【作者】 郭晓纲；

【导师】 陈君柱；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 研究背景炎症因子对心肌细胞外基质（extracellular matrix,ECM）的重构有重要的病理生理学作用。在调节ECM降解和合成中,基质金属蛋白酶（matrixmetalloproteinases,MMPs）是主要的酶类。心肌组织中MMP-2是最为广泛分布的MMP,MMP-2组织抑制剂（type 2 tissue inhibitor of matrixmetalloproteinases,TIMP-2）是MMP-2的内源性抑制剂,与MMP-2一起参与调节ECM的动态平衡。现有资料表明小鼠MMP-2、MMP-9基因敲除后,明显延缓了左室重构,心肌失代偿和心力衰竭的进展。白介素-1β（interleukin—1β,IL-1β）是心力衰竭中主要的炎症因子,它可影响心脏成纤维细胞血管紧张素受体1A的表达;诱导MMP-9、MMP-2的表达和活性增高,参与ECM的重构。一氧化氮（nitric oxide,NO）是体内重要的细胞内信号分子,它的合成受一氧化氮合酶（nitric oxide synthase,NOS）的调控。其中诱导型NOS（inducible NOS,iNOS）在正常情况下不表达,内毒素或炎症因子刺激时可被迅速诱导,促进NO的大量生成,从而诱发各种病理生理过程,其中包括促进心脏成纤维细胞MMP-2合成和活性增加。研究表明心脏成纤维细胞iNOS-NO系统也是IL-1β作用的靶点,但目前对于IL-1β是否能通过iNOS-NO系统影响心脏成纤维细胞MMPs以及TIMPs的表达还无相关报道。血管紧张素转换酶抑制剂（angiotensin-converting enzyme inhibitor,ACEI）是治疗慢性心力衰竭的基石药物,它具有明显的抗心肌重构作用,但详细机制仍有待进一步揭示。在盐敏感的Dah1大鼠上发现ACEI能抑制心肌iNOS的表达,延缓心肌重构进程和心力衰竭的发生,同样在自发性大鼠模型上ACEI减轻了MMPs的基因表达和活性增高,并缩小了左室内径。上述ACEI的抗心肌重构作用显然有别于传统机制,即通过阻滞组织和循环血管紧张素Ⅱ生成,抑制缓激肽降解发挥抗心肌重构作用,但目前对此机理研究鲜有报道。针对上述问题本研究拟探索炎症因子IL-1β是否能通过iNOS-NO系统影响心脏成纤维细胞MMPs/TIMPs的平衡;ACEI是否对心脏成纤维细胞MMPs/TIMP-2的失衡有纠正作用。目的（1）观察IL-1β对人心脏成纤维细胞MMP-2、MMP-9、TIMP-2的作用及相关机理;（2）观察ACEI制剂咪达普利拉（imidaprilat）对心脏成纤维细胞MMP-2、MMP-9和TIMP-2的影响及可能机理;（3）观察咪达普利拉对IL-1β诱导地心脏成纤维细胞MMP-2、MMP-9和TIMP-2的影响及可能机理。方法细胞培养将人心脏成纤维细胞以5×10⁴/孔的密度接种于6孔培养板内,待实验选用的3-6代细胞群80-90%融合后用无血清完全培养基（CS-C medium,CSM）继续培养,24h后用PBS洗涤细胞2次并接受各种干预措施。RT-PCR待实验细胞接受干预12h后,提取细胞总RNA进行逆转录。根据基因库内GAPDH、MMP-2、TIMP-2、MMP-9基因全序列设计相应引物,进行PCR扩增。以特异性MMP-2、MMP-9和TIMP-2扩增条带灰度与对应GAPDH扩增条带灰度比值对mRNA表达量进行半定量分析。酶谱分析细胞约80-90%融合并用CSM孵育24h后,接受含IL-1β（1ng/ml～10ng/ml）和/或咪达普利拉(10^-9M～10^-6M)的CSM继续孵育24h。完成各种干预后,收集培养上清。通过凝胶酶谱进行MMP-2、MMP-9的活性检测,上样量通过对应细胞蛋白含量进行标化。MMPs显示为位于蓝色背景上的透亮带,采用条带密度分析仪进行图像分析并测定吸光度值。NO测定采用Griess法测定样品中的亚硝基（nitrite,NO₂）含量来衡量细胞上清中NO水平。取培养上清100μl与等量的Griess试剂（Griess试剂A和Griess试剂B各一份）,室温反应10分钟后,用酶联仪测A540nm波长下吸光度值。用NANO₂作标准曲线。Western blot细胞接受各种刺激24h后,收集细胞总蛋白,采用Western blot法测定细胞iNOS蛋白的表达,用条带密度分析仪进行图像分析并测定吸光度值。数据分析每组实验重复4次。数据以均数±标准差表示。利用SPSS 11.5软件处理数据,多组间均数的比较采用方差分析,有显著差异者用LSD检验。两组间比较采用t检验。P＜0.05为差异有统计学意义。结果1.通过凝胶酶谱分析表明人心脏成纤维细胞能分泌MMP-2,但我们的实验系统难以发现心脏成纤维细胞MMP-9的分泌。通过RT-PCR检测到MMP-2、TIMP-2的mRNA表达,但试用各种循环参数也难以发现MMP-9的mRNA表达,而同样的引物能在阳性对照（人巨噬细胞）上发现MMP-9的mRNA表达。2.人心脏成纤维细胞接受IL-1β刺激24h后,酶谱分析结果表明IL-1β促进了细胞MMP-2的活性增加,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）,而10ng/ml IL-1β刺激组与4ng/mlIL-1β刺激组相比,两者间MMP-2活性无明显差异（P＞0.05）。3.IL-1β干预心脏成纤维细胞12h后,通过RT-PCR结果表明:与正常对照组相比,4ng/ml IL-1β组MMP-2 mRNA表达明显增加（P＜0.01）;10ng/ml IL-1β组MMP-2 mRNA表达与4ng/ml IL-1β组MMP-2 mRNA表达相比无明显差异（P＞0.05）。4.心脏成纤维细胞接受IL-1β刺激24h后,通过酶谱分析结果表明IL-1β时间依赖性地促进心脏成纤维细胞MMP-2的活性增加,刺激24h作用达到高峰,与对照组相比有显著统计学差异（P＜0.01）。5.心脏成纤维细胞接受IL-1β刺激24h后,提取其上清,通过Griess法检测表明IL-1β（4ng/ml）可以明显促进细胞NO水平升高（P＜0.01）,而iNOS抑制剂N^G-甲基—L-精氨酸(N^G-methyl L-Arginine,L-NMMA,10^-3M)显著抑制了IL-1β诱导的MMP-2 mRNA表达（P＜0.01）、MMP-2活性（P＜0.01）以及NO的升高（P＜0.01）。通过Westen 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明显促进细胞iNOS的蛋白合成。6.通过酶谱分析和RT-PCR结果表明咪达普利拉在实验所选浓度范围内对心脏成纤维细胞MMP-2的活性（P＞0.05）和mRNA表达（P＞0.05）无明显影响。7.心脏成纤维细胞接受咪达普利拉(10^-9～10^-6M)和IL-1β（4ng/ml）联合作用后,通过酶谱分析结果表明咪达普利拉可以浓度依赖性地抑制IL-1β诱导地细胞MMP-2的活性增高（P＜0.01）。8.心脏成纤维细胞在咪达普利拉(10^-7M)和IL-1β（4ng/ml）联合作用后,通过Griess法发现咪达普利拉抑制了IL-1β诱导地细胞上清NO水平的上升（P＜0.01）,通过Westen blot发现咪达普利拉抑制了IL-1β诱导地细胞iNOS蛋白的增加（P＜0.01）;而SNP顿挫了咪达普利拉的作用,包括对IL-1β诱导地MMP-2 mRNA表达（P＜0.01）、MMP-2活性（P＜0.01）的抑制和对IL-1β诱导地iNOS蛋白表达的抑制（P＜0.01）。9.心脏成纤维细胞接受IL-1β（4ng/ml）和咪达普利拉(10^-7M)刺激12h后,通过RT-PCR发现它们对细胞TIMP-2 mRNA表达无明显影响（P＞0.05）。10.通过体外鳌合实验发现咪达普利拉(10^-7M,主要实验浓度)未能通过对鳌合作用竞争性抑制MMP-2的活性（P＞0.05）,而EDTA明显抑制MMP-2的活性（P＜0.01）。结论（1）IL-1β能促进心脏成纤维细胞MMP-2的mRNA表达和MMP-2活性增加,并提示其作用与细胞iNOS-NO水平相关。（2）咪达普利拉对未受干预的心脏成纤维细胞MMP-2 mRNA表达和活性无明显影响,但它能抑制IL-1β诱导地心脏成纤维细胞MMP-2 mRNA表达和活性的增加,其机理可能咪达普利拉抑制了IL-1β诱导地心脏成纤维细胞iNOS蛋白的合成以及NO水平升高,进而影响MMP-2表达与活性。（3）IL-1β和咪达普利拉对心脏成纤维细胞TIMP-2 mRNA表达无明显作用。更多还原

