基于EB病毒-易感基因间表观遗传对话探讨解毒祛瘀滋阴方治疗SLE作用机理

Systemic lupus erythematosus(SLE) is an autoimmune disease with multi-systemic organs injury and bad prognosis. EB virus, as a trigger, maybe result in SLE relative susceptible genes abnormal expression through epigenetic regulation (DNA methylation, modifications of histone, microRNAs) . Our previous studies confirmed that Jieduquyuziyin decocotion can obviously lighten SLE disease activity and improve patients' life quality. The main contents including: Firstly, the relationship between gene expression of latent membrane protein 1 of SLE patients with positive EB virus maker and susceptible genes was detected. Secondly, T and B lymphocytes of SLE patients with negative EB virus maker were transfected by lentivirus with LMP-1gene and cultured with different drugs. Expression of epigenetic regulation relative genes (DNA methylation, modifications of histone, microRNAs) and susceptible genes were detected. Finally, lentivirus with LMP-1gene were injected into MRL/lpr mice and confirmed whether susceptible gene expression were affected through epigenetic mechanism or not. The effects of Jieduquyuziyin decocotion on them were also detected. The aim of this study was to investigate the mechanism of Jieduquyuziyin decocotion treating SLE based on epigenetic dialogue between EB virus and susceptible genes.

系统性红斑狼疮（SLE）是一种多器官受累、预后差的疾病。EB病毒感染作为激发因素，通过表观遗传途径（DNA甲基化、组蛋白修饰、MicroRNA），引起SLE易感基因（HLA-DRB1、BLK、TNFSF4、STAT4、ETS1、TET3）表达异常，诱导SLE发病。解毒祛瘀滋阴方可缓解SLE病情，提高生存质量。本项目先检测EBV（+）SLE患者潜伏膜蛋白1（LMP-1）和易感基因表达的相关性；再将携带LMP-1基因慢病毒感染EBV（-）SLE患者T、B细胞，干预培养后检测易感基因表达量和表观遗传调控水平（DNA甲基化、组蛋白修饰、miRNA）；最后将LMP-1慢病毒质粒注射到MRL/lpr狼疮鼠体内，验证EBV是否通过表观遗传途径影响易感基因表达，同时观察中药对其干预作用。 从“EBV与SLE易感基因间表观遗传对话”研究解毒祛瘀滋阴方治疗SLE作用机理。

系统性红斑狼疮（SLE）是一种多器官受累、预后差的疾病。EB病毒感染作为激发因素，通过表观遗传途径（DNA甲基化和组蛋白修饰）引起SLE易感基因（HLA-DRB1、STAT4、ETS1、TET3等）表达异常，诱导SLE发病。本项目先检测EBV（+）SLE患者潜伏膜蛋白1（LMP-1）和易感基因表达的相关性；再将携带LMP-1基因慢病毒感染T细胞，检测易感基因表达量；最后将LMP-1慢病毒质粒注射到MRL/lpr狼疮鼠体内，验证EBV是否通过表观遗传途径影响易感基因表达，同时观察中药解毒祛瘀滋阴方对其干预作用。研究发现：解毒祛瘀滋阴方可明显缓解狼疮小鼠皮肤损害和肾脏损伤，其作用机制可能与其上调CD4 +T细胞基因组 DNA 甲基化水平、抑制 DNA 甲基化敏感基因表达有关。SLE患者LMP-1表达量比健康对照者明显升高，SLE相关易感基因HLA-DRB1、BLK、ARID5B表达水平明显升高。使用含LMP-1表达质粒的慢病毒转染H9细胞后，细胞内LMP-1表达量变化具有显著意义。SLE易感基因HLA-DRB1、STAT4、ETS1、TET3、ARID5B、TET3、DRAM1、CDKN1B等易感基因的相对表达量均有上升，其中HLA-DRB1、BLK、ARID5B基因的相对表达量差异显著。与阴性质粒转染组相比，LMP-1 过表达质粒转染组细胞中ARID5B、p-P65 的相对表达量均明显增加；而用NF-κB 抑制剂BAY117082 处理LMP-1 过表达质粒转染组后，NF-κB 通路被抑制，导致细胞中ARID5B、p-P65 的相对表达量下降。组蛋白修饰相关基因EZH2实验组相对表达量与阴性对照组相比增加最为显著。实验发现EB病毒如何在小鼠体内稳定表达非常棘手。首先将LMP-1表达质粒的慢病毒直接通过尾静脉注射到小鼠体内，发现含LMP-1基因质粒的慢病毒在小鼠体内存活率很低，原因估计是被小鼠体内免疫系统清除掉。通过Knock-in技术将LMP-1基因敲入到小鼠胚胎基因组DNA中，然后进行孵育繁殖，发现病毒基因对胚胎毒性较大，小鼠无法成功繁育。在前两次实验失败的基础上，我们采取靶向Knock-in技术，将LMP-1基因靶向整合到小鼠脾脏T淋巴细胞中，这样既可以减少EB病毒对小鼠胚胎的毒性，也可以提高EB病毒基因LMP-1在小鼠体内的表达，目前已经孵育出F0代小鼠。