趋化因子CCL2在狼疮肾表达的调控机制研究

狼疮肾炎(lupus nephritis，LN)是SLE最常见的临床表现，严重影响SLE的预后。大量基础和临床研究证实，趋化因子CCL2是LN的关键致病因子。为进一步明确CCL2在LN系膜细胞表达的分子调控机制，我们根据人基因库资料分析CCL2启动子上的转录因子应答元件(RE), 构建系列的野生及突变型报告载体，应用荧光酶报告载体、突变载体、凝胶迁移实验及染色质免疫沉淀分析确定所筛选靶RE。利用腺病毒表达载体及siRNA转染技术抑制或上调靶分子表达，探讨所筛选的目的基因对CCL2表达细胞株中CCL2 mRNA及蛋白质表达的影响。运用实时定量RT-PCR、ELISA、免疫组化等方法分析目的基因的高表达或基因沉默对LN系膜细胞和人肾系膜细胞株表达CCLE的影响，及对MRL/lpr小鼠发生肾损伤的影响，从而深入探讨CCL2在LN 表达的调控分子机制，为LN的基础及临床研究提供新的理论和实验基础。

Lupus nephritis(LN) is the most important predictive factor of poor prognosis in SLE. A large of basic and clinical studies have proven that, chemokine CCL2 is a key pathogenic factor in LN. This study is designed to inestigate that how the expression of CCL2 is regulated by the transcription factors in LN mesangial cells. The transcription factor response components (RE) in CCL2 promoter will be searched by using TESS software.The RE involved in induction of CCL2 expression will be identified by using a series of wild and mutant type of CCL2-leciferase reporter vector in electrophoretic mobility shift assays and Chromatin Immunoprecipitation. In order to elucidate the effects of the target gene on transcriptional activity,the expression of CCL2 in LN mesangial cells will be assessed by real-time quantitative RT-PCR, ELISA, and immunohistochemical method after up-regulation or inhibition of the target gene by transfection of targeting expression vector or siRNA into the cells, and incidence of LN in SLE-prone mouse model after adminstration of the targeting expression vector or siRNA will be also investigated. This study will clarify the molecular mechanisms of regulation of CCL2 expression in LN, and thus will provide valuable informations for understanding of the pathogenesis of LN