ASPP2抑制HBx引起细胞自噬的实验研究

The experiment research about using ASPP2 to inhibit the autophagy caused by HBx

目的观察ASPP2对HBx引起的细胞自噬的改变。方法运用Hbx转染Hep G 2细胞,ASPP2过表达与对照情况下,采用免疫印迹技术检测内源性自噬相关蛋白LC3Ⅱ/LC3Ⅰ和P62的表达,采用免疫荧光方法检测LC3荧光颗粒表达水平,采用RT-PCR技术检测自噬相关基因Beclin-1的m RNA转录水平。结果免疫印迹技术显示转染HBx质粒比转染空载组的LC3Ⅱ量增多,但是在加入ASPP2腺病毒组比空载组的LC3Ⅱ表达量减少。免疫荧光显示转染HBx质粒比转染空载组的LC3荧光颗粒多,但是在共转染ASPP2质粒组比空载组的LC3荧光颗粒减少。RT-PCR方法显示转染HBX质粒比转染空载组的表达增多,但是在加入ASPP2腺病毒组比空载组表达量减少。结论ASPP2可以抑制HBx引起的细胞自噬。

Objective To investigate the effect of ASPP2 on autophagy caused by HBx. Methods In condion of over-expession of ASPP2 and control, Hep G 2 cells were transfected with HBx plasmid, endogenous LC3 protein were detected by western blotting. LC3 punctas in HBV infected cells were counted by immunofluorescence. m RNA levels of autophagy related gene Beclin-1 were tested by realtime-PCR（RT-PCR）. Results Immunoblotting showed that the amount of LC3 was increased in the transfected HBx plasmid compared with the transfected no-load group, but the expression of LC3 II was lower in the Aspp2 adenovirus group than in the empty group. Immunofluorescence showed that the transfected HBx plasmid was more abundant than the LC3 fluorescent particles in the transfected no-load group, but decreased in the co-transfected ASPP2 plasmid group than the empty group. RT-PCR showed that the expression of HBx was higher than that of transfection group（P〈0.05）. Conclusion ASPP2 could inhibit the autophagy caused by HBx.