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1、第31卷第5期中華中醫(yī)藥學(xué)刊Vol．31No．52013年5月CHINESEARCHIVESOFTRADITIONALCHINESEMEDICINEMay2013原代大鼠肝細(xì)胞的分離和培養(yǎng)12111蔣永生，程向東，劉文洪，項(xiàng)海，姚立(1．浙江中醫(yī)藥大學(xué)，浙江杭州310053;2．浙江省腫瘤醫(yī)院，浙江杭州310022)摘要:目的:探討原代大鼠肝細(xì)胞分離及培養(yǎng)的簡易方法，建立穩(wěn)定簡便的大鼠原代肝細(xì)胞分離培養(yǎng)方法。方法:采用改良原位兩步循環(huán)灌流法及低速離心法分離原代大鼠肝細(xì)胞，常規(guī)DMEM:高糖培養(yǎng)基培養(yǎng)原代大鼠肝細(xì)胞，測定1周內(nèi)生
2、長情況，倒置顯微鏡及掃描電鏡觀察肝細(xì)胞形態(tài)變化。結(jié)果:倒置顯微鏡下觀察肝細(xì)胞在接種后3h基本完成貼壁，貼壁的細(xì)胞呈卵圓形，24h后胞體變大變平，隨后細(xì)胞相互靠攏呈島狀或條索狀連接，3d后掃描電鏡下細(xì)胞呈現(xiàn)良好形態(tài)。結(jié)論:用改良原位兩步循環(huán)灌注法及低速離心法成功分離并培養(yǎng)原代大鼠肝細(xì)胞，可操作性強(qiáng)，可在具備基本細(xì)胞培養(yǎng)條件的實(shí)驗(yàn)室推廣。關(guān)鍵詞:大鼠;原代肝細(xì)胞;分離;培養(yǎng)中圖分類號:R－332文獻(xiàn)標(biāo)志碼:A文章編號: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肝臟是人
8、體重要的實(shí)質(zhì)性器官，很多疾病都會(huì)累及肝型膠原酶，EGTA，牛血清白蛋白，HEPES，自制鼠尾膠原，［1］臟。體外肝細(xì)胞培養(yǎng)可以用于探索疾病的發(fā)病機(jī)制，研PVDF中空膜。［2－3］究藥物的毒理性，是體外藥物試驗(yàn)的“金標(biāo)準(zhǔn)”。肝細(xì)主要儀器:CO2恒溫細(xì)胞培養(yǎng)箱;臺(tái)式高速冷凍離心［
