Tim-3调控单核/巨噬细胞系统调节急性肾损伤及机制研究

Clinically, acute kidney injury （AKI）is a common and serious disease with high mortality. Renal ischemia-reperfusion injury (IRI) is the major cause of AKI, but its molecular mechanism remains unclear. Immunologic mechanisms in the inflammatory response to ischemic-reperfusion (I/R) have attracted increasing attention. Recent studies have shown that Tim-3 is a new immune regulatory molecule, which is involved in the development of many inflammation-related diseases. Our previous study showed that Tim-3 expression of mononuclear cells in patients with AKI was significantly higher than those in normal control subjects. I/R could induce the up-regulation of Tim-3 mRNA and protein expression in mouse kidney, TIM-3 was predominantly expressed on CD11b+ monocyte/macrophage lineage, and antibody blockade of Tim-3 promoted the renal tubulointerstitial injury induced by IRI. These results suggest that Tim-3 may play an important role in renal IRI through regulating monunuclear phagocyte system function. Our study will reveal the role of Tim-3 in IRI through clinical samples, animal experiments, cell model, and many methods including gene chip, gene transfection and gene knock-out, and furthermore, investigate the molecular mechanism of mononuclear phagocyte system regulated by Tim-3. These studies will provide new and valuable detection indicators for diagnosis and prognosis of ischemic-reperfusion AKI, and obtain new target sites for clinical treatment.

炎症免疫损伤是肾缺血再灌注损伤（IRI）的重要原因。Tim-3是近期发现的一种新型免疫调节分子，参与多种炎症相关性疾病的发生。我们前期研究发现，缺血再灌注(I/R)急性肾损伤（AKI）患者外周血单核细胞Tim-3表达水平显著高于正常人群；I/R可诱导鼠肾组织内Tim-3mRNA及蛋白质表达明显上调；Tim-3蛋白优势表达于CD11b+细胞（活化的单核/巨噬细胞）；抗Tim-3抗体阻断加重I/R鼠急性肾小管间质损伤。由此推测，Tim-3可能通过调控单核/巨噬细胞系统功能在肾IRI中发挥重要调节作用。本研究拟通过临床标本、动物实验、体外细胞模型，利用基因芯片、RNA干扰、基因转染等分子生物学方法揭示Tim-3在肾IRI中的作用，并进一步探究Tim-3调控单核/巨噬细胞系统的分子机制。此研究将为缺血再灌注AKI的早期诊断及预后评价提供新的有价值的检测指标，为其临床治疗寻找新的作用靶点。

目的：观察Tim-3对缺血再灌注肾损伤的影响，并探讨单核/巨噬细胞在此过程中的作用。方法：炎症因子芯片法筛选缺血再灌注患者炎症因子及相关通路。建立缺血再灌注小鼠模型，Tim-3单克隆抗体阻断Tim-3信号通路，应用免疫组化、Real time PCR、流式细胞术等方法观察肾损伤、凋亡、Tim-3表达，探讨Tim-3在缺血再灌注肾损伤中的作用；免疫荧光结合共聚焦显微镜观察肾小管间质单核/巨噬细胞浸润数量，ELISA法检测单核/巨噬细胞相关炎症因子表达，探讨Tim-3在IRI中调控单核/巨噬细胞的作用。构建Tim-3过表达及Tim-3敲除RAW264.7细胞模型，RT-PCR和ELISA检测各组细胞巨噬细胞相关炎症因子表达；Western blot方法检测NF-κB通路、MAPK通路等中的关键分子；通路阻断剂阻断上述通路，ELISA法检测下游炎症因子TNF-a表达以验证上述信号通路在Tim-3调控缺氧缺血巨噬细胞功能中的作用。结果：芯片结果显示：缺血再灌注组与模型组相比较，44个基因出现差异表达。缺血再灌注（I/R）组动物Tim-3在肾组织中表达较对照组明显升高。肾组织病理学及血肾功结果显示I/R组鼠肾损伤积分、血肌酐较对照组明显升高，Tim-3阻断显著降低I/R组升高的肌酐水平，改善组织损伤。TUNEL、WB检测显示I/R组肾小管间质细胞凋亡率、capase-3、Bax/bcl-2表达较对照组明显升高， Tim-3 mAb组上述指标较I/R组明显降低。免疫荧光结果显示，I/R组肾小管间质F4/80+表达较对照组明显升高，I/R+Tim-3 mAb组F4/80+较I/R组表达下调；F4/80+与Tim-3表达部位一致。ELISA检测结果显示，I/R组动物炎症因子水平均较对照组升高，Tim-3mAb明显下调I/R引起的炎症因子升高。Tim3过表达组细胞炎症因子表达明显升高；Tim-3敲除组各炎症因子表达明显下调。western blot结果显示，缺氧复氧刺激使MAPK、NF-κB通路中的关键分子表达较对照组明显升高；Tim-3过表达促进IRI引起的MAPK、NF-κB等信号通路活化；MAPK、NF-κB等通路抑制剂明显降低IRI诱导的TNF-ɑ表达（p＜0.05）。结论：Tim-3通过单核/巨噬细胞促进缺血再灌注肾损伤。