and Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agriculturaland Aquatic Products, Food Safety Key Laboratory of Liaoning Province, College of Food and Technology,Bohai University, Jinzhou 121013, China)Abstract: Broth has a palatable flavor, rich nutrition and health benefits. Beginning with an overview of the nutritional value and basic classification of meat broth, this paper is focused on the formation mechanism of flavor compounds in broth, the factors influencing it, the pattern of changes in flavor compounds during storage and processing, and the traditional analytical methods for identifying broth flavor compounds. In addition, the key flavor components are briefly described. It is expected that this review will provide a theoretical basis for the processing and quality improvement of traditional Chinese meat soup and research on flavor compounds in broth.Keywords: broth; characteristic flavor compounds; formation mechanism; mechanism; analytical methods DOI:10.7506/rlyj1001-8123-20210106-004中图分类号：TS251.1 文献标志码：A 文章编号：1001-8123（2021）01-0066-08引文格式：关海宁, 徐筱君, 孙薇婷, 等. 肉汤中特征风味体系的形成机理及分析方法研究进展[J]. 肉类研究, 2021, 35(1): 66-73. DOI:10.7506/rlyj1001-8123-20210106-004. GUAN Haining, XU Xiaojun, SUN Weiting, et al. Recent progress in the formation mechanism and analytical methods for characteristic flavor compounds in broth[J]. Meat Research, 2021, 35(1): 66-73. DOI:10.7506/rlyj1001-8123-20210106-004. 收稿日期：2021-01-06基金项目：辽宁省教育厅科学技术研究项目（LJ2020010）；渤海大学博士科研启动基金项目（05013/0520bs006）；2020科技特派专项行动计划项目（2020JH5/10400009）；辽宁省重点研发计划项目（2017205003）第一作者简介：关海宁（1980—）（ORCID: 0000-0002-2232-1564），男，副教授，博士，研究方向为功能性成分分析及肉制品加工技术。

肌氨肽苷注射液是哺乳动物肌肉和心脏为原料的提取物，主要成分降钙素基因相关肽是生物体内具有代表性的一种活性多肽，具有强大的扩张血管作用，其扩张冠状动脉的能力是硝酸甘油的240倍。

临床可用于脑功能紊乱，脑卒中、脑供血不足所致脑功能减退，周围神经疾病［1，2］。

肌氨肽苷注射液制备提取工艺中，采用三氯乙酸蛋白沉淀法。

三氯乙酸是生物样本沉淀蛋白质的常用物质［3］，由于三氯乙酸及降解产物三氯甲烷对人体和动物具有较大的毒性和潜在的致癌作用，肌氨肽苷注射液中三氯乙酸残留量的质量控制，对人体健康和用药安全具有重要的意义。

目前文献报道三氯乙酸的测定方法有：气相色谱法、气相色谱⁃质谱法、分光光度法、离子色谱法、离子色谱⁃质谱联用法、超高效液相色谱⁃质谱法、衍生化气相色谱法等，不同方法优缺点各异。

本研究建立的电子俘获检测器（ECD ）检测器自动顶空气相色谱法，灵敏度高、专属性好、操作简单，可同时测定三氯乙酸与三氯甲烷的总量，并可满足肌氨肽苷注射液中三氯乙酸含量不超0.1μg /m l 的检测要求。

1仪器及试药1.1仪器：3400气相色谱仪带ECD 检测器（瓦里安）；7694E 顶空进样器（安捷伦）；Chromeleon7色谱工作站（戴安）；DB⁃WAX 石英毛细管柱（安捷伦，60m×0.53mm×1.0μm ）；BP211D 型电子天平（德国赛多利斯）；HY⁃2调速多用振荡器（江苏金坛市中大仪器厂）。

1.2试药：三氯乙酸（分析纯，≥99.0%，天津市凯通化学试剂有限公司）；肌氨肽苷注射液（河北医科大学生物医学工程中心）；甲醇（色谱纯，迪马科技有限公司）；二氯甲烷、氢氧化钠（分析纯，国药集团化学试剂有限公司）；实验用水均为纯化水（河北华维纯净水有限公司）。

1.3溶液配制：空白溶液：纯化水。

三氯乙酸（对照品）溶液：快速称取经干燥器干燥的三氯乙酸20g ，加少量纯化水溶解，于50m l 容量瓶中用纯化水定容至刻度，配置成20g /L 的贮备液，以氢氧化钠标准滴定液标定，置于4℃冰箱中保存，临用时按需逐级稀释为对照品工作液。