胎牛小肠上皮干细胞的分离和培养并诱导其向肝细胞样细胞分化

Isolation and culture of fetal bovine intestine-derived epithelial stem cells and the differentiation into hepatocyte-like cells

目的建立胎牛小肠上皮干细胞（IESC）分离和培养的体系,并检测其表面标志物及向肝细胞样细胞分化的潜能。方法取3~5月龄胎牛,分离小肠组织,用胶原酶消化获得IESC,在DMEM/F12培养基中培养,观察其形态学特征。传代培养、生长曲线分析其增殖能力,并探索体外分化潜能。反转录PCR检测IESC标志基因Bmi1、Hes1、Lgr5和细胞角蛋白19（CK19）的mRNA表达,免疫细胞化学染色检测CK19、Bmi1、LGR5蛋白的表达。经成纤维细胞生长因子4（FGF-4）和肝细胞生长因子（HGF）诱导后,通过糖原染色和反转录PCR法观察向肝细胞样细胞的分化情况。结果 IESC可在体外扩增培养,表达CK19、Bmi1、Hes1、Lgr5的mRNA和CK19、Bmi1、LGR5蛋白,诱导后细胞糖原染色阳性,表达甲胎蛋白（AFP）和白蛋白（ALB）的mRNA。结论成功建立了IESC的分离培养方法,并成功诱导IESC向肝细胞样细胞分化。

Objective To establish the culture system of fetal bovine intestinal epithelial stem cells （IESCs） in vitro, identify specific markers of the cell lines and analyze the differentiation potential into hepatocyte-like cells. Methods IESCs were isolated from the 3- to 5-month fetal bovine intestine by the digestion of collagenase I, and cultured in the DMEM/F12 medium. The cell morphology was observed, and the proliferation ability and multiple differentiation potential were demonstrated by subculturing and its growth curve. The mRNA expressions of the surface markers Bmit, Hest, Lgr5 and cytokeratin 19 （CK19） were determined by reverse transcription PCR （RT-PCR）, and the protein levels of Bmil, LGR5 and CK19 were detected by immunofluorescence cytochemistry. Under the induction of fibroblast growth factor 4 （FGF-4） and hepatocyte growth factor （ HGF）, the cell differentiation into hepatocyte-like cells was assayed by the glycogen staining and RT-PCR. Results IESCs cultured in vitro expressed Bmil, Hes1, Lgr5 and CK19 mRNAs, and CKt9, Bmil and LGR5 proteins. The differentiated cells were positively stained by glycogen, and RT-PCR showed that the cells expressed α-fetoprotein （AFP） and albumin （ALB） mRNAs. Conclusion The culture system of IESCs in vitro is successfully established, and the cells are differentiated into hepatocyte-like cells.