【摘要】 目的 探讨人外周血来源间充质干细胞(mesenchymal stem cells,MSCs)的体外分离、培养及诱导分化成有功能肝样细胞的可行性。方法 静脉采集13例成人外周血,分别应用单纯密度梯度离心法及人间充质干细胞富集试剂+密度梯度离心两步法分离MSCs;将得到的MSCs经贴壁纯化扩增,以流式细胞仪检测细胞表面抗原后,经肝细胞生长因子(HGF)、成纤维细胞生长因子-4(FGF-4)及抑瘤素M(OSM)诱导分化成肝样细胞;以RT-PCR、免疫荧光染色及Western blot检测所得细胞中白蛋白(ALB)、甲胎蛋白(AFP)在基因及蛋白水平上的表达情况;电镜检测所得细胞的超微结构。结果 外周血应用单纯密度梯度离心法无法培养得到MSCs。人间充质干细胞富集试剂+密度梯度离心两步法可分离并培养获得梭形细胞,经细胞表面抗原检测证实为MSCs;所得细胞经过纯化扩增和诱导分化后,RT-PCR、免疫荧光及Western blot均证实细胞在基因及蛋白水平上存在ALB和AFP的表达;电镜证实其具有类肝细胞的超微结构。结论 人外周血中存在MSCs并可使用非侵袭性的人间充质干细胞富集试剂+密度梯度离心两步法方法提取分离,所得MSCs具诱导分化成有功能肝样细胞的能力,此方法能够为以后的肝组织工程研究提供种子细胞来源。更多还原

【Abstract】 Objective To culture and amplify mesenchymal stem cells from human peripheral blood,and study the possibility of differentiating them into hepatocyte-like cells in vitro and observe the function of hepatocyte-like cells.Methods Human peripheral blood was obtained sterilely by ulnar vein from 13 donors.MSCs were isolated by ficoll or human mesenchymal stem cell enrichment cocktail.Mature cells were removed by the difference of average adhesion time.Purified cells induced by recombinant HGF,FGF-4 and OSM,were identified by the specific biological functions with biochemical assays in vitro.Results We could isolate and culture MSCs from peripheral blood by cocktail procedures rather than ficoll procedures.The expression of cell surface antigens resulted in the MSCs phenotype.In differentiated MSCs,gene expression of ALB and AFP were found by RT-PCR analysis,ALB and AFP protein were confirmed to express by immunofluorescence and Western blot.The ultrastruture of the cells in transmission electron microscope showed the induced cells were hepatocyte-like cells.Conclusion The existence of MSCs in human peripheral blood is confirmed by our study.These cells can be isolated by non-invasive method and differentiate into heppatocyte-like cells.This method provides us a potential seed cells source for liver tissue engineering.更多还原