【摘要】 目的评估肝细胞生长因子(HGF)和成纤维细胞生长因子4(FGF4)在诱导骨髓间充质干细胞向肝细胞分化中的作用。方法CCl4经腹腔注射复制小鼠肝损伤模型,48h后处死小鼠取肝组织,分离肝脏组织制备肝损伤条件培养液进行培养。以正常小鼠肝组织制备的培养液作为对照组。ELISA法测定培养0、1.5、3、6、12、24、48h后肝组织培养液中的HGF和FGF4含量。用肝损伤条件培养液培养骨髓间充质干细胞,分别加入HGF抗体和(或)FGF4抗体,以封闭培养液中的HGF和(或)FGF4因子,未添加细胞因子抗体的培养液作为对照组;ELISA法检测肝细胞特异性白蛋白(ALB)水平,免疫荧光法检测细胞内ALB、甲胎蛋白(AFP)、细胞角蛋白19(CK19)的表达,评价骨髓间充质干细胞向肝细胞的分化状况。结果与对照组比较,肝损伤小鼠肝细胞培养液中HGF和FGF4表达量均升高(P<0.05);在培养3h时HGF、FGF4上升达峰值,随后下降;FGF4在培养12h达低谷后再次升高。抑制HGF和(或)FGF4第14天后,ELISA和荧光标记染色法检测均显示ALB表达下降,与对照组比较差异显著(P<0.05);荧光标记染色显示封闭后AFP及CK-19表达均有下降(P<0.05)。结论HGF、FGF4是诱导骨髓间充质干细胞向肝细胞分化的重要因子,且可能有其他因子参与诱导肝细胞的定向分化。更多还原

【Abstract】 Objective To evaluate the effect of HGF and FGF4 on inducing differentiation of bone marrow stromal stem cells (BMSSCs) to hepatocytes. Method Culture solution of injured liver was prepared with male ICR mouse liver injured by CCl4 (peritoneal injection). HGF and FGF4 released from the culture solution were measured by using ELISA at the time points of 0,1.5,3,6,12,24,and 48h after liver injury. BMSSCs were cultivated with the culture solution of injured liver,and anti-HGF and anti-FGF4 antibodies were added separately or simultaneously to block the effects of HGF and FGF4 in the process of inducing BMSSCs differentiation to hepatocytes. The normal culture medium and the culture solution of injured liver without antibodies were employed as control. The levels of ALB,AFP and CK-19 were measured by ELISA and immunofluorescence test,and the differentiation of BMSSCs to hepatocytes was evaluated. Result Compared to control group,HGF and FGF4 level increased to the peak at 3h after liver injury,and then decreased. However,the FGF4 level increased again at 12h after liver injury. On the day 14,ALB level in HGF and/or FGF4 blocked group was detected to decline significantly by ELISA and immunofluorescence test compared to control group (P<0.05). AFP and CK-19 levels presented significant difference between experimental group and control group (P<0.05). Conclusion It is proved by HGF and FGF4 blocking experiment that HGF and FGF4 were the important factors in the process of differentiation from BMSSCs to hepatocytes. Moreover,there might be other relative factors which can induce differentiation of BMSSCs to hepatocytes.更多还原