【Abstract】 BackgroundInflammatory factor plays an important role in myocardial extracellular matrix （ECM）remodeling.The synthase and degradation of ECM are regulated by matrix metalloproteinases（MMPs）.Among MMPs,MMP-2 has the widest distribution in the myocardium and type 2 tissue inhibitor of matrix metalloproteinase（TIMP-2）is an endogenous inhibitor of MMP-2.Inhibition or knockout of MMP-2 and MMP-9 in mouse suppressed ventricular remodeling,myocardial dysfunction and progression of heart failureInterleukin-1β（IL-1β）is a key pro-inflammatory cytokine during heart failure. It was testified that IL-1βstimulated the expression and transcription of type 1A angiotensin receptor in rat cardiac fibroblasts and induced protein synthase and activity of MMP-9 and MMP-2 of cardiac fibroblasts,leading to ECM remodeling. Nitric oxide（NO）is an important cellular messenger,produced by nitric oxide synthases.Inducible NOS（iNOS）,one of NOS,isn’t usually expressed in normal tissue,however,LPS or pro-inflammatory cytokines,such as IL-1β,can induce expression of iNOS that generates large quantities of NO,resulting in cardiac pathophysiological change including stimulating the expression and activity of MMP-2.It has been observed that IL-1βcould affect the iNOS-NOS system in cardiac fibroblasts.But up to now,there are no evidences whether IL-1βcan affect MMPs or TIMPs by intracellular iNOS-NOS pathway of cardiac fibroblasts.Angiotensin converting enzyme inhibitor（ACEI）is widely used in the treatment of heart failure because of the attenuation of ventricular remodeling. However,the respond mechanisms remain to be elucidated.Recently,Kobayasi et al found that ACEI depressed the ventricular remodeling and heart failure by inhibiting expression of iNOS protein in myocardium of salt-sensitive Dahl rat,meanwhile, ACEI suppressed the gene expression and activity of MMPs at the predilatation stage and prevented the transition to congestive heart failure in spontaneously hypertensive rat.These effects of ACEI against remodeling are difference from traditional mechanisms:blocking the release of tissue and circulatory angiotensinⅡand decreasing the breakdown of bradykinin.But it is little report about how ACEI regulates ventricular remodeling except traditional mechanisms.Corresponding questions mentioned,we are ready to explore whether IL-1βhas effects on MMPs /TIMPs balance by iNOS-NOS of cardiac fibroblasts;whether ACEI can regulate the unbalance of MMPs/TIMPs by iNOS-NOS system.Objective（1）Observe effects of IL-1βon the transcription and activity of MMP-2,MMP-9 and the transcription of TIMP-2 in cultured human cardiac fibroblasts;（2）Investigate effects of imidaprilat,an ACEI,on MMP-2,MMP-9 and TIMP-2 in cultured human cardiac fibroblasts;（3）Observe effects of imidaprilat,an ACEI,on MMP-2,MMP-9 and TIMP-2 in cultured human cardiac fibroblasts induced