基质金属蛋白酶及其抑制剂对心肌重构的影响
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摘要
背景及目的
基质金属蛋白酶(Matrix metalloproteinase, MMPs)是由许多结构和功能相似的中性蛋白酶组成的多基因内源性锌依赖性酶家族,它由血管平滑肌细胞、单核细胞、内皮细胞等多种细胞产生,可降解除多糖以外所有的细胞外基质(Extracellular matrix, ECM)成分。目前已发现的MMPs家族成员有20多种,根据其结构及功能的不同可分为四大类：第一类为胶原酶(MMP-1,8,13等),其主要水解底物是纤维类胶原,即Ⅰ、Ⅱ、Ⅲ型胶原；第二类为明胶酶(MMP-2,9),主要迅速水解变性胶原蛋白及和明胶蛋白；第三类为基质水解酶类(MMP-3,10,11等),水解Ⅲ,Ⅳ型胶原和蛋白多糖,明胶和糖蛋白；第四类为结构与功能比较特殊的MMPs (MMP-7,12,19,20,23,26等),其除能降解几种ECM成分外,还能激活其他MMPs。基质金属蛋白酶抑制剂(Tissue inhibitors of metalloproteinase, TIMPs)是MMPs的内源性特异性抑制物,分泌MMPs的细胞同时也分TIMPs, TIMPs家族里共有四个成员,分别为TIMP-1、TIMP-2、TIMP-3和TIMP-4。TIMPs主要具有两方面功能：它们对不同的MMPs具有不同的抑制强度,通常TIMPs以1：1的分子比例与激活的MMPs形成高亲和力复合物,并通过封闭MMPs的催化区,从而抑制ECM的降解。此外,TIMPs还有促进有丝分裂、抑制血管生成和诱导细胞死亡的作用。
心肌组织中三分之二的细胞是非心肌细胞,即细胞外基质,其对心肌细胞不仅具有结构上的支持和保护作用,而且,在心肌细胞间信息传递、舒缩功能的协调中都发挥重要的作用。主要功能有：①为心肌细胞、血管和淋巴管的相互联系和排列提供支持,从而决定心肌的僵硬度和结构；②防止心肌细胞过度伸长；③将心肌细胞产生的压力传至心室腔；④防止心肌细胞过度回缩；⑤提供心肌舒张时的阻力；⑥给心肌提供张力强度以防止其断裂；⑦防止心肌细胞和肌纤维滑脱。细胞外基质中心肌胶原蛋白占主要位置,成人心肌组织中的胶原至少包括Ⅰ、Ⅲ、Ⅳ,Ⅴ和Ⅵ型。其中最重要的是Ⅰ型(ColⅠ)和Ⅲ型胶原,是构成间质的主要成分。各种胶原成分共同构成胶原纤维网络,将心肌细胞连成一体,能够直接影响心脏的结构和功能。在心功能衰竭的动物模型以及此类病人体内,均可见到心脏间质胶原类型含量及其性质的改变,且此改变往往对其预后具有决定性意义,这证明心脏间质的重要性。心肌胶原也处于不断变化与代谢中,其每天的更新率约0.6%。MMPs/TIMPs平衡对于维持心肌间质胶原合成与降解代谢的动态平衡具有重要的作用。在某些疾病状态,如心肌梗死后、高血压等,心肌胶原的数量及分布发生了改变(即心肌重构),引起心肌间质纤维化,限制心肌活动,降低心室的顺应性,影响心肌的收缩及舒张功能,是引起心力衰竭的重要原因。另外,胶原沉积可限制除极波通过心肌的速度,对心肌组织的电稳定性产生负面影响,从而可导致心律失常。胶原在成年心脏是持续合成及降解的,并保持动态的平衡,随年龄的增长胶原合成和降解的平衡发生偏移,胶原逐渐累积。许多酶类可以降解ECM成分中的胶原蛋白,因此,了解修饰心肌ECM结构的酶系统对于了解心肌重构具有重要的意义,而MMPS对于ECM的重构有着重要的作用。
目前针对MMPs在心房颤动及心肌梗死的发病、发展中所起的作用及治疗研究尚不多,研究主要集中在以下几个方面：①合成MMPs的特殊抑制剂,但主要集中于防治肿瘤的侵袭和转移；②利用重组蛋白或病毒载体基因转运技术给予内源性抗蛋白酶剂,或提高体内TIMPs的分泌；③根据MMPs的表达及酶活性调控机制,开发具有调控MMPs/TIMPs平衡的药物。在本研究中,我们收集了50例风湿性心脏病患者以及40例冠心病患者的临床资料及心肌标本,进行免疫组化染色检测及实时荧光定量PCR反应,研究MMPs及TIMPs与心肌重构的相关性,进一步深入分析心肌重构发病机制以及与临床特征的关系。
材料与方法
1.病人及标本收集：选取2007年9月至2009年5月期间在我院接受人工心脏二尖瓣置换术的风湿性心脏病患者共50例,男28例,女22例,年龄26-66岁,分为窦性心律组(SR)20例,心房纤颤组(AF)30例。有下列情况之一者不纳入本研究：①年龄小于20岁或大于66岁者；②合并有感染性心内膜炎者；③合并有高血压、糖尿病、冠心病、甲状腺功能亢进、心肌病、慢性肺源性心脏病或严重肝肾功能不全者；④非风湿性心脏病而接受心脏瓣膜置换术者；⑤风湿性心脏病非二尖瓣瓣膜置换术者；⑥临床有风湿活动征象或发热者。并同期选取接受冠状动脉搭桥手术的冠心病患者共40例,男25例,女15例,年龄40-65岁,分为心肌梗死组(MI)22例,不稳定型心绞痛组(UA)18例。术前行冠状动脉造影检查,造影结果显示在MI组中,9例为左主干病变超过50%(41%),8例为三支冠脉血管病变(36%),5例为双支冠脉血管病变(23%)；UA组中8例为左主干病变超过50%(44%),6例为三支冠脉血管病变(33%),4例为左前降支近段严重狭窄(23%)。所有患者均符合行冠状动脉搭桥术手术指证,并排除合并肝肾功能不全、肺纤维化、骨代谢性疾病、严重糖尿病、全身免疫性疾病、感染、风湿性疾病及恶性肿瘤等。登记各研究对象的相关临床资料,包括年龄、性别、身高、体重、合并的其他疾病、用药情况等,并于术前行心电图、超声心动图、胸片等检查。所有患者心功能均为Ⅱ-Ⅲ级(NYHA分级)。术前所有患者均签署知情同意书。在开胸建立体外循环之前取患者右心耳标本约100mg,除去血液和脂肪组织后立刻放入液氮中保存备用。
2.免疫组化染色检测：采用链霉菌亲生物素蛋白-过氧化物酶连接法(streptavidin peroxidase conjugated method, SP法),滴加适量稀释的小鼠抗人MMP-1、MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4单克隆抗体与特异性抗原结合形成抗原-抗体复合物,之后再使用生物素标记的二抗结合后用DAB显色剂显色,阳性结果为胞浆中出现棕黄色颗粒,表明在心肌中为弥漫阳性表达。在光镜下观察,并在同等光照条件下拍摄数码照片,运用Image-pro plus软件进行图像定量分析,计算阳性单位(PU值)。
3.荧光定量PCR反应：采用Trizol一步法提取组织RNA,测定各RNA样品的OD260、OD280,并计算RNA浓度,将RNA反转录合成cDNA,反转录步骤严格按TaKaRa PrimeScriptTM RT reagent Kit (Perfect Real Time)试剂盒说明进行,进行常规PCR反应,设计并合成引物,分别利用内参(GAPDH)引物和各指标引物进行常规PCR反应,以检验模板cDNA与引物,再进行荧光定量PCR反应,反应参数等均按TaKaRa SYBR(?) Premix Ex TagTM (Perfect Real Time)试剂盒说明进行。数据按照以下公式分析,计算每个样品中各指标相对于GAPDH的相对百分含量,即(指标／GAPDH)*100%
4.统计分析：所有数据应用SPSS13.0统计软件包进行分析,指标以均数±标准差表示,组间差异采用两独立样本t检验,以p<0.05为差异有显著性。
结果
1.风湿性心脏病患者：SR与AF两组的年龄、身高、体重等临床指标比较差异无显著性,两组患者超声心动图检查显示AF组左心房内径与右心房内径显著大于SR组(p<0.01),差异有显著性,左室舒张末期内径、左室射血分数等指标差异无显著性。免疫组织化学染色检测：阳性结果为心房肌细胞胞浆中出现棕黄色颗粒。SR组心房肌细胞胞浆可见分布均匀,稀疏的浅褐色颗粒,AF组心房肌细胞胞浆内可见浓密的不规则分布的棕黄色颗粒。运用Image-proplus软件进行图像定量分析,计算PU值显示：与SR组相比较,AF组除MMP-1外,MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达均明显增高(均p<0.01),差异具有显著性。MMP-1在两组中比较虽差异无显著性,但总体水平较SR组增加。实时荧光定量PCR反应：AF组心肌组织MMP-3、MMP-7、MMP-9和TIMP-1、TIMP-2、TIMP-3、TIMP-4表达较SR组明显增高(均p<0.01),差异具有显著性。MMP-1在两组中比较虽差异无显著性,但总体水平较SR组增加。
