酶消化法分离培养原代肺动脉平滑肌细胞及免疫组化鉴定.pdf

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1、78MedicalScienceJournalofCentralSouthChina，January2015，Vo1．43，No．1DOI：10．15972／j．cnki．43—1509／r．2015．O1．020·技术与方法·酶消化法分离培养原代肺动脉平滑肌细胞及免疫组化鉴定王爱平，李严兵，谢巍，曹宇辉，彭田红，周小兵，龚邵新(1．南华大学医学院应用解剖研究所，湖南衡阳421001；2．南华大学附属第一医院病理科)摘要：目的建立体外胶原酶消化法分离培养大鼠肺动脉平滑肌细胞(PASMCs)及免疫鉴定的方法。方法采用I型胶原酶对PASMC

2、s进行体外培养。在无茵环境下，分离提取雄性SpragueDawley(sD)大鼠肺动脉主干，剥离外膜、剔除内皮细胞，经酶消化法，体外培养PASMCs。倒置相差显微镜观察PASMCs生长状态及特点；台盼兰测定细胞活力；免疫细胞染色法进行平滑肌O／．一肌动蛋白(—SMactin)鉴定。结果酶消化法分离培养的PASMCs3d后，可见细胞贴壁呈梭形生长，6d后生长迅速呈典型的峰一谷状生长，9d后达90％融合。通过形态学观察及免疫荧光染色法鉴定表明：培养的细胞为PASMCs；细胞存活率为98．5％；原代培养8～10d后即可传代，3—10代细胞生长

3、迅速、细胞形态不易发生改变，可用于进行细胞实验。结论采用I型胶原酶消化法简单易行、可靠、PASMCs生长迅速、传代周期短的特点；尤为重要的是，培养的原代PASMCs可用于肺动脉高压和肺血管重构的研究工作。关键词：酶消化法；大鼠肺动脉平滑肌细胞；免疫细胞化学；SD大鼠中图分类号：R563文献标识码：AMethodsofCultureofRatPulmonaryArterySmoothMuscleCellsandIdentificationWANGAiping，LIYanbing，XIEWei，etal(Instituteofapplied

4、anatomy，schoolofmedicine，SouthChinauniversity)Abstract：ObjectiveTosetupthemethodsofratpulmonaryarterysmoothmusclecells(PASMCs)isolation，cultureandimmunologicalidentificationinvitro．MethodsThePASMCsculturedinvitrowithtypeIcollagenase．Be—forethePASMCscultured，maleSDratpulm

5、onarytrunkseparatedweresubjectedtooutermembranepeelandendothelialcellsremovebyenzymaticdigestioninasterileenvironment．WeobservedthestatusandcharacteristicsofPASMCswithinvertephasecontrastmicroscope，determinedthecellviabilitywithtrypanbluestaining，andindentifiedthe0【-smoo

6、thmuscleactin(0【一SMactin)withimmuncytochemistrystaining．ResultsTheculturedPASMCssubjectedtoenzymaticdigestionandisolationpresentedshutdeshapeatd3，typicalpeak—valley—likegrowthatd6，and9o％confluenceatd9．Themorphologicalobservationandimmuncytochemistrystainingidentification

7、showedthat：thecellsculturedwerePASMCs：cel1survivalrateupto98．5％：theprimaryculturescallbepassagedatd8～dl0andcellscanbeusedforcellexperimentsat3thgenerationto10thforstablemorphologyandfastgrowth．ConclufionsThemethodtypeIcollage·nasedigestioniseasyoperationandreliable，thepr

8、imaryculturedPASMCspresentedfastgrowtandshortcellcyclecanbeusedforpulmonaryarterialhypertensionandpulmo