高糖和血管紧张素Ⅱ对人肾小管上皮细胞Toll样受体4信号表达及炎性和纤维化因子的影响  

Effect of high glucose or anglotensin II on the expression of ton-like receptor 4 signal pathway,inflammatory and fibrotic factors in human tubular epithelial cells

作　　者：熊梅梅[1,4] 吕柳青 肖红波[3] 程玉花[1] 吕金雷[1] 王瑜[1] 陈钦开[1] 

机构地区：[1]南昌大学一附院肾内科,南昌330006 [2]湖北省黄石市二医院内科,435000 [3]北京大学深圳医院肾内科,深圳518036 [4]赣南医学院附一医院肾内科

出　　处：《中华肾脏病杂志》2016年第1期43-49,共7页Chinese Journal of Nephrology

基　　金：基金项目：国家自然科学基金（81060063）;江西省教育厅科学基金（JJJ11354）;江西省自然科学青年基金（20132BAB215004）

摘　　要：目的通过研究高糖和血管紧张素Ⅱ（AngII）环境下人肾小管上皮细胞Toll样受体4（TLR4）表达及其相关的炎性和纤维化因子的变化及TLR4siRNA的干扰作用，从天然免疫角度探讨糖尿病。肾病（DN）发病机制和靶向干预措施。方法设计并合成3对针对人TLR4基因的特异性siRNA片段，以带有红色荧光的BLOCK-ITAlexaFluor作为阴性对照，荧光显微镜下观察细胞转染效率，实时定量PCR检测TLR4mRNA的表达变化。确定TLR4siRNA基因沉默效果较佳后再进一步实验。实验分2部分进行，第1部分细胞分3组：正常糖对照组（NG，5．5mmol／L葡萄糖）、甘露醇组（M，5．5mmol／L葡萄糖＋19．5mmol／L甘露醇）和高糖组（HG，25mmol／L葡萄糖），初步验证高糖、高渗的影响。第2部分7组：NG组、HG组、AngⅡ（10-7mmol／L）组、AnglI＋空载体对照组、HG＋空载体对照组、AnglI＋TLR4siRNA转染组及HG＋TLR4siRNA转染组。采用实时定量PCR法检测TLR4、髓样分化因子88（MyD88）、热休克蛋白47（HSP47）mRNA的表达；Western印迹法检测TLR4、MyD88、HSP47、核因子KB（NF．KB）、Ⅳ型胶原蛋白（ColⅣ）的蛋白表达；ELISA法检测细胞上清液单核细胞趋化蛋白1（MCP-1）、白细胞介素6（IL-6）的水平。结果与NG组比较，甘露醇组TLR4、MyD88蛋白表达差异均无统计学意义（P〉0．05）；HG、AngⅡ组TLR4、MyDS8、HSP47mRNA及TLR4、MyD88、NF．KB、HSP47、ColⅣ蛋白表达均显著上调（p〈0．01），细胞上清液MCP-1、IL-6水平亦升高（P〈0．01）；TLR4siRNA转染后，与HG组或AngU组相比上述指标均显著下调（P〈0．01）；空载体转染后，与HG组或AngⅡ组相比上述指标差异无统计学意义（P〉0．05）。结论高糖或血管紧张素Ⅱ均刺激人肾小管上皮细胞TLR4信号并引起天然免疫应答，导致炎性和纤维化因子上调。特异性基因沉默可通过阻断由高糖或AngⅡ诱导的TLR4信�Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) or high glucose on the toll-like receptor 4 (TLR4) expression,inflammatory cytokines and fibrotic factors in human tubular epithelial cells (HK-2),revealing the innate immune-related pathogenesis of diabetic nephropathy (DN) which may have clinical implications.Methods Three TLR4 siRNA sequences were designed and synthetized.After transfection,the most effective siRNA was selected to use for further expriments.The experiment consisted of 2 parts.Part 1:Cells were divided into three groups:normal-glucose group (NG,5.5mmol/L glucose),mannose group (M,5.5 mmol/L glucose + 19.5 mmol/L mannose),High-glucose group (HG,25 mmol/L glucose),preliminary validated the effects of high glucose and high osmotic pressure.Part 2:Cells were divided into seven groups:NG group,HG group,Ang Ⅱ group,Ang Ⅱ + negative group,HG+ negative group,Ang Ⅱ + siRNA group and HG+ siRNA group.Real time PCR was used to analyze the mRNA expression of TLR4,myeloid differentiation factor 88 (MyD88),heat shock protein 47 (HSP47).Western blotting was used to observe the protein expression of TLR4,MyD88,HSP47,NF-κB,type Ⅳ collagen (ColⅣ).ELISA was used to detect the expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6).Results Compared with NG group,TLR4,MyD88,HSP47 mRNA and TLR4,MyD88,NF-κB,ColⅣ,HSP47 protein were highly expressed under high glucose or Ang Ⅱconditions (P ＜ 0.01),and the expression levels of MCP-1 and IL-6 also increased significantly (P ＜ 0.01).Compared with HG or Ang Ⅱ group,the above indicators were obviously inhibited in the TLR4 siRNA groups (P＜0.01).Comparison between blank vector transfected groups and HG group as well as Ang Ⅱ group indicated no statistic significance (P ＞ 0.05).Conclusions Both Ang Ⅱ and high glucose stimulate TLR4 expression,which result in the up-regulation of inflammatory and fibrotic factors in HK-2.Specific target of TLR4 gene silencing can block the TLR4 pathway that is activated by high

关 键 词：血管紧张素Ⅱ TOLL样受体4 葡萄糖 肾小管上皮细胞 天然免疫应答 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]