2.冠心病患者：MI组与UA组两组的年龄、身高、体重等临床指标比较差异无显著性,两组患者超声心动图检查显示：MI组左右心房内径及左室舒张末期内径显著大于UA组(均p<0.01),差异有显著性,左室射血分数低于UA组,差异有显著性(p<0.05)。免疫组织化学染色检测：阳性结果为心房肌细胞胞浆中出现棕黄色颗粒。UA组心房肌细胞胞浆可见分布均匀,稀疏的浅褐色颗粒,MI组心房肌细胞胞浆内可见浓密的不规则分布的棕黄色颗粒。运用Image-pro plus软件进行图像定量分析,计算PU值显示：与UA组相比较,MI组除MMP-1、MMP-7外,MMP-3、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达均明显增高,差异具有显著性(p<0.01,p<0.01,p<0.05,p<0.05,p<0.01,p<0.01)。MMP-1、MMP-7在两组中比较虽差异无显著性,但总体水平较UA组增加。实时荧光定量PCR反应：MI组心肌组织MMP-3、MMP-9和TIMP-1、TIMP-2、TIMP-3、TIMP-4表达较UA组明显增高,差异具有显著性(均p<0.01),MMP-1、MMP-7在两组中比较差异无显著性,但总体水平较UA组增加。
讨论
心肌细胞外基质是由纤维胶原、基质膜蛋白、蛋白聚糖和具有生物活性的信号分子所组成的,是心肌细胞结构的重要组成成分,且具有细胞间信息传递、协调舒缩功能、维持心肌细胞及心肌纤维的排列、限制心肌细胞过度拉长的作用。心肌细胞外基质的改变还可造成心肌收缩和有效的射血之间失去协调性,并且失去了对心脏几何形状的支持作用,致使泵功能受损。
MMPs是一组与锌结合的分解细胞外基质的蛋白酶家族,至今发现已有20余种,按作用底物分为胶原酶、明胶酶、基质水解酶、膜型酶等4种,参与胚胎正常发育及组织重塑,参与人体许多病理和生理过程。TIMPs是MMPs的内源性特异性抑制剂,分为4型,二者之间的相互作用及动态平衡中维持心脏正常形状和心功能中起着重要的作用。已有研究表明,MMPs及TIMPs的表达及活性异常参与心肌结构的重构。
目前国内外对MMPs及TIMPs在心血管疾病中的表达水平报道不一,且多数研究集中在检测血清中二者的表达水平或利用动物模型来推测MMPs及TIMPs在人类心脏疾病患者心肌内的表达。在我们的研究中使用人类心肌活检的方式,分别选取四类基质金属蛋白酶中的各一种及所有的基质金属蛋白酶抑制剂来进行探讨基质金属蛋白酶及其抑制剂在风湿性心脏病房颤患者及心肌梗死患者心肌的表达情况,具有全面性及概括性。由于风湿性心脏病瓣膜病变的不同及心力衰竭的程度会影响心房纤维化程度和房颤的发生,为此本研究所选病例均为二尖瓣病变患者,且各组中瓣膜病变差异无显著性,各组病例中左室射血分数差异无统计学意义。超声心动图结果显示,风湿性心脏病房颤患者与窦性心律患者相比较,左右心房内径明显扩大,差异具有显著性。说明房颤导致心肌组织发生了结构变化,房腔扩大,心肌僵硬度增加。在对冠心病患者的研究中发现,心肌梗死患者与不稳定型心绞痛患者相比较,左右心房内径及左室舒张内径明显扩大,左室射血分数降低,差异具有显著性,说明心肌梗死患者心肌组织发生了结构变化,心房腔及心室腔均扩大,心肌僵硬度增加,心肌顺应性下降,一心功能受损。本研究使用免疫组织化学染色法,并使用PU值定量计算各MMPs及TIMPs的表达。再使用实时荧光定量PCR方法更准确的测量各MMPs及TMPs的表达,结果显示在风湿性心脏病房颤患者心肌组织中MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达明显增加,虽然MMP-1表达在两组患者中差异无显著性,但总体数值仍然是呈增加趋势,在心肌梗死患者心肌组织中MMP-3、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达明显增加,虽然MMP-1、MMP-7表达在两组患者中差异无显著性,但总体数值仍然是呈增加趋势。提示在风湿性心脏病心房纤颤及冠心病心肌梗死患者的心肌组织中,MMP-1、MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4的表达增高,使得心肌细胞外基质降解,导致心肌细胞拉长,心房壁变薄、心房扩大,正常细胞成分减少,基质成分增加,心肌广泛纤维化,最终促进心房组织重构的发生与维持。说明MMPs表达活性增强可使ECM合成与降解失衡,MMPs、TIMPs两者参与心肌和血管重构,在心血管疾病的发病过程中起重要作用。因此,调节MMPs的活性可能是心血管疾病治疗的一个新方向,对其进一步研究将促进临床的早日应用。
总之,MMPs、TIMPs二者水平的改变直接影响和改变胶原的新陈代谢,决定着心肌不同程度的重构。通过调节MMPs、TIMPs的表达和活性来阻止甚至逆转心肌重构可能成为临床上重要的治疗和预防手段。
Background and purpose:
MMPs is a multi-gene regulation of endogenous zinc-dependent enzyme family which is composition of neutral protease that similar to the structure and function and is secreted by vascular smooth muscle cells, monocytes and endothelial cells, and its function is to degrade all extracellular matrix components(ECM), excluding polysaccharide. MMPs has been found that there are more than 20 kinds, according to its structure and function can be divided into four different categories:①collagenase (MMP-1, MMP-8, MMP-13):the main substrate is hydrolyzed collagen fiber, namely,Ⅰ,Ⅱ,Ⅲcollagen;②gelatinase (MMP-2, MMP-9):digest denatured collagen and gelatin quickly;③stromelysin (MMP-3, MMP-10, MMP-11): hydrolysisⅢ,Ⅳcollagen and proteoglycan, gelatin and sugar protein;⑤The fourth category is the structure and function of more specific MMPs (MMP-7,12,19, 20,23,26, etc.), which can degrade several ECM components, inter alia, but also activate other MMPs. Tissue inhibitors of metalloproteinase (TIMPs) are specific endogenous inhibitor of MMPs, MMPs secreting cells also secrete TIMPs, TIMPs are four kinds:TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMPs have two main functions: the inhibition of MMPs activity, TIMPs and activated MMPs are usually to form a high-affinity 1:1 complex and through the closure of the catalytic domain of MMPs to inhibit the degradation of ECM. In addition, TIMPs also promote mitosis, inhibit angiogenesis and inducing cell death role.
