MASP-2效应酶介导感染性肉芽肿形成、成熟的免疫调节机制研究

Granulomatous diseases are a group of disorders characterized by a particular type of immune response termed granulomatous inflammation, The most important factor of granulomatous diseases is organisms invading. Granuloma formation become a critical step in the development of granulomatous inflammation via creating the pathological condition suitable for infection transmission and the mechanism are still not well understood. MASP-2, the effector enzyme of lectin pathway of complement activation, is the most critical enzyme in early stage of infection. In the preliminary experiment with infectious granuloma rabbit model, MASP-2 can promote the formation of infectious granuloma, liquefaction and healing process. we hypothesize that the macrophages and T cells, the most basic cell types and population in the infectious granuloma, are regulated by means of MASP-2 dependent pathway, which effect the function of macrophages and T cells and accelerate the granuloma formation process in early stage of infection. Therefor, the MASP-2 gene knockout mice (MASP-2-/- or MASP-2high/MASP-2low) were infected with Mycobacterium tuberculosis. Then the bacterial load and pathological changes of granuloma were observed, and the level of cytokine in serum or tissue (spleen/ lung) were examined with immunohistochemistry, RT-PCR and ELISA. Meanwhile, the CD69+, CD4+ and CD8+ cells number in infectious granuloma were detected with immunohistochemistrys. In this study, we evaluate the role of MASP-2 effector enzyme in infectious granulomatous diseases and discover a new clue or means to prevent or therapy granulomatous diseases.

肉芽肿病是以肉芽肿性炎症为共同病理特征的疾病，多由感染引起，肉芽肿的形成是导致炎症损伤加重最关键的一步，但其形成机制仍不清楚。而MASP-2效应酶是感染早期即被激活的最关键补体成分，在预实验中发现MASP-2能促进肉芽肿的形成、液化和愈合，推测通过触发MASP-2依赖性肉芽肿的形成途径，促进巨噬细胞、T细胞的募集和活化，加速肉芽肿的形成和发展。因此，拟以结核菌感染性肉芽肿小鼠（MASP-2-/-或MASP-2high/MASP-2low）模型为基础，采用RT-PCR、免疫组化、ELISA等手段检测肉芽肿组织CD68+、CD4+、CD8+细胞和相关细胞因子mRNA的表达水平、载菌量及血清细胞因子含量，研究MASP-2对感染性肉芽肿形成、成熟过程的影响，为肉芽肿性疾病的免疫学防治提供新的思路和依据。

肉芽肿病是以肉芽肿性炎症为共同病理特征的疾病，尚未阐明肉芽肿形成、发生发展的机制，缺乏有效的治疗肉芽肿性疾病的方法。本项目以病理结构特征最典型的结核性肉芽肿为实验模型，完成凝集素激活途径最关键效应酶MASP-2对结核性肉芽肿形成的调节及其免疫机制的研究。①成功构建人MASP-2重组腺病毒（rAd-hMASP-2），在哺乳动物细胞可高水平地表达MASP-2。②建立家兔皮肤结核模型动态观察MASP-2对结核肉芽肿的影响，发现MASP-2显著增大肉芽肿的体积、降低肉芽肿组织的载菌量，促进结核肉芽肿形成、液化和愈合过程。③小鼠肺部感染BCG（5×107CFU）模型的实验研究发现：MASP-2能够增加肺部肉芽肿的面积（P=0.023），明显降低肺部的细菌载量（3.31×105 CFU/g），提高小鼠BCG感染的生存率（80%）；MASP-2大量表达并裂解C4，导致小鼠肺组织C4b沉积显著增多（P<0.01），大量CD11b+细胞如单核-巨噬细胞、粒细胞的浸润（P<0.01），并诱导单核-巨噬细胞、粒细胞、淋巴细胞分泌TNF-α、IFN-γ、CCL2、IL-6、IL-8、IL-12等细胞因子（P<0.01），继而促进巨噬细胞、T细胞的募集和活化，增加肺组织淋巴细胞（p=0.038）、CD3+T细胞的数量（p=0.037），尤其CD3+CD8+T细胞亚群数量较对照组明显升高（p=0.038），表明机体存在MASP-2依赖性肉芽肿形成的新途径，MASP-2能够促进巨噬细胞、T细胞趋化、活化，降低肉芽肿组织的载菌量和提高小鼠生存，加速肉芽肿的形成及愈合，为MASP-2应用于肉芽肿性疾病的的免疫学治疗提供了实验依据和新思路。④成功构建MASP-2活性结构域CCP1-CCP2-SP，CCP2-SP和SP腺病毒穿梭质粒。⑤检测痰菌阳性肺结核患者血浆IL-10的水平，肺结核患者血浆IL-10水平显著高于健康对照组（8.58 ± 3.04 pg ml）（P <0.01），IL-10的变化参与结核病的损伤过程。