大鼠心梗模型中管家基因的选择及ADAMTSs在心梗后心肌中的表达及机制
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摘要
研究背景及目的
基因表达分析在许多生命科学研究领域里的重要性日益增加,其研究的深入将为探索疾病相关基因、了解基因表达调控、解析生命奥秘、从而最终为人类服务大有裨益。反转录实时荧光聚合酶链反应是对特定信使RNA进行定量研究的最灵敏方法。为了分析特定信使RNA含量的差别,要用内部参照基因来进行定量。管家基因有几百种,目前,作为内参使用最广泛的管家基因是3磷酸甘油醛脱氢酶、β肌动蛋白、18SrRNA和28SrRNA。教条地使用一种管家基因或盲目参照不同实验对象及条件所使用的管家基因作为内参,一方面可能使基因表达的微小差异难以发现,另一方面可能导致错误甚至相反的结论。大鼠是一种常用制作心梗模型的实验动物,对于冠心病相关基因的研究使用大鼠做动物模型是许多实验室的首选。然而,心梗后大鼠内参基因选择问题还较少研究报道。
心肌梗死是威胁人类生命的重大疾病,心肌梗死动物模型对于研究人类冠心病的发病机制、病理生理改变以及对治疗方法的评估都具有重大意义。长期以来实验室研究是用结扎大鼠冠状动脉的方法来制备模型,但在实践中,由于麻醉和人工呼吸的使用不当、肺损伤以及左冠状动脉定位错误等各种原因,造成动物死亡或制模失败。因此提高存活率及制模成功率是解决心肌梗死动物模型的一个核心问题。我们对制作大鼠心梗模型的方法加以改进,明显提高了大鼠的存活率,较好地解决了大鼠心梗模型的制备问题。
急性心梗是引起人类死亡与残疾的主要疾病之一。心梗后心室重塑可导致心衰与死亡率的增加。在心室重塑过程中包括细胞外基质(ECM)分子的集聚和降解的动态变化。许多生物物质包括蛋白酶,蛋白酶抑制剂及生长因子等对ECM的重建起作用。其中基质金属蛋白酶(MMPS)表达的增加与激活也牵涉其中。除基质金属蛋白酶外,还有一些蛋白酶家族可降解细胞外基质。ADAMTS是一类分泌性金属蛋白酶,其主要结构包括解聚素,基质金属蛋白酶和血小板反应素基序。在人类中其家族已发现19个成员,涉及裂解多种蛋白聚糖,胶原的代谢,抗血管新生,VWF多聚体的降解,以及胚胎器官发育,生殖等多种功能。其中某些成员可与细胞外基质结合发挥作用。在颈动脉粥样硬化斑块及冠脉不稳定斑块中的富巨噬细胞区域ADAMTS4与8的表达增加,在动脉粥样硬化发展过程中,ADAMTS4的表达上调。Versican是聚集蛋白聚糖家族成员之一,在组织中广泛存在,参与伤口愈合及组织重塑过程。在冠脉结扎致心梗大鼠模型中其在心肌中表达并短暂升高,提示其参与了心梗后的炎症反应。ADAMTS4降解血管壁里的蛋白多聚糖versican,其作用位点在V1/V0versican的Glu1428-Ala1429。ADAMTS4能被内源性的抑制剂TIMPs阻断,ADAMTS4和ADAMTS5均能被TIMP-3有效抑制,而对TIMP-1,2,4却基本上不敏感。
近年来ADAMTS在炎症及动脉粥样硬化中的作用得到关注。免疫组化分析示ADAMTS1、4、5、8在人类颈动脉病变及冠脉粥样硬化斑块处表达,ADAMTS4、5、8与巨噬细胞共存而ADAMTS1与内皮细胞及平滑肌细胞共存。Versican在组织中广泛分布,亦存在于心脏中。versican在心梗后心脏中表达增高且其来源于渗出的单核细胞,提示其参与心梗后炎症反应。Aggrecan是一个软骨特异性的硫酸软骨素蛋白多聚糖,是ADAMTS的作用底物。ADAMTS4及5表现为较强的降解aggrecan活性。ADAMTS4在心梗后心脏中的具体表达部位,是否与versican在同一部位表达以及心梗后是否涉及aggrecan的表达,目前尚不明确。
在剪切应力作用下,人静脉内皮细胞及心脏微血管内皮细胞的ADAMTS1mRNA表达上调。在动脉粥样硬化斑块内膜及增殖/迁移的主动脉血管平滑肌细胞中,ADAMTS1的mRNA表达增高。应用INF-γ、TNF-α及IL-1β刺激巨噬细胞发现INF-Y刺激后ADAMTS4、7、8、9表达增加,ADAMTS1及17表达下降,而ADAMTS2、5、10不受其影响,TNF-α刺激后ADAMTS4、7、8表达增加,ADAMTS9轻微升高,IL-1β刺激对ADAMTS4、7、8表达无影响,而ADAMTS1及9在早期升高,说明ADAMTS家族中不同成员表达及调控机制存在差异。
以往研究提示,在动脉硬化发展及动脉粥样硬化斑块不稳定的过程中,ADAMTS4扮演着重要角色；ADAMTS4可以作为一种预示斑块不稳定及急性冠脉综合征严重程度的指标。经皮冠脉介入治疗(PCI)过程可以看作为机械挤压诱导斑块破裂的模型,因此研究PCI过程中的炎症反应或许可以为斑块不稳定机制提供线索。许多临床研究报道了冠心病患者PCI后的炎症反应。既然ADAMTS4已被证明是炎症调节酶且与动脉硬化的发展及斑块不稳定相关,我们猜测在冠心病患者冠脉循环中ADAMTS4水平可能升高且受PCI手术影响。
