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胎肝间充质干细胞和皮肤成纤维细胞有限细胞系的建立及肝向分化研究

ISSN号：1674-1927
期刊名称：《中华生物医学工程杂志》
时间：0
分类：R622.1[医药卫生—整形外科;医药卫生—临床医学;医药卫生—外科学]
作者机构：[1]天津市人工细胞重点实验室,天津市再生医学与疾病生物治疗工程研究中心,天津市肝胆疾病研究所,天津市第三中心医院,300170
相关基金：2005年天津市高新技术产业化专项资金（津发改高技[2006]26号）

作者：朱争艳[1], 杜智[1], 骆莹[1], 黎娇[1], 王鹏[1], 刘彤[1], 高英堂[1]

关键词：胚胎, 间质干细胞, 成纤维细胞, 类肝细胞, 诱导分化, Embryo, Msesenchymal stem cell, Fibroblasts , Hepatocyte-like cell, Hepatic culture medium

中文摘要：

目的通过体外长期培养胎肝间充值干细胞和皮肤成纤维细胞，建立两种细胞长期培养体系并比较此两种细胞的表型特征及向肝细胞分化的潜能。方法从人胎肝中分离贴壁生长的间充质干细胞，同时取同一个体的皮肤组织，分离培养成纤维细胞。采用免疫细胞化学法及流式细胞术检测上述两种细胞的CD34，CD90，CD105等细胞表型；染色体分析及软琼脂克隆形成实验鉴定细胞的生物学特性。利用肝细胞生长因子（HGF），成纤维细胞生长因子4（FGF4）、抑瘤素M（OsM）等组成的肝细胞培养基对P3—30细胞进行肝细胞诱导分化。采用免疫细胞化学方法对诱导和未诱导的细胞进行白蛋白（ALB）、CK18、CK19等免疫学检测。PAS法糖原染色，RT-PCR法检测甲胎蛋白（AFP）和ALB mRNA表达。结果胎肝间充质干细胞高表达CD90，而皮肤成纤维细胞几乎不表达。在肝细胞条件培养基中培养，成纤维细胞转化为类肝细胞的比例为（5．1±0．2）％，而胎肝间充质干细胞转化为类肝细胞的比例为（10．3±0.3）％，两组差异有统计学意义（P〈0．01）。此两种细胞均可在体外长期稳定培养，传50余代，保持正常核型，无致瘤性，但难以无限传代。结论成体组织中体外能够长期培养的间充质干细胞可能是有一定寿命的。特定成体组织内的间充质干细胞可能具有更高的分化为所在组织实质细胞的潜力。

英文摘要：

Objective To establish a long-term culture system of fetal liver mesenchymal stem cells and skin fibroblasts, and to compare the phenotyping and the hepatocyte differentiation between two types of cells. Methods Fetus liver-derived mesenchymal stem cells were selected by adherence culture, and fibroblasts were isolated and cultured from skin tissue of the same individual. Immunocytochemistry （ICC） and flow cytometry （FCM） were used in detection of the expressions of CD34, CD90 and CD105, and chromosome analysis and soft agar colony formation test in study of biological characteristics in the two types of cells. The induced differentiation of the P3-30 cell into hepatocyte was performed with hepatic culture medium （HCM） containing hepatocyte growth factor （HGF） , fibroblast growth factor 4 （FGF4） and oncostatin M （OSM）. Albumin （ALB）, CK18 and C K 19 in the induced and non-induced cells were detected by ICC, glycogen stained by PAS, and the expressions of AFP and ALB mRNA by RT-PCR. Results CD90 was highly expressed in fetal liver mesenchymal stem cells but barely detectable in skin fibroblasts. Culture in conditioned medium led to hepatocyte-like differentiation in approximately （5.1 ±0.2）% of fibroblasts versus （ 10.3±0.3）% of fetal liver mesenchymal stem cells （P〈0.01）. Both of the two types of cells could be cultured in vitro for about 50 passages that appeared karyotypically normal, non-oncogenic but not immortal. Conclusion Mesenchymal stem cells of adult tissues derived from any adult tissue may have a limited life- time over a long-term culture in vitro, and some from certain adult tissues may show a stronger potential of differentiation to parenchyma cells of the origin.