人骨髓间充质干细胞向肝细胞诱导分化的进一步研究

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）。分别进行培养,诱导前通过流式细胞仪分析细胞表面标志CD44、CD90的表达率对培养的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情况。结果：本文通过建立从人骨髓中分离、培养、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。

The further study of human bone marrow-derived mesenchymal stem cells differentiation into hepatocytes

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 divided 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 cytometry 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.