HBx介导的内质网应激和细胞凋亡在环磷酰胺调控HBV复制和肝细胞损伤的分子机制研究

Some clinical trials have shown that acute hepatitis due to reactivation of HBV has always been a recognized complication in patients with chronic HBV infection who received the immunosuppressive or cytotoxic (cyclophosphamide, CTX) therapy. The abrupt rise or reappearance of HBV DNA in the serum of patients with foregoing resolved or inactive HBV infection may cause severe liver damage and even liver failure or death. However, the molecular mechanism of reactivation which was caused by cytotoxic chemotherapy such as CTX still remains unclear. Through former researches, CTX could upregulated the expression of HBx and then substantially increased HBV replication by inducing AP-1 expression. Based on the previous experimental results, we speculate that the chemotherapeutic regimens, such as CTX, which may directly increase HBV replication in vitro, which could lead to acute hepatocellular damage due to HBx mediated ERS and apoptosis. The PCR, ChIP, LSCM and siRNA method was used in cell experiments, animal experiments and clinical trials. We will further clarify that CTX could promote the expression of HBx which could subsequently trigger the ERS and apoptosis signal path, is the major cause of CTX-induced hepatocellular damage. This research will illuminate the molecular mechanism of reactivation of HBV, and may offer new insights into HBV-associated pathogenesis and may be conducive to develop an effective therapy against the HBV replication. Finally, we will estimate the effect of HBx-ERS-Apoptosis pathway on both cell fate and HBV replication. This research may offer a thorough comprehension among the HBV infection, gene expression and cellular response after drug stimulation.

多项临床实验证实环磷酰胺(CTX)等化疗药物可诱导HBV再激活，导致急性肝炎或肝衰竭的发生，使死亡率大增，然而HBV再激活分子机制尚不清楚。通过前期研究发现，CTX可上调HBx和AP-1的表达，继而增强HBV启动子活性，在转录水平调控HBV；同时抑制细胞增殖并阻滞于G1期及诱导细胞内质网应激-自噬和凋亡等。基于前期实验结果，我们推测"CTX通过HBx介导的内质网应激和细胞凋亡途径，是造成HBV再激活和肝细胞损伤的主要分子基础"这一科学假说。本课题将通过PCR、CHIP、LSCM和siRNA等技术在细胞、动物和临床实验中证实CTX通过HBx介导的内质网应激和细胞凋亡来诱导HBV再激活和肝细胞损伤。本课题将化疗药物诱导HBV再激活临床现象与病毒复制等基础研究紧密结合，从药物、病毒和细胞全面探索HBx-ERS-Apoptosis通路对病毒复制及细胞命运的影响；并为全面解析该临床现象提供新的思路。

HBV感染是威胁人类健康的重要公共卫生问题，据WHO报道，全球约2.57亿慢性HBV感染者，每年死于HBV感染相关疾病的人数约88.7万，其中肝硬化和原发性肝细胞癌病死率分别占52%和38%。我国肿瘤发病率高且呈逐年上升趋势，故肿瘤合并HBV感染患者的人群数量是极其庞大的。肿瘤患者使用化疗药物等干预措施时则可能会打破病毒和生物体之间的平衡，重新出现病毒复制水平显著升高，我们将这种现象称之为HBV再激活。大量瞬时活化的 HBV则可能导致患者出现严重的肝功能损伤甚至是肝衰竭的现象，从而导致治疗减缓或是中断，继而延误肿瘤患者的有效治疗，必将严重影响其预后甚至是危及其生命。多项临床实验证实环磷酰胺(CTX)等化疗药物可诱导HBV再激活，导致急性肝炎或肝衰竭的发生，使死亡率大增，然而HBV再激活分子机制尚不清楚。. 我们希望明确CTX诱导HBV再激活的分子机制并最终全面解析上调HBV复制的关键核苷酸信息以及相关转录因子等，阐明CTX诱导HBV再激活的具体分子机制；同时验证CTX是否通过HBx介导的内质网应激和细胞凋亡途径诱导稳定表达HBV细胞的凋亡，并最终导致急性肝细胞损伤及肝衰竭。. 本研究发现在HBV稳定表达的细胞模型和HBV转基因小鼠模型中CTX均可显著促进HBV的转录和复制，透导HBV再激活。其通过上调转录因子AP1的表达，活化了HBV Cp的转录活性，进而增强了HBV的转录和复制。CTX可上调HBx和AP-1的表达，继而增强HBV启动子活性，在转录水平调控HBV;同时抑制细胞增殖并阻港于G1期及诱导细胞内质网应激-自噬和凋亡等。通过PCR、CHIP、LSCM和siRNA等技术在细胞、动物和临床实验中证实CTX通过HBx介导的内质网应激和细胞凋亡来诱导HBV再激活和肝细胞损伤。. 本课题将化疗药物诱导HBV再激活临床现象与病毒复制等基础研究紧密结合，从药物、病毒和细胞全面探索HBx-ERS-Apoptosis通路对病毒复制及细胞命运的影响；并为全面解析该临床现象提供新的思路。化疗药物诱导HBV再激活的关键标志物有望开发出新型抗病毒药物，并实现临床转化。