Connective tissue growth factor synergistically with transforming growth factor β1 to promote renal fibrosis

目的探讨结缔组织生长因子(CTGF)和转化生长因子β1(TGF-β1)对肾脏成纤维细胞合成和分泌基质金属蛋白酶-2(MMP-2)和细胞表型转化的影响.方法将大鼠肾成纤维细胞分为对照组、CTGF刺激组、TGF-β1刺激组、CTGF加TGF-β1联合刺激组,以明胶酶谱法和Western印迹方法分别检测细胞上清中MMP-2活性和蛋白的变化,实时定量-聚合酶链反应(PCR)方法检测细胞中MMP-2 mRNA的水平.Western印迹方法检测成肌纤维细胞标志蛋白α-平滑肌肌动蛋白(α-SMA)的表达水平和细胞上清中细胞外基质成分纤黏连蛋白(FN)的水平.结果 MMP-2的活性和蛋白水平在刺激24 h时,各组之间无明显差异;刺激48 h,100 ng/ml CTGF组和5 ng/ml TGF-β1组明显属于对照组(P<0.05);而不同浓度的CTGF加TGF-β1联合刺激组分别低于CTGF刺激组、TGF-β1刺激组,其中100 ng/ml CTGF 加5 ng/ml TGF-β1、50 ng/ml CTGF 加5 ng/ml TGF-β1均分别明显低于CTGF刺激组、TGF-β1刺激组(P<0.05).上述各组细胞刺激12 h,MMP-2 mRNA水平在100 ng/ml CTGF组和5 ng/ml TGF-β1组均明显高于对照组(1.72,1.68 vs 1.29, P<0.01),而100 ng/ml CTGF加5 ng/ml TGF-β1联合组明显低于CTGF刺激组、TGF-β1刺激组(0.67 vs 1.72,1.68,P<0.01).CTGF单独刺激对细胞表达α-SMA无明显影响,上清中FN水平与对照组相比无明显变化(P>0.05),而TGF-β1单独刺激组两者水平分别显著高于对照组(P<0.01),并且在 CTGF加TGF-β1联合组两者水平较TGF-β1刺激组又有明显升高(P<0.05).结论 CTGF和TGF-β1可以协同下调肾成纤维细胞产生MMP-2,并促进成肌纤维细胞生成.To investigate the influence of CTGF and TGF-beta(1) on the synthesis and secretion of matrix metalloproteinase-2 (MMP-2) and myofibrotic activation in renal fibroblasts.Equal numbers of renal fibroblasts (NRK-49F) were planted and divided into vechile, CTGF treated alone, TGF-beta(1) treated alone, and CTGF plus TGF-beta(1) treated groups. Gelatin zymography and Western-blot analysis were used for assay of the MMP-2 activity and protein level in the supernatant cultured medium, respectively. The levels of MMP-2 mRNA were assessed by real time-PCR. Western-blot analysis was carried out to measure the expression of alpha-smooth muscle actin (alpha-SMA), a maker protein of myofibroblast in cells, and the levels of extracellular matrix (ECM) component Fibronectin in supernantant medium.The activity and protein level of MMP-2 were no significant difference between the groups when cells were stimulated for 24 hours. While cells were stimulated for 48 hours, 100 ng/ml CTGF and 5 ng/ml TGF- beta(1) induce a increase in MMP-2 activity and protein levels compared with vechile, respectively (P < 0.05); different dose of CTGF plus TGF-beta(1) had the tendency to suppress MMP-2 activity and protein level, and a significant decrease was seen in 50 ng/ml CTGF plus 5 ng/ml TGF-beta(1) group, 100 ng/ml CTGF plus 5 ng/ml TGF-beta(1) group compared with CTGF group and TGF-beta(1) group, respectively (P < 0.05). When cells were stimulated for 12 hours, the levels of MMP-2 mRNA were increased significantly in 100 ng/ml CTGF group and 5 ng/ml TGF-beta(1) group compared with vechile respectively beta(1.72), 1.68 vs 1.29, (P < 0.01), decreased significantly in CTGF plus TGF-beta(1) group compared with CTGF group and TGF-beta(1) group, respectively (0.67 vs 1.72, 1.68, P < 0.01). 100 ng/ml CTGF had no prominent effect on the expression of alpha-SMA in cells and FN in supernatant medium (P > 0.05), whereas 5 ng/ml TGF-beta(1) significantly stimulated both the expression of alpha-SMA and FN (P < 0.05), and CTGF plus TGF-beta(1) induced more alpha-SMA and FN compared with TGF-beta(1) (P < 0.05).CTGF synergistically with TGF-beta(1) to induce the formation of myofibroblasts and down-regulate the production of MMP-2 in renal fibroblast.国家自然科学基金; 面向21世纪教育振兴行动计划(985计划); 高等学校博士学科点专项科研项目PubMed中文核心期刊要目总览(PKU)中国科学引文数据库(CSCD)07569-5738