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目的 探索人骨髓来源多能成体祖细胞 (hMAPCs)在肝细胞生长因子 (HGF) +成纤维生长因子 4 (FGF 4 )诱导下分化为肝细胞的可行性 ,为肝组织工程提供新的种子细胞来源。方法 取健康成人适量骨髓 ,采用梯度密度离心法分离获取单个核细胞并行贴壁培养 ,获取骨髓间充质干细胞(MSCs) ,将MSCs通过CD4 5、GlyA免疫微磁珠负分选得到hMAPCs;将获取的hMAPCs用HGF(2 0ng/ml) +FGF 4(10ng/ml)进行诱导分化 ,L 0 2人肝细胞株为阳性对照 ,未加任何诱导因素的hMAPCs为阴性对照 ;免疫细胞化学染色鉴定不同诱导分化阶段细胞的ALB、AFP、CK 18、CK 19等肝细胞特征的表型变化 ,并计数阳性细胞比率 ;Westernblot检测诱导分化第 2 1天、35天后细胞的ALB表达。结果 ALB、CK 18在诱导组中基本为阳性着色 ,CK 19均为阴性着色 ,AFP仅在诱导第 7天有阳性着色。在诱导分化第 2 1天、35天 ,ALB均有阳性表达。结论 hMAPCs在一定诱导条件下具有向肝样细胞分化的潜能
Objective To explore the feasibility of differentiating hMAPCs into hepatocytes under the induction of hepatocyte growth factor (HGF) and fibroblast growth factor 4 (FGF 4) and to provide new seed cells for liver tissue engineering source. Methods Bone marrow was collected from healthy adult. The cells were separated by gradient density centrifugation and cultured in adherent culture. BMSCs were obtained by MSCs. The MSCs were negatively sorted to hMAPCs by CD4. The obtained hMAPCs were induced to differentiate with HGF (20ng / ml) + FGF4 (10ng / ml). L0 2 human hepatocyte cell line was positive control, hMAPCs without any inducing factor were negative control. Immunocytochemical staining The phenotypic changes of hepatocytes such as ALB, AFP, CK 18 and CK 19 were observed in different stages of differentiation, and the percentage of positive cells was counted. The expression of ALB was detected by Western blot after the day 2 and day 35. Results ALB and CK 18 were basically positive staining in the induction group, CK 19 were negative staining, AFP only positive staining on the 7th day of induction. In the induction of differentiation on the first day, 35 days, ALB were positive. Conclusion hMAPCs have the potential to differentiate into hepatocyte-like cells under certain inducing conditions