本研究用反转录实时荧光聚合酶链反应方法比较了四种管家基因在心梗大鼠心脏中的表达情况,采用GeNorm算法分析了哪些管家基因最适于心梗后大鼠基因表达研究。改进了大鼠心梗模型的制作方法,提高了手术成功率及大鼠存活率。探讨了ADAMTS4、ADAMTS8、versican、TIMP3在大鼠心梗后心脏中表达的动态变化、分布区域及可能机制,进一步认识急性心梗后局部ECM分子的变化及相互作用,为治疗提供新的理论依据。检测了IL-1β刺激内皮细胞后ADAMTSs的表达趋势。通过从冠状静脉窦取血检测了冠脉ADAMTS4及hs-CRP水平,并评估了PCI对ADAMTS4及hs-CRP水平的影响。本学位论文的主要研究内容及实验结果如下：
一、大鼠心梗模型中管家基因的选择
我们采用结扎冠脉前降支的方法制作心梗模型,结合实际工作,分析并挑选出使用广泛的4个标准管家基因：3磷酸甘油醛脱氢酶(GAPDH)、核糖体蛋白L13A(RPL13A)、β-肌动蛋白(ACTB)和结合区连接蛋白(ARBP),用反转录实时荧光聚合酶链反应方法比较了它们在心梗大鼠心脏中的表达情况,采用GeNorm算法分析哪些管家基因最适于心梗后大鼠基因表达的研究。结果示RPL13A、GAPDH、 ARBP及ACTB基因的M值分别为0.812、0.721、0.812和1.2,分析得出GAPDH及ARBP是在心梗模型中表达最稳定的基因。
二、大鼠心肌梗死模型制备的改进
选取健康雄性Wistar大鼠100只(体重230-270g),分为模型组(n=80)和假手术组(n=20)。3%戊巴比妥钠(40mg/kg)腹腔注射麻醉,气管切开,小动物呼吸机辅助呼吸,采用容量控制模式,潮气量3ml/100g,呼吸频率60-70次/min,吸呼比1：1。左侧胸部开胸,由第4肋间入胸,用乳突牵开器牵开肋骨,暴露心脏。在肺动脉圆锥和左心耳之间距主动脉根部约3mm处结扎左冠状动脉前降支。观察心电图变化,以肢体导联2个以上导联ST段抬高,或者肉眼观察结扎处下方大面积心肌表面变得苍白,周围瘀血征出现,室壁搏动减弱为结扎成功标准。假手术组大鼠只穿线不结扎。逐层关胸,撤除呼吸机,拔除气管插管。为防止气道狭窄及分泌物导致窒息,不缝合气管及颈部切口。术中至术后12h采用40W灯泡照射保暖,术后4h内注意观察气管切口处分泌物,及时用吸痰管清除。结果示心肌梗死模型制作的成功率为98.6%。模型组术后3周成活率为88.75%,假手术组存活率为100%。
通过对大鼠心梗模型制作方法的改进,提高了手术成功率及大鼠存活率。
三、大鼠心梗后心肌中ADAMTS4、ADAMTS8、versican、TIMP-3基因表达的动态变化
选择250-300g雄性Wistar大鼠。按前述方法制作心梗模型,设假手术组。分别于手术后0、3、6、12、24小时及3、7、14、21天(n=5)过量麻醉处死大鼠,迅速开胸摘取心脏,将结扎点下方梗死部位心肌分离切割,放入-70℃冰箱备用。用实时荧光定量RT-PCR方法分析梗死心肌中ADAMTS4、ADAMTS8、versican及TIMP-3mRNA表达,ELISA法检测(?)ADAMTS4的蛋白表达。结果示ADAMTS4表达在心梗后6及12小时明显升高(p<0.05),随后快速下降。而ADAMTS8则在心梗后6小时开始升高,至24小时达峰值,3天时仍持续在高水平(p<0.05),然后缓慢下降。心梗后versican mRNA水平显著增高,至3天时达峰值,且升高持续时间较长。TIMP3表达水平降低。ADAMTS4蛋白水平在心梗后6小时(p=0.026)、12小时(p=0.003)、24小时(p=0.002)及3天(p=0.009)显著增高。
ADAMTS4、ADAMTS8, versican及TIMP-3在心梗大鼠心脏中表达,且表现为不同的动态变化趋势。ADAMTS4、versican及TIMP-3的表达之间存在相互关系。
四、AAMTS4在梗死大鼠心脏血管内皮细胞及心肌细胞中表达
选择250-300g雄性Wistar大鼠制作心梗模型,设假手术组。手术后3天处死大鼠,取左心室制作冰冻切片。切片厚度为5um。免疫组织化学方法检测ADAMTS4、versican及aggrecan蛋白表达。对于阴性对照组,我们以等比稀释度用兔IgG处理切片。结果示ADAMTS4在梗死边缘区域心肌细胞中表达,在梗死区域及远离梗死区的正常心肌未见ADAMTS4表达。在梗死周围区域毛细血管内皮细胞ADAMTS4有弱表达,versican强表达。Aggrecan在心梗组及假手术组的心肌组织及血管内膜均未见表达。
ADAMTS4与versican共同表达于内皮细胞提示二者存在某种联系,其动态变化可能在心梗后基质重塑中起作用。
五、白介素1β刺激内皮细胞后ADAMTSs的表达
选取人脐静脉血管内皮细胞,以传代培养法培养细胞。分别以2.5ng/ml,5ng/ml,10ng/ml,20ng/ml浓度的IL-1β刺激内皮细胞,24h后收集细胞提取总RNA及蛋白。用10ng/ml IL-1β刺激内皮细胞,分别于刺激后2hh、6h、12h、24h、48h收集细胞提取总RNA及蛋白,设空白对照组,每组5个样本。RT-PCR测定ADAMTS1、4、8、9、15mRNA表达,Western-blot测定ADAMTS4蛋白表达。结果示IL-1β刺激内皮细胞后ADAMTS1、4、8、9、15mRNA表达均增高,但随时间变化趋势不同,ADAMTS4蛋白表达增高。