by IL-1βand the possible mechanism involved.MethodsCulture of primary human cardiac fibroblasts Primary human cardiac fibroblasts were purchased at passage 2 from Cell Systems.It was plated in 6-well tissue culture plates（5×10⁴ cells/well）.All cells were harvested at passage 3 to 6 and used at 80% to 90%confluence for experiments.The culture medium was replaced with CS-C medium（CSM）without serum and growth factor for 24h.Cells were washed two times with PBS and then cells were cultured in CSM in the absence or the presence of various treatments.RT-PCR Subconfluent human cardiac fibroblasts were cultured in CSM in the absence or the presence of various treatments 12h later.Total RNA was isolated from cultured cells and one microgram of total RNA was reversely transcripted with Super ScriptⅡ^TM-reverse transcriptase.PCR amplification of cDNA was done using the primers of MMP-9,MMP-2,TIMP-2 and GAPDH respectively that were designed according to the sequence in GENEBANK.The resulting densities of the MMP-2, TIMP-2 and MMP-9 bands were expressed relative to the corresponding density of the GAPDH band from the same RNA sample.Zymography Subconfluent cells were serum deprived and treated with IL-1β（1ng/ml～10ng/ml）and/or imidaprilat(10^-9M～10^-6M)for 24h.Medium samples were harvested and the activity of MMPs was analyzed by zymography.Clear zones against the blue background indicated the presence of gelatinase.To quantify the amount of gelatinase production,the stained zymograms were scanned on a densitograph.NO assessment Assessment of NO production in the culture medium was performed using the Griess reagent that measures the level of nitrite（NO₂）,100μl Griess reagent（a mixture of one part of Griess reagent A and one part of Griess reagent B）was added to 100μl of sample or standard（sodium nitrite served as the standard）in each well of a 96-well plate.After incubation at room temperature for 10 min,the optical density at 540 nm（OD540）was measured and the amount of nitrite was calculated from a standard curve.Western blot Cardiac fibroblasts were treated for 24h,the protein of cells were collected.The expression of iNOS protein level was detected by Western blot analysis.Statistical analysis Each experiment was repeated four times.Data are presented as means±standard deviation（SD）.Statistical analysis was performed with the Statistical Package for the Social Sciences（SPSS version 11.5）.Differences among all data were analyzed for significance by one-way analysis of variance（ANOVA） followed by unpaired Student t test.A probability level of P＜0.05 were considered significant.Results（1）MMP-2 can be detected in human cardiac fibroblasts by zymography and RT-PCR.TIMP-2 can be also detected by RT-PCR;however,it was difficulty to detect MMP-9 in human cardiac fibroblasts even if we tried to maximal sample volumn during zymography and adjusted many protocols of PCR.（2）After cardiac fibroblasts were incubated with IL-βfor 24h,MMP activities in supernatant were measured using zymogrpahy.We observed that IL-1βincreased MMP-2 activity.4ng/ml IL-1βincreased more evidently MMP activity（foldchange versus control,170.24±13.12%,P＜0.01）.There was no significant difference between MMP-2 activity in 4ng/ml IL-1βgroup and 10ng/ml IL-1βgroup（P＞0.05）.