论文部分内容阅读
目的:利用体外培养人肾小球系膜细胞观察凝血酶对系膜细胞表达MMP-9 mRNA的诱导作用及其可能的细胞内信号传导机制。方法:体外分离并培养人肾小球系膜细胞,以不同浓度凝血酶(500,1500和4500u/L)刺激后采用Northern杂交及凝胶阻滞试验(EMSA)分别观察MMP-9 mRNA表达水平及转录因子AP-1活性变化;同时观察了凝血酶特异性抑活物(水蛭素)、AP-1药理阻断剂(curcumin)和c-fos反义寡核苷酸对凝血酶上述作用的影响。结果:Northern分析表明凝血酶可以剂量依赖性地提高人系膜细胞表达MMP-9 mRNA,与对照组相比,分别提高1.1,3.3和4.8倍;EMSA实验结果发现凝血酶在促进MMP-9 mRNA表达的同时,系膜细胞AP-1的结合能力也相应地提高,与对照组相比分别增加2.5,4.9和6.1倍,与MMP-9 mRNA呈平行性变化;凝血酶抑活物水蛭素可明显地抑制凝血酶上调MMP-9mRNA及AP-1的DNA结合活性;同时AP-1药理阻断剂curcumin和c-fos反义寡核苷酸在有效抑制AP-1结合活性的同时,也明显地抑制MMP-9 mRNA表达水平。结论:凝血酶在体外培养的人肾小球系膜细胞具有上调MMP-9 mRNA表达的作用,转录因子AP-1介导了凝血酶的这一作用。
OBJECTIVE: To investigate the effect of thrombin on the expression of MMP-9 mRNA in mesangial cells and its possible mechanism of intracellular signal transduction by culturing human mesangial cells in vitro. Methods: Human glomerular mesangial cells were isolated and cultured in vitro. The expression of MMP-9 mRNA was detected by Northern blot and EMSA after stimulation with different concentrations of thrombin (500, 1500 and 4500u / L) And the activity of transcription factor AP-1. The effects of thrombin-specific inhibitor (hirudin), AP-1 pharmacological inhibitor (curcumin) and c-fos antisense oligonucleotide on thrombin influences. Results: Northern analysis showed that thrombin can increase the expression of MMP-9 mRNA in human mesangial cells in a dose-dependent manner, which were increased by 1.1, 3.3 and 4.8 times compared with the control group respectively. The results of EMSA showed that thrombin could promote the expression of MMP-9 mRNA Meanwhile, the binding capacity of AP-1 in mesangial cells also increased correspondingly, which were increased by 2.5, 4.9 and 6.1 times compared with the control group, respectively, which showed a parallel change with MMP-9 mRNA. Thrombin inhibitor hirudin Obviously inhibited the up-regulation of DNA-binding activity of MMP-9 mRNA and AP-1 by thrombin; meanwhile, AP-1 pharmacological inhibitors curcumin and c-fos antisense oligonucleotide effectively inhibited AP-1 binding activity as well as obvious Inhibit the expression of MMP-9 mRNA. Conclusion: Thrombin can induce MMP-9 mRNA expression in human glomerular mesangial cells cultured in vitro. The transcription factor AP-1 mediates the effect of thrombin.