原代大鼠肝细胞的分离和培养_蒋永生

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1、第31卷第5期中华中医药学刊Vol．31No．52013年5月CHINESEARCHIVESOFTRADITIONALCHINESEMEDICINEMay2013原代大鼠肝细胞的分离和培养12111蒋永生，程向东，刘文洪，项海，姚立(1．浙江中医药大学，浙江杭州310053;2．浙江省肿瘤医院，浙江杭州310022)摘要:目的:探讨原代大鼠肝细胞分离及培养的简易方法，建立稳定简便的大鼠原代肝细胞分离培养方法。方法:采用改良原位两步循环灌流法及低速离心法分离原代大鼠肝细胞，常规DMEM:高糖培养基培养原代大鼠肝细胞，测定1周内生

2、长情况，倒置显微镜及扫描电镜观察肝细胞形态变化。结果:倒置显微镜下观察肝细胞在接种后3h基本完成贴壁，贴壁的细胞呈卵圆形，24h后胞体变大变平，随后细胞相互靠拢呈岛状或条索状连接，3d后扫描电镜下细胞呈现良好形态。结论:用改良原位两步循环灌注法及低速离心法成功分离并培养原代大鼠肝细胞，可操作性强，可在具备基本细胞培养条件的实验室推广。关键词:大鼠;原代肝细胞;分离;培养中图分类号:R－332文献标志码:A文章编号:1673－7717(2013)05－1111－02IsolationandCultureofPrimaryRats

3、Hepatocytes12111JIANGYongsheng，CHENGXiangdong，LIUWenhong，XIANGHai，YAOLi(1．ZhejiangChineseMedicalUniversity，Hangzhou310053，Zhejiang，China;2．ZhejiangCancerHospital，Hangzhou310022，Zhejiang，China)Abstract:Objective:Toinvestigateasimpleandfeasiblemethodforprimaryratshepa

4、tocytesisolationandculture，andtoestablishastableandeasymethodsofisolationandcultureofprimaryratshepatocytes．Methods:Hepatocyteswereisolatedbyusingamodifiedtwo－stepin－situ－circulatingperfusionmethodfollowedbylow－speedcentrifugation．ThehepatocyteswereculturedinDulbecc

5、o＇smodifiedEagle＇smediumwithhighlevelofglucose．Theviabilitiesofcellswereevaluatedwithinoneweek．Cellularmorphologicalchangeswereobservedbyusinganinvertedmicroscopeandscanningelectronmicroscope(SEM)．Results:Theelementaryadherencecompletionofthehepaticcellshadbeenposte

6、dinoculationafter3hunderinvertedmicroscope，thecellsofadherenceofferredovate．Thehepatocytesenlargedandflattenedround，attachedtothesurfaceofcollagen－coateddishes24hafterseedingandthenconnectedwithsurroundingcells，formingislandsortrabs．After3days，cellswerewellundertheS

7、EM．Conclusions:Primaryratshepatocytescanbesuccessfullyi-solatedandculturedusingamodifiedtwo－stepin－situ－circulatingperfusionmethod，whichishighlyfeasible．Thismethodisavailabletoperforminlabswithbasiccellcultureconditions．Keywords:rat;hepatocytes;isolation;culture肝脏是人

8、体重要的实质性器官，很多疾病都会累及肝型胶原酶，EGTA，牛血清白蛋白，HEPES，自制鼠尾胶原，［1］脏。体外肝细胞培养可以用于探索疾病的发病机制，研PVDF中空膜。［2－3］究药物的毒理性，是体外药物试验的“金标准”。肝细主要仪器:CO2恒温细胞培养箱;台式高速冷冻离心［