肝星狀細(xì)胞具有向肝細(xì)胞樣細(xì)胞分化的干細(xì)胞潛能

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1、肝星狀細(xì)胞具有向肝細(xì)胞樣細(xì)胞分化的干細(xì)胞潛能羅茜，王斌，雷增杰，李青，李硯，熊吉，陳瀟迪，張霹雲(yún)，魏艷玲，王軍，陳東風(fēng)(400042重慶，第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所消化內(nèi)科)[摘要]目的探討肝星狀細(xì)胞是否能體外誘導(dǎo)分化為肝細(xì)胞樣細(xì)胞以及在這過程中某些干細(xì)胞標(biāo)記物的表達(dá)和變化。方法采用標(biāo)準(zhǔn)膠原酶灌注聯(lián)合percoll密度梯度離心法提取并純化原代大鼠肝星狀細(xì)胞，免疫熒光鑒定細(xì)胞表型；將原代肝星狀細(xì)胞分為對(duì)照組和實(shí)驗(yàn)組進(jìn)行體外培養(yǎng)，對(duì)照組肝星狀細(xì)胞不加任何細(xì)胞因子培養(yǎng)24h，實(shí)驗(yàn)組肝星狀細(xì)胞加入bFGF20μg/L、FGF420μg/L、HGF2

2、0μg/L、IL-61μg/L細(xì)胞因子誘導(dǎo)7d；倒置相差顯微鏡下觀察誘導(dǎo)細(xì)胞形態(tài)的變化，并通過Real-timePCR和細(xì)胞免疫熒光技術(shù)檢測(cè)肝星狀細(xì)胞誘導(dǎo)前后肝細(xì)胞ALB、AFPmRNA和蛋白的表達(dá)情況，以及誘導(dǎo)后干細(xì)胞標(biāo)記物mRNA和蛋白水平的表達(dá)及變化。結(jié)果①通過膠原酶灌注聯(lián)合percoll密度梯度離心法獲得的大鼠肝星狀細(xì)胞，其特異性蛋白Desmin和GFAP表達(dá)陽性率≥95%。②實(shí)驗(yàn)組大鼠肝星狀細(xì)胞經(jīng)過bFGF、FGF4、HGF、IL-6等細(xì)胞因子誘導(dǎo)7d后，與對(duì)照組細(xì)胞相比細(xì)胞形態(tài)由星形變?yōu)轭悎A形或多角形，轉(zhuǎn)錄水平出現(xiàn)肝細(xì)胞特異性標(biāo)記物AL

3、B的表達(dá)（P＜0.01）和AFP的表達(dá)（P＜0.05），同時(shí)實(shí)驗(yàn)組ALB和AFP蛋白表達(dá)呈陽性，而對(duì)照組則呈陰性。③實(shí)驗(yàn)組大鼠肝星狀細(xì)胞經(jīng)過bFGF、FGF4、HGF、IL-6等細(xì)胞因子誘導(dǎo)7d后，與對(duì)照組相比干細(xì)胞標(biāo)記物P75NTR轉(zhuǎn)錄水平表達(dá)下降（P＜0.05），CD90、c-kit、sox-2、oct-4等轉(zhuǎn)錄水平表達(dá)顯著下降（P＜0.01），并伴有干細(xì)胞標(biāo)記物蛋白水平的表達(dá)下調(diào)或無表達(dá)。結(jié)論初步證明肝星狀細(xì)胞可能具有潛在的干細(xì)胞特性，在體外可誘導(dǎo)分化形成肝細(xì)胞樣細(xì)胞，在誘導(dǎo)過程中肝星狀細(xì)胞的干細(xì)胞標(biāo)記物明顯下調(diào)。[關(guān)鍵詞]肝星狀細(xì)胞；干細(xì)胞

4、；細(xì)胞分離；誘導(dǎo)；分化[中圖法分類號(hào)]R365;R33-33;R333.4;R392.12[文獻(xiàn)標(biāo)志碼]AHSCsmightpossessstemcellcapabilitiesofdifferentiationtohepatocyte-likecellsinvitroLuoXi,WangBin,Leizengjie,LiQing,LiYan,XiongJi,ChenXiaodi,ZhangPiyun,WeiYanlin,WangJun,ChenDongfeng（DepartmentofGastroenterology,InstituteofSur

5、geryResearch,DapingHospital,ThirdMilitaryMedicalUniversity,Chongqing,400042,China）[Abstract]ObjectiveToinvestigatewhetherhepaticstellatecells(HSCs)candifferentiatetohepatocyte-likecellsinvitroortheexpressionofsomestemcellmarkerseverchangedduringtheinduction.MethodsPrimaryratHS

6、CswereisolatedbythestandardcollagenaseperfusionandpurifiedbydensitygradientcentrifugationthenidentifiedwithfluorescentImmunocytochemistry.Thecellsweredividedintotwogroups,inthecontrolgroupHSCswereculturedwithoutanycytokinesinductionfor24h,whileintheexperimentalgroupHSCsweretre

7、atedcytokinesbFGF(20ug/L),FGF4(20ug/L),HGF(20ug/L)andIL-6(1ug/L)for7days.ThemorphologicalchangesofHSCswereexaminedwithinvertedphasecontrastmicroscope,andthehepatocyte-likephenotypesweredeterminedbywhetherthesecellsobtainedthecharacteristicsofhepatocytethrough7daysinductionbyme

8、ansofrealtime-PCRandImmunocytochemistry,alsomarkersforstemcel