Two-thirds of myocardial cells in non-cardiac cells, namely, extracellular matrix, its myocardial cells not only has the structural support and protection, and in information transmission between myocardial cells, the coordination of both systolic and diastolic function play an important role. Main functions are:①to provide support to the myocardial cells, blood vessels and lymphatic vessels intercomnected and arranged, determine myocardial stiffness and structure;②to prevent excessive elongation of myocardial cells;③the pressure of cardiac cells will be transmitted to ventricular cavity;④to prevent excessive retraction of myocardial cells;⑤provide resistance when myocardial relaxation;⑥to provide tensile strength to the heart in order to prevent its fracture;⑦to prevent cardiac cells and muscle fibers slippage. Myocardial extracellular matrix of collagen accounts for the main location of the adult myocardial tissue includes at least collagenⅠ,Ⅲ,Ⅳ,ⅤandⅥtype. Two of the most important collagen are the typeⅠ(ColⅠ) and typeⅢ. Collagen fiber network composed by various components of collagen facilitate the integration between myocardial cells, and it can directly influence the structure and function of the heart. In animal models of heart failure and heart failure patient may see the change of the content of cardiac interstitial collagen and the nature, and this change often determines the prognosis, and this prove the importance of myocardial interstitial. Myocardial collagen is also in the ever-changing and metabolism, its daily update rate is about 0.6%. The balance between MMPs and TIMPs plays an important role in the metabolism of dynamic balance for the maintenance of myocardial interstitial collagen synthesis and degradation.
In some disease states, such as myocardial infarction, high blood pressure and so on, the number and distribution of myocardial collagen has changed (ie, cardiac remodeling), causing myocardial interstitial fibrosis, limiting cardiac activity and reduce ventricular compliance, impact the systolic and diastolic function of myocardial. It is an important reason for causing heart failure. In addition, collagen deposition may limit the cardiac depolarization waves through the speed of the electrical stability of the myocardium have a negative impact, which can lead to cardiac arrhythmia. Collagen in the adult heart is a continuous synthesis and degradation and to maintain dynamic balance, age-related increase of collagen synthesis and degradation of the balance of offset, a gradual accumulation of collagen. Many enzymes can degrade extracellular matrix components of the collagen protein, therefore, understanding the modification of myocardial extracellular matrix structure of the enzyme system can understand the cardiac remodeling, while the matrix metalloproteinase play an important role on the reconstruction of extracellular matrix.
At present, the research of the MMPs in the pathogenesis and treatment of atrial fibrillation and myocardial infarction was few, and research focused on the following aspects:①a special inhibitor of synthetic MMPs, the main focus on the prevention and treatment of invasive and transfer of tumors;②the use of recombinant protein or viral vector gene transfer technology for the endogenous anti-protease agent, or enhance the secretion of TIMPs in vivo;③According to the expression of MMPs and the enzyme activity of regulatory mechanisms, development the drug to control the balance between MMPs and TIMPs. In this study, we collected clinical data and myocardial specimens of 50 cases of rheumatic heart disease patients and 40 cases of coronary heart disease patients, and immunohistochemical staining and fluorescence quantitative PCR reaction for myocardial specimens, to study the MMPs and TIMPs associated with myocardial remodeling, and analysis of the pathophysiology mechanisms of myocardial remodeling and the clinical features of myocardial remodeling.
Materials and methods:
1. Patients and sample collection:Selection 50 cases of rheumatic heart disease patients for artificial mitral valve replacement surgery from September 2007 to May 2009 in our hospital,28 males and 22 females, aged from 26 to 66 years old, the 50 cases is divided into 20 cases of sinus rhythm group (SR) and 30 cases of atrial fibrillation group (AF). One of the following conditions are not included in this study:①younger than 20 years old or more than 66 years of age;②complicated with infective endocarditis;③complicated with hypertension, diabetes, coronary heart disease, hyperthyroidism, myocardial disease, chronic pulmonary heart disease or severe liver and kidney dysfunction;④non-rheumatic heart disease undergoing heart valve replacement surgery;⑤rheumatic heart disease were non-mitral valve replacement surgery;⑥The clinical signs of rheumatism activity or fever. And over the same period selected 40 cases of coronary heart disease patients who received coronary artery bypass surgery,25 males and 15 females, aged from 40 to 65 years old, those were divided into 22 cases of myocardial infarction group(MI) and 18 cases of unstable angina group(UA). Preoperative coronary angiography, imaging results showed that in the MI group,9 cases of left main coronary vascular disease more than 50% (41%),8 cases of three coronary vascular disease (36%),5 cases of double coronary vascular disease (23%); UA group,8 cases of left main coronary vascular disease more than 50% (44%),6 cases for three coronary vascular disease (33%),4 cases of severe proximal left anterior descending artery stenosis (23%). All patients were consistent with coronary artery bypass graft surgery to testify. One of the following conditions are not included in this study:merger liver, kidney dysfunction, pulmonary fibrosis, bone metabolic diseases, severe diabetes, systemic autoimmune disease, infection, rheumatic diseases and malignant tumors and so on. Registration clinical data of all research subjects, including age, sex, height, weight, combined with other diseases, drug use, and electrocardiogram, echocardiography and chest X-ray in preoperatively. All patients cardiac function wereⅡtoⅢgrade (NYHA classification). All patients were preoperatively to sign informed consent. Taken 100mg specimens from the right atrial appendage before cardiopulmonary bypass surgery, quickly placed it in liquid nitrogen to save after removed blood and fatty tissue.
2. Immunohistochemical staining:using streptavidin peroxidase conjugated method(SP immunohistochemical staining method), dropping amount of dilution of mouse anti-human MMP-1, MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 monoclonal antibody with specific antigen to form antigen-antibody complex, followed by the use of biotin-labeled anti-2, combined with the DAB color reagent, the positive results appear brown cytoplasmic granules, Showing that for the diffuse positive expression of myocardial. To use the light microscope observation and take digital photographs in the same lighting conditions,and then using Image-pro plus image analysis software to calculate the PU values.
