转化生长因子β1信号通路对胚胎肝前体细胞分化的诱导作用

Effect of the transforming growth factor-β1 signaling pathway in inducing the differentiation of hepatic progenitor cells

目的探讨TGFβ1对胚胎肝前体细胞(HPC)分化的影响。方法体外培养HPC-E14.5细胞株并以细胞免疫荧光染色法鉴定。将细胞分为对照组(HPC)、HPC+TGFβ1组和HPC+SB431542组。HPC+TGFβ1组滴加外源性TGFβ1,HPC+SB431542组加入TGFβ1受体抑制剂SB431542,处理72 h后通过qRT-PCR法和Western Blot法分别检测AFP、Alb、细胞角蛋白19(CK19)mRNA及蛋白的表达情况;糖原染色法检测细胞分化后的功能。计量资料多组间比较采用单因素方差分析,进一步两两比较采用Dunnett’s t检验。结果qRT-PCR检测结果:对照组的AFP mRNA表达量高于其他两组(F=128.937,P<0.01),HPC+TGFβ1组的CK19 mRNA表达量高于其他两组(F=414.467,P<0.01),HPC+SB431542组的Alb mRNA表达量最高(F=794.929,P<0.01)。Western Blot法检测结果:对照组的AFP蛋白表达量高于其他两组(F=269.000,P<0.01),HPC+TGFβ1组的CK19蛋白表达量高于其他两组(F=93.010,P<0.01),HPC+SB431542组的Alb蛋白表达水平均较其他两组明显升高(F=1086.000,P<0.01)。糖原染色法结果:对照组的糖原染色表达阳性面积比例小于其他两组(F=79.251,P<0.05)。结论TGFβ1信号诱导HPC向有功能的胆管细胞分化,而阻断TGFβ1信号通路后HPC具有向成熟肝细胞分化的趋势,分化后的胆管细胞和肝细胞具有正常的糖原储存和合成功能,这些细胞可能能够参与代谢活动。

Objective To investigate the effect of the transforming growth factor-β1(TGF-β1)signaling pathway on the differentiation of hepatic progenitor cells(HPCs).Methods HPC-E14.5 cells were cultured in vitro and immunofluorescent staining was performed for identification.The cells were divided into control group(HPCs),HPCs+TGF-β1 group,and HPCs+SB431542 group.The cells in the HPCs+TGF-β1 group were treated with exogenous TGF-β1,and those in the HPCs+SB431542 group were treated with the TGF-β1 receptor inhibitor SB431542;after 72 hours of treatment,qRT-PCR and Western blot were used to measure the mRNA and protein expression of the stem cell marker alpha-fetoprotein(AFP),the hepatocyte marker albumin(Alb),and the cholangiocyte marker cytokeratin 19(CK19),and periodic acid-Schiff staining was used to evaluate function after differentiation.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the Dunnett’s t-test was used for further comparison between two groups.Results The results of qRT-PCR showed that the control group had significantly higher mRNA expression of AFP than the other two groups(F=128.937,P<0.01),the HPCs+TGF-β1 group had significantly higher mRNA expression of CK19 than the other two groups(F=414.467,P<0.01),and the HPCs+SB431542 group had the highest mRNA expression of Alb(F=794.929,P<0.01).The results of Western blot showed that the control group had significantly higher protein expression of AFP than the other two groups(F=269.000,P<0.01),the HPCs+TGF-β1 group had significantly higher protein expression of CK19 than the other two groups(F=93.010,P<0.01),and the HPCs+SB431542 group had significantly higher protein expression of Alb than the other two groups(F=1086.000,P<0.01).Periodic acid-Schiff staining showed that the control group had a significantly smaller positive staining area than the other two groups(F=79.251,P<0.05).Conclusion The TGF-β1 signaling pathway induces HPC differentiation into functional cholangiocytes;however,HPCs tend to differentiate into mature hepatocytes after the TGF-β1 signaling pathway is blocked.Such cholangiocytes and hepatocytes have the normal function of glycogen storage and synthesis and may participate in metabolic activities.