miR-326靶向负调控IgG FcγRⅢ在糖尿病肾病免疫损伤中的作用及机制研究

Humoral immunity is a new research area in the pathogenesis of diabetic nephropathy (DN). Our previous study found that: the expression of IgG FcγRⅢof peripheral blood leukocyte and mesangial area of kidney tissue in DN patients was increased compared with normal control; and upregulation of FcγRⅢ of mesangial cells cultured in vitro was induced by high glucose. It implied that FcγR Ⅲ may be involved in the pathogenesis of DN and may be its interventional target. Further miRNAs gene chip detection, miRNAs target gene prediction and dual luciferase reporter gene system validated the negative regulationship between significantly downregulated miR-326 and upregulated FcγR Ⅲ in kidney tissue of DN patients. All these revealed that significantly downregulated miR-326 in DN may decreased the translational inhibition of FcγR Ⅲof mesangial cell so as to increase the expression of FcγR Ⅲ.Stimulated by oxidative modified low density lipoprotein immune complexes, NFκB and TGF-β/Smad signaling pathways was activited and involved in immune injury and fibrosis in DN. This study will further explore miR-326/FcγR Ⅲ/NFκB and miR-326/FcγR Ⅲ/TGF-β pathway in the DN mice model and mesangial cells to provide new prevention and treatment targets and strategies of DN.

体液免疫是糖尿病肾病（DN）发病机制研究的新领域。我们的前期研究发现：DN患者外周血白细胞及肾组织系膜区IgG FcγRⅢ表达较正常对照增加；高糖可以诱导体外培养系膜细胞 FcγRⅢ表达上调。以上提示FcγRⅢ可能参与DN发病机制并成为其干预靶点。进一步经miRNAs基因芯片检测、miRNAs靶基因预测及双荧光素酶报告基因系统验证了DN患者肾组织中表达明显下调的miR-326与上调的FcγRⅢ的靶定负调控关系。由此推测，在DN中，表达下调的miR-326降低了对系膜细胞表面FcγRⅢ的翻译抑制，使得FcγRⅢ表达上调，与沉积的氧化修饰低密度脂蛋白免疫复合物结合，从而启动了NFκB及TGF-β/Smad信号通路，参与DN免疫损伤和纤维化过程。本研究将进一步探讨DN以及系膜细胞中的miR-326/FcγRⅢ/NFκB和miR-326/FcγRⅢ/TGF-β通路，为DN防治提供新靶点、新策略。

本课题的科学假说是：在糖尿病肾病发生发展过程中，表达下调的miR-326降低了对肾脏系膜细胞表面IgG FcγRⅢ的翻译抑制，使得IgG FcγRⅢ表达上调，与沉积的氧化修饰低密度脂蛋白免疫复合物结合，从而启动了NFκB及TGF-β/Smad信号通路，参与肾脏免疫损伤和纤维化过程。为验证此研究假说，课题组从动物模型和细胞培养两方面进行验证。在体内研究中，我们利用FcgRⅢ基因敲除小鼠和FcgRⅡb基因敲除小鼠，通过高脂喂养+STZ联合诱导糖尿病肾病，成功分离肾小球，检测oxLDL表达及炎症和纤维化指标表达，并分别检测IgG FcγRⅠ、FcgRⅡb及FcgRⅢ表达；体外实验利用小鼠系膜细胞，分别用siRNA-FcgRⅢ, siRNA-FcgRⅡ以及siRNA- FcgRⅠ沉默受体，然后在高糖和/或oxLDL刺激下，检测炎症及纤维化指标以及IgG FcγRⅠ、FcgRⅡb及FcgRⅢ的表达。本课题高质量成功分离肾小球，同时，研究发现：oxLDL在糖尿病肾病状态下在肾小球的沉积增加，参与糖尿病肾病发生发展；三种不同的IgG FcγR在糖尿病肾病的发生发展过程中起不同的作用；FcgRⅡb基因敲除加重了肾脏炎症和纤维化，而FcgRⅢ基因敲除并没有表现出肾脏保护作用，可能与升高表达的FcγRⅠ有关。