维生素D受体调控狼疮肾炎中MCP-1表达的作用及分子机制研究

Lupus nephritis (LN) is the most common clinical manifestation of SLE. Studies have proved that chemokine MCP-1 is a key pathogenic factor in LN. Meanwhile, Vitamin D receptor (VDR) has been showed to play an important role in regulating MCP-1 expression. In our preliminary study, the downregulated expression of VDR accompanied by high level of the MCP-1 was found in patients with LN. This study is designed to investigate the effect of VDR on MCP-1 expression by renal mesangial cells, and to probe whether the effect of VDR on MCP-1 expression is mediated by its response elements (VDRE) in MCP-1 promoter region. The effect of VDR on MCP-1 expression will be assessed by detction of VDR and MCP-1 expression in renal tissue from patients with LN, and in vitro cell culture studies. Identification of functional VDER in MCP-1 promoter region will be carried out by analysis of DNA sequence of MCP-1 promoter region to probe putative VDRE, and by using a series of wild and mutant type of MCP-1 leciferase reporter vector in electrophoretic mobility shift assays and Chromatin immunoprecipitation. In order to elucidate the effects of VDR complex on transcriptional activity,the expression of MCP-1 in renal mesangial cells will be assessed by real-time RT-PCR and immunohistochemical method after up-regulation or inhibition of the target gene by transfection of target gene expression vector or siRNA into the cells. This study will clarify the role and the molecular mechanisms of VDR in regulation of MCP-1 expression in LN, and thus will provide valuable information for understanding the pathogenesis of LN.

狼疮性肾炎（LN）是SLE最常见的临床表现。研究证实， MCP-1是LN的关键致病因子，另有研究则显示VDR在调控MCP-1表达中起重要作用，我们前期的研究发现，LN患者VDR表达伴随MCP-1水平升高而下调。为探讨VDR在调控MCP-1表达中的作用及分子机制，我们对VDR和MCP-1在LN肾组织中的表达进行同步检测，并采用体外细胞培养试验以明确VDR调控MCP-1表达的作用。同时，根据人基因库资料分析MCP-1启动子上的VDR应答元件(VDRE), 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VDRE的活性功能。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，运用qPCR等方法分析基因高表达或沉默对肾系膜细胞表达MCP-1的影响，从而深入探讨VDR在调控LN 表达MCP-1的分子机制，为LN病机提供新的理论和实验基础。