此结果提示ADAMTSs参与炎症反应,具体机制有待进一步阐明。
六、冠心病患者冠脉血中ADAMTS4水平的变化及经皮冠脉介入治疗对冠脉ADAMTS4水平的影响
根据冠脉造影结果,将81例患者分为对照组、简单病变组及复杂病变组,其中35例患者接受了PCI治疗。血液检测标本从冠状静脉窦获得。ADAMTS4及hs-CRP值分别通过ELISA及免疫浊度法检测。结果示复杂病变组ADAMTS4及hs-CRP值明显高于对照组及简单病变组(P<0.001)。所有研究对象及冠心病患者的ADAMTS4水平均与hs-CRP水平相关(r,=0.73, r2=0.763, P<0.01)。接受了PCI治疗的患者ADAMTS4值高于未接受PCI者(P<0.001), hs-CRP亦表现出相似结果(P=0.025)。
冠心病患者冠脉ADAMTS4及hs-CRP水平升高,且随病变复杂程度增加而升高。PCI后冠脉ADAMTS4及hs-CRP升高,其可能由PCI过程中球囊扩张或支架植入的机械挤压导致冠脉斑块破裂释放而出。
Background and objective
The importance of gene expression analysis in many areas of life science research is growing. Its in-depth study will benefit to explore disease-related genes, understand gene regulation, resolve the mysteries of life, and ultimately to service to humanity. Real-time fluorescence reverse transcription-polymerase chain reaction is the most sensitive method of studying specific messenger RNA quantitatively. To analyze the differences of the specific messenger RNA content, the internal reference gene is used to quantify. There are hundreds of housekeeping genes. At present, the most widely used internal reference housekeeping genes are GAPDH, β-actin,18SrRNA and28SrRNA. Using of one housekeeping gene singly or selection housekeeping genes blindly in accordance with different experimental subjects may make the small differences in gene expression difficult to find, on the other hand it may lead to errors or even opposite conclusions. Rat is a commonly used animal to be made myocardial infarction model. It is the first choice for many laboratories to use rats as animal model for coronary heart disease-related genes studies. However, there are fewer studies about the selection of reference genes in rats after myocardial infarction.
Myocardial infarction is a major disease to threat human life. Animal model of myocardial infarction is significant greatly for studying pathogenesis and pathophysiology of human coronary heart disease and evaluation of treatment methods. The method of coronary artery ligation is used to prepare the model ever since a long time ago. In practice, many reasons result in animal death or making model failure just as anesthesia, using