3. Fluorescence quantitative PCR reactions:using Trizol one-step method extracting organizations RNA, measuring OD260 and OD280 of the RNA samples, and calculating the concentration of RNA, then RNA reverse transcription synthesis of cDNA, reverse transcription steps in strict accordance with TaKaRa SYBR(?) Premix Ex TaqTM RT reagent Kit (Perfect Real Time) instructions to carry out,and primers were designed and synthesized, and then use of restricted reference materials (GAPDH) primers and the various indicators primers to carry out conventional PCR reaction to test the template cDNA and primers, and then do fluorescence quantitative PCR reaction,the parameters according TaKaRa SYBR(?) Premix Ex TaqTM (Perfect Real Time) kit instructions. Analysis of the data in accordance with the following formula to calculate each target relative to the percentage of GAPDH levels in each sample, namely, (target/GAPDH)* 100%
4. Statistical Analysis:All data applied SPSS 13.0 statistical package for analysis, Indicators to mean±standard deviation expressed, difference between the groups used t tests,p<0.05 mean the significant difference.
Results:
1. Rheumatic heart disease:Clinical data such as age, height and weight were no significant difference between the SR group and AF group, the left atrial diameter and right atrial diameter of the AF group were significantly greater than those of the SR group (p<0.01), the difference was significant, but the left ventricular diastolic diameter and left ventricular ejection fraction were no significant differences. Immunohistochemical staining:positive results was that the cytoplasm appeared brown granules. SR group atrial cytoplasm can be seen evenly distributed sparse brownish particles, AF groups atrial cytoplasm can be seen the irregular distribution of thick brown-yellow granules. Using Image-proplus image analysis software to calculate the PU values, it shown that the expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 were significantly increased in AF group (all p<0.01), excluding MMP-1, the difference was significant. The difference of MMP-1 of the two groups was not statistically significant, but the level of AF group was more than that of the SR group. FQ-PCR reaction:the AF group atrium expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 were significantly increased (all p<0.01), the difference was significant. The difference of MMP-1 of the two groups was not statistically significant, but the level of AF group was more than that of the SR group.
2. Coronary heart disease:Clinical data such as age, height and weight were no significant difference between the MI group and UA group, Two groups of patients with echocardiography shown that the left atrial diameter and right atrial diameter and left ventricular diameter of the MI group were significantly greater than those of the UA group (p<0.01), but the left ventricular ejection fraction was significantly less than that of the UA group (p<0.05). Immunohistochemical staining:positive results was that the cytoplasm appeared brown granules. UA group atrial cytoplasm can be seen evenly distributed sparse brownish particles, MI group atrial cytoplasm can be seen the irregular distribution of thick brown-yellow granules. Using Image-proplus image analysis software to calculate the PU values, it shown that the expression of MMP-3, MMP-9 and TIMP-1 TIMP-2、TIMP-3、TIMP-4 were significantly increased in MI group(respectively, p<0.01, p<0.01, p<0.05, p<0.05,p<0.01, p<0.01), excluding MMP-1 and MMP-7. The difference of MMP-1 and MMP-7 of the two groups was not statistically significant, but the level of MI group was more than those of the UA group. FQ-PCR reaction:The MI group myocardial expression of MMP-3, MMP-9 and TIMP-1 TIMP-2、TIMP-3、TIMP-4 were significantly increased (all p<0.01), The difference of MMP-1 and MMP-7 of the two groups was not statistically significant, but the level of MI group was more than those of the UA group.
Discussion:
Cardiac extracellular matrix is composed by collagen fiber, matrix membrane protein, proteoglycan, and the signaling molecules with biological activity, it is an important component of myocardial structure and has important physiological function such as cell information transmission and coordination the role of systolic and diastolic function. It can maintain the myocardial cells and myocardial fibers arranged, to limit the role of myocardial cells over-stretched. Myocardial extracellular matrix can also cause changes in cardiac contraction and ejection of effective coordination, and lost the support of the role of the heart geometry, resulting in impaired pump function.
MMPs are a zinc binding protease family with the decomposition of extracellular matrix, it has been found more than 20 kinds, according to the substrate, it is divided into four types including collagenase, gelatinase, matrix soluble enzymes and membrane-type enzymes. It is involved in normal embryonic development and tissue remodeling and involved in many of the body pathological and physiological processes. TIMPs are specific inhibitors of endogenous MMPs, it is divided into four types, the interaction and the dynamic balance between TIMPs and MMPs play an important role to maintain the normal heart shape and heart function. There studies have shown that expression and abnormalities activity of MMPs and TIMPs involved in the atrial structural remodeling.
At present, different levels of MMPs and TIMPs expression in cardiovascular disease have been proposted at home and abroad.Most studies have focused on the expression of both serum levels or the use of animal models to speculate MMPs and TIMPs in human heart disease in patients with myocardial expression. In our study, we use the way of human myocardial biopsy.This study selected four types of MMPs and all kinds of TIMPs to explore the expression of matrix metalloproteinases and their inhibitors in patients with atrial fibrillation and myocardial infarction, so the study was comprehensive and general. Due to the different and the degree of heart failure of rheumatic heart valve disease will affect the degree of atrial fibrosis and the incidence of atrial fibrillation, for which the election in this study were all patients with mitral valve disease, and valvular disease in all groups was no significant difference nature of cases in each group and no significant difference in left ventricular ejection fraction. The results shown that the right and left atrial diameter of rheumatic heart disease patients with atrial fibrillation expanded significantly compared to sinus rhythm patients, the difference was significant. To show that atrial fibrillation leading to myocardial structural changes have taken place, the atrial cavity expansion increased myocardial stiffness.The right and left atrial diameter and left ventricular diameter of myocardial infarction patients expanded significantly compared to angina pectoris patients, the difference was significant. These indicated myocardial structural remodeling has taken place of myocardial infarction patients and the cavity expansion and myocardial stiffness increased, which leading to-impair the cardiac function. This study used immunohistochemical staining and the PU values which can quantitatively calculat the expression of MMPs and TIMPs, and then used fluorescence quantitative PCR method to accurate measure the expression of MMPs and TIMPs. The results shown that the expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 of atrial fibrillation patients of Rheumatic heart disease were significantly increased, while MMP-1 expression in the two groups was no significant difference, but the overall value was still an increasing trend, and the expression of MMP-3, MMP-9 and TIMP-1、TIMP-2、TIMP-3、TIMP-4 of myocardial infarction patients were significantly increased, although the expression of MMP-1 and MMP-7 in the two groups were no significant difference, but the overall value was still an increasing trend, these suggest that the increase expression of MMP-1, MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 cause myocardial extracellular matrix degradation, myocardial cell stretch, atrial wall thinning, atrial expansion, normal cell components decreased, matrix components increased and myocardial extensive fibrosis, and then promote the occurrence of atrial remodeling and maintain it. It shows increased activity of MMPs expression can imbalance of ECM synthesis and degradation, both involved in cardiac and vascular remodeling and ventricular dilatation. It plays an important role in the pathogenesis of cardiovascular disease. Therefore, regulating the activity of MMPs may be the treatment of cardiovascular disease, a new direction for further research will contribute to its clinical application as soon as possible.
In summary, the change of the level of MMPs and TIMPs determines varying degrees of myocardial remodeling, and directly affect and change of collagen metabolism. By regulating expression and activity of the MMPs and TIMPs to prevent or even reverse the cardiac remodeling may be an important clinically mean of treatment and prevention.
