人脐带间充质干细胞与肝细胞共培养可分化为肝样细胞***☆

摘要

背景：文献报道，从骨髓与脐带中分离获得的间充质干细胞可在体外连续传代培养，仍保持干细胞的特性，并在多种细胞因子的“鸡尾酒式”诱导下分化为肝细胞样细胞。目的：进一步验证人脐带间充质干细胞在体外正常人肝细胞共培养体系下是否可分化为肝细胞并探讨其分化方法。方法：采用贴壁法，从脐带中分离培养间充质干细胞，流式细胞仪检测脐带间充质干细胞表面标志。人肝细胞 LO2细胞与人脐带间充质干细胞建立共培养体系，不添加外源诱导因子，分别于第7，14，21天，通过RT-PCR 法检测肝细胞特异标志物甲胎蛋白、白蛋白、人细胞角蛋白19 mRNA的表达，糖原染色进行功能鉴定。结果与结论：从人脐带中可分离得到贴壁生长的间充质干细胞，其中CD29+细胞比例为96.02%，CD105+细胞比例为96.6%，CD34-细胞比例为99.65%，CD105+CD29+双阳性细胞比例为94.84%。与LO2细胞共培养后第7天仅有甲胎蛋白阳性表达；第14天表达白蛋白、人细胞角蛋白19，第21天时，LO2与人脐带间充质干细胞共培养组未出现甲胎蛋白表达；人细胞角蛋白19和白蛋白的表达比第14天略有增强。共培养21 d后，糖原染色呈阳性。结果证实，无需额外添加外源诱导因子，脐带间充质干细胞可在人正常肝细胞共培养的微环境中，向正常肝细胞分化。%BACKGROUND:The studies have shown that the mesenchymal stem cel s derived from bone marrow and umbilical cord can be continuously cultured in vitro, and maintain the characteristics of stem cel s. The mesenchymal stem cel s can differentiate into hepatocyte-like cel s after“cocktail”induction by various cytokines. OBJECTIVE:To further identify whether umbilical cord-derived mesenchymal stem cel s in vitro co-cultured with normal hepatocytes can differentiate into hepatocyte-like cel s, and to investigate the differentiation method. METHODS:Mesenchymal stem cel s were isolated from human umbilical cord with adherent method, and the surface markers of umbilical cord-derived mesenchymal stem cel s were detected with flow cytometry. The umbilical cord-derived mesenchymal stem cel s were co-cultured with liver LO2 cel s without adding exogenous inducers. The expressions of alpha-fetoprotein, albumin and human cytokeratin 19 mRNA of hepatocyte specific markers were detected with reverse transcription PCR at 7, 14 and 21 days after culture, and periodic acid-Schiff staining was used to identify the functions. RESULTS AND CONCLUSION:Mesenchymal stem cel s could isolated from human umbilical cord successful y, showing fibroblastic morphology and adherent cel characterization. Among these cel s, 96.02%cel s were CD29 positive cel s and 96.6%cel s were CD105 positive cel s. The percentage of CD34 negative cel s was 99.65%. The percentage of CD105+CD29+double positive cel s was 94.84%. The mRNA of alpha-fetoprotein was found on the 7th day after co-cultured with LO2 cel s, and the mRNA of albumin and human cytokeratin 19 were found on the 14th day. After co-cultured for 21 days, the alpha-fetoprotein mRNA could not be observed in the co-culture group. The expressions of albumin and human cytokeratin 19 were increased at 14 days. After co-cultured for 21 days, the glycogen staining was positive. Umbilical cord-derived mesenchymal stem cel s can differentiate into hepatocyte-like cel s after co-cultured with normal hepatocytes.