高糖刺激下大鼠肾小球系膜细胞MMP-2，TIMP-2，MT1MMP 和CTGF的表达及意义  

Expression of MMP-2, TIMP-2, CTGF and MT1-MMP in rat glomerular mesangial cells stimulated by high-glucose and its significance

作　　者：姚芳[1] 李志红[2] 闫喆[1] 刘青娟[1] 段惠军[1] 郄涛[2] 

机构地区：[1]河北医科大学基础医学院病理教研室,石家庄050017 [2]保定市第一中心医院内分泌科,河北保定071000

出　　处：《解放军医学杂志》2013年第10期811-817,共7页Medical Journal of Chinese People's Liberation Army

基　　金：河北省教育厅青年基金(2010150)~~

摘　　要：目的观察高糖刺激的大鼠肾小球系膜细胞基质金属蛋白酶-2(MMP-2)及其组织抑制物-2(TIMP-2)、膜型基质金属蛋白酶-1(MT1-MMP)和结缔组织生长因子(CTGF)的动态变化以探讨糖尿病肾病(DN)的发病机制。方法体外培养的大鼠HBZY-1肾小球系膜细胞分为低糖(5.5mmol/L葡萄糖)组、高糖(30mmol/L葡萄糖)组和渗透压对照(5.5mmol/L葡萄糖+24.5mmol/L甘露醇)组,24、48、72、96h后采用RT-PCR及Western blotting法分别检测MMP-2、TIMP-2、MT1-MMP及CTGF的mRNA及蛋白表达情况,酶联免疫吸附法(ELISA)检测培养上清中Ⅳ型胶原的含量。结果 Western blotting结果显示,与低糖组相比,高糖组MMP-2的表达在24h时略有升高,较低糖组增加10%±4%(P<0.05),至48h时则较低糖组减少42%±2%,其后随时间延长表达持续降低,至96h时较低糖组减少78%±2%;MT1-MMP表达在24h开始下降并随时间呈下降趋势,与低糖组相比较,在刺激的24h,高糖组MT1-MMP表达下降了29%±3%,随后持续下降,至96h则下降了78%±9%(P<0.01)。高糖组各时间点TIMP-2和CTGF表达均较低糖组增高,其中CTGF的表达在高糖刺激的24h即显著增高,为低糖组的201%±24%,随后持续增高,至培养96h为低糖组的484%±51%(P<0.01);TIMP-2的表达在24h时较低糖组增加55%±3%,且随时间延长呈增高趋势(P<0.01)。MMP-2、TIMP-2、MT1-MMP和CTGF的mRNA表达与相应蛋白的表达趋势基本一致。与低糖组相比,高糖组细胞上清中的Ⅳ型胶原于24h即有增加,且持续增高至96h(P<0.05)。低糖组和渗透压对照组组内、组间的各指标差异均无统计学意义。结论尽管高糖刺激早期可小幅诱导MMP-2的表达增强,但长期高糖刺激则可抑制MMP-2和MT1-MMP的表达及活化,同时促进系膜细胞TIMP-2和CTGF的表达。DN中肾小球细胞外基质的积聚可能是由于上述细胞因子和蛋白酶引起细胞外基质代谢失衡所致。Objective To observe the dynamic changes of matrix metalloproteinase-2 （MMP-2）, tissue inhibitor of metalloproteinase-2 （TIMP-2）, membrane-type 1 matrix metalloproteinase （MT1-MMP） and connective tissue growth factor （CTGF） expression in high glucose-stimulated glomerular mesangial cells （GMCs） in rats, and investigate the mechanism of the pathogenesis of diabetic nephropathy. Methods Rat HBZY-1 GMCs were cultured and divided into 3 groups： low concentration （5.5mmol/L） D-glucose （LG） group, high concentration （30mmol/L） D-glucose （HG） group and 24.5mmol/L mannitol plus 5.5mmol/L D-glucose （LG＋M） group （served as osmotic pressure control）. The mRNA and protein expressions of MMP-2, TIMP-2, MT1-MMP and CTGF were detected with semi-quantitative RT-PCR and Western blotting, and the secreted collagen Ⅳ in supernatants of the GMCs was detected by ELISA after cultured for 24, 48, 72 and 96h. Results Compared with LG group, after exposure to high glucose for 24h, MMP-2 expression was slightly increased （increased by 10%±4%） in GMCs in HG group （P〈0.05）. But when the exposure time last from 48h to 96h, the expression of MMP-2 was decreased by 42%±2% to 78%±2% （P〈0.01）. Compared with LG group, high-glucose incubation resulted in down-regulation of MT1-MMP （decreased by 29%±3% at 24h to 78%±9% at 96h, P〈0.01） whereas up-regulation of TIMP-2 （increased by 55%±3% at 24h, P〈0.01） and CTGF （increased by 201%±24% at 24h to 484%±51% at 96h, P〈0.01）. RT-PCR revealed consistent dynamic changes of MMP-2, TIMP-2, MT1-MMP and CTGF with their protein changes listed above. Compared with LG group, the secreted collagen Ⅳ in supernatants of HG group was increased by 201%±24% at 24h to 1232%±198% at 96h （P〈0.01）. There was no significant difference of above indexes between LG group and LG＋M group. Conclusions High glucose may induce and activate MMP-2 transiently, but can inhibit the expression of MMP-2 and MT1-MMP for a long term where

关 键 词：糖尿病肾病 肾小球系膜细胞 基质金属蛋白酶-2 基质金属蛋白酶组织抑制物-2 结缔组织生长因子 膜型基质金属蛋白酶-1 

分 类 号：R587.1[医药卫生—内分泌]