小鼠胚胎肝祖细胞的分离和扩增培养

目的从胎龄14.5 d的C57小鼠胚胎肝脏中分离、培养小鼠胚胎肝祖细胞(m HPCs),并将其诱导分化为胆管细胞。方法用荧光激活细胞筛选法(FACS)分选DLK1表面抗原阳性的小鼠胚胎肝细胞,并和小鼠胚胎成纤维细胞(MEFs)Transwell共培养或者单独培养。细胞免疫荧光检测刚分选和共培养4和6 d的DLK1~+细胞的甲胎蛋白(AFP)、白蛋白(ALB)及细胞角蛋白19(CK19)抗原的表达。结果体外共培养时,大部分DLK1~+细胞分裂增殖明显,呈葡萄状聚集生长。第4天,部分细胞开始贴壁增殖,形态开始变成梭形。结果显示,分选的DLK1~+细胞表达AFP和少量ALB,但不表达CK19;在共培养的第4天其开始表达CK19,和微弱表达ALB;第6天,其高表达CK19,而几乎不表达的ALB。结论应用FACS技术成功从E14.5胎肝细胞中分选出DLK1~+细胞并鉴定其大部分为mHPCs,并可在体外与MEFs Transwell共培养的条件下,诱导培养其分化为胆管细胞。

Isolation and amplification of hepatic progenitor cells from fetal mouse in vitro

Objective To isolate and cultivate mouse hepatic progenitor cells (mHPCs) from E14.5 mouse fetal liver in vitro and induce mHPCs differentiation into cholangiocytes.Methods Isolation of mHPCs from mouse fetal liver was based on the cell surface antigen delta-like protein 1/preadipocyte factor 1 (Dlk/Pref-1) by a fluorescence-activated cell sorter (FACS).Then mHPCs isolated were co-cultured with/without mouse embryonic fibroblasts (MEFs) by using Transwell.The cell antigen alpha-fetoprotein (AFP), albumin (ALB) and cytokeratin19 (CK19) expression in freshly isolated DLK1+cells or co-cultured for 4 days and 6 days were observed with immunocytochemical method.Results When co-cultured with MEFs, the division and proliferation were observed in most of DLK1+ cells and grape-like aggregation was formed.Cells began to adhere to growth and began to become spindle-shaped on 4th day.The DLK1+cells isolated freshly by FACS were expressed AFP and low levels of ALB but not expressed CK19.But, these cells expressed CK 19 and weak expression of ALB on 4th day.In addition, the expression of CK19 increased and the expression of ALB almost not detected on 6th day.Conclusions Most of DLK1+ cells, isolated from E14.5 fetal livers by FACS, are proved to be mHPCs.Furthermore, these cells can proliferate quickly and differentiate into cholangiocytes by co-culture with MEFs.