着色性干皮病基因D和p53对HBV复制的影响

发布时间：2019-01-14 08:24

【摘要】：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20μmol/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、pEGFPN2/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P0.01)。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P0.01)。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P0.01)。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P0.01)。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。
[Abstract]:Objective: to investigate the effect of human xeroderma pigmentosa gene D (XPD) and p53 on the replication of hepatitis B virus (HBV) (HBV). Methods: recombinant plasmid pEGFP-N2/XPD and empty plasmid pEGFP-N2 were transfected into human hepatoma cell line HepG2.2.15 cells by liposome transfection. The cells were incubated with 20 渭 mol/L pifithrin- 伪 (p53 inhibitor) for 24 h on the second day after transfection. The experiment was divided into blank control group, pEGFP-N2 group, pEGFPN2/XPD group, pEGFP-N2/XPD pifithrin- 伪 group and pifithrin- 伪 group. RT-PCR method was used to detect the expression of (HBsAg), hepatitis B antigen (HBeAg) and hepatitis B virus X protein (HBx) mRNA, ELISA method was used to detect the content of HBsAg and HBeAg in the supernatant. Fluorescence quantitative PCR assay was used to detect the changes of HBV-DNA content in supernatant and bDNA method was used to detect the content of HBV-DNA in the core granules. Results: the results of RT-PCR showed that transfection of recombinant plasmid pEGFP-N2/XPD could increase the expression of XPD mRNA and decrease the expression of HBsAg,HBeAg and HBx mRNA. However, pifithrin- 伪 could inhibit this effect of XPD (P0.01). ELISA results showed that the increase of XPD expression could significantly reduce the contents of HBsAg and HBeAg in the supernatant, while pifithrin- 伪 could inhibit the effect of XPD (P0.01). The results of fluorescence quantitative PCR showed that the increased expression of XPD could significantly decrease the content of HBV-DNA in the supernatant, while pifithrin- 伪 could inhibit the effect of XPD (P0.01). BDNA results showed that, P < 0.05). The increased expression of XPD significantly reduced the content of HBV-DNA in the core granules, while pifithrin- 伪 inhibited the effect of XPD (all P0.01). Conclusion: XPD can inhibit HBV replication through p53 pathway, so XPD and p53 may be the targets of antiviral therapy.
【作者单位】：南昌大学第二附属医院消化内科;
【基金】：国家自然科学基金资助项目(No.81300348) 江西省科技厅社会发展领域项目(No.20141BBG70038) 江西省卫生厅科技计划项目(No.20141070)
【分类号】：R758.58;R512.62

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