多重耐药相关蛋白与HBV对核苷类似物抗病毒药物耐药

It is known that HBV drug resistant related mutations are only responsible for part of chronic hepatitis B patients resistance to nucleot(s)ide analogues treatment. It is unclear whether there are any other mechanisms involved in HBV resistance to nucleot(s)ide analogues, especially for some patients who have primary resistance to nucleot(s)ide analogues. In this study we will investigate the effect of multi-drug resistance protein 4,5,7, and 8, which are expressed on human hepatocyto membrane, on nucleot(s)ide analogues' uptaking and metabolism for exploring the new mechanisms of HBV drug resistance. The expression of MRP in liver tissue and PBMC as well as their correlation will be determined by RT-PCR and immunohistochemistry from the samples obtained from both chronic HBV infection patients and chronic HBV replication mice, which are established by hydrodynamic injection of HBV wild type 、entecavir mutant and multi-drug resistant plasmids into mice. To investigate the effect of MRP on nucleot(s)ide analogues' uptaking and metabolism and anti-viral response in vitro, MRP expression plasmids or MRP siRNA will be transfected into stable-tranfected HBV wild type、entecavir mutant and multi-drug resistant mutant cell lines and the cells will be treated with nucleot(s)ide analogues. To investigate the effect of MRP on nuclot(s)ide analogues' anti-viral response in vivo, MRP expression plamids/HBV-wild type and HBV-mutant plasmids or MRP shRNA/HBV-wild type and HBV-mutant plasmids will be injected into mice by hydrodynamic injection and the mice will be administrated with nucleot(s)ide analogues. This study will provide experimental and theoretical foundation for exploring new individual anti-HBV therapy evaluation marker and improving the treatment efficacy of nucleot(s)ide analogues to HBV。

迄今，HBV耐药变异仅能解释部分乙肝患者中对核苷类似物抗病毒治疗耐药的机制。仍有相当一部分患者的耐药机制特别是原发性耐药的机制并不清楚。本研究首次从宿主肝细胞膜上表达的多重耐药相关蛋白4,5,7,8（Multi-drug resistance protein, MRP）对核苷类似物的吸收和代谢的角度来探讨HBV药物耐受的新机制。利用乙肝患者和高压尾静脉注射建立的慢性HBV感染小鼠模型，确定MRP在肝脏和PBMC中的表达及相关性; 应用HBV野生株、ETV耐药突变株和多药耐药突变株建立的稳定复制细胞系或持续复制的小鼠模型，通过转染MRP表达质粒上调或siRNA/MRP抑制剂下调MRP在细胞系或小鼠肝脏中的表达，给予不同核苷类似物处理后，观察MRP表达水平对核苷类似物吸收和代谢及抗病毒效应的影响。这将为探索新的个体化治疗评估指标，提高核苷类似物抗HBV药物的疗效奠定实验和理论基础。

HBV耐药突变和治疗依从性差被认为是核苷酸类似物（NA）耐药的两大因素。但是，病毒突变并不能完全解释NA耐药发生的现象。研究显示多重耐药相关蛋白4（MRP4）与抗逆转录病毒治疗的NA耐药相关，MRP4是否与慢性乙型病毒性肝炎（CHB）的NA耐药相关尚不清楚。采用实时定量PCR、western blot及免疫组化方法检测MRP4在健康对照及慢乙肝外周血单个核细胞（PBMCs）、肝组织和肝癌细胞系中的表达。经MRP抑制剂或干扰RNA慢病毒下调MRP4表达，经MRP4过表达慢病毒上调MRP4表达，并通过定量PCR和DNA测序检测HBVDNA水平和进行HBV突变分析。经高液相质谱联用方法检测在有或没有MRP4抑制剂条件下细胞内外的NAs浓度。在NA耐药（原发性耐药，部分病毒血应答及病毒血突破）患者的PBMCs中的MRP4表达明显升高，且MRP4在PBMCs中的蛋白表达与其在对应的肝癌旁组织中的表达具有相关性。抑制或敲除MRP4体外增加了NAs的药物敏感性（除对恩替卡韦耐药的HepG2.A64细胞系），而过表达MRP4则降低了NAs的药物敏感性。MRP抑制剂可增加细胞内的NAs浓度，而降低细胞外的NAs水平。MRP4高表达可能是HBV对NA耐药的一个宿主因素，并有望成为抑制HBV耐药的一个靶向蛋白。