血小板反应蛋白1通过活化转化生长因子β1促进人间皮细胞间皮间叶转化  

Thrombospondin-1 enhances mesothelial-to-mesenchymal transition in human mesothelial cells through activating transforming growth factor-β1

作　　者：张珍[1] 姜娜[1] 邵兴华[1] 井冉[1] 车霞静[1] 庞慧华[1] 牟姗[1] 倪兆慧[1] 

机构地区：[1]上海交通大学医学院附属仁济医院肾脏科,上海200127

出　　处：《上海医学》2015年第7期590-595,F0004,共7页Shanghai Medical Journal

基　　金：国家自然科学基金(81200553);"十二五"国家科技支撑计划(2011BAI10B08);上海市高级中西医结合人才培养项目(ZYSNXD012-RC-ZXY017);上海高校青年教师培养资助计划(ZZjdyx13049)资助项目

摘　　要：目的探讨血小板反应蛋白1(TSP-1)对TGF-β1活性的调控在人间皮细胞间皮间叶转化(MMT)中的作用及其机制。方法体外培养人间皮细胞株(Met-5A细胞),与无血清培养基组(对照组)比较,测定外源性TSP-1刺激下(以5ng/mL的TSP-1刺激为TSP-1小剂量组,以10ng/mL的TSP-1刺激为TSP-1大剂量组)Met-5A细胞中TGF-β1的表达和活性,Smad通路活化,以及MMT标志因子纤维连接蛋白(FN)、人Ⅲ型胶原蛋白(CollⅢ)、α平滑肌肌动蛋白(α-SMA)、Snail的表达。同时观察5ng/mL的TSP-1和10\mg/mL的TGF-β1中和抗体刺激下(中和组)Met-5A细胞中Smad通路活化和MMT发生的变化。结果 TSP-1小剂量组和TSP-1大剂量组对Met-5A细胞中TGF-β1表达量和活性的上调作用均呈时间依赖性(P值均<0.05),但两组间同时间点的差异均无统计学意义(P值均>0.05)。故之后的实验均应用5ng/mL的TSP-1刺激细胞。TSP-1小剂量组在刺激24、48、72h时Met-5A细胞中FN、CollⅢ、Snail和α-SMA mRNA的相对表达量均显著高于对照组同时间点(P值均<0.05),FN、CollⅢ、Snail和α-SMA蛋白的相对表达量均显著高于对照组刺激0h时(P值均<0.05)。TSP-1小剂量组在刺激10、30、60、120min时Met-5A细胞中p-Smad2/3/Smad2/3蛋白的相对表达量均显著高于对照组刺激0h时(P值均<0.05)。TSP-1小剂量组在刺激24、48、72h时Met-5A细胞中的FN、CollⅢ、Snail和α-SMA mRNA相对表达量均显著低于对照组刺激0h时和中和组同时间点(P值均<0.05)。结论 TSP-1通过活化TGF-β1/Smad信号通路促进人间皮细胞MMT的发生。Objective To investigate the effect of thrombospondin-1 （TSP-1） on mesothelial-to- mesenchymal transition （MMT） in human mesothelial cells through regulation of transforming growth factor-β1 （TGF-β1） bioactivity. Methods Human mesothelial cells （Met-5A cells） were cultured in vitro with or without serum medium （control group）. Then the cells were treated with exogenous TSP-1 （5 ng/mL TSP-1 as low-dose group and 10 ng/mL as high-dose group） in the presence or absence of a neutralizing antibody to TGF-β1 （neutralizing group）. TGF-β1 secretion and bioactivity were measured by enzyme-linked immunosorbent assay （ELISA） and MLEC bioassay, respectively. The expression of MMT molecules （fibronectin [FN], Coll Ⅲ, α- smooth muscle actin [SMA], Snail） and activation of Smad signaling were assessed by quantitative real-time polymerase chain reaction （PCR） or Western blotting analysis. Results In the TSP-1 low-dose group and high- dose group, TGF-β1 expression and bioactivity were all elevated significantly in a time-dependent manner （all P〈0.05）, but there was no significant difference between the two groups （all P〉0.05）. Therefore, 5 ng/mL TSP-1 was applied to stimulate cells in the following experiments. The relative expressions of FN/GAPDH,Coll Ⅲ/GAPDH, Snail/GAPDH and α-SMA/GAPDH in the TSP-1 low-dose group were significantly higher than those in the control group at 24, 48 and 72 h after TSP-1 treatment （all P〈0.05）. The relative expressions of FN/β1-actin, Coll Ⅲ/β1-actin, Snail/β1-actin and α-SMA/β1-actin in the TSP-1 low-close group at 24, 48 and 72 h after TSP-1 treatment were significantly higher than baseline in the control group （all P〈0.05）. The relative expression of p-Smad2/3/Smad2/3 at 10, 30, 60 and 120 min after TSP-1 treatment and the relative expression of FN/GAPDH, Coll Ⅲ/GAPDH, Snail/GAPDH and α-SMA/GAPDH at 24, 48 and 72 h after TSP-1 treatment in the TSP-1 low-dose group were significantly higher than baseline in the

关 键 词：血小板反应蛋白1 人间皮细胞 间皮间叶转化 转化生长因子Β1 

分 类 号：R459.5[医药卫生—治疗学] R572.2[医药卫生—临床医学]