过氧化物酶体增殖物激活受体γ激动剂对转化生长因子β1诱导体外血管外膜成纤维细胞表型的改变

Effect of peroxisome proliferators-activated receptor gamma agonist on phenotypic transition of adventitia fibroblasts induced by transforming growth factor beta 1 in vitro

背景:近年来的研究表明,血管外膜成纤维细胞在血管损伤后新生内膜的增生中起重要作用,但对于引起血管外膜成纤维细胞表型转化的机制尚不十分清楚。目的:观察过氧化物酶体增殖物激活受体(peroxisome proliferators-activated receptor,PPAR)γ激动剂对转化生长因子β1诱导体外血管外膜成纤维细胞表型改变,同时检测对Ⅰ胶原蛋白表达的作用。设计、时间及地点:随机分组设计,对比观察,于2007-07/2008-10在上海交通大学医学院附属第九人民医院组织工程实验室完成。材料:鼠龄2个月的健康雄性SD大鼠,体质量约120g,用于体外培养大鼠胸主动脉外膜成纤维细胞;转化生长因子β1由美国GenWay Biotech公司提供;罗格列酮粉剂为北京高盟化工有限公司产品。方法:实验分为3组:转化生长因子β1诱导组:以不同质量浓度的转化生长因子β1(3,5,10,15μg/L)诱导成纤维细胞48h;罗格列酮干预组:用不同浓度的PPAR-γ激动剂罗格列酮(5,10,30,50μmol/L)干预成纤维细胞45min后,加入转化生长因子β110μg/L共同培养48h;对照组:不添加任何干预措施。主要观察指标:①免疫组织化学α-平滑肌肌动蛋白检测不同浓度转化生长因子β1、罗格列酮干预后成纤维细胞的表型改变。②蛋白免疫印迹检测成纤维细胞中平滑肌α2肌动蛋白及Ⅰ型胶原蛋白的表达。结果:不同质量浓度转化生长因子β1诱导成纤维细胞48h后,与对照组相比,5μg/L转化生长因子β1可明显刺激成纤维细胞表型改变及细胞中平滑肌α2肌动蛋白和Ⅰ型胶原蛋白的表达,10μg/L转化生长因子β1刺激细胞表型改变及平滑肌α2肌动蛋白和Ⅰ型胶原蛋白的表达最明显(P<0.05),随后转化生长因子β1再增加至15μg/L,与10μg/L转化生长因子β1浓度组相比,其刺激细胞表型改变及平滑肌α2肌动蛋白和Ⅰ型胶原蛋白的表达不再增加(P>0.05)。不同浓度罗格列酮干预10μg/L转化生长因子β1诱导的成纤维细胞48h后,与对照组相比,30μmol/L可明显抑制成纤维细胞表型改变及细胞中平滑肌α2肌动蛋白和Ⅰ型胶原蛋白的表达(P<0.05)。结论:PPAR-γ激动剂罗格列酮能抑制转化生长因子β1诱导体外血管外膜成纤维细胞表型改变及平滑肌α2肌动蛋白和Ⅰ型胶原蛋白的表达。

BACKGROUND： Previous studies demonstrated that adventitia fibroblasts exhibit important role in the hyperplasia of newly born endomembrane after blood vessel injury. However, the mechanism regarding phenotypic transition of adventitia fibroblasts in vitro remains unclear. OBJECTIVE： To observe effect of peroxisome proliferators-activated receptor γ agonist （PPAR γ ） on phenotypic transition of adventitia fibroblasts induced by transforming growth factor β1 （TGF- β1） in vitro, as well as the expression of collagen protein I. DESIGN, TIME AND SETTING： A randomized grouping control experiment was performed at the Tissue Engineering Laboratory of Ninth Affiliated Hospital of Shanghai Jiao Tong Medical College between July 2007 and October 2008. MATERIALS： The aorta thoracica＇s adventitial fibroblasts were cultured from Sprague Dawley rats, with 2-month-old, weighing 120 g. The TGF-β1 was supplied by American GenWay Biotech Company. The rosiglitazone power was purchased from Beijing Comens Chemical Co., Ltd. METHODS： The experiment was divided into 3 groups： In the TGF- ~ 1 group, fibroblasts was induced with TGF-β1 with different concentrations （3, 5, 10, 15 μg/L） for 48 hours; in the rosiglitazone group, fibrobMsts was induced with rosigtitazone （5, 10, 30, 50 μmol/L） for 45 minutes followed by co-culture with 10μg/L TGF- β1 for 48 hours. There was no intervention measure in the control group. MAIN OUTCOME MEASURES： The phenotypic transition of fibroblasts was measured by α-smooth muscle actin, the expression of smooth muscle α 2-actin, as well as collagen type I was determined by protein immunoblotting. RESULTS： Compared with the control group, 5 μg/L TGF- β1 could significantly stimulate the changes of phenotypic transition of fibroblasts as well as the expression of smooth muscle α2-actin and collagen type I, which reach a peak level when treated with 10 μg/L TGF-β1 （P 〈 0.05）. The expression of smooth muscle α 2-actin and collagen type I would not increase with the increasing of TGF- β1 to 15 μg/L, compared with 10 μg/L TGF- β1 group （P〉 0.05）. Compared with control group, 30 μmol/L rosiglitazone could significantly inhibit changes of phenotypic transition of fibroblasts as well as the expression of smooth muscle 2-actin and collagen type [ （P〈 0.05）. CONCLUSION： PPAR-γ agonist rosiglitazone can inhibit the phenotypic transition of adventitia fibroblasts induced by TGF- β1, as well as the expression of smooth muscle α 2-actin and collagen type I.