Numb基因通过TLR4-NF-κB-细胞因子通路参与痛风发病机制的研究

Genetic factors play a key role in the pathogenesis of gout, however, the confirmed pathogenic genes are poorly understood and only a few family genes are reported. In our previous genome-wide association study, including 82 people in 4 generations (ten of them were gout patients), we have found a new risky gene of gout, which mechanism needs to be confirmed. Numb, expressed in macrophage, has an effect on the release of inflammatory cytokines. We speculated that Numb mutation may mediates the incidence of gout through the Toll-like receptor 4 - NF -κB - cytokine pathway. This study will further collect 500 cases of familial gout to verify the association between Numb and gout. We also will collect human monocytes containing Numb mutations to investigate the effect of Numb gene on monocytes function and the possible mechanism. Finally, we’ll construct the Numb over-express and down-express THP-1 cell lines to explore its functions and molecular pathways in macrophage. This study will provide new ideas for understanding the pathogenesis of gout.

遗传因素是痛风发病的主要因素，但目前已知的痛风致病基因甚少，而基于家系的致病基因更鲜有报道。本课题组前期对一个原发性痛风大家系（5代82人，10名患者）进行全基因组重测序分析，发现一个全新的痛风致病基因Numb，但其作用机制还有待探讨。Numb基因在单核细胞内表达且影响炎性因子的释放，结合前期研究我们推测，其可能通过TLR4-NF-κB-细胞因子途径影响单核细胞功能进而参与痛风发病。本项目拟进一步检测500余例家系痛风患者Numb基因外显子，深入研究Numb基因与痛风的关联。收集含有Numb基因突变的人单核细胞，研究Numb基因突变对痛风患者单核细胞功能的影响。构建Numb基因突变和Numb基因沉默的单核细胞系，在细胞水平明确Numb基因是否通过TLR4-NF-κB-细胞因子途径影响单核细胞功能进而参与痛风发病。该研究将对痛风的发病机制提供新的思路。

课题组前期在原发性痛风大家系中发现位于Numb基因编码区的错义突变G1712A，据了解尚没有Numb基因变异与高尿酸血症和痛风相关的报道。进一步研究发现G1712A突变显著减少Numb表达,并且该突变明显增加尿酸盐晶体刺激下单核细胞分泌IL-1β。已有研究表明痛风炎症反应主要是因为单核巨噬细胞在尿酸盐晶体刺激下激活TLR4-NF-κB通路，因此我们推测G1712A突变通过TLR4-NF-κB途经影响单核细胞功能进而参与痛风发病。本研究主要在细胞水平观察G1712A突变对单核细胞功能及对TLR4-NF-κB通路的影响，并明确相关机制。结果表明：①G1712A突变增加Numb与内吞相关蛋白AP2的亲和力；②Numb促进细胞质膜尿酸盐重吸收转运体URAT1降解，但G1712A突变对Numb这一功能没有显著影响；③携带G1712A突变的痛风患者单核细胞在尿酸盐晶体刺激下分泌IL-1β显著高于不携带G1712A突变的痛风患者来源单核细胞；④ Numb敲低的单核细胞在尿酸盐晶体的刺激下，炎性因子表达水平高于对照组。这些研究结果提示Numb突变通过NF-κB通路影响痛风炎症的产生。本研究确定新的痛风致病基因，并在此基础上发现调控痛风炎症的相关通路，从而为痛风的防治提供新的分子靶点。