诱骗受体3对肝细胞凋亡的影响

Effect of decoy receptor 3 gene on hepatocyte apoptosis

目的了解诱骗受体3(DcR3)对肝细胞凋亡的影响,探讨DcR3在此过程中的可能作用机制。方法体外培养人肝细胞细胞株,分别设置pEF1α-DcR3转染组、pEF1α-IRES转染组和阴性对照组。构建pEF1α-IRES-DsRed-Express2-DcR3真核表达载体,转染人肝细胞36 h,采用实时荧光定量PCR检测DcR3、Fas蛋白配体(Fas L)、α-平滑肌肌动蛋白(α-SMA)和转化生长因子(TGF)β1的mRNA表达水平;Western Blot法检测DcR3蛋白表达变化;流式细胞术检测细胞凋亡情况。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t法。结果 pEF1α-DcR3转染人肝细胞36 h后,DcR3的mRNA表达量和蛋白表达水平与pEF1α-IRES转染组和阴性对照组相比,显著升高(F值分别为33 169.5、141.54,P值均<0.01);pEF1α-DcR3转染组与其他两组对比,Fas L、α-SMA和TGFβ1的mRNA表达水平显著降低(F值分别为269 451.8、20 790.4、8067.8,P值均<0.01),表明DcR3可以抑制Fas L、α-SMA和TGFβ1的表达;转染pEF1α-DcR3的肝细胞凋亡率较pEF1α-IRES组和阴性对照组显著降低(F=558.63,P值均<0.01)。结论 DcR3能够抑制人肝细胞凋亡,下调Fas L、α-SMA和TGFβ1 mRNA的表达水平。

Objective To investigate the effect of decoy receptor 3（ DcR3） gene on hepatocyte apoptosis,as well as the possible mechanism of action of DcR3 in this process. Methods The human liver cell lines were cultured in vitro,and p EF1α- DcR3 transfection group,p EF1α- IRES transfection group,and negative control group were established. The p EF1α- IRES- DsRed- Express2- DcR3 eukaryotic expression vector was constructed and transfected into human liver cell lines for 36 hours. qRT- PCR was used to measure the mRNA expression of DcR3,Fas ligand（ Fas L）,α- smooth muscle actin（ α- SMA）,and transforming growth factor- β1（ TGF- β1）,Western blot was used to measure the change in the protein expression of DcR3,and flow cytometry was used to measure apoptosis. An analysis of variance was used for comparison of continuous data between groups,and the least significant difference t- test was used for comparison between any two groups. Results After human liver cell lines were transfected with p EF1α- DcR3 for 36 hours,the p EF1α- DcR3 transfection group showed significant increases in the mRNA and protein expression of DcR3 compared with the p EF1α- IRES transfection group and negative control group（ F = 33 169. 5and 141. 54,all P〈0. 01）. Compared with the other two groups,the p EF1α- DcR3 transfection group showed significant reductions in the mRNA expression of Fas L,α- SMA,and TGF- β1（ F = 269 451. 8,20 790. 4,and 8067. 8,all P〈0. 01）,which suggested that DcR3 inhibited the expression of Fas L,α- SMA,and TGF- β1. Compared with the p EF1α- IRES transfection group and negative control group,the p EF1α- DcR3 transfection group showed a significant reduction in apoptosis rate（ F = 558. 63,all P〈0. 01）. Conclusion DcR3 can inhibit hepatocyte apoptosis and downregulate the mRNA expression of Fas L,α- SMA,and TGF- β1.