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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19mRNA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21d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: It has been reported in the literature that mesenchymal stem cells isolated from bone marrow and umbilical cord can be continuously subcultured in vitro and still retain the characteristics of stem cells, and differentiate into hepatocyte-like under the induction of “cocktail” of various cytokines cell. Objective: To further verify whether human umbilical cord mesenchymal stem cells can differentiate into hepatocytes under normal human hepatocyte co-culture system and to explore their differentiation methods. Methods: Adherent method was used to separate and culture mesenchymal stem cells from umbilical cord. Flow cytometry was used to detect the surface markers of umbilical cord mesenchymal stem cells. Human hepatocyte LO2 cells and human umbilical cord mesenchymal stem cells co-culture system was established, without adding exogenous inducer, on day 7, 14, 21, respectively, by RT-PCR detection of hepatocyte-specific markers alpha-fetoprotein Protein, human cytokeratin 19 mRNA expression, glycogen staining for functional identification. RESULTS AND CONCLUSION: Adherent MSCs could be isolated from human umbilical cord. The percentage of CD29 + cells was 96.02%, the percentage of CD105 + cells was 96.6%, the percentage of CD34- cells was 99.65%, the percentage of CD105 + CD29 + double positive cells 94.84%. After coculture with LO2 cells, only α-fetoprotein was expressed on the 7th day; albumin and cytokeratin 19 were expressed on the 14th day; on day 21, there was no alpha-fetoprotein on the co-culture of LO2 and human umbilical cord mesenchymal stem cells Expression; human cytokeratin 19 and albumin expression slightly enhanced than the 14th day. After co-cultured for 21 days, glycogen staining was positive. The results confirmed that umbilical cord mesenchymal stem cells can differentiate into normal hepatocytes in the microenvironment of normal human hepatocytes co-cultured without adding exogenous inducer.