【作者】 李曼丽；

【导师】 甘华；

【作者基本信息】 重庆医科大学 ， 内科学， 2008， 博士

【摘要】 研究背景:TLR4是研究最多的Toll样受体,主要配体为LPS,呼吸道合胞病毒蛋白和脂磷壁酸。TLR4还能识别内源性配体如HSP,硫酸乙酰肝素,纤连蛋白额外域A等。TLR4有两条信号通路:MyD88依赖的信号通路及非MyD88依赖的信号通路。其信号通路激活后能启动炎症因子的转录和合成,与炎症因子的产生密切相关。许多研究表明,TLR4的表达也影响炎症因子的产生。影响TLR4表达的因素众多,但高糖对TLR4表达调节作用的研究,目前国内外均未见文献报道。来自临床和实验室的众多证据表明,炎症在糖尿病肾病的发生发展中具有重要作用。在糖尿病肾病发病机制中,高血糖作为起始因素,促进肾脏固有细胞产生炎症介质,参与糖尿病肾病炎症的发生,相关信号途径的研究有蛋白激酶C,NF-κB,p38MAPK和氧化应激。高糖促进炎症因子表达与TLR4及其信号通路关系的研究国内外均未见报道。目的:(1)探讨高糖对肾小管上皮细胞TLR4表达及其信号通路的影响;(2)探讨高糖对内皮细胞TLR4表达的影响,高糖与LPS共同刺激下对内皮细胞TLR4的表达及MCP-1产生的影响;(3)探讨糖尿病肾病患者外周血单核细胞TLR4的表达及其与血浆MCP-1浓度的关系,以进一步了解TLR4与糖尿病肾病炎症的关系。方法:(1)RT-PCR分析TLR4mRNA的表达;(2)Western Blot和流式细胞术分析TLR4蛋白的表达;(3)Western Blot和免疫沉淀法分析TLR4与MyD88的相互作用,以了解TLR4信号通路是否被激活;(4)ELISA分析MCP-1的浓度。结果:(1)高糖可使小鼠近曲肾小管上皮细胞(MCT细胞)TLR4蛋白的表达上调,高糖环境下MyD88依赖的TLR4信号通路被激活;(2)高糖可使HUVEC细胞TLR4mRNA及其蛋白的表达增加,高糖与LPS共同刺激下,HUVEC细胞TLR4蛋白表达进一步增加,HUVEC细胞TLR4的表达上调与其MCP-1产生增加有关;(3)糖尿病肾病尿毒症患者外周血单核细胞TLR4mRNA及蛋白表达较健康人显著增加,外周血单核细胞TLR4蛋白的表达与血浆MCP-1浓度呈正相关。结论:(1)高糖刺激小鼠近曲肾小管上皮细胞TLR4表达增加,高糖环境下,MyD88依赖的TLR4信号通路被激活。(2)高糖刺激人脐静脉内皮细胞TLR4表达增加,并协同LPS促进内皮细胞表达TLR4及分泌MCP-1。内皮细胞TLR4的表达上调与其MCP-1产生增加有关。(3)糖尿病肾病尿毒症患者外周血单核细胞TLR4表达上调,外周血单核细胞TLR4的表达与血浆MCP-1浓度呈正相关。高糖可促进肾脏固有细胞TLR4的表达, TLR4与糖尿病肾病炎症有关。更多还原

【Abstract】 Background: Among the 10 human TLRs, TLR4 is the most thoroughly studied. It recognizes LPS as well as other pathogen associated molecular pattern (PAMP). TLR4 has both a MyD88-dependent and–independent pathway. Endogenous ligands, such as HSP, heparan sulfate are found to activate TLR4 and cause the production of inflammatory factors.A lot of studies have shown that the expression of TLR4 is correlated with the production of inflammatory factors. Many factors affect the expression of TLR4.Evidences from patients and the laboratories have shown that inflammation might play a critical role in the pathogenesis of diabetic nephropathy. High glucose is involved in the inflammatory mechanism of diabetic nephropathy by promoting renal innate cells to secret inflammatory factors through PKC, p38MAPK and oxidative stress. However, it is not known if TLR4 and the signal pathway are related with high glucose induced inflammation. Object: (1)to explore the effect of high glucose on the expression of TLR4 and its’pathway in MCT cells; (2)to explore the effect of high glucose on the expression of TLR4, the co-effect of high glucose and LPS on the expression of TLR4 and the production of MCP-1; (3)to explore the relation between the expression of TLR4 on the peripheral blood monocytes from patients with diabetic nephropathy and the concentration of plasma MCP-1.Methods: (1)the expression of TLR4mRNA is analyzed with RT-PCR; (2)the expression of TRL4 protein is analyzed with flow cytomety and western blot; (3) the correlation of TLR4 with MyD88 is analyzed with Western Blot and immunoprecipitation; (4) ELISA is undertaken to measure the excretion of MCP-1 and the concentration of plasma MCP-1.Results: (1) high glucose upregulated the expression of TLR4 protein and activated MyD88-dependent pathyway in MCT cells; (2) high glucose upregulated the expression of TLR4 in HUVEC cells and induced the production of MCP-1. The expression of TLR4 and the production of MCP-1 were further increased when co-stimulated with LPS; (3) the expression level of TLR4 was significantly increased on the peripheral blood monocytes from patients with diabetic nephropathy compared with the healthy people; the expression of TLR4 was positively correlated with the concentration of plasma MCP-1.Conclusion: (1) high glucose upregulated the expression of TLR4 and activated MyD88-dependent pathyway in proximal tubular eplithelial cells;(2) high glucose upregulated the expression of TLR4 in human umbilical venous endothelial cells, further increasing the expression of TLR4 and the production of MCP-1 when co-stimulated with LPS. The enhanced expression of TLR4 is related with the production of MCP-1;(3) the expression of TLR4 on the peripheral blood monocytes from the patients with diabetic nephropathy is increased compared with those from the health control, the expression of TLR4 on the peripheral blood monocytes is positively related with the concentration of plasma MCP-1.High glucose upregulated the expression of TLR4 in renal innate cells. TLR4 is involved in the inflammation in diabetic nephropathy.更多还原