發(fā)布時間：2018-03-08 05:37

  本文選題：糖尿病腎病　切入點：巨噬細胞M1/M2表型　出處：《東南大學》2016年碩士論文　論文類型：學位論文

【摘要】：目的：異質性和可塑性是巨噬細胞的重要特征。目前認為,經典激活巨噬細胞(M1)主要發(fā)揮組織炎癥以及損傷作用,而替代激活巨噬細胞(M2)則參與抗炎以及組織修復進程。腎組織中巨噬細胞浸潤是糖尿病腎病(DN)重要病理學特征,然而巨噬細胞M1/M2表型轉化在DN發(fā)生、發(fā)展中的具體作用尚未明確闡明。髓系細胞觸發(fā)受體TREM-1表達于巨噬細胞表面,參與觸發(fā)以及擴大炎癥反應。近期研究發(fā)現(xiàn),在UUO模型中,TREM-1能夠調節(jié)巨噬細胞表型,參與疾病的進展,但TREM-1在DN中是否具有類似作用尚未明確。本研究旨在探討各病理階段糖尿病腎病患者腎組織巨噬細胞表型特征及其與組織功能學改變關系,并通過細胞培養(yǎng)技術探討TREM-1在高糖誘導的巨噬細胞M1/M2表型轉化中的作用。方法：選取東南大學附屬中大醫(yī)院腎臟病研究所2011-2015年通過腎活檢診斷為DN的46例腎組織作為實驗組并收集腎活檢前的臨床資料包括血肌酐、24小時尿蛋白定量。按照DN病理分型標準將腎組織病理損害分為Ⅰ、Ⅱa、Ⅱb、Ⅲ、Ⅳ期。另取4例因腎外傷或腎腫瘤切除的患腎中的正常腎組織作為對照組。PAS染色觀察腎臟病理改變；免疫組化法檢測腎組織CD68+(巨噬細胞標志物),MR+(M2標志物)巨噬細胞數(shù)量以及TREM-1表達：免疫熒光法檢測腎組織(iNOS+CD68)+(M1標志物)巨噬細胞表達。體外實驗采用高濃度葡萄糖(25mM)干預小鼠巨噬細胞系(RAW264.7), RT-PCR和Western blot分別檢測M1標志物iNOS, M2標志物MR以及TREM-1的表達,并通過siRNA沉默TREM-1,再次用上述方法檢測M1和M2各標志物表達水平。結果：(1)CD68、M1巨噬細胞在腎小球和腎間質均有浸潤,而M2巨噬細胞主要浸潤于腎間質。與對照組相比,DN組腎組織CD68、M1、M2巨噬細胞浸潤數(shù)量明顯增加(P0.05),且呈病程依賴性(P0.05)。(2)DN患者腎小球和腎間質CD68、M1、M2浸潤數(shù)量分別與24小時尿蛋白定量以及血肌酐成正相關。(3)進一步探討DN各個病理階段腎組織巨噬細胞M1/M2活化表型發(fā)現(xiàn),DN早期(Ⅰ+Ⅱa)階段,腎組織中浸潤巨噬細胞主要是M1表型(P0.05),此時M1與M2比值達到最大值,相反,在DN晚期(Ⅲ)階段,腎組織中浸潤巨噬細胞主要是M2表型(P0.05),此時M1與M2比值達到最低值。(4)DN組TREM-1主要表達于腎間質,并且隨著疾病進展,表達不斷增加(P0.05)。(5)體外研究發(fā)現(xiàn)與對照組相比,25mmol/L葡萄糖干預RAW264.7細胞24h后,RT-PCR和Western blot結果均顯示巨噬細細胞TREM-1以及M1標志物iNOS表達上調(P0.05),而M2標志物MR的表達則顯著下降(P0.05)。siRNA沉默TREM-1后,高糖的上述作用均被抑制(iNOS mRNA:高糖組與基因敲除組比較5.048±0.645 vs 2.260±0.062, P0.05; MR mRNA:高糖組與基因敲除組比較1.042±0.036vs 2.214±0.083,P0.05)。結論：1. 巨噬細胞在DN患者腎組織中浸潤顯著增加,其浸潤程度同DN分期相關。2. 腎組織中M1型與M2型巨噬細胞活化狀態(tài)與DN進展密切相關。3. TREM-1在高糖誘導的巨噬細胞M1/M2表型轉化中起重要作用。
[Abstract]:Objective: heterogeneity and plasticity are important characteristics of macrophages. Renal macrophage infiltration is an important pathological feature of diabetic nephropathy (DN). However, macrophage M1 / M2 phenotypic transformation occurs in DN. The specific role of development has not been clearly elucidated. Myeloid cell trigger receptor TREM-1 is expressed on macrophage surface, involved in triggering and expanding inflammatory response. Recent studies have found that TREM-1 can regulate macrophage phenotype in UUO model. The purpose of this study was to investigate the phenotypic characteristics of renal macrophages in diabetic nephropathy patients at different stages and their relationship with histopathological changes. The role of TREM-1 in the phenotypic transformation of macrophages M1 / M2 induced by high glucose was investigated by cell culture technique. Methods: 46 cases of DN diagnosed by renal biopsy from 2011 to 2015 in the Institute of Kidney Disease, affiliated Chinese University of Southeast University, were selected. The clinical data before renal biopsy were collected as experimental group, including 24 hour urinary protein quantification of serum creatinine. According to DN pathological classification criteria, renal tissue pathological damage was divided into two groups. Stage 鈪，

本文編號：1582658