respirator inadequately, lung injury and the left coronary artery positioning erroneously. Therefore, it is a core issue of improving survival and the success rate of making model to solve the question of animal model of myocardial infarction. We consummate the method of making rat model of myocardial infarction and improve the survival rate of rats significantly.
Acute myocardial infarction is a leading cause of death and disability in world. Ventricular remodeling after myocardial infarction can lead to heart failure and mortality. The dynamic change of extracellular matrix (ECM) accumulation and degradation is included in the process of ventricular remodeling. Many biological substances such as proteases, protease inhibitors and growth factors play a part in ECM reconstruction. The increased expression and activation of matrix metalloproteinases (MMPS) is also involved. In addition to matrix metalloproteinases, there are other protease families having the capacity to degrade the ECM. ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) is a group of secreted proteases that now incorporates19genes in humans, involving of cracking a variety of proteoglycans, collagen metabolism, anti-angiogenesis, VWF polymer degradation, the embryonic organ development, reproduction and other functions. Some of ADAMTS can bind to the ECM. ADAMTS4and8have been shown inflammatory regulated enzymes expressed in macrophage-rich areas of carotid atherosclerosis plaque and coronary instable plaque. ADAMTS4expression increases in the development of atherosclerosis process. Versican is a member of the aggrecan family, spread in the organization widely and participate in the process of wound healing and tissue remodeling. Versican expressed and incrased temporally in the myocardium of coronary artery ligation-induced myocardial infarction rat model, suggesting their involvement in the inflammatory response after myocardial infarction. ADAMTS4degrade versican of the vessel wall at V1/V0versican's Glu1428-Ala1429site. ADAMTS4can be blocked by the endogenous inhibitors TIMPs. ADAMTS4and ADAMTS5can be effectively suppressed by TIMP-3, while not be sensitive to TIMP-1,2and4.
