结缔组织生长因子协同转化生长因子β1的促肾纤维化效应

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 2mRNA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 2 4h时 ,各组之间无明显差异 ;刺激 4 8h ,10 0ng/mlCTGF组和 5ng/mlTGF β1组明显属于对照组 (P <0 0 5 ) ;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 10 0ng/mlCTGF加 5ng/mlTGF β1、5 0ng/mlCTGF加 5ng/mlTGF β1均分别明显低于CTGF刺激组、TGF β1刺激组 (P <0 0 5 )。上述各组细胞刺激 12h ,MMP 2mRNA水平在 10 0ng/mlCTGF组和 5ng/mlTGF β1组均明显高于对照组 (1 72 ,1 6 8vs 1 2 9,P <0 0 1) ,而 10 0ng/mlCTGF加 5ng/mlTGF β1联合组明显低于CTGF刺激组、TGF β1刺激组 (0 6 7v

Connective tissue growth factor synergistically with transforming growth factor beta 1 to promote renal fibrosis

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: CTGF synergistically with TGF-beta(1) to induce the formation of myofibroblasts and down-regulate the production of MMP-2 in renal fibroblast.