VEGF对上皮间充质转化的抑制与Smad信号途径及SnoN表达的变化  

Smad signal transduction pathway involved in supression of VEGF on TGF-β1 induced EMT of HK-2 cells

作　　者：连耀国[1] 张颖娟[1] 郑法雷[1] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院肾内科,100730

出　　处：《北京医学》2012年第4期299-304,共6页Beijing Medical Journal

基　　金：国家自然科学基金(30570854)

摘　　要：目的探讨血管内皮生长因子(VEGF)抑制转化生长因子-β1(TGF-β1)诱导HK-2细胞发生肾小管上皮-间充质细胞转化(EMT)的过程中对Smad2/3,Smad7信号途径以及SnoN表达的影响。方法体外培养的HK-2细胞分为:①正常对照组;②TGF-β1(5μg/L)阳性对照组;③VEGF165(100μg/L)作用组;④TGF-β1(5μg/L)+VEGF165(100μg/L)共同作用组。采用WesternBlot法和RT-PCR分别检测各组细胞p-Smad2/3和Smad2/3(共同作用30和60min),α-平滑肌肌动蛋白(α-SMA)、Smad7、SnoN的表达水平(共同作用48h)。结果 TGF-β1组α-SMA蛋白和mRNA表达与正常对照组比较明显增强,TGF-β1与VEGF165共同作用组α-SMA蛋白和mRNA表达与TGF-β1单独作用组比较明显减弱(P<0.05)。体外HK-2细胞,TGF-β1组刺激30、60min,与正常对照组比较,(p-Smad2/3)/(Smad2/3)比值明显升高,TGF-β1+VEGF165组与TGF-β1单独作用组相比明显下降,且以30min时下降明显。TGF-β1组作用48h与正常对照组比较,Smad7蛋白和mRNA表达明显下降,VEGF165+TGF-β1组较TGF-β1单独作用组Smad7表达明显升高(P<0.05)。VEGF165组与正常对照组相比,α-SMA、(p-Smad2/3)/(Smad2/3)、Smad7蛋白和mRNA表达的差异无统计学意义(P﹥0.05)。TGF-β1组SnoN蛋白表达与正常对照组比较明显增强(P<0.05),TGF-β1与VEGF165共同作用组SnoN蛋白表达与TGF-β1单独作用组相比差异无统计学意义(P﹥0.05),各组SnoNmRNA表达差异均无统计学意义(P﹥0.05)。结论 VEGF165抑制TGF-β1诱导HK-2细胞EMT的机制可能与直接抑制Smad2/3磷酸化、上调Smad7信号表达有关,而与调节SnoN表达无关。Objective To examine the relationship between the inhibitory effect of VEGF on EMT of HK-2 cells and the influence of changes in the expressions of SnoN and Smad pathway on the effect of VEGF mentioned above. Methods The cultured HK-2 ceils were divided into four groups： （1）without treatment group, （2）treated with TGF-151 （5 μg/L） alone as positive control group, （3） treated with VEGF165 （100 μg/L） alone group, （4）treated with TGF-151 （5μg/L） and VEGF165 （100 μg/L） as the co-treatment group. The α-SMA, p-Smad 2/3 and Smad 2/3, Smad 7, SnoN expressions were assessed with western blot and RT-PCR, respectively. Results By western blot and RT-PCR analysis, it＇s found α-SMA expression was significantly increased in HK-2 cells treated with TGF-151 （5 μg/L） compared with no treatment （P 〈 0.05）, and the ratio of p-Smad 2/3 and Smad 2/3 were significantly increased in both 30 min and 60 min （P 〈 0.05）. These phosphorylation marker mentioned above decreased in HK-2 cells co-treatment group compared with the positive controls, especially at 30 min （P 〈 0.05）. But Smad 7 expression was exactly opposite to that of these phosphorylation marker mentioned above. VEGF165 dramatically abrogated TGF-151 induced α-SMA expression and phosphorylation of Smad 2/3 and restored Smad 7 expression＇in HK-2 cells （P 〈 0.05）. However, VEGF165 itself failed to induce phosphorylation of Smad 2/3 and the expression of Smad 7. SnoN protein expression significantly increased in HK-2 ceils treated with TGF-β1 （5 αg/L） compared with no treatment （P 〈 0.05） patients, but was not different in HK-2 cells co-treatment patients compared with positive controls （P 〉 0.05）. SnoN mRNA expression was not significantly different in all groups （P 〉 0.05）. Conclusions The results documented that VEGF165 may partially inhibit TGF-β1-induced EMT in HK-2 cells in vitro, and this effect is related to suppress of phosphorylation of Smad 2/3 directly, and up-regulates S

关 键 词：血管内皮生长因子 上皮-间充质转化 转化生长因子-Β1 SMAD2/3 

分 类 号：R363[医药卫生—病理学]