In recent years the role of ADAMTS in inflammation and atherosclerosis is attentioned. Immunohistochemical analysis showed that ADAMTS1,4,5and8expressed in the human carotid artery plaque and coronary atherosclerotic plaque. ADAMTS4,5and8co-exist with macrophages and ADAMTS1co-exist with smooth muscle cells and endothelial cells. Versican is widely distributed in the organization and also present in the heart. Versican expression increased in the myocardial infarction heart and came from the mononuclear cells, suggesting their participation in the inflammatory response after myocardial infarction. Aggrecan is a cartilage-specific protein of chondroitin sulfate polysaccharide and is a substrate of ADAMTS. ADAMTS4and5showed strong activity in the degradation of aggrecan. It is not clear about the expression position of ADAMTS4in the heart after myocardial infarction. Whether versican express in the same area of ADAMTS4and whether aggrecan express in the heart after myocardial infarction are unclear. The shear stress up regulated the ADAMTS1mRNA expression in the human vein endothelial cells and cardiac microvascular endothelial cells. In the atherosclerotic plaque intima and proliferation/migration vascular smooth muscle cells, ADAMTS1mRNA expression increased. Stimulation macrophages with INF-γ, TNF-α and IL-1γ showed ADAMTS4,7,8,9expression increased, ADAMTS1and17expression decreased, while ADAMTS2,5,10is not affected after stimulation with INF-γ. ADAMTS4,7and8expressions increased, ADAMTS9slightly elevated after stimulation with TNF-α. ADAMTS4,7and8expressions were not affected, ADAMTS1and9increased in the early phase after stimulation with IL-1β. All of these indicate there are different expression and regulation mechanisms of ADAMTS family members. Previous observations suggest that (a) ADAMTS4plays an important role in the development of atherosclerosis and instability of atherosclerotic plaques;(b) ADAMTS4may serve as a marker of plaque destabilization and for predicting the severity of ACS. Percutaneous coronary intervention (PCI) procedure can be regarded as a model for mechanical induced plaque rupture. Thus studies on the inflammatory reaction during PCI could give information on mechanisms of plaque destabilization. Many clinical studies have evaluated the inflammatory response after PCI in patients with CAD. Since ADAMTS4has been proved to be inflammatory regulated enzymes and involved in the development of atherosclerosis and instability of atherosclerotic plaques, we speculated ADAMTS4level would elevate in the coronary circulation of patients with CAD and be influenced by PCI.
In this study, we compared four housekeeping genes'expression in hearts of myocardial infarction rats with real-time fluorescence reverse transcription-polymerase chain reaction method and analyzed which is the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm algorithms. We consummate the method of making rat model of myocardial infarction and improve the operation success rate and survival rate of rats. The dynamic expression changes and distribution area of ADAMTS4, ADAMTS8, versican, TIMP3and the possible mechanisms were studied. The changes and interactions of local ECM molecules after acute myocardial infarction were further understanded and a new theoretical basis for treatment was provided. ADAMTSs expression trend in the endothelial cells after stimulation withlL-1β were detected. We detected coronary ADAMTS4and high-sensitivity C-reactive levels using coronary sinus sampling and assessed the effect of PCI on ADAMTS4and hs-CRP levels. The main research contents and results are as follows:
1. Selection of housekeeping genes in rat model of myocardial infarction We made myocardial infarction models using the anterior descending coronary artery ligation method,combined with practical work, analyzed and selected the four widely used standard housekeeping genes:
glyceraldehydes-3-phosphate dehydrogenase (GAPDH), ribosomal protein L13A (RPL13A), beta-actin (ACTB) and acidic ribosomal phosphoprotein PO (ARBP). We compared their expression in the rat heart after myocardial infarction using real-time fluorescent reverse transcription polymerase chain reaction, analyzed which is the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm algorithms. The results showed the M values of RPL13A, GAPDH, ARBP and ACTB gene were0.812,0.721,0.812and1.2respectively. The results suggest that GAPDH and ARBP are the most stable genes for the rat myocardial infarction model.
