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目的探讨乙型肝炎病毒(hepatitis B virus,HBV)X基因(HBX)促肿瘤坏死因子相关凋亡诱导配体(TNF relatedapoptosis inducing ligand,TRAIL)蛋白诱导人肝癌细胞Huh7凋亡的机制。方法应用脂质体转染法将pcDNA3.1-HBX、pcDNA3.1分别转染Huh7细胞,建立稳定表达HBX的细胞模型Huh7-HBX和空载体对照细胞模型Huh7-3.1;运用TRAIL蛋白分别作用Huh7-HBX细胞、Huh7-3.1及Huh7细胞后,采用CCK8、流式细胞术检测细胞增殖和凋亡情况;Western blot检测死亡受体4(deathreceptor 4,DR4)和死亡受体5(death receptor 5,DR5)的表达情况;分光光度法检测细胞caspase8和caspase3活性。结果 RT-PCR及Western blot证实成功构建细胞株Huh7-HBX;CCK8和流式细胞术检测结果均显示Huh7-HBX细胞的凋亡率明显高于Huh7-3.1及Huh7(P<0.05);Western blot显示Huh7-HBX细胞的DR4、DR5的表达量明显高于Huh7-3.1及Huh7细胞(P<0.05);caspase活性检测结果显示,Huh7-HBX细胞caspase8及caspase3的活性明显高于Huh7-3.1及Huh7(P<0.05)。结论 HBX能够通过上调Huh-7细胞表面死亡受体DR4、DR5的表达,进而促进TRAIL蛋白诱导的细胞凋亡,为进一步研究HBX的促凋亡机制提供实验依据。
Objective To investigate the mechanism of hepatitis B virus (HBV) X gene (HBX) inducing apoptosis of Huh7 cells induced by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). Methods Huh7 cells were transfected with pcDNA3.1-HBX and pcDNA3.1 respectively by lipofection method. Huh7-HBX, a cell model stably expressing HBX, and Huh7-3.1, an empty vector control cell model, were established. Huh7 -HBX cells, Huh7-3.1 and Huh7 cells, CCK8 was used to detect the cell proliferation and apoptosis. Flow cytometry was used to detect the expression of death receptor 4 (DR4) and death receptor 5 DR5). The activity of caspase8 and caspase3 were detected by spectrophotometry. Results Huh7-HBX was successfully constructed by RT-PCR and Western blot. The results of CCK8 and flow cytometry showed that the apoptosis rate of Huh7-HBX cells was significantly higher than that of Huh7-3.1 and Huh7 (P <0.05) The expression of DR4 and DR5 in Huh7-HBX cells was significantly higher than that in Huh7-3.1 and Huh7 cells (P <0.05). The results of caspase activity showed that the activity of caspase8 and caspase3 in Huh7-HBX cells was significantly higher than that of Huh7-3.1 and Huh7 (P <0.05). Conclusions HBX could up-regulate the expression of DR4 and DR5 on Huh-7 cells, and further promote the apoptosis induced by TRAIL protein, which may provide an experimental basis for further study on the mechanism of apoptosis induced by HBX.