Cdc42对炎症性肠病中Th17细胞和潘氏细胞作用的研究

Inflammatory bowel disease (IBD) is a kind of nonresolving, non-specificity chronic inflammation,which includes Crohn's Disease (CD) and Ulcerative Colitis (UC). Mucosal immune takes critical part in the development of IBD. We have performed a Proteomic analysis to suggest that there are 30 different proteins changed significantly between UC patients and control. Among them, 16 proteins were up-regulated whereas 14 proteins were down-regulated. Cdc42 spot of UC was highly expressed compared with control. We still use western blot to confirm this founding. In addition, we found that, in RAW264.7 cell line, the activation of p38MAPK pathway was increased as Cdc42 up-regulated. Moreover, Cdc42 was suppressed with the down-regulation of p38MAPK pathway by adding SB203580, a specific inhabitor of P38. Cdc42, the upstream regulatory molecule of p38, is a member of Rho GTPase family. After been activated, It can gather at the membrane of cells in order to turn GDP into GTP. Activated Cdc42 regulates the polarity of cells and participates in the positive selection and maturation of lymphocytes. So we infer that Cdc42 affects the development of inflammation through the regulation of mucosal lymphocytes. Paneth cells influence mucosal immunity seriously. The absence of them has close connection with the occurrence of CD. In order to investigate the relationship between Cdc42 and IBD, we designed experiences on IL-10 knockout mice to test changes of amount and distribution of Cdc42 in Paneth cells and Th17 cells separated from lamina propria lymphocytes. Peripheral blood lymphocytes of IBD patients and control are also be concluded to carry out this test. The number and proportion of lamina propria and spleen Th17 cells of Cdc42 knockout mice will also be analyzed. After the positive results are received, we still plan to analyze the same indexes when the IL-10 knockout mice is treated with allicin, an anti-inflammatory drug extracted form garlics.

炎症性肠病（IBD）是一种肠道非可控性、非特异性慢性炎症，粘膜免疫在其中发挥了重要作用。前期我们通过蛋白组学方法，建立了UC差异蛋白表达谱，发现Rho GTP酶家族中Cdc42在UC患者肠粘膜高表达，且活性与p38MAPK信号通路正相关。Cdc42是MAPK通路的上游信号分子，激活后在细胞膜富集，使GDP转化为GTP。它可以调控细胞极性，淋巴细胞的阳性选择和成熟也需要Cdc42的参与。因此我们推断Cdc42通过对肠粘膜淋巴细胞的调节影响炎症进程。粘膜免疫中另一类重要的细胞为潘氏细胞，它的缺乏与CD的发生紧密相关。本项目拟在IBD动物模型及人外周血中进一步证实Cdc42含量及分布的变化，通过检测外周血淋巴细胞、肠粘膜固有层淋巴细胞中Th17细胞以及潘氏细胞内Cdc42的变化,并在Cdc42基因敲除模型小鼠中检测对Th17细胞的影响，探索Cdc42与IBD的关系。

炎症性肠病（IBD）是一种肠道非特异性慢性炎症，粘膜免疫在其中发挥了重要作用。前期我们通过蛋白组学方法，建立了UC差异蛋白表达谱，发现Rho GTP酶家族中Cdc42在UC患者肠粘膜高表达。Cdc42可以调控细胞极性，淋巴细胞的阳性选择和成熟也需要Cdc42的参与。因此我们推断Cdc42通过对肠粘膜淋巴细胞的调节影响炎症进程。进而开展了CDC42在炎症性肠病中TH17细胞作用的研究。首先基于2012年中华医学会消化病学分会炎症性肠病学组发表的《炎症性肠病诊断与治疗的共识意见》中推荐的诊断标准纳入南方医院2013年10月-2014年10月住院治疗的活动期UC患者，CD患者以及健康对照者，并收集IBD患者及健康对照者外周血，检测血浆中的细胞因子IL-17A，分离出外周血中单个核细胞（PBMCs），进一步纯化分离出TH17细胞进行相关检测。结果提示活动期IBD患者与健康对照者相比，血浆中IL-17A水平增加、Th17细胞比例升高，PBMCs中Cdc42蛋白表达下降；Cdc42和IL-17A的mRNA水平也下降。分选人外周血Th17细胞中Cdc42 mRNA水平，活动期IBD患者也显著低于健康对照者。其次通过免疫磁珠分选，体外分离出IBD患者及健康对照组外周血naïve cd4+ T细胞，并进行体外培养。在加入CDC42特异性抑制剂ML141后上清中的IL-17升高，提示抑制Cdc42功能后可以促使Naive CD4+ T 细胞向Th17 细胞方向分化。然后利用DSS建立小鼠肠炎模型。DSS组与空白对照组小鼠相比，脾脏及肠系膜淋巴结中Th17 、Th1细胞比例增高,Th2、Treg细胞无明显变化；DSS小鼠与正常小鼠相比，脾脏和淋巴结Cdc42 mRNA水平下降，蛋白表达增高。进一步在体外进行CDC42和TH17细胞功能相关的实验。抑制Cdc42对CD4+T细胞的增殖及凋亡无影响；并且能够促进Th17 、Th1、Th2、Treg细胞群的分化，且促进Th17细胞向病理性Th17细胞分化，结果提示了抑制Cdc42功能，促进Th17细胞分化，可促进DSS小鼠肠道炎症加重。

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