2. Improvement of the rat myocardial infarction model
One hundred male Wistar rats (weight230-270g) were divided into model group (n=80) and sham-operatered group (n=20). The rats were anaesthetised with sodium pentobarbital (40mg/kg, intraperitoneally), operated tracheostomy, then ventilated with an automatic breathing apparatus using the volume control mode(tidal volume,3ml/100g, respiratory rate,60-70cycles/min, respiratory ratio,1:1). An left anterior thoracotomy was performed at the fourth intercostal space and rib was retracted with the mastoid retractor for exposing the heart. The left anterior descending coronary artery was ligated approximately3mm from its origin with the use of6-0silk to induce MI. The induction of MI was confirmed by a cardiac surface color change from reddish to a pale color and by ST-segment elevation documented by continuous electrocardiographic monitoring or left ventricular motion weakened. Sham-operated rats underwent similar surgery but without coronary artery ligation. Then closed thoracic cavity layer by layer, removed ventilator and tracheal intubation. To prevent the airway stegnosis and secretions lead to suffocation, the trachea and the neck incision were not closed. Warmed the rats with40W light bulb12h after surgery, observed the airway secretions4h after surgery and removed with suction tube in time. The results showed the success rate of making myocardial infarction model was98.6%, the survival rate of model group3weeks after surgery was88.75%and survival rate of sham-operated group was100%. We consummate the method of making rat model of myocardial infarction and improve the operation success rate and survival rate of rats.
3. The dynamic expression of ADAMTS4, ADAMTS8, versican and TIMP-3genes in rat myocardium after myocardial infarction
Chose250-300g male Wistar rats and made myocardial infarction model by the method mentioned above. pats were sacrificed anesthesia overdose at3h,6h,12h,24h,3days,7days,14days, or21days (n=5) after surgery and removed the heart rapidly. The infarcted myocardium below the ligature points were separated and put into the-70℃refrigerator. The ADAMTS4, ADAMTS8, versican and TIMP-3mRNA expression were analyzed by real-time fluorescence quantitative RT-PCR. ADAMTS4protein expression was analyzed by ELISA assay. The results showed ADAMTS4mRNA expression was significantly increased in the infarcted hearts6h and12h after MI compared with the level in sham-operated hearts (p<0.05), then decreased rapidly. While the relative level of ADAMTS8increased at6h, peaked at24h, remained high at3days(p<0.05),then decreased gradually. After myocardial infarction versican mRNA levels were significantly increased, peaked at3days and remain high for a long time. TIMP3expression levels decreased. ADAMTS4protein levels was significantly increased at6hours (p=0.026),12hours (p=0.003),24hours (p=0.002) and3days (p=0.009) after myocardial infarction.
ADAMTS4, ADAMTS8, versican, and TIMP-3mRNA expressed in rat heart after myocardial infarction and presented different dynamic trends. There are interrelationship among the expression of ADAMTS4, versican, and TIMP-3.
4. ADAMTS4expressed in endothelial cells and myocardial cells of the rat infarcted heart
Male Wistar rats (250-300g) were chosed to make myocardial infarction model. Rats were killed3days after surgery and the left ventricle were taken to produce frozen section with slice thickness5um. ADAMTS4, versican and aggrecan protein expression were detected by immunohistochemistry method. For the negative control group, we use the same dilution of rabbit IgG to handle slices. The results showed ADAMTS4expressed in myocardial cells at marginal zone of the infarcted area and did not express at normal myocardium and area away from infracted myocardium. At marginal zone of the infarcted area ADAMTS4expressed weakly and versican had strong expression in capillary endothelial cells. Aggrecan showed no expression in the cardiac tissue and vascular endothelium of MI group and sham-operation group. ADAMTS4and versican expression co-existed in endothelial cells hinted a link between them. The dynamic change may play a role in matrix remodeling after myocardial infarction.
