肿瘤坏死因子相关凋亡诱导配体对乙型肝炎病毒启动子的调控作用

The Effect of Tumor Necrosis Factor-related Apoptosis-inducing Ligand on the Self-promoter of Hepatitis B Virus

目的肿瘤坏死因子相关凋亡诱导配体(TRAIL)能够诱导乙型肝炎病毒(HBV)感染细胞发生凋亡,但抑制病毒复制的具体机制不清楚,研究通过非凋亡浓度TRAIL对4种HBV启动子调控作用的研究,探讨HBV复制的可能调控机制。方法采用噻唑蓝法和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记荧光法,检测不同浓度TRAIL作用后人肝癌细胞株HepG2的存活率。使用HBV的4种启动子重组质粒,4种启动子分别控制乙肝表面抗原、X抗原、核心抗原、PS1抗原基因的转录与表达。将受HBV上述4种启动子调控的荧光素酶报道基因表达质粒(SpLUC、XpLUC、CpLUC、PS1pLUC)转染HepG2细胞,6 h后按300、30、3 ng/mL浓度梯度加入可溶性TRAIL,采用双荧光报道基因分析系统检测细胞化学发光值,计算相对荧光素酶活性。结果 300 ng/mL是可溶性TRAIL诱导HepG2细胞凋亡的浓度阈值。采用远低于凋亡阈值浓度(30 ng/mL)的TRAIL可明显上调对HBV的Sp启动子活性(P<0.001),另3种质粒的相对荧光素酶活性在加入TRAIL后改变不大。结论 TRAIL仅对HBV的Sp启动子活性具有上调作用,其生物学意义值得进一步研究。

Objective To investigate if tumor necrosis factor-related apoptosis-inducing ligand（TRAIL） has an effect on the self-promoter of hepatitis B virus（HBV） by studying the infl uences of TRAIL on four kinds of HBV selfpromoters. Methods After being treated with soluble TRAIL,the survival rate and apoptosis of HepG2 cells were measured by MTT assay and TUNEL assay.HepG2 cells were transfected with reported Luciferase vector CpLUC, XpLUC,SpLUC,and PS1pLUC respectively,and then treated with TRAIL of different concentration six hours later.DualGloR Luciferase Assay System was performed to detect the luminescence,and the relative luciferase activity was calculated. Results The data showed over 300 ng/mL of TRAIL could lead to obvious apoptosis in HepG2 cells.The relative luciferase activity of SpLUC increased more than 100% with the concentration of 30 ng/mL TRAIL（P 0.001）,while those of other self-promoters only changed a little.Conclusion TRAIL can enhance the activity of HBV Sp self-promoter, and its biological significance is worthy to be studied further.