基质金属蛋白酶(Matrix metalloproteinase, MMPs)是由许多结构和功能相似的中性蛋白酶组成的多基因内源性锌依赖性酶家族,它由血管平滑肌细胞、单核细胞、内皮细胞等多种细胞产生,可降解除多糖以外所有的细胞外基质(Extracellular matrix, ECM)成分。目前已发现的MMPs家族成员有20多种,根据其结构及功能的不同可分为四大类：第一类为胶原酶(MMP-1,8,13等),其主要水解底物是纤维类胶原,即Ⅰ、Ⅱ、Ⅲ型胶原；第二类为明胶酶(MMP-2,9),主要迅速水解变性胶原蛋白及和明胶蛋白；第三类为基质水解酶类(MMP-3,10,11等),水解Ⅲ,Ⅳ型胶原和蛋白多糖,明胶和糖蛋白；第四类为结构与功能比较特殊的MMPs (MMP-7,12,19,20,23,26等),其除能降解几种ECM成分外,还能激活其他MMPs。基质金属蛋白酶抑制剂(Tissue inhibitors of metalloproteinase, TIMPs)是MMPs的内源性特异性抑制物,分泌MMPs的细胞同时也分TIMPs, TIMPs家族里共有四个成员,分别为TIMP-1、TIMP-2、TIMP-3和TIMP-4。TIMPs主要具有两方面功能：它们对不同的MMPs具有不同的抑制强度,通常TIMPs以1：1的分子比例与激活的MMPs形成高亲和力复合物,并通过封闭MMPs的催化区,从而抑制ECM的降解。此外,TIMPs还有促进有丝分裂、抑制血管生成和诱导细胞死亡的作用。
心肌组织中三分之二的细胞是非心肌细胞,即细胞外基质,其对心肌细胞不仅具有结构上的支持和保护作用,而且,在心肌细胞间信息传递、舒缩功能的协调中都发挥重要的作用。主要功能有：①为心肌细胞、血管和淋巴管的相互联系和排列提供支持,从而决定心肌的僵硬度和结构；②防止心肌细胞过度伸长；③将心肌细胞产生的压力传至心室腔；④防止心肌细胞过度回缩；⑤提供心肌舒张时的阻力；⑥给心肌提供张力强度以防止其断裂；⑦防止心肌细胞和肌纤维滑脱。细胞外基质中心肌胶原蛋白占主要位置,成人心肌组织中的胶原至少包括Ⅰ、Ⅲ、Ⅳ,Ⅴ和Ⅵ型。其中最重要的是Ⅰ型(ColⅠ)和Ⅲ型胶原,是构成间质的主要成分。各种胶原成分共同构成胶原纤维网络,将心肌细胞连成一体,能够直接影响心脏的结构和功能。在心功能衰竭的动物模型以及此类病人体内,均可见到心脏间质胶原类型含量及其性质的改变,且此改变往往对其预后具有决定性意义,这证明心脏间质的重要性。心肌胶原也处于不断变化与代谢中,其每天的更新率约0.6%。MMPs/TIMPs平衡对于维持心肌间质胶原合成与降解代谢的动态平衡具有重要的作用。在某些疾病状态,如心肌梗死后、高血压等,心肌胶原的数量及分布发生了改变(即心肌重构),引起心肌间质纤维化,限制心肌活动,降低心室的顺应性,影响心肌的收缩及舒张功能,是引起心力衰竭的重要原因。另外,胶原沉积可限制除极波通过心肌的速度,对心肌组织的电稳定性产生负面影响,从而可导致心律失常。胶原在成年心脏是持续合成及降解的,并保持动态的平衡,随年龄的增长胶原合成和降解的平衡发生偏移,胶原逐渐累积。许多酶类可以降解ECM成分中的胶原蛋白,因此,了解修饰心肌ECM结构的酶系统对于了解心肌重构具有重要的意义,而MMPS对于ECM的重构有着重要的作用。
目前针对MMPs在心房颤动及心肌梗死的发病、发展中所起的作用及治疗研究尚不多,研究主要集中在以下几个方面：①合成MMPs的特殊抑制剂,但主要集中于防治肿瘤的侵袭和转移；②利用重组蛋白或病毒载体基因转运技术给予内源性抗蛋白酶剂,或提高体内TIMPs的分泌；③根据MMPs的表达及酶活性调控机制,开发具有调控MMPs/TIMPs平衡的药物。在本研究中,我们收集了50例风湿性心脏病患者以及40例冠心病患者的临床资料及心肌标本,进行免疫组化染色检测及实时荧光定量PCR反应,研究MMPs及TIMPs与心肌重构的相关性,进一步深入分析心肌重构发病机制以及与临床特征的关系。
材料与方法
1.病人及标本收集：选取2007年9月至2009年5月期间在我院接受人工心脏二尖瓣置换术的风湿性心脏病患者共50例,男28例,女22例,年龄26-66岁,分为窦性心律组(SR)20例,心房纤颤组(AF)30例。有下列情况之一者不纳入本研究：①年龄小于20岁或大于66岁者；②合并有感染性心内膜炎者；③合并有高血压、糖尿病、冠心病、甲状腺功能亢进、心肌病、慢性肺源性心脏病或严重肝肾功能不全者；④非风湿性心脏病而接受心脏瓣膜置换术者；⑤风湿性心脏病非二尖瓣瓣膜置换术者；⑥临床有风湿活动征象或发热者。并同期选取接受冠状动脉搭桥手术的冠心病患者共40例,男25例,女15例,年龄40-65岁,分为心肌梗死组(MI)22例,不稳定型心绞痛组(UA)18例。术前行冠状动脉造影检查,造影结果显示在MI组中,9例为左主干病变超过50%(41%),8例为三支冠脉血管病变(36%),5例为双支冠脉血管病变(23%)；UA组中8例为左主干病变超过50%(44%),6例为三支冠脉血管病变(33%),4例为左前降支近段严重狭窄(23%)。所有患者均符合行冠状动脉搭桥术手术指证,并排除合并肝肾功能不全、肺纤维化、骨代谢性疾病、严重糖尿病、全身免疫性疾病、感染、风湿性疾病及恶性肿瘤等。登记各研究对象的相关临床资料,包括年龄、性别、身高、体重、合并的其他疾病、用药情况等,并于术前行心电图、超声心动图、胸片等检查。所有患者心功能均为Ⅱ-Ⅲ级(NYHA分级)。术前所有患者均签署知情同意书。在开胸建立体外循环之前取患者右心耳标本约100mg,除去血液和脂肪组织后立刻放入液氮中保存备用。
2.免疫组化染色检测：采用链霉菌亲生物素蛋白-过氧化物酶连接法(streptavidin peroxidase conjugated method, SP法),滴加适量稀释的小鼠抗人MMP-1、MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4单克隆抗体与特异性抗原结合形成抗原-抗体复合物,之后再使用生物素标记的二抗结合后用DAB显色剂显色,阳性结果为胞浆中出现棕黄色颗粒,表明在心肌中为弥漫阳性表达。在光镜下观察,并在同等光照条件下拍摄数码照片,运用Image-pro plus软件进行图像定量分析,计算阳性单位(PU值)。
3.荧光定量PCR反应：采用Trizol一步法提取组织RNA,测定各RNA样品的OD260、OD280,并计算RNA浓度,将RNA反转录合成cDNA,反转录步骤严格按TaKaRa PrimeScriptTM RT reagent Kit (Perfect Real Time)试剂盒说明进行,进行常规PCR反应,设计并合成引物,分别利用内参(GAPDH)引物和各指标引物进行常规PCR反应,以检验模板cDNA与引物,再进行荧光定量PCR反应,反应参数等均按TaKaRa SYBR(?) Premix Ex TagTM (Perfect Real Time)试剂盒说明进行。数据按照以下公式分析,计算每个样品中各指标相对于GAPDH的相对百分含量,即(指标／GAPDH)*100%
4.统计分析：所有数据应用SPSS13.0统计软件包进行分析,指标以均数±标准差表示,组间差异采用两独立样本t检验,以p<0.05为差异有显著性。
结果
1.风湿性心脏病患者：SR与AF两组的年龄、身高、体重等临床指标比较差异无显著性,两组患者超声心动图检查显示AF组左心房内径与右心房内径显著大于SR组(p<0.01),差异有显著性,左室舒张末期内径、左室射血分数等指标差异无显著性。免疫组织化学染色检测：阳性结果为心房肌细胞胞浆中出现棕黄色颗粒。SR组心房肌细胞胞浆可见分布均匀,稀疏的浅褐色颗粒,AF组心房肌细胞胞浆内可见浓密的不规则分布的棕黄色颗粒。运用Image-proplus软件进行图像定量分析,计算PU值显示：与SR组相比较,AF组除MMP-1外,MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达均明显增高(均p<0.01),差异具有显著性。MMP-1在两组中比较虽差异无显著性,但总体水平较SR组增加。实时荧光定量PCR反应：AF组心肌组织MMP-3、MMP-7、MMP-9和TIMP-1、TIMP-2、TIMP-3、TIMP-4表达较SR组明显增高(均p<0.01),差异具有显著性。MMP-1在两组中比较虽差异无显著性,但总体水平较SR组增加。
