高純度原代大鼠肝細(xì)胞的分離與培養(yǎng)

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1、·822·　第16卷　第11期醫(yī)學(xué)研究生學(xué)報(bào)Vol.16No.112003年11月JournalofMedicalPostgraduatesNov.2003·論　　著·高純度原代大鼠肝細(xì)胞的分離與培養(yǎng)X王新穎,　李維勤,　李　寧,　黎介壽(南京軍區(qū)南京總醫(yī)院解放軍普通外科研究所,江蘇南京210002)摘要:目的:分離及培養(yǎng)高純度的原代大鼠肝細(xì)胞?！》椒?用含EGTA的D2Hanks液及含DNaseⅠ的膠原酶消化液分兩步原位灌注大鼠肝臟,分離細(xì)胞經(jīng)3次低速離心(50g,5min)后獲得純化的肝細(xì)胞。肝細(xì)胞存活率檢測用錐蟲藍(lán)拒染法,純度檢測用蘇木精2伊紅染色。采

2、用加入特殊營養(yǎng)成分的RPMI1640培養(yǎng)液培養(yǎng),用倒置相差顯8微鏡觀察細(xì)胞活力?！〗Y(jié)果:每只180～200g的大鼠平均可獲(1.0～1.5)×10的細(xì)胞,存活率>50%,純度高于98%。培養(yǎng)1、3、5天肝細(xì)胞的存活率分別為100%、94.5%、89.5%,形態(tài)良好?！〗Y(jié)論:改良原位灌注消化法分離肝細(xì)胞產(chǎn)率較高,活性較好,加入特殊營養(yǎng)成分的RPMI1640培養(yǎng)液培養(yǎng)能保持肝細(xì)胞良好的形態(tài)。關(guān)鍵詞:肝細(xì)胞;　分離;　培養(yǎng);　大鼠中圖分類號(hào):Q813.11　　　文獻(xiàn)標(biāo)識(shí)碼:A　　　文章編號(hào):100828199(2003)1120822203Separationan

3、dcultureofhigh2purityprimaryrathepatocyteWANGXin2ying,LIWei2qin,LINing,LIJie2shou(ResearchInstituteofGeneralSurgery,NanjingGeneralHospitalofNanjingCommand,Nanjing210002,Jiangsu,China)Abstract:Objectives:Toestablishamethodofseparationandcultureofhigh2purityprimaryrathepatocyte.Metho

4、ds:TheperfusionsolutionsusedinthefirstandthesecondstepwereD2Hank’ssolutioncontainingEGTA,andthedigestivesolutioncontainedDNaseIandcollagenaserespectively.Purifiedhepatocyteswereseparatedfromthedissociativecellsbylow2speedcentrifugation(50g,5min)3times.Thesurvivalratewasmeasuredbyty

5、panblueexclusionandthepuritywasmeasuredbyroutinehematoxylinand2eosincytochemistry.SeparatedhepatocyteswereculturedinRPMI1640cul2turemediumwithsomespecialnutrientelements.Throughouttheinvitroculture,wekeptcontinu2ousobservationoftheshapechangesofthehepatocyteandmeasuredthesurvivalra

6、tewithinthefirst58days.Results:(1.0～1.5)×10cellswerepreparedfroma180-200gratonaverage,thesurvivalratewashigherthan50%andthehepatocytepuritywashigherthan98%.Microscopically,thehepa2tocyteswerenormalinshapeandviability.Conclusions:Themodifiedinsituperfusiondigestionmethodandthecultur

7、einRPMI1640andspecialnutrientsareadvantageoustogethighviabilityandpurityhepatocytewithnormalshape.Keywords:Hepatocyte;Separation;Culture;Rat雖然目前國內(nèi)外在這些領(lǐng)域的研究中應(yīng)用大鼠體外0引　　言肝細(xì)胞已非常普遍,但由于肝細(xì)胞體外培養(yǎng)增生困體外培養(yǎng)的大鼠原代肝細(xì)胞是研究毒理學(xué)、藥難,而且難以長期維持功能。因此,如何獲得高存活理學(xué)、生物化學(xué)及致癌作用的有效手段,同時(shí)也是研[1～2]率、高產(chǎn)率的原代肝細(xì)胞仍然是人們關(guān)心的問題。

8、究感染狀況下肝急性相反應(yīng)良好的體外模型。X收稿日期: