HBx基因通过调节凋亡相关蛋白Bax/Bcl-2比值促进人肾小管上皮细胞凋亡

HBx Gene Promotes Apoptosis in HK-2 Cells Through Regulating Apoptosis-related Protein Ratio of Bax/Bcl-2

目的探讨HBV x基因(HBx)对人肾小管上皮细胞株(HK-2)增殖和凋亡的影响及机制。方法采用脂质体转染法,将质粒pCDNA3.1(+)HBx转染至HK-2细胞株。细胞实验分5组,对照组、转染空质粒pCDNA3.1(+)组、转染pCDNA3.1(+)HBx24 h组、转染pCDNA3.1(+)HBx 48 h组、转染pCDNA3.1(+)HBx 72 h组。CCK8方法检测HBx对各组细胞增殖抑制率的影响;用透射电镜及AV/PI双染流式细胞仪检测细胞凋亡率。应用免疫细胞化学及免疫印迹检测各组细胞凋亡相关蛋白Bax及Bcl-2水平。结果经酶切鉴定及测序结果显示pCDNA3.1-HBx构建成功。与对照组相比,空质粒组细胞存活率及凋亡率无明显差异,凋亡相关蛋白Bax/Bcl-2比值无明显差异(P>0.05)。转染HBx基因24 h后,细胞增殖明显受抑,凋亡率显著增加,Bax/Bcl-2比值开始增高。至72 h,细胞增殖抑制率最高达到(35.27±1.10)%,细胞凋亡率达到最高为(23.80±2.16)%,Bax/Bcl-2比值显著增高。与对照组及其他各组比较,具有显著性差异(P<0.05)。结论 HBx基因可抑制HK-2细胞增殖并通过提高凋亡相关蛋白Bax/Bcl-2比值诱导其凋亡。

Objective To discuss the influence ofHBV X gene （HBx） on the proliferation and apoptosis of human renal tubular epithehal cell line （ HK - 2）. Methods The experiment was divided into five groups ： the control group, cells transfected with empty plasmid PCDNA3.1 （ ＋ ）, PCD- NA3.1 （＋） HBx 24 h, PCDNA3.1 （＋） HBx 48 h and PCDNA3.1 （＋） HBx 72 h, respectively. The CCK8 method was used to detect the influence of HBx to the inhibition rates of cell proliferation in each group, and transmission electron microscopy and AV/PI double staining flow cytometry were used to detect apoptosis rates. With immunocytochemistry and Western blot tests were used to measure the cell apoptosis related proteins Bax and Bcl -2 levels. Results Restriction enzyme digestion and sequencing results show that the PCDNA3.1-HBx was constructed successfully. Compared with the contro! group, the viabihty and apoptosis rate of the cells in the empty plasmid group had no significant difference（P 〉 0.05 ）, the same to the cell apoptosis related protein ratio of Bax/Bel-2 （ P 〉 0.05 ）. After tiansferring HBx gene for 24 hours, cell proliferation was inhibited, the apoptosis rate increased significantly and the Bax/Bcl-2 ratio started to increase. After 72 hours, the inhibition rate of cell proliferation was the highest （ 35.27 ± 1.10）%, the apoptosis rate was （23.80 ±2.16）%, and the Bax/Bel-2 ratio increased significantly. Compared to the control group and the other groups, there was a significant difference（P 〈 0.05 ）. Conclusion HBx gene could inhibit the HK-2 cell proliferation and induce apoptosis through increasing the cell apoptosis related protein ratio of Bax/Bcl-2.