2.冠心病患者：MI组与UA组两组的年龄、身高、体重等临床指标比较差异无显著性,两组患者超声心动图检查显示：MI组左右心房内径及左室舒张末期内径显著大于UA组(均p<0.01),差异有显著性,左室射血分数低于UA组,差异有显著性(p<0.05)。免疫组织化学染色检测：阳性结果为心房肌细胞胞浆中出现棕黄色颗粒。UA组心房肌细胞胞浆可见分布均匀,稀疏的浅褐色颗粒,MI组心房肌细胞胞浆内可见浓密的不规则分布的棕黄色颗粒。运用Image-pro plus软件进行图像定量分析,计算PU值显示：与UA组相比较,MI组除MMP-1、MMP-7外,MMP-3、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达均明显增高,差异具有显著性(p<0.01,p<0.01,p<0.05,p<0.05,p<0.01,p<0.01)。MMP-1、MMP-7在两组中比较虽差异无显著性,但总体水平较UA组增加。实时荧光定量PCR反应：MI组心肌组织MMP-3、MMP-9和TIMP-1、TIMP-2、TIMP-3、TIMP-4表达较UA组明显增高,差异具有显著性(均p<0.01),MMP-1、MMP-7在两组中比较差异无显著性,但总体水平较UA组增加。
讨论
心肌细胞外基质是由纤维胶原、基质膜蛋白、蛋白聚糖和具有生物活性的信号分子所组成的,是心肌细胞结构的重要组成成分,且具有细胞间信息传递、协调舒缩功能、维持心肌细胞及心肌纤维的排列、限制心肌细胞过度拉长的作用。心肌细胞外基质的改变还可造成心肌收缩和有效的射血之间失去协调性,并且失去了对心脏几何形状的支持作用,致使泵功能受损。
MMPs是一组与锌结合的分解细胞外基质的蛋白酶家族,至今发现已有20余种,按作用底物分为胶原酶、明胶酶、基质水解酶、膜型酶等4种,参与胚胎正常发育及组织重塑,参与人体许多病理和生理过程。TIMPs是MMPs的内源性特异性抑制剂,分为4型,二者之间的相互作用及动态平衡中维持心脏正常形状和心功能中起着重要的作用。已有研究表明,MMPs及TIMPs的表达及活性异常参与心肌结构的重构。
目前国内外对MMPs及TIMPs在心血管疾病中的表达水平报道不一,且多数研究集中在检测血清中二者的表达水平或利用动物模型来推测MMPs及TIMPs在人类心脏疾病患者心肌内的表达。在我们的研究中使用人类心肌活检的方式,分别选取四类基质金属蛋白酶中的各一种及所有的基质金属蛋白酶抑制剂来进行探讨基质金属蛋白酶及其抑制剂在风湿性心脏病房颤患者及心肌梗死患者心肌的表达情况,具有全面性及概括性。由于风湿性心脏病瓣膜病变的不同及心力衰竭的程度会影响心房纤维化程度和房颤的发生,为此本研究所选病例均为二尖瓣病变患者,且各组中瓣膜病变差异无显著性,各组病例中左室射血分数差异无统计学意义。超声心动图结果显示,风湿性心脏病房颤患者与窦性心律患者相比较,左右心房内径明显扩大,差异具有显著性。说明房颤导致心肌组织发生了结构变化,房腔扩大,心肌僵硬度增加。在对冠心病患者的研究中发现,心肌梗死患者与不稳定型心绞痛患者相比较,左右心房内径及左室舒张内径明显扩大,左室射血分数降低,差异具有显著性,说明心肌梗死患者心肌组织发生了结构变化,心房腔及心室腔均扩大,心肌僵硬度增加,心肌顺应性下降,一心功能受损。本研究使用免疫组织化学染色法,并使用PU值定量计算各MMPs及TIMPs的表达。再使用实时荧光定量PCR方法更准确的测量各MMPs及TMPs的表达,结果显示在风湿性心脏病房颤患者心肌组织中MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达明显增加,虽然MMP-1表达在两组患者中差异无显著性,但总体数值仍然是呈增加趋势,在心肌梗死患者心肌组织中MMP-3、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4表达明显增加,虽然MMP-1、MMP-7表达在两组患者中差异无显著性,但总体数值仍然是呈增加趋势。提示在风湿性心脏病心房纤颤及冠心病心肌梗死患者的心肌组织中,MMP-1、MMP-3、MMP-7、MMP-9及TIMP-1、TIMP-2、TIMP-3、TIMP-4的表达增高,使得心肌细胞外基质降解,导致心肌细胞拉长,心房壁变薄、心房扩大,正常细胞成分减少,基质成分增加,心肌广泛纤维化,最终促进心房组织重构的发生与维持。说明MMPs表达活性增强可使ECM合成与降解失衡,MMPs、TIMPs两者参与心肌和血管重构,在心血管疾病的发病过程中起重要作用。因此,调节MMPs的活性可能是心血管疾病治疗的一个新方向,对其进一步研究将促进临床的早日应用。
总之,MMPs、TIMPs二者水平的改变直接影响和改变胶原的新陈代谢,决定着心肌不同程度的重构。通过调节MMPs、TIMPs的表达和活性来阻止甚至逆转心肌重构可能成为临床上重要的治疗和预防手段。
Background and purpose:
MMPs is a multi-gene regulation of endogenous zinc-dependent enzyme family which is composition of neutral protease that similar to the structure and function and is secreted by vascular smooth muscle cells, monocytes and endothelial cells, and its function is to degrade all extracellular matrix components(ECM), excluding polysaccharide. MMPs has been found that there are more than 20 kinds, according to its structure and function can be divided into four different categories:①collagenase (MMP-1, MMP-8, MMP-13):the main substrate is hydrolyzed collagen fiber, namely,Ⅰ,Ⅱ,Ⅲcollagen;②gelatinase (MMP-2, MMP-9):digest denatured collagen and gelatin quickly;③stromelysin (MMP-3, MMP-10, MMP-11): hydrolysisⅢ,Ⅳcollagen and proteoglycan, gelatin and sugar protein;⑤The fourth category is the structure and function of more specific MMPs (MMP-7,12,19, 20,23,26, etc.), which can degrade several ECM components, inter alia, but also activate other MMPs. Tissue inhibitors of metalloproteinase (TIMPs) are specific endogenous inhibitor of MMPs, MMPs secreting cells also secrete TIMPs, TIMPs are four kinds:TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMPs have two main functions: the inhibition of MMPs activity, TIMPs and activated MMPs are usually to form a high-affinity 1:1 complex and through the closure of the catalytic domain of MMPs to inhibit the degradation of ECM. In addition, TIMPs also promote mitosis, inhibit angiogenesis and inducing cell death role.
