DNMT3a介导的GSK3β甲基化对调节性B细胞的诱导分化及其在乙肝肝纤维化进程中的作用

Liver fibrosis is the intermediate link and reversible stage during the development of chronic liver disease to cirrhosis. Pathological immune response is an important factor in the regulation of liver fibrosis. Our previous study found that the number of regulatory B cells (Bregs) is increased in the process of HBV-related liver fibrosis and affect the process of liver fibrosis by down-regulating the function of virus-specific T cells. However, the regulatory mechanism of Bregs’ proliferation and differentiation remains unclear. Recently, it has been reported that glycogen synthase kinase-3 (GSK3)β plays an important regulatory role in the homeostasis of B cells. It has been found in our pre-experiment that the expression of GSK3β decreased and DNA methyltransferase 3a (DNMT3a) increased in peripheral blood B cells in patients with chronic hepatitis B (CHB) and early cirrhosis than that in healthy control; Furthermore, by use of methylation chip, we found that methylation level of GSK3β promoter elevated in CHB patients. In this study, human primary CD19+B cells and animal models of HBV-related liver fibrosis were employed to explore the regulatory role of DNMT3a-mediated GSK3β methylation in the expression of transcription factors c-Maf, GATA3, CREB and cytokine IL-10, in order to confirm the new mechanism that GSK3β can participate in the process of HBV-related liver fibrosis by regulating the proliferation and differentiation of Bregs. The results of this study will lay a solid theoretical foundation for the targeted treatment of HBV-related liver fibrosis.

病理状态下的免疫应答是调控肝纤维化进程的重要因素。我们前期研究发现乙肝肝纤维化进程中调节性B细胞（Bregs）数量增多并通过下调病毒特异性T细胞功能影响肝纤维化进程，但Bregs增殖分化的调控机制尚不明确。新近报道糖原合成酶激酶-3（GSK3）β对B细胞的稳态维持具有重要调控作用。我们预实验发现慢乙肝及早期肝硬化患者较正常人群外周血B细胞中GSK3β表达下调而DNA甲基转移酶3a（DNMT3a）表达上调；进一步甲基化芯片显示慢乙肝患者B细胞中GSK3β启动子甲基化水平增高。本课题拟以人原代CD19+B细胞和乙肝肝纤维化动物模型为研究对象，探究DNMT3a介导的GSK3β甲基化对B细胞中c-Maf、GATA3、CREB等关键转录因子及IL-10表达的调控，以期证实GSK3β能通过调控Bregs分化增殖参与乙肝肝纤维化进程这一新机制。本项研究结果将为靶向治疗乙肝肝纤维化打下坚实的理论基础。

项目背景：病理状态下的免疫应答是调控肝纤维化进程的重要因素。我们前期研究发现乙肝肝纤维化 进程中调节性B细胞（Bregs）数量增多并影响肝纤维化进程，但Bregs增殖分化的调控机制尚不明确。新近报道糖原合成酶激酶-3（GSK3）β对B细胞的稳态 维持具有重要调控作用。我们预实验发现慢乙肝及早期肝硬化患者较正常人群外周血B细胞中GSK3β表达下调而DNA甲基转移酶3a（DNMT3a）表达上调。本课题拟以人原代CD19+B细胞和乙肝肝纤维化动物模型为研究对象，探究DNMT3a介导的GSK3β甲基化对B细胞中IL-10表达的调控，以期证实GSK3β能通过调控Bregs分化增殖参与乙肝肝纤维化进程这一新机制。. 研究方法：从血液样本中分离外周血单个核细胞(PBMC)，并使用CD19 mAb包被的微珠纯化B细胞。将DNMT3a和GSK3β的质粒转染到B细胞中，通过流式细胞仪检测和分析外周Breg（CD19+CD24hiCD38hi细胞、CD19+IL-10+细胞）的数量。通过甲基化特异性PCR(MSP)分析GSK3β启动子的甲基化状态。通过qRT-PCR和蛋白质印迹检测特定基因和蛋白质的表达。将含有pCDNA3.1-HBV1.3和抗小鼠CD137激动剂单克隆抗体的质粒注射到小鼠C57BL/6J中，构建HBV相关肝纤维化动物模型。注射后6周处死所有小鼠，采集肝组织样本，用HE、SiriusRed和Masson’s染色，以评估肝纤维化程度。. 研究结果：体外研究证实B细胞中DNMT3a过表达促进GSK3β启动子区甲基化状态而抑制其基因表达。GSK3β表达水平或者活性被抑制，将导致B细胞向Breg细胞分化，表现为IL-10相关转录因子表达上调，CD19+CD24hiCD38hi和CD19+IL-10+细胞比值增加。体内研究进一步证实，HBV感染后GSK3β基因敲除小鼠较野生型小鼠肝组织中促纤维化因子表达更低，肝纤维化程度进展更缓慢。. 研究结论及科学意义：DNMT3a通过DNA甲基化抑制GSK3β的表达，进而促进Breg细胞的分化和IL-10的分泌。靶向GSK3β是乙肝相关肝纤维化的潜在疗法。