着色性干皮病基因D和p53对HBV复制的影响

 目的:探讨人着色性干皮病基因D(XPD) 和p53对乙型肝炎病毒（HBV）复制的影响。方法:使用脂质体转染法把重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2转染进入人肝癌细胞株HepG2.2.15细胞，转染后的第2天用20 μmol/L pifithrin-α（p53抑制剂）孵育细胞24 h。实验共分为空白对照组、pEGFP-N2组、pEGFP-N2/XPD组、pEGFP-N2/XPD+pifithrin-α组和pifithrin-α组。使用RT-PCR法检测XPD、乙型肝炎表面抗原（HBsAg）、乙型肝炎e抗原（HBeAg）及乙型肝炎病毒X蛋白（HBx） mRNA表达的变化；使用ELISA法检测培养上清液中HBsAg和HBeAg含量的变化；用荧光定量PCR法检测培养上清液中HBV-DNA含量的变化；用bDNA法检测细胞内核心颗粒中HBV-DNA含量的变化。结果:RT-PCR结果显示，重组质粒pEGFP-N2/XPD的转染可使得XPD mRNA表达增高，XPD表达增高能使得HBsAg、HBeAg和HBx mRNA表达明显减少，而pifithrin-α能抑制XPD的这一作用（均P<0.01）。ELISA结果显示，XPD表达增高能使得培养上清液中HBsAg和HBeAg含量明显减少，而pifithrin-α能抑制XPD的这一作用（均P<0.01）。荧光定量PCR结果显示，XPD表达增高能使得培养上清液中HBV-DNA含量明显减少，而pifithrin-α能抑制XPD的这一作用（均P<0.01）。bDNA结果显示，XPD表达增高使得细胞内核心颗粒中HBV-DNA含量明显减少，而pifithrin-α能抑制XPD的这一作用（均P<0.01）。结论: XPD能通过p53通路抑制HBV复制，所以XPD和p53可能成为乙型肝炎抗病毒治疗的作用靶点。

Effects of xeroderma pigmentosum group D and p53 on replication of HBV

AIM:To investigate the effects of xeroderma pigmentosum D (XPD) and p53 on the replication of hepatitis B virus (HBV). METHODS:Recombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 cells by liposome. On the next day, these cells were incubated with pifithrin-α, a p53 inhibitor, at a concentration of 20 μmol/L for 24 h. The cells were divided into 5 groups: blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD+pifithrin-α group and pifithrin-α group. The mRNA expression of XPD, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus X protein (HBx) was detected by RT-PCR. The content of HBsAg and HBeAg in the supernatants of culture medium  was measured by ELISA. The content of HBV-DNA in the supernatants of culture medium was  examined by fluorescence quantitative PCR. Using the method of bDNA, the content of HBV-DNA in the core particles was assessed. RESULTS:The expression  of XPD mRNA  was elevated by the transfection of recombinant plasmid pEGFP-N2/XPD. The increase in XPD expression significantly down-regulated the mRNA expression of HBsAg, HBeAg and HBx. The content of HBsAg and HBeAg in the supernatants of culture medium was significantly  decreased by the increase in XPD expression.  The results of fluorescence quantitative PCR showed that the content of HBV-DNA in the supernatants of culture medium was significantly down-regulated by the increase in XPD expression. bDNA results showed that the content of HBV-DNA in the core particles was  significantly decreased by the increase in XPD expression.Pifithrin-α abolished the above-mentioned effects of XPD (all P<0.01). CONCLUSION: XPD inhibits the replication of HBV through p53 pathway. Therefore, XPD and p53 may be the targets for antiviral therapy of hepatitis B.