5. The expression of ADAMTSs in endothelial cells stimulated with interleukin-1β
Selected human umbilical vein endothelial cells to subculture. Stimulated endothelial cells with2.5ng/ml,5ng/ml,10ng/ml,20ng/ml IL-1β respectively (n=5) and collected cells to extract total RNA and protein24hours after stimulation. Stimulated endothelial cells with10ng/ml IL-1β and collected cells to extract total RNA and protein respectively2h,6h,12h,24h,48h (n=5) after stimulation. Blank control group were set up. ADAMTS1,4,8,9,15mRNA expression were analyzed by RT-PCR and protein expression by Western-blot. The results showed after stimulation with IL-1β ADAMTS1,4,8,9,15mRNA expression were all increased, but the trends with time was different, ADAMTS4protein expression increased.
The results suggest that ADAMTSs involved in inflammation, the specific mechanism remains to be further clarified.
6. The change of coronary level of ADAMTS4and effect of percutaneous coronary intervention on ADAMTS4in patients with coronary artery disease
The81subjects were classified according to their coronary angiographic findings into control, simple, and complex groups. PCI was performed in35patients. Blood samples were taken from the coronary sinus. ADAMTS4and hs-CRP levels were measured by ELISA and turbidometry respectively. Results:ADAMTS4and hs-CRP showed higher level in complex group than in control and simple group (P<0.001). The ADAMTS4level showed a positive correlation with the hs-CRP level whether from all patients or from patients with CAD (r1=0.73, r2=0.763, P<0.01). The ADAMTS4value was higher in patients who underwent PCI than in those who did not (P<0.001), and the same was found regarding CRP (P=0.025).
Coronary ADAMTS4and hs-CRP levels elevated in patients with CAD and increased with the complexity of the lesions. The marked increase of ADAMTS4and hs-CRP after PCI in patients with CAD can be attributed to their release from the coronary atheroma secondary to the direct mechanical effect applied on the atheroma itself by balloon inflation and stent deployment.
