诱导型一氧化氮合酶参与白细胞介素-1β对人心脏成纤维细胞基质金属蛋白酶-2作用的实验研究  

Interleukin-1β upregulates matrix metalloproteinase-2 expression and activity in cardiac fibroblasts via nitric oxide synthase pathway

作　　者：郭晓纲[1] 陈君柱[1] 朱建华[1] 张芙荣[1] 邱原刚[1] 赵莉莉[1] 尚云鹏[1] 

机构地区：[1]浙江大学医学院附属第一医院心内科,杭州310003

出　　处：《中华心血管病杂志》2009年第1期63-68,共6页Chinese Journal of Cardiology

基　　金：基金项目：浙江省科学技术厅钱江人才计划资助项目（2006R10024）;浙江省医药卫生科学研究基金资助项目（20078052）

摘　　要：目的观察白细胞介素-1β（IL-1β）对人心脏成纤维细胞基质金属蛋白酶-2（MMP-2）的作用及相关机制。方法将3—6代人心脏成纤维细胞接种于6孔培养板内接受各种干预措施。待实验细胞接受刺激12h后，用RT-PCR法观察MMP-2的mRNA表达量。细胞接受各种刺激24h后，收集细胞总蛋白和培养上清，通过凝胶酶谱法进行上清MMP-2的活性检测，采用Western blot法测定细胞诱导型一氧化氮合酶（iNOS）蛋白的表达，采用Griess法衡量细胞上清中一氧化氮（NO）水平。结果IL-1β作用人心脏成纤维细胞24h后，促进了细胞MMP-2的活性增加，其中4ng／ml的IL-1β刺激作用达到高峰，与对照组相比活性增加了（170．24±13．12）％（P〈0．01），并且该浓度IL-1β能时间依赖性地促进心脏成纤维细胞MMP-2的活性增加。IL-1β作用心脏成纤维细胞12h后，与正常对照组相比4ng／ml和10ng/ml IL-1β组MMP-2mRNA表达明显增加（P〈0．01）。IL-1β（4ng／ml）可以明显促进细胞NO水平升高（P〈0．01），而NOS抑制剂N^G-甲基-L-精氨酸（10^-3mol／L）显著抑制了IL-1β诱导的MMP-2mRNA表达（P〈0．01）、MMP-2活性（P〈0．01）以及NO水平的升高（P〈0．01）。通过Western blot发现在生理水平的心脏成纤维细胞上未能检测到iNOS蛋白的表达，而不同浓度的IL-1β明显促进了细胞iNOS的蛋白水平的升高。结论IL-1β能促进人心脏成纤维细胞MMP-2的mRNA表达和活性，并提示其作用与细胞iNOS-NO水平有关。Objective To investigate the effect of interleukin-1β （IL-1β） on expression and activity of matrix metalloproteinase-2 （MMP-2） of cultured human cardiac fibroblasts and related signaling pathway. Methods Primary human cardiac fibroblasts seeded in 6-well tissue culture plates and cultured to 80% to 90% confluence were harvested at passage 3 to 6 and exposed to IL-1β at various concentrations for 24 h, culture supernatant and cell protein were obtained. MMP-2 mRNA was determined by RT-PCR. The activity of MMP-2 was analyzed by zymography and the expression of inducible nitric oxide synthese （iNOS） protein level was detected by Western blot analysis. Assessment of NO production in the culture supernatant was performed using the Griess method. Results IL-1β （4 ng,/ml） significantly increased MMP-2 activity of cultured fibroblasts in a time-dependent manner. MMP-2 mRNA expression was significantly upregulated by IL-1β （4 ng,/ml and 10 ng/ml, all P 〈 0.01）. Moreover, IL-1β also significantly increased NO production in supernatant （P 〈 0. 01 ） and these effects could be significantly blocked by cotreatment with L-NMMA （10^-3mol/L, all P 〈0. 01 ）. Western blot analysis showed that iNOS could not be detected in unstimulated human cardiac fibroblasts but could be detected in cardiac fibroblasts exposed to IL-1β.Conclusion IL-1β increased MMP-2 activity and transcription of human cardiac fibroblasts via iNOS-NO pathway.

关 键 词：心脏 成纤维细胞 白细胞介素-1Β 一氧化氮合酶 基质金属蛋白酶类 

分 类 号：R686[医药卫生—骨科学]