Objective: To explore the available method of human bone marrow-derived mesenchymal stem cells (BM-MSCs) differentiation into hepatocytes and indentify the induced cells.Methods: BM-MSCs were isolated from the healthy adult, used gradient centrifugation to separate BM-MSCs and cultured through adherence, amplification to the 3rd generation which were then djvided into three groups. Group A: hepatocyte growth factor + epidermal growth factor; Group B:hepatocyte growth factor + fibroblast growth factor; Group C: hepatocyte growth factor + epidermal growth factor + fibroblast growth factor. All groups were cultured separately. Before the induction;CD44 and CD90 were examined by flow cytornetry to analyze the purity of BM-MSCs; after the induction, RT-PCR, immunohistochemistry, glycogen staining was applied to identify the cells differentiation. Results: BM-MSCs were isolated from human bone marrow via culture, amplification,passage, purified, orientation-induced differentiation into hepatocytes successfully. Image analysis indicates that the Group C was most visible expression of ALB. Conclusion: The results proved that three kinds of combinations may succeed in inducing BM-MSCs into hepatocytes which express specific markers such as ALB, AFP and glycogen, and group C is more prominent.%目的:探索骨髓间充质干细胞(BM-MSCs)向肝细胞诱导的最有效方法,并对诱导后的细胞进行鉴定.方法:取健康成人的适量骨髓,采用梯度密度离心法分离BM-MSCs并行贴壁培养,扩增至第3代,取第3代细胞分3组分别进行诱导.A组:肝细胞生长因子(HGF)+表皮生长因子(EGF);B组:肝细胞生长因子(HGF)+成纤维生长因子(FGF);C组:肝细胞生长因子(HGF)+表皮生长因子(HGF)+成纤维生长因子(HGF).分别进行倍养,诱导前通过流式细胞仪分析细胞表面标志CD44、CD90的表达率对倍养的BM-MSCs进行鉴定,诱导后通过RT-PCR、免疫组织化学法、糖原染色检测鉴定其诱导分化倩况.结果:本文通过建立从人骨髓中分离、倍养、扩增、传代、纯化BM-MSCs,定向诱导分化,使其成功向肝样细胞转化,图像分析表明C组鼠抗人白蛋白单克隆抗体(ALB)表达最为明显.结论:证明3种组合均可成功将骨髓间充质干细胞向肝样细胞诱导分化,均能分泌肝细胞特异性标记如鼠抗人甲胎蛋白单克隆抗体(AFP)、ALB、糖原,其中C组效果最为突出.
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