microRNA-33/TRAF3信号通路在实验性自身免疫性重症肌无力发病中的作用机制研究

Using miRNA chip technology and real-time PCR analysis ,we recently found that miR33 was significantly up-regulated in dendritic cells (DC) and B cells from EAMG rats.Western blot analysis confirmed that TRAF3 , the target gene of miR-33, reduced significantly in DC and B cells. TRAF3 plays an important role in the maintenance of immune homeostasis. First, TRAf3 can significantly inhibit the secretion of IL-12 and IL-6 by DC and B cells whose innate immune receptor were activated. Importantly, recent studies show that IL-6 and IL-12 are crucial induction factor which promote the Tfh cell（specialized providers of B-cell help）differentiation. Second, TRAf3 can significantly inhibit CD40(B cell)/CD40L(Tfh) interaction mediated antibodies production by B cells.Based on the above results, we speculate that in the specific inflammatory microenvironment, miR-33 in DC and B cells increased and degradated TRAF3, resulting in the loss of immune homeostasis and so as to enable the Tfh differentiation and B cells activation, and high affinity antibodies production，constituting an important molecular mechanism in EAMG pathogenesis. We intended to apply overexpression , RNA Interference and rescue exprement involved in immune molecules, cells, tissues and animal levels，to investigate the role of miR-33/TRAF3 in EAMG, , and offer a new target for the treatment of MG.

近期我们通过microRNA芯片及荧光定量PCR分析发现miR33在EAMG大鼠DC及B细胞表达显著上调，免疫印迹证实miR-33的靶基因TRAF3在DC、B细胞显著减低。TRAF3在维持免疫稳态中发挥重要功能：1可以显著抑制DC和B细胞天然免疫受体活化而诱导的IL-6，IL-12分泌，而IL-6、IL-12是诱导Tfh细胞（辅助B细胞活化的关键Th细胞）分化的关键因子。2显著抑制Tfh/CD40L-Bcell/CD40信号介导的B细胞抗体产生。据此我们提出假说：特定炎症微环境下，miR33在DC、B细胞显著上调并降解TRAF3基因使免疫稳态被打破，导致Tfh分化并辅助B细胞活化、产生高亲和力抗体，是EAMG发病的重要分子机制之一。本项目旨在利用过表达、RNA干扰及恢复实验等方法，从免疫分子、细胞、组织及动物水平多层次评估miR-33/TRAF3是否参与EAMG发病，为治疗MG提供新靶点。

microRNA作为一种非编码性单链小分子RNA可以通过调节相关靶基因的表达而影响免疫细胞的分化及免疫应答过程。我们前期研究发现miR33及其靶基因TRAF3（负调控B细胞活化的重要蛋白）在EAMG大鼠B细胞异常表达。我们拟探讨miR33/TRAF3在MG/EAMG中的作用机制。我们的研究结果显示：（1）与大鼠研究结果一致，miR33/TRAF3在MG 患者B细胞呈反向表达。MG患者miR33表达较健康对照明显增高，其中全身型（GMG）患者miR33表达水平较眼肌型（OMG）患者明显增高。同时，GMG/OMG患者B细胞TRAF3蛋白水平较健康对照明显降低，其中GMG TRAF3蛋白的表达水平较OMG患者明显降低。（2）GMG/OMG患者B细胞miR33表达水平与QMG评分、CD19+CD27+CD38+抗体分泌型细胞频率均呈正相关性；B细胞miR33表达水平与TRAF3表达负相关；B细胞TRAF3表达水平与QMG评分、CD19+CD27+CD38+抗体分泌型B细胞频率均呈负相关性。（3）探讨了各种刺激条件对MG B细胞miR33表达的影响： CD40L , IL-21, anti-IgM , CpG ODN, IL-6 ，BAFF 刺激24到72小时，在各个刺激条件的三个时间点，miR33 都有不同程度的升高，其中TLR9激动剂CpG ODN(1 mg/ml)刺激72小时miR33表达显著升高。（4） CpG ODN刺激B细胞导致miR-33表达增高而TRAF3表达下调, 增加CD19+CD27+CD38+抗体分泌型细胞频率和培养上清IgG水平；转染miR33 inhibitor后可以上调TRAF3表达,减低CD19+CD27+CD38+抗体分泌型B细胞频率和培养上清IgG水平。（5）为抑制大鼠体内miR-33表达，尝试了miR-33反义寡核苷酸，质粒和慢病毒表达的miR-33海绵几种抑制物，发现慢病毒miR-33海绵抑制效果最为明显。总之，我们在重肌动物模型和患者均发现miR33/TRAF3异常表达，TLR9活化是引起miR33过表达进而抑制TRAF3、促使抗体分泌型细胞分化的重要始动因素之一。进一步观察miR-33慢病毒海绵对EAMG的治疗效果，为MG治疗新途径提供理论基础。