结缔组织生长因子与转化生长因子β1协同促进肾纤维化
[Connective tissue growth factor synergistically with transforming growth factor beta 1 to promote renal fibrosis].
作者信息
Yang Min, Huang Hai-chang, Li Jing-zi, Wang Hai-yan
机构信息
Institute of Nephrology & Renal Division, Peking University First Hospital, Beijing 100034, China.
出版信息
Zhonghua Yi Xue Za Zhi. 2004 Apr 2;84(7):569-73.
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.
目的
探讨结缔组织生长因子（CTGF）和转化生长因子-β1（TGF-β1）对肾成纤维细胞基质金属蛋白酶-2（MMP-2）合成与分泌及肌成纤维细胞活化的影响。
方法
将等量的肾成纤维细胞（NRK-49F）接种并分为空白对照组、单独CTGF处理组、单独TGF-β1处理组以及CTGF加TGF-β1处理组。分别采用明胶酶谱法和蛋白质免疫印迹法检测培养上清中MMP-2的活性和蛋白水平。通过实时荧光定量聚合酶链反应（real time-PCR）评估MMP-2 mRNA水平。采用蛋白质免疫印迹法检测细胞中肌成纤维细胞标志物α-平滑肌肌动蛋白（α-SMA）的表达以及培养上清中细胞外基质（ECM）成分纤连蛋白的水平。
结果
细胞刺激24小时时，各组间MMP-2的活性和蛋白水平无显著差异。细胞刺激48小时时，100 ng/ml CTGF和5 ng/ml TGF-β1分别诱导MMP-2活性和蛋白水平较空白对照组升高（P<0.05）；不同剂量的CTGF加TGF-β1有抑制MMP-2活性和蛋白水平的趋势，50 ng/ml CTGF加5 ng/ml TGF-β1组、100 ng/ml CTGF加5 ng/ml TGF-β1组与CTGF组和TGF-β1组相比，MMP-2活性和蛋白水平显著降低（P<0.05）。细胞刺激12小时时，100 ng/ml CTGF组和5 ng/ml TGF-β1组MMP-2 mRNA水平较空白对照组显著升高（分别为1.72、1.68 vs 1.29，P<0.01），CTGF加TGF-β1组与CTGF组和TGF-β1组相比显著降低（0.67 vs 1.72、1.68，P<0.01）。100 ng/ml CTGF对细胞中α-SMA的表达及培养上清中FN无显著影响（P>0.05），而5 ng/ml TGF-β1显著刺激α-SMA和FN的表达（P<......
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