Two-thirds of myocardial cells in non-cardiac cells, namely, extracellular matrix, its myocardial cells not only has the structural support and protection, and in information transmission between myocardial cells, the coordination of both systolic and diastolic function play an important role. Main functions are:①to provide support to the myocardial cells, blood vessels and lymphatic vessels intercomnected and arranged, determine myocardial stiffness and structure;②to prevent excessive elongation of myocardial cells;③the pressure of cardiac cells will be transmitted to ventricular cavity;④to prevent excessive retraction of myocardial cells;⑤provide resistance when myocardial relaxation;⑥to provide tensile strength to the heart in order to prevent its fracture;⑦to prevent cardiac cells and muscle fibers slippage. Myocardial extracellular matrix of collagen accounts for the main location of the adult myocardial tissue includes at least collagenⅠ,Ⅲ,Ⅳ,ⅤandⅥtype. Two of the most important collagen are the typeⅠ(ColⅠ) and typeⅢ. Collagen fiber network composed by various components of collagen facilitate the integration between myocardial cells, and it can directly influence the structure and function of the heart. In animal models of heart failure and heart failure patient may see the change of the content of cardiac interstitial collagen and the nature, and this change often determines the prognosis, and this prove the importance of myocardial interstitial. Myocardial collagen is also in the ever-changing and metabolism, its daily update rate is about 0.6%. The balance between MMPs and TIMPs plays an important role in the metabolism of dynamic balance for the maintenance of myocardial interstitial collagen synthesis and degradation.
In some disease states, such as myocardial infarction, high blood pressure and so on, the number and distribution of myocardial collagen has changed (ie, cardiac remodeling), causing myocardial interstitial fibrosis, limiting cardiac activity and reduce ventricular compliance, impact the systolic and diastolic function of myocardial. It is an important reason for causing heart failure. In addition, collagen deposition may limit the cardiac depolarization waves through the speed of the electrical stability of the myocardium have a negative impact, which can lead to cardiac arrhythmia. Collagen in the adult heart is a continuous synthesis and degradation and to maintain dynamic balance, age-related increase of collagen synthesis and degradation of the balance of offset, a gradual accumulation of collagen. Many enzymes can degrade extracellular matrix components of the collagen protein, therefore, understanding the modification of myocardial extracellular matrix structure of the enzyme system can understand the cardiac remodeling, while the matrix metalloproteinase play an important role on the reconstruction of extracellular matrix.
At present, the research of the MMPs in the pathogenesis and treatment of atrial fibrillation and myocardial infarction was few, and research focused on the following aspects:①a special inhibitor of synthetic MMPs, the main focus on the prevention and treatment of invasive and transfer of tumors;②the use of recombinant protein or viral vector gene transfer technology for the endogenous anti-protease agent, or enhance the secretion of TIMPs in vivo;③According to the expression of MMPs and the enzyme activity of regulatory mechanisms, development the drug to control the balance between MMPs and TIMPs. In this study, we collected clinical data and myocardial specimens of 50 cases of rheumatic heart disease patients and 40 cases of coronary heart disease patients, and immunohistochemical staining and fluorescence quantitative PCR reaction for myocardial specimens, to study the MMPs and TIMPs associated with myocardial remodeling, and analysis of the pathophysiology mechanisms of myocardial remodeling and the clinical features of myocardial remodeling.
Materials and methods:
1. Patients and sample collection:Selection 50 cases of rheumatic heart disease patients for artificial mitral valve replacement surgery from September 2007 to May 2009 in our hospital,28 males and 22 females, aged from 26 to 66 years old, the 50 cases is divided into 20 cases of sinus rhythm group (SR) and 30 cases of atrial fibrillation group (AF). One of the following conditions are not included in this study:①younger than 20 years old or more than 66 years of age;②complicated with infective endocarditis;③complicated with hypertension, diabetes, coronary heart disease, hyperthyroidism, myocardial disease, chronic pulmonary heart disease or severe liver and kidney dysfunction;④non-rheumatic heart disease undergoing heart valve replacement surgery;⑤rheumatic heart disease were non-mitral valve replacement surgery;⑥The clinical signs of rheumatism activity or fever. And over the same period selected 40 cases of coronary heart disease patients who received coronary artery bypass surgery,25 males and 15 females, aged from 40 to 65 years old, those were divided into 22 cases of myocardial infarction group(MI) and 18 cases of unstable angina group(UA). Preoperative coronary angiography, imaging results showed that in the MI group,9 cases of left main coronary vascular disease more than 50% (41%),8 cases of three coronary vascular disease (36%),5 cases of double coronary vascular disease (23%); UA group,8 cases of left main coronary vascular disease more than 50% (44%),6 cases for three coronary vascular disease (33%),4 cases of severe proximal left anterior descending artery stenosis (23%). All patients were consistent with coronary artery bypass graft surgery to testify. One of the following conditions are not included in this study:merger liver, kidney dysfunction, pulmonary fibrosis, bone metabolic diseases, severe diabetes, systemic autoimmune disease, infection, rheumatic diseases and malignant tumors and so on. Registration clinical data of all research subjects, including age, sex, height, weight, combined with other diseases, drug use, and electrocardiogram, echocardiography and chest X-ray in preoperatively. All patients cardiac function wereⅡtoⅢgrade (NYHA classification). All patients were preoperatively to sign informed consent. Taken 100mg specimens from the right atrial appendage before cardiopulmonary bypass surgery, quickly placed it in liquid nitrogen to save after removed blood and fatty tissue.
2. Immunohistochemical staining:using streptavidin peroxidase conjugated method(SP immunohistochemical staining method), dropping amount of dilution of mouse anti-human MMP-1, MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 monoclonal antibody with specific antigen to form antigen-antibody complex, followed by the use of biotin-labeled anti-2, combined with the DAB color reagent, the positive results appear brown cytoplasmic granules, Showing that for the diffuse positive expression of myocardial. To use the light microscope observation and take digital photographs in the same lighting conditions,and then using Image-pro plus image analysis software to calculate the PU values.