（3）RT-PCR analysis demonstrated that expression of MMP-2 mRNA was increased after 12h incubation of IL-1β.The significant change in MMP-2 mRNA compared with control group was detectable at 4ng/ml concentration of IL-1β.There was no significant difference between MMP-2 mRNA expressions in 4ng/ml IL-1βgroup and 10ng/ml IL-1βgroup（P＞0.05）（4）After cardiac fibroblasts were incubated with 4ng/ml IL-βfor 0.5h～24h,MMP-2 activity in conditioned media was measured by zymogrpahy.We observed that IL-1βstimulated MMP-2 activity through a time-dependent method and the effect of IL-1βon MMP-2 activity reached maximum after 24h incubation （P＜0.01）.（5）After cardiac fibroblasts were incubated with 4ng/ml IL-βfor 24h,the level of NO in conditioned media was measured by Griess method.The result showed that IL-1βincreased NO accumulation in supematant（P＜0.01）;however, inclusion of N^G-methyl L-Arginine(L-NMMA,10^-3M),a competitive inhibitor of NO synthase（NOS）,significantly prevented the effect of IL-1βon cardiac fibroblasts including the increasing of NO（P＜0.01）and the increasing of MMP-2 activity（P＜0.01）and transcription（P＜0.01）.Western blot analysis showed that iNOS did not detected in human cardiac fibroblasts;however,IL-1βstimulated significantly the expression of iNOS protein in cardiac fibroblasts.（6）Using RT-PCR and zymography,we found that imidaprilat alone with experimental concentration had no effect on the activity（P＞0.05）and transcription(（P＞0.05）of MMP-2 in cardiac fibroblasts.（7）After human cardiac fibroblasts were incubated with 4ng/ml IL-βand imidaprilat with different concentrations for 24h,It was demonstrated that imidaprilat(10^-9～10^-6M)significantly inhibited the increasing of MMP-2 activity induced by IL-1β（4ng/ml）in a dose-dependent manner.（8）After human cardiac fibroblasts were incubated with 4ng/ml IL-βand imidaprilat (10^-7M)for 24h,Griess method showed that imidaprilat inhibited the accumulation of supematant NO induced by IL-1β（P＜0.01）and Western blot testified imidaprilat inhibited expression of iNOS protein in cardiac fibroblasts induced by IL-1β（P＜0.01）.Sodium nitroprusside(SNP,10^-5M),an exogenous NO donor,blunted the inhibition effect of imidaprilat on IL-1β-induced MMP-2 transcription（P＜0.01）and activity（P＜0.01）.（9）RT-PCR analysis of TIMP-2 showed that after 12h incubation of IL-1β（4ng/ml） or imidaprilat(10^-7M),neither of them could significantly change expression of TIMP-2 mRNA in human cardiac fibroblasts（P＞0.05）.（10）We demonstrated that imidaprilat(10^-7M)didn’t affect MMP-2 activity in vitro system due to forming chelating complexes（P＞0.05）,however EDTA chelated metal ion and resulting in inactivity of MMP-2（P＜0.01）.Conclusions1.IL-1βstimulated MMP-2 activity and transcription of human cardiac fibroblasts and iNOS-NO system was possibly involved with effects of IL-1β.2.Imidaprilat alone had no significant effects on MMP-2 transcription and activity of human cardiac fibroblasts without medications,but it significantly inhibited the increasing of MMP-2 mRNA expression and activity induced by IL-1β.Inhibition to iNOS-NOS system induced by IL-1βwas possibly related with effects of imidaprilat on MMP-2 induced by IL-1β.3.There were no significant effects of IL-1βor imidaprilat on TIMP-2 mRNA expression in human cardiac fibroblasts.更多还原