内皮素对黑素瘤A375细胞转化生长因子-β1及磷酸化Smad3蛋白表达的调节  

Regulatory effect of endothelin on the expression of transformation growth factor-beta 1 and phosphorylated Smad 3 in A375 cells in vitro

作　　者：黄巍[1] 方险峰[1] 杨凌云[1] 陶娟[1] 林能兴[1] 陈宏翔[1] 刘业强[1] 李延[1] 杨井[1] 李艳秋[1] 涂亚庭[1] 黄长征[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院皮肤科,武汉430022

出　　处：《中华皮肤科杂志》2009年第7期463-466,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金（30671891）

摘　　要：目的探讨内皮素家族（ET）对恶性黑素瘤（MM）A375细胞转化生长因子betal（TGF—β1）表达的差异性调节及对TGF—β信号通路蛋白Smad 3磷酸化水平的影响。方法ELISA、RT—PCR技术测定不同浓度ET-1、ET-3（0、0．1、1、10、100nmol/L）及其与内皮素受体阻断剂BQ123、BQ788联合干预下MM细胞TGF—β1在蛋白质和mRNA水平的表达。Western印迹法检测联合干预下A375细胞Smad 3蛋白磷酸化的表达。结果ET-1对A375细胞（10^5细胞）TGF—β1蛋白表达的影响呈浓度依赖性上调，100nmol／L浓度下达1289．38±89．42ng/L（P〈0．05）；ET-3的作用与此相反，100nmol／L浓度下表达仅为85．09±9．37ng/L（P〈0．05）。BQ123明显阻断ET-1上调TGF—β1的效应（P〈0．05），BQ788对此差异无统计学意义（P〉0．05）；联合干预下，在TGF—β1 mRNA水平也表现出相同的趋势。Western印迹法检测磷酸化Smad3蛋白（P—Smad3）表达显示，ET-1显著上调P—Smad3的表达（P〈0．05），BQ123干预下其表达水平接近对照组（P〉0．05），BQ788不能阻断其对P—Smad3表达的上调（P〉0．05）；ET-3则明显抑制P-Smad3的表达（P〈0．05）。结论ET-1可诱导MMA375细胞TGF-β1的高表达，ET-3有相反的作用。ET受体-A介导ET-1诱导的TGF—β通路信号蛋白Smad3的磷酸化。Objective To investigate the effects ofendothelin-1 （ET-1） and endothelin-3 （ET-3） on the expression of transformation growth factor-beta 1 （TGF-β1） and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group （no stimulation）, ET-1 group （stimulated with ET-1）, ET-1 ＋ BQ123 group （treated with ET-1 and BQ123）, ET-1 ＋ BQ788 group （treated with ET-1 and BQ788）, ET-3 group （stimulated with ET-3）, to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 （P-Smad 3 ）. Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ±89.42 ng/L per 10^5 ceils and 85.09 ± 9.37 ng/L per 10^5 cells, respectively, significantly different from that in unstimulated cells （both P 〈 0.05）. BQ123 significantly blocked the up-regulatory effect of ET-1 on the expression TGF-β1 protein（P 〈 0.05 ）, but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1 significantly elevated the expression of P-Smad 3 in A375 cells （P 〈 0.05 ）, and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells （P 〈 0.05）. Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.

关 键 词：黑色素瘤 内皮缩血管肽类 受体 内皮素A 转化生长因子Β1 

分 类 号：R285.5[医药卫生—中药学]