3. Fluorescence quantitative PCR reactions:using Trizol one-step method extracting organizations RNA, measuring OD260 and OD280 of the RNA samples, and calculating the concentration of RNA, then RNA reverse transcription synthesis of cDNA, reverse transcription steps in strict accordance with TaKaRa SYBR(?) Premix Ex TaqTM RT reagent Kit (Perfect Real Time) instructions to carry out,and primers were designed and synthesized, and then use of restricted reference materials (GAPDH) primers and the various indicators primers to carry out conventional PCR reaction to test the template cDNA and primers, and then do fluorescence quantitative PCR reaction,the parameters according TaKaRa SYBR(?) Premix Ex TaqTM (Perfect Real Time) kit instructions. Analysis of the data in accordance with the following formula to calculate each target relative to the percentage of GAPDH levels in each sample, namely, (target/GAPDH)* 100%
4. Statistical Analysis:All data applied SPSS 13.0 statistical package for analysis, Indicators to mean±standard deviation expressed, difference between the groups used t tests,p<0.05 mean the significant difference.
Results:
1. Rheumatic heart disease:Clinical data such as age, height and weight were no significant difference between the SR group and AF group, the left atrial diameter and right atrial diameter of the AF group were significantly greater than those of the SR group (p<0.01), the difference was significant, but the left ventricular diastolic diameter and left ventricular ejection fraction were no significant differences. Immunohistochemical staining:positive results was that the cytoplasm appeared brown granules. SR group atrial cytoplasm can be seen evenly distributed sparse brownish particles, AF groups atrial cytoplasm can be seen the irregular distribution of thick brown-yellow granules. Using Image-proplus image analysis software to calculate the PU values, it shown that the expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 were significantly increased in AF group (all p<0.01), excluding MMP-1, the difference was significant. The difference of MMP-1 of the two groups was not statistically significant, but the level of AF group was more than that of the SR group. FQ-PCR reaction:the AF group atrium expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 were significantly increased (all p<0.01), the difference was significant. The difference of MMP-1 of the two groups was not statistically significant, but the level of AF group was more than that of the SR group.
2. Coronary heart disease:Clinical data such as age, height and weight were no significant difference between the MI group and UA group, Two groups of patients with echocardiography shown that the left atrial diameter and right atrial diameter and left ventricular diameter of the MI group were significantly greater than those of the UA group (p<0.01), but the left ventricular ejection fraction was significantly less than that of the UA group (p<0.05). Immunohistochemical staining:positive results was that the cytoplasm appeared brown granules. UA group atrial cytoplasm can be seen evenly distributed sparse brownish particles, MI group atrial cytoplasm can be seen the irregular distribution of thick brown-yellow granules. Using Image-proplus image analysis software to calculate the PU values, it shown that the expression of MMP-3, MMP-9 and TIMP-1 TIMP-2、TIMP-3、TIMP-4 were significantly increased in MI group(respectively, p<0.01, p<0.01, p<0.05, p<0.05,p<0.01, p<0.01), excluding MMP-1 and MMP-7. The difference of MMP-1 and MMP-7 of the two groups was not statistically significant, but the level of MI group was more than those of the UA group. FQ-PCR reaction:The MI group myocardial expression of MMP-3, MMP-9 and TIMP-1 TIMP-2、TIMP-3、TIMP-4 were significantly increased (all p<0.01), The difference of MMP-1 and MMP-7 of the two groups was not statistically significant, but the level of MI group was more than those of the UA group.
Discussion:
Cardiac extracellular matrix is composed by collagen fiber, matrix membrane protein, proteoglycan, and the signaling molecules with biological activity, it is an important component of myocardial structure and has important physiological function such as cell information transmission and coordination the role of systolic and diastolic function. It can maintain the myocardial cells and myocardial fibers arranged, to limit the role of myocardial cells over-stretched. Myocardial extracellular matrix can also cause changes in cardiac contraction and ejection of effective coordination, and lost the support of the role of the heart geometry, resulting in impaired pump function.
MMPs are a zinc binding protease family with the decomposition of extracellular matrix, it has been found more than 20 kinds, according to the substrate, it is divided into four types including collagenase, gelatinase, matrix soluble enzymes and membrane-type enzymes. It is involved in normal embryonic development and tissue remodeling and involved in many of the body pathological and physiological processes. TIMPs are specific inhibitors of endogenous MMPs, it is divided into four types, the interaction and the dynamic balance between TIMPs and MMPs play an important role to maintain the normal heart shape and heart function. There studies have shown that expression and abnormalities activity of MMPs and TIMPs involved in the atrial structural remodeling.
At present, different levels of MMPs and TIMPs expression in cardiovascular disease have been proposted at home and abroad.Most studies have focused on the expression of both serum levels or the use of animal models to speculate MMPs and TIMPs in human heart disease in patients with myocardial expression. In our study, we use the way of human myocardial biopsy.This study selected four types of MMPs and all kinds of TIMPs to explore the expression of matrix metalloproteinases and their inhibitors in patients with atrial fibrillation and myocardial infarction, so the study was comprehensive and general. Due to the different and the degree of heart failure of rheumatic heart valve disease will affect the degree of atrial fibrosis and the incidence of atrial fibrillation, for which the election in this study were all patients with mitral valve disease, and valvular disease in all groups was no significant difference nature of cases in each group and no significant difference in left ventricular ejection fraction. The results shown that the right and left atrial diameter of rheumatic heart disease patients with atrial fibrillation expanded significantly compared to sinus rhythm patients, the difference was significant. To show that atrial fibrillation leading to myocardial structural changes have taken place, the atrial cavity expansion increased myocardial stiffness.The right and left atrial diameter and left ventricular diameter of myocardial infarction patients expanded significantly compared to angina pectoris patients, the difference was significant. These indicated myocardial structural remodeling has taken place of myocardial infarction patients and the cavity expansion and myocardial stiffness increased, which leading to-impair the cardiac function. This study used immunohistochemical staining and the PU values which can quantitatively calculat the expression of MMPs and TIMPs, and then used fluorescence quantitative PCR method to accurate measure the expression of MMPs and TIMPs. The results shown that the expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 of atrial fibrillation patients of Rheumatic heart disease were significantly increased, while MMP-1 expression in the two groups was no significant difference, but the overall value was still an increasing trend, and the expression of MMP-3, MMP-9 and TIMP-1、TIMP-2、TIMP-3、TIMP-4 of myocardial infarction patients were significantly increased, although the expression of MMP-1 and MMP-7 in the two groups were no significant difference, but the overall value was still an increasing trend, these suggest that the increase expression of MMP-1, MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 cause myocardial extracellular matrix degradation, myocardial cell stretch, atrial wall thinning, atrial expansion, normal cell components decreased, matrix components increased and myocardial extensive fibrosis, and then promote the occurrence of atrial remodeling and maintain it. It shows increased activity of MMPs expression can imbalance of ECM synthesis and degradation, both involved in cardiac and vascular remodeling and ventricular dilatation. It plays an important role in the pathogenesis of cardiovascular disease. Therefore, regulating the activity of MMPs may be the treatment of cardiovascular disease, a new direction for further research will contribute to its clinical application as soon as possible.
In summary, the change of the level of MMPs and TIMPs determines varying degrees of myocardial remodeling, and directly affect and change of collagen metabolism. By regulating expression and activity of the MMPs and TIMPs to prevent or even reverse the cardiac remodeling may be an important clinically mean of treatment and prevention.
引文
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