HGF+FGF-4体外定向诱导人骨髓来源的多能成体祖细胞向肝样细胞分化的特征性表型鉴定

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(FGF4)诱导F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CD5、GIyA免疫微磁珠负分选得到hMAPCs；将获取的hMAPCs用HGF(20ng／m1) IFGF-4(10ng／m1)进行诱导分化，L-02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；Western blot检测诱导分化第21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21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。

Identification of characteristic proteins of hepatocytes of multipotent adult progenitor cells from human bone marrow differentiating into hepatocyte-like cells with hepatocyte growth factor and fibroblast growth factor-4 in vitro

Objective To investigate the possibility of differentiation of multipotent adult progenitor cells from human bone marrow(hMAPCs) into hepatocytes with hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) in vitro to find a new cell source for liver tissue engineering. Methods (1) Harvest of the hMAPCs: The bone marrow mononuclear cells were obtained from bone marrow obtained from volunteers by gradient centrifuging. The adherent mononuclear cells were cultured. The mesenchymal stem cells(MSCs) were isolated and collected by magnetic activated cell sorting(MACS) through depletion selection by the use of CD45 and GlyA microbeads. (2) The hMAPCs were induced to differentiate with HGF(20ng/ml) +FGF-4(10ng/ml). L-02 human hepatocytes (cell lines) were used as the control, with undifferentiated hMAPCs serving as the contrast. (3)The expression of albumin (ALB), alpha fetoprotein (AFP), cytokeratin-18(CK-18), cytokeratin-19(CK-19) was detected with immunocytochemistry to identify the characters of the differentiated cells at different time, and to determine the ratio of positive cells. (4) ALB expression of the differentiated cells at different time was detected by Western blot on day 21 and 25. Results (1) The result of immunocytochemistry: The differentiated hMAPCs expressed the late markers (ALB, CK-18) of hepatocyte at all the time but did not express CK-19. The differentiated hMAPCs expressed AFP on day 7. (2) The result of Western blot assay: the differentiated cells expressed the late markers (ALB) of hepatocyte on day 21 and 35. Conclusions hMAPCs can differentiate into hepatocyte-like cells under specific inducing conditions.