微粒（Microparticles）在狼疮肾炎发病中的作用及其潜在信号通路的研究

Lupus nephritis (LN) is one of the most serious manifestations of systemic lupus erythematosus (SLE). Ten-20% patients with LN finally develop renal failure and need hemodialysis or kidney transplantation. Up to now, the underlying mechanisms in the pathogenesis of LN are still poorly understood. Recently, microparticles（MPs）become a renewed interest in pathogenesis of LN. MPs, released by activated or apoptotic cells are membrane-bound vesicles ranging from 200 to 1,000 nm in diameter. Internally, MPs, carrying a broad variety of DNA and RNA are immunoactivate. It was reported that MPs were sufficient to break immune tolerance and trigger autoimmune response. The concentration and composition of MPs in LN patients differed markedly from those in healthy subjects. However, the role of MPs in the pathogenesis of LN is still poorly defined. In this study, we mainly illustrate the pathogenic role of MPs in the development of LN in mice by using ELISA, immunofluorescence staining and flow cytometry analysis. Furthermore, the immunoactivity of MPs is testified on the cultured splenocytes of mice. The proliferation and activation of immune cells are detected by MTT and flow cytometry analysis, respectively. After defining the immune activity of MPs, we want to clarify the potential signaling pathways of MPs, especially the TLR7/9 signaling pathway. To implicate the role of TLR7/9 on the activity of MPs,the specific antagonists of TLR7/9 are used to block TLR7/9. The binding capability of MPs to TLR7/9, the activity of B cells and pDC, and the expression and activity of key proteins of TLR7/9 signaling pathway are detected by Western-blot and Real-time PCR. The results will firstly demonstrate the role of MPs in the pathogenesis of lupus nephritis and its potential signaling pathway. The data will provide new ideas for the treatment of lupus nephritis.

狼疮肾炎是系统红斑狼疮常见并发症，10-20%狼疮肾炎病人最终发展成肾衰竭，需血液透析或肾脏移植维持生命。微粒（MPs）是细胞活化或凋亡时脱落的直径为0.2-10μm的小颗粒物质，形成的球型空间结构中包含多种自身抗原如DNA和RNA。研究表明MPs具有免疫刺激活性，可打破免疫耐受触发自身免疫应答，但MPs在狼疮肾炎发病中的作用还未见报道。本课题在狼疮肾炎小鼠模型及体外培养淋巴细胞的平台上综合利用ELISA、免疫荧光及流式细胞术等方法探讨MPs在狼疮肾炎发病中的作用。进一步我们将考察TLR7/9信号通路在MPs发挥免疫刺激活性中的作用。通过特异性封闭TLR7/9，比较封闭前后免疫细胞活化情况、MPs与TLR7/9结合量及TLR7/9通路中关键信号蛋白的变化论证MPs是否依赖TLR7/9发挥免疫激活作用。本研究将首次论证MPs在狼疮肾炎发病中的作用及其潜在信号通路，为治疗狼疮肾炎提供新思路。

微粒是细胞活化或凋亡时脱落的小颗粒物质，形成的球形空间结构中包含多种自身抗原如DNA，RNA。已有的研究证实不同细胞来源的微粒具有多种作用。血小板来源的微粒可促进CD4+T细胞向Treg细胞转化，并分泌抑制性细胞因子TGF-β；可抑制活化的T细胞分泌TNF-α和IL-10；可影响巨噬细胞和DC细胞的成熟等。中性粒细胞来源的微粒可抑制DC细胞的吞噬活性，促进TGF-β分泌；抑制IL-8，IL-10，IL-12, TNF-α等细胞因子分泌。本课题主要研究了MPs对巨噬细胞、PBMC以及Th1/Th2细胞的作用，进一步研究了MPs在体内对小鼠狼疮肾炎以及小鼠类风湿关节炎的作用。研究结果显示SLE和RA血浆中的MPs可以明显抑制活化的巨噬细胞释放TNF-α以及IL-10；可以抑制活化的CD4+ T细胞产生TNF-α，促进其产生IL-6以及IL-10，纠正Th1/Th2失衡的状况。此外，将MPs应用到小鼠体内，发现MPs可以降低狼疮肾炎小鼠血清中抗核抗体滴度，减少肾小球免疫复合物沉积，减轻肾炎病变，对于小鼠狼疮肾炎有一定缓解作用；对于II型胶原诱导产生的小鼠类风湿关节炎也有缓解作用，可以降低血清中抗II型胶原抗体浓度，减少小鼠足爪肿胀程度，减轻足部软组织炎症；减轻足部关节软骨破坏和血管翳形成。研究结果为进一步加深对自身免疫性疾病如系统红斑狼疮以及类风湿关节炎发病机制的认识，为研发治疗自身免疫性疾病的新型药物提供了初步的实验数据。