糖尿病肾病肾组织巨噬细胞表型特征及TREM-1在其中的作用机制探讨

发布时间：2018-03-08 05:37

  本文选题：糖尿病肾病　切入点：巨噬细胞M1/M2表型　出处：《东南大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：异质性和可塑性是巨噬细胞的重要特征。目前认为,经典激活巨噬细胞(M1)主要发挥组织炎症以及损伤作用,而替代激活巨噬细胞(M2)则参与抗炎以及组织修复进程。肾组织中巨噬细胞浸润是糖尿病肾病(DN)重要病理学特征,然而巨噬细胞M1/M2表型转化在DN发生、发展中的具体作用尚未明确阐明。髓系细胞触发受体TREM-1表达于巨噬细胞表面,参与触发以及扩大炎症反应。近期研究发现,在UUO模型中,TREM-1能够调节巨噬细胞表型,参与疾病的进展,但TREM-1在DN中是否具有类似作用尚未明确。本研究旨在探讨各病理阶段糖尿病肾病患者肾组织巨噬细胞表型特征及其与组织功能学改变关系,并通过细胞培养技术探讨TREM-1在高糖诱导的巨噬细胞M1/M2表型转化中的作用。方法：选取东南大学附属中大医院肾脏病研究所2011-2015年通过肾活检诊断为DN的46例肾组织作为实验组并收集肾活检前的临床资料包括血肌酐、24小时尿蛋白定量。按照DN病理分型标准将肾组织病理损害分为Ⅰ、Ⅱa、Ⅱb、Ⅲ、Ⅳ期。另取4例因肾外伤或肾肿瘤切除的患肾中的正常肾组织作为对照组。PAS染色观察肾脏病理改变；免疫组化法检测肾组织CD68+(巨噬细胞标志物),MR+(M2标志物)巨噬细胞数量以及TREM-1表达：免疫荧光法检测肾组织(iNOS+CD68)+(M1标志物)巨噬细胞表达。体外实验采用高浓度葡萄糖(25mM)干预小鼠巨噬细胞系(RAW264.7), RT-PCR和Western blot分别检测M1标志物iNOS, M2标志物MR以及TREM-1的表达,并通过siRNA沉默TREM-1,再次用上述方法检测M1和M2各标志物表达水平。结果：(1)CD68、M1巨噬细胞在肾小球和肾间质均有浸润,而M2巨噬细胞主要浸润于肾间质。与对照组相比,DN组肾组织CD68、M1、M2巨噬细胞浸润数量明显增加(P0.05),且呈病程依赖性(P0.05)。(2)DN患者肾小球和肾间质CD68、M1、M2浸润数量分别与24小时尿蛋白定量以及血肌酐成正相关。(3)进一步探讨DN各个病理阶段肾组织巨噬细胞M1/M2活化表型发现,DN早期(Ⅰ+Ⅱa)阶段,肾组织中浸润巨噬细胞主要是M1表型(P0.05),此时M1与M2比值达到最大值,相反,在DN晚期(Ⅲ)阶段,肾组织中浸润巨噬细胞主要是M2表型(P0.05),此时M1与M2比值达到最低值。(4)DN组TREM-1主要表达于肾间质,并且随着疾病进展,表达不断增加(P0.05)。(5)体外研究发现与对照组相比,25mmol/L葡萄糖干预RAW264.7细胞24h后,RT-PCR和Western blot结果均显示巨噬细细胞TREM-1以及M1标志物iNOS表达上调(P0.05),而M2标志物MR的表达则显著下降(P0.05)。siRNA沉默TREM-1后,高糖的上述作用均被抑制(iNOS mRNA:高糖组与基因敲除组比较5.048±0.645 vs 2.260±0.062, P0.05; MR mRNA:高糖组与基因敲除组比较1.042±0.036vs 2.214±0.083,P0.05)。结论：1. 巨噬细胞在DN患者肾组织中浸润显著增加,其浸润程度同DN分期相关。2. 肾组织中M1型与M2型巨噬细胞活化状态与DN进展密切相关。3. TREM-1在高糖诱导的巨噬细胞M1/M2表型转化中起重要作用。
[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，

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