体外诱导小鼠胚胎干细胞分化为肝细胞的研究

Induced differentiation mouse ES cells into hepatocytes

目的 基于肝脏发育生物学进展，建立有效诱导胚胎干细胞（ESC）向肝细胞分化的体外培养体系。方法 小鼠ESC细胞系E14，在内含1000U／ml的rmLIF的无饲养层高糖DMEM培养基中培养，去除rmLIF后用悬滴培养法制备成拟胚体。依次加入10^-7M的Dexamethasone、10μg／L的FGF-4、25μg／L的HGF等诱导因子，继续培养1～2周。然后取分化细胞，行细胞形态学观察、免疫细胞化学染色检测白蛋白和CK8／18的表达、RT-PCR检测白蛋白和转甲状腺蛋白等肝细胞特征性蛋白的mRNA转录水平。糖原染色、尿素合成功能检测则对其功能进行检验。结果 ESC体外培养生长旺盛，呈”巢状”克隆性增殖。去除rmLIF后悬浮培养，一般2～3d可制备成拟胚体。序贯加入Dex、FGF-4和HGF等诱导因子进行分化培养后，约于ED12可见分散、克隆样增殖的小上皮样细胞集落，ED19左右有较多细胞分化为多角形的肝细胞样细胞。免疫组化染色，见胞浆内出现棕黄色颗粒，表达肝细胞特有的白蛋白以及CK8／18等蛋白。RT-PCR显示有白蛋白、转甲状腺蛋白等的mRNA转录。糖原染色见胞浆内存在红色颗粒，呈阳性反应；尿素合成功能检测显示，培养液内可见尿素氮的逐日升高，说明细胞具有肝细胞特有的糖原合成、尿素合成和分泌功能。结论 加入FGF-4、HGF和Dex等肝脏胚胎发育时期的关键性调控因子的体外培养体系可以诱导胚胎干细胞分化为肝细胞样细胞。

Objective To induce ES cells to differentiate into hepatocytes. Methods E14 Mouse ES cell line was cultured in Dulbecco＇s modified Eagle＇s medium containing 1000 U/ml rmLIF and 10% FCS. After forming embryonic bodies （EBs） by the hanging drop culture method without rmLIF, 10-TM Dexamethasone, 10 μg /L FGF-4 and 25 μg /L HGF were added for 1 to 2 weeks. Morphological examination, immunocytochemical staining of albumin and CK8/18 were carried out, and mRNA level of albumin and transthyretin were assayed by RT-PCR. Glycogen storage and urea synthesis of the ceils were tested with PAS staining and colorimetric assay respectively. Results ES ceils proliferated and grew as small clusters in the culture system. After eliminating rmLIF, embryonic bodies （Ebs） start to form. After adding Dex, FGF-4 and HGF orderly, small epithelioid cell clusters were observed at E12. On E19, a large number of hepatocyte-like cells were observed, which stained positive for albumin and CK8/18, expressed mRNA of albumin and transthyretinThese cells have also acquired functional characteristics of hepatocytes： they synthesized and secreted urea, and stored glycogen. Conclusions Our in vitro culture system supplemented with Dex, FGF-4 and HGF, could effectively induce ES cells to differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes.