过表达Wnt3基因抑制肝前体细胞凋亡

Overexpression of Wnt3 inhibits apoptosis of hepatic progenitor cells in vitro

目的利用腺病毒载体转染体外培养的肝前体细胞,研究过表达Wnt3对肝前体细胞凋亡的影响。方法用携带Wnt3的腺病毒载体(Ad-Wnt3)转染肝前体细胞,同时设立空载体腺病毒转染组(Ad-GFP)为对照。转染72 h后,用荧光显微镜观察Hoechst33342染色后细胞凋亡情况;流式细胞术检测Annexin-PE/7-ADD法标记的细胞凋亡率;RT-PCR和Western blot分别检测细胞凋亡相关蛋白Bax、Bcl-2和Bcl-xl的mRNA及蛋白表达水平。结果 qRT-PCR和Western blot结果显示,Wnt3在肝前体细胞中特异性表达,表明腺病毒系统Ad-Wnt3能高效转染肝前体细胞。与对照组相比较,肝前体细胞转染Wnt3后,肝前体细胞的凋亡水平明显下降,凋亡相关蛋白Bcl-2和Bcl-xl的mRNA及蛋白表达水平明显升高(P<0.05)。Bax的mRNA及蛋白表达水平则明显下降(P<0.05)。结论在肝前体细胞中,Wnt3基因过表达能抑制肝前体细胞凋亡,其机制可能是通过Bcl-2家族起作用。

Objective To investigate the effects of adenoviral vector-mediated over-expression of Wnt3 on the apoptosis of hepatic progenitor cells in vitro. Methods Hepatic progenitor ceils transfected with Ad-GFP-Wnt3 vector or the control vector Ad-GFP were examined for cell apoptosis under fluorescence microscopy with Hoechst 33342 staining, and the proportion of apoptotic cells were determined by flow cytometric analysis with Annexin-PE/7-ADD staining. The mRNA and protein expressions of Bax, Bcl-2 and Bcl-xl in the cells were detected by real-time PCR and Western blotting, respectively. Results Real-time PCR and Western blotting showed a high expression of Wnt3 in Ad-GFP-Wnt3-transfected hepatic progenitor ceils, which exhibited significantly decreased cell apoptosis as compared with the control group. The expressions of Bcl-2 and Bcl-xl mRNA and proteins increased significantly while Bax expression decreased obviously in Ad-GFP-Wnt3-transfected cells （P〈 0.05）. Conclusion Adenoviral vector-mediated over-expression of Wnt3 can suppress apoptosis of hepatic progenitor cells possibly through the Bcl-2 pathway.