摘要目的 探讨转化生长因子β1(TGF-β1)对羊膜WISH细胞中基质金属蛋白酶9(MMP-9)及其组织抑制物1(TIMP-1)和核因子κB(NF-κB)表达的影响及其调控机制.方法 在羊膜细胞系WISH细胞中分别加入不同浓度(0、2、10、20 ng/ml)的TGF-β1,培养24 h后,分别应用逆转录(RT)PCR技术检测WISH细胞中TIMP-1及MMP-9 mRNA的表达水平,蛋白印迹法检测TIMP-1、MMP-9及NF-κB蛋白的表达.结果 (1)TIMP-1 mRNA及蛋白表达:经不同浓度TGF-β1(0、2、10、20 ng/ml)处理后,WISH细胞中TIMP-1 mRNA的表达水平随TGF-β1浓度的增加而升高,分别为0.413±0.036、0.623±0.058、1.392±0.124及1.387±0.102,其中2、10及20 ng/ml浓度时TIMP-1 mRNA表达水平分别与0 ng/ml浓度比较,差异均有统计学意义(P ＜0.05)；WISH细胞中TIMP-1蛋白表达水平随TGF-β1浓度的增加而升高,分别为0.357±0.031、0.596±0.048、1.243±0.097及1.359±0.121,2、10及20 ng/ml浓度的TGF-β1处理后,WISH细胞中TIMP-1蛋白表达水平分别与0 ng/ml浓度时比较,差异有统计学意义(P＜0.05).(2) MMP-9 mRNA及蛋白表达:经不同浓度的TGF-β1(0、2、10、20 ng/ml)处理后,WISH细胞中MMP-9 mRNA表达水平随TGF-β1浓度的增加而降低,分别为1.325±0.056、0.987±0.081、0.610±0.034及0.347±0.023,其中2、10及20 ng/ml浓度时MMP-9 mRNA表达水平分别与0 ng/ml浓度时比较,差异有统计学意义(P＜0.05)；WISH细胞中MMP-9蛋白表达水平随TGF-β1浓度的增加而降低,分别为1.119±0.064、1.008±0.052、0.578±0.041及0.401±0.015,2、10及20 ng/ml浓度时MMP-9蛋白的表达水平分别与0 ng/ml浓度时比较,差异有统计学意义(P＜0.05).(3) NF-κB蛋白表达:经2、10、20 ng/ml浓度时TGF-β1处理后,WISH细胞中NF-κB蛋白的表达水平分别为1.116±0.045、0.796±0.041及0.359±0.021,分别与0 ng/ml浓度时(1.423±0.065)比较,差异均有统计学意义(P＜0.05).结论 羊膜WISH细胞中TGF-β1浓度降低,可引起羊膜细胞中TIMP-1 mRNA及蛋白表达水平下降,MMP-9 mRNA及蛋白表达水平升高,导致两者在羊膜组织中的比例失衡,此与胎膜破裂的发生机制有关；TGF-β1通过抑制羊膜细胞中NF-κB通路而调控MMP-9的表达,从而对胎膜破裂有一定的保护作用.

abstractsObjective To investigate the effect of transforming growth factor β1 (TGF-β1) on the expression of matrix metalloproteinase 9 (MMP-9),tissue inhibitor of metalloproteinase 1 (TIMP-1),nuclear factor kappa B(NF-κB) and the possible signalling pathways in human amniotic cells WISH.Methods The WISH cell line was cultured.WISH cells were added with TGF-β1 of different concentrations (0,2,10 and 20 ng/ml,respectively) for 24 hours.Then,reverse transcription (RT) PCR and western blotting were used to analyze the protein and mRNA expression of TIMP-1 and MMP-9; and the expression of NF-κB was analyzed by western blot.Results (1) The profile of TIMP-1 mRNA (0.413 ±0.036,0.623 ±0.058,1.392 ±0.124,1.387 ±0.102) in WISH cells elevated when the concentration of TGF-β1 increased (0,2,10,20 ng/ml).In accordance with TIMP-1 mRNA,the expression of TIMP-1 also elevated with the increase of TGF-β1 (0.357 ± 0.031,0.596 ± 0.048,1.243 ± 0.097 and 1.359 ± 0.121,respectively).And when 2,10 or 20 ng/ml of TGF-β1 was added,the TIMP-1 mRNA and protein were significantly higher than the TIMP-1 mRNA and protein when no TGF-β1 was added(P ＜ 0.05).(2)In contrast with TIMP-1,MMP-9 mRNA (1.325 ±0.056,0.987 ±0.081,0.610 ±0.034,0.347 ±0.023) in WISH cells decreased when the concentration of TGF-β1 increased (0,2,10,20 ng/ml).The MMP-9 protein (1.119 ±0.064,1.008 ±0.052,0.578 ±0.041,0.401 ±0.015) also decreased with the increase of TGF-β1.And when 2,10 or 20 ng/ml of TGF-β1 was added,the MMP-9 mRNA and protein were significantly lower than the MMP-9 mRNA and protein when no TGF-β1 was added (P ＜ 0.05).(3) The NF-κB protein (1.423 ±0.065,1.116 ± 0.045,0.796 ± 0.041,0.359 ± 0.021) was significandy reduced with the increase of TGF-β1 (0,2,10,20 ng/ml; P ＜ 0.05).Conclusions The mRNA and protein expression of TIMP-1 decreased when TGF-β1 was low in WISH cells,whereas those of MMP-9 elevated when TGF-β1 was low.The unbalance of TIMP-1 and MMP-9 was related to the pathology of the premature rupture of membrane.And the NF-κB singalling pathway might be an important mechanism in the regulation of TIMP-1 and MMP-9 system.