原代小鼠肝脏细胞的高效分离纯化与培养-论文.pdf

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1、现代生物医学进展www．shengwuyixue．comProgressinModernBiome~cineVoL14NO．6FEB．2014DOI：10．13241／j．cnki．pmb．2014．06．002原代小鼠肝脏细胞的高效分离纯化与培养木张钰付亮鲁超锁涛宋陆军△(复旦大学附属中山医院普外科上海200032)摘要目的：建立一种能稳定获得高活力和高纯度原代小鼠肝脏细胞的分离、纯化及培养方法。方法：应用改良的Seglen二步法原位灌注和机械离心分离肝脏细胞，并用改良的高糖DMEM培养基进行培养。台盼蓝拒染法检测接种时肝脏细胞的存活率，倒置显微

2、镜动态观察肝脏细胞形态变化，应用免疫荧光技术对肝脏细胞进行Albumin染色。结果：每只小鼠可获取肝脏细胞的总产量平均为1．35~10／g体重，肝脏细胞存活率>90％。倒置显微镜下观察贴壁前肝细胞直径为35．14ixm~4．35txm，肝脏细胞在接种后3h基本完成贴壁；肝脏细胞接种后24h，所有肝脏细胞均强阳性表达成熟肝脏细胞标志物Albumin，肝细胞纯度>95％。结论：改良的分离纯化及培养方法能稳定获得高产量、高活率及高纯度的小鼠肝脏细胞。关键词：肝脏细胞；原代细胞培养；分离；纯化；小鼠中图分类号：Q95．3，Q813．11，R322．47文献

3、标识码：A文章编号：1673—6273(2014)06．1005—04Isolation．PurificationandPrimaryCu1mreOfMouseHepatocyteswithHighViabi1itv术ZHANGYu,FULiang,LUChao,SUOTao,SONGLu-jun(DepartmentofGeneralSurgery,ZhongshanHospital，ShanghaiFudanUniversity,Shanghai,200032,Chma)ABSTRACTObjective：Astablemethodwasest

4、ablishedforisolationandpfima~cultureofmousehepatocyteswithhighpurityandviability．Methods：TheC57BL／6mousehepatocyteswereisolatedbyamodifiedtwo-stepcollagenaseperfusionandlow—speedmechanicalcentrifugation．ThehepatocyteswereculturedinmodifiedDMEMwithhighlevelofglucose．Theviabilit

5、iesofcellswereevaluatedbyTrypanblueexclusionbeforecellswereseeded．Cellularmorphologicalchangeswereobservedusingaphasecontrastmicroscopy．TheAlbuminandCK19stainingwereperformedonhepatocytesbyimmunofluorescence．Results：Thetotalcellyieldofhepatocyteswas1．35~10。cells／gwetweight．Tot

6、alcellviabilitywasmorethan90％．Thediameterofhepatocyteswas35．14ktm~4．35Ixmbeforeadherence．Thehepatocyteswithenlargedandflattenedroundshapewereattachedtothesurfaceofcollagen—coateddishes3hoursafterseeding．Albuminwaspositivelyexpressedinallhepatocytesat24hatteratachment，andthepur

7、ityofhepatocyteswasmorethan95％．Conclusion：Mousehepatocyteswithhighquantity，purityandsurvivalratewereobtainedbytheimprovedmethodforisolation，purificationandculture．Keywords：Hepatocytes；Primarycellculture；Isolation；Purification；MouseChineseLibraryClassification(CLC)：Q95-3，Q813．1

8、l，R322．47Documentcode：AArticleID：1673．6273(2014)06．1005．04大，将