摘要：
BACKGROUND:Umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells in vitro and in vivo.However,the exact mechanism is still unknown. Existing studies have shown that the Wnt/β-catenin signaling pathway is closely related to this process. OBJECTIVE: To explore the effect of Wnt/β-catenin signaling pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells and its potential molecular mechanism. METHODS: Human umbilical cord mesenchymal stem cells were extracted from the neonatal umbilical cord by tissue adherent method. After being cultured and purified, the umbilical cord mesenchymal stem cells at passages 4-6 were divided into four groups: control group (DMEM culture group), hepatocyte-like differentiation group, activator Wnt3a group (adding 20 μg/L Wnt3a, an activator of Wnt/β-catenin signaling pathway, under the differentiation condition), and inhibitor Dkk-1 group (adding 20 μg/L Dkk-1, an inhibitor of Wnt/β-catenin signaling pathway, under the differentiation condition). Induced cells were collected respectively on days 7, 14, 21, 28. Their mRNA and protein expressions of α-fetoprotein (AFP), albumin (ALB), hepatocyte nuclear factor 4α (HNF4α) and Cytokeratin-19 (CK-19) in the cells were detected by real-time quantitative PCR and western blot respectively. Meanwhile, Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test were applied to detect the function of hepatocyte-like cells. RESULTS AND CONCLUSION: Compared with the control group, expressions of AFP and HNF4α mRNA and protein as well as ALB mRNA were significantly up-regulated in the hepatocyte-like differentiation group, activator Wnt3a group and inhibitor Dkk-1 group (P < 0.05). Whereas, there was a decrease in the CK-19 expression at mRNA and protein levels (P < 0.01) in these three groups. Compared with the hepatocyte-like differentiation group, the mRNA and protein expressions of AFP and HNF4α, and the mRNA expression of ALB were significantly down-regulated in the activator Wnt3a group (P < 0.05). Compared with hepatocyte-like differentiation group and activator Wnt3a group, the inhibitor Dkk-1 group had higher expression of AFP, HNF4α mRNA and their proteins as well as the mRNA expression of ALB (P <0.05). Findings from the Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test showed more positive cells in the inhibitor Dkk-1 group than in the hepatocyte-like differentiation group and least positive cells in the activator Wnt3a group. Overall, these findings suggest that the inhibition of Wnt/β-catenin signaling pathway promotes the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells;conversely,the cell differentiation can be inhibited via the Wnt/β-catenin pathway.%背景:脐带间充质干细胞可在体内外诱导分化为肝样细胞,然而确切机制目前尚不清楚,研究表明Wnt/β-catenin信号通路与之密切相关.目的:探讨Wnt/β-catenin信号通路对脐带间充质干细胞肝向分化的影响及其机制.方法:采用组织块贴壁法分离培养人脐带间充质干细胞,将培养至第4-6代脐带间充质干细胞分成4组,分别为对照组,常规诱导肝向分化组,激活剂Wnt3a组(在常规诱导肝向分化基础上加入20 μg/L Wnt/β-catenin信号通路激活剂Wnt3a),抑制剂Dkk-1组(在常规诱导肝向分化基础上加入20 μg/L Wnt/β-catenin信号通路抑制剂Dkk-1).分别在诱导第7,14,21,28天收集细胞,采用荧光定量PCR和Western blot检测肝细胞分化相关基因的mRNA和蛋白表达.运用PAS染色、LDL摄取实验、ICG吸收实验在诱导第28天时进行肝样细胞功能检测.结果与结论:①与对照组相比,常规诱导组、激活剂Wnt3a组、抑制剂Dkk-1组中除了CK-19 mRNA及蛋白表达均下调外(P < 0.01),AFP、ALB、HNF4α mRNA和AFP、HNF4α蛋白表达均显著上调(P < 0.05);②与常规诱导组相比,激活剂Wnt3a组AFP、ALB、HNF4α mRNA及AFP、HNF4α蛋白表达均显著下调(P < 0.05);③与常规诱导组、激活剂Wnt3a组相比,抑制剂Dkk-1组AFP、ALB、HNF4α mRNA及AFP、HNF4α蛋白表达显著增高(P < 0.05);④抑制剂Dkk-1组PAS染色、LDL摄取实验、ICG吸收实验中检测到的阳性细胞最多,常规诱导组其次,阳性细胞最少的是激活剂Wnt3a组;⑤以上结果表明,Wnt/β-catenin信号通路受抑制时可促进脐带间充质干细胞肝向分化,反之,则抑制脐带间充质干细胞肝向分化.
