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目的探讨wnt3对小鼠肝前体细胞(14-19)发生上皮-间质转化的影响。方法分别将空载体Ad-GFP和表达wnt3的腺病毒Ad-GFP-wnt3转入小鼠肝前体细胞中,显微镜下观察细胞形态变化。细胞划痕实验和细胞迁移实验观察14-19细胞迁移能力。实时荧光定量PCR和蛋白质印迹分析分别检测14-19细胞中上皮标记物和间质标记物的表达改变。结果显微镜下可见高表达wnt3的14-19细胞由不规则多边形变为长梭形。细胞划痕实验和细胞迁移实验结果显示,高表达wnt3的14-19细胞迁移能力明显提高,与空载体转染细胞相比差异具有统计学意义(P<0.01)。实时荧光定量PCR和蛋白质印迹结果显示,间质标记物N-cadherin、vimentin和twist1的mRNA和蛋白水平表达上调,相反上皮标记物E-cadherin的mRNA水平和蛋白水平表达下调,与空载体转染细胞相比差异具有统计学意义(P<0.01)。结论 Wnt3能够促使小鼠肝前体细胞发生上皮-间质转化,提示其可能参与了肝纤维化的发展进程。
Objective To investigate the effect of wnt3 on epithelial-mesenchymal transition in mouse hepatic precursor cells (14-19). Methods Ad-GFP vector and Ad-GFP-wnt3 expressing wnt3 were transfected into mouse liver precursor cells, and the morphological changes were observed under microscope. Cell scratch assay and cell migration assay 14-19 cell migration ability. Real-time quantitative PCR and Western blot analysis were used to detect the changes of epithelial markers and interstitial markers in 14-19 cells respectively. Results Microscopically, 14-19 cells with high expression of wnt3 changed from irregular polygons to long fusions. The results of cell scratch assay and cell migration showed that the migration ability of 14-19 cells with high expression of wnt3 was significantly increased compared with that of empty vector transfected cells (P <0.01). Real-time quantitative PCR and Western blotting showed that the mRNA and protein expression of interstitial markers N-cadherin, vimentin and twist1 were upregulated. On the contrary, mRNA and protein expression of epithelial marker E-cadherin was down-regulated and transfected with empty vector The difference between cells was statistically significant (P <0.01). Conclusion Wnt3 can induce epithelial-mesenchymal transition in mouse hepatic precursor cells, suggesting that Wnt3 may be involved in the development of hepatic fibrosis.