基因表达分析在许多生命科学研究领域里的重要性日益增加,其研究的深入将为探索疾病相关基因、了解基因表达调控、解析生命奥秘、从而最终为人类服务大有裨益。反转录实时荧光聚合酶链反应是对特定信使RNA进行定量研究的最灵敏方法。为了分析特定信使RNA含量的差别,要用内部参照基因来进行定量。管家基因有几百种,目前,作为内参使用最广泛的管家基因是3磷酸甘油醛脱氢酶、β肌动蛋白、18SrRNA和28SrRNA。教条地使用一种管家基因或盲目参照不同实验对象及条件所使用的管家基因作为内参,一方面可能使基因表达的微小差异难以发现,另一方面可能导致错误甚至相反的结论。大鼠是一种常用制作心梗模型的实验动物,对于冠心病相关基因的研究使用大鼠做动物模型是许多实验室的首选。然而,心梗后大鼠内参基因选择问题还较少研究报道。
心肌梗死是威胁人类生命的重大疾病,心肌梗死动物模型对于研究人类冠心病的发病机制、病理生理改变以及对治疗方法的评估都具有重大意义。长期以来实验室研究是用结扎大鼠冠状动脉的方法来制备模型,但在实践中,由于麻醉和人工呼吸的使用不当、肺损伤以及左冠状动脉定位错误等各种原因,造成动物死亡或制模失败。因此提高存活率及制模成功率是解决心肌梗死动物模型的一个核心问题。我们对制作大鼠心梗模型的方法加以改进,明显提高了大鼠的存活率,较好地解决了大鼠心梗模型的制备问题。
急性心梗是引起人类死亡与残疾的主要疾病之一。心梗后心室重塑可导致心衰与死亡率的增加。在心室重塑过程中包括细胞外基质(ECM)分子的集聚和降解的动态变化。许多生物物质包括蛋白酶,蛋白酶抑制剂及生长因子等对ECM的重建起作用。其中基质金属蛋白酶(MMPS)表达的增加与激活也牵涉其中。除基质金属蛋白酶外,还有一些蛋白酶家族可降解细胞外基质。ADAMTS是一类分泌性金属蛋白酶,其主要结构包括解聚素,基质金属蛋白酶和血小板反应素基序。在人类中其家族已发现19个成员,涉及裂解多种蛋白聚糖,胶原的代谢,抗血管新生,VWF多聚体的降解,以及胚胎器官发育,生殖等多种功能。其中某些成员可与细胞外基质结合发挥作用。在颈动脉粥样硬化斑块及冠脉不稳定斑块中的富巨噬细胞区域ADAMTS4与8的表达增加,在动脉粥样硬化发展过程中,ADAMTS4的表达上调。Versican是聚集蛋白聚糖家族成员之一,在组织中广泛存在,参与伤口愈合及组织重塑过程。在冠脉结扎致心梗大鼠模型中其在心肌中表达并短暂升高,提示其参与了心梗后的炎症反应。ADAMTS4降解血管壁里的蛋白多聚糖versican,其作用位点在V1/V0versican的Glu1428-Ala1429。ADAMTS4能被内源性的抑制剂TIMPs阻断,ADAMTS4和ADAMTS5均能被TIMP-3有效抑制,而对TIMP-1,2,4却基本上不敏感。
近年来ADAMTS在炎症及动脉粥样硬化中的作用得到关注。免疫组化分析示ADAMTS1、4、5、8在人类颈动脉病变及冠脉粥样硬化斑块处表达,ADAMTS4、5、8与巨噬细胞共存而ADAMTS1与内皮细胞及平滑肌细胞共存。Versican在组织中广泛分布,亦存在于心脏中。versican在心梗后心脏中表达增高且其来源于渗出的单核细胞,提示其参与心梗后炎症反应。Aggrecan是一个软骨特异性的硫酸软骨素蛋白多聚糖,是ADAMTS的作用底物。ADAMTS4及5表现为较强的降解aggrecan活性。ADAMTS4在心梗后心脏中的具体表达部位,是否与versican在同一部位表达以及心梗后是否涉及aggrecan的表达,目前尚不明确。
在剪切应力作用下,人静脉内皮细胞及心脏微血管内皮细胞的ADAMTS1mRNA表达上调。在动脉粥样硬化斑块内膜及增殖/迁移的主动脉血管平滑肌细胞中,ADAMTS1的mRNA表达增高。应用INF-γ、TNF-α及IL-1β刺激巨噬细胞发现INF-Y刺激后ADAMTS4、7、8、9表达增加,ADAMTS1及17表达下降,而ADAMTS2、5、10不受其影响,TNF-α刺激后ADAMTS4、7、8表达增加,ADAMTS9轻微升高,IL-1β刺激对ADAMTS4、7、8表达无影响,而ADAMTS1及9在早期升高,说明ADAMTS家族中不同成员表达及调控机制存在差异。
以往研究提示,在动脉硬化发展及动脉粥样硬化斑块不稳定的过程中,ADAMTS4扮演着重要角色；ADAMTS4可以作为一种预示斑块不稳定及急性冠脉综合征严重程度的指标。经皮冠脉介入治疗(PCI)过程可以看作为机械挤压诱导斑块破裂的模型,因此研究PCI过程中的炎症反应或许可以为斑块不稳定机制提供线索。许多临床研究报道了冠心病患者PCI后的炎症反应。既然ADAMTS4已被证明是炎症调节酶且与动脉硬化的发展及斑块不稳定相关,我们猜测在冠心病患者冠脉循环中ADAMTS4水平可能升高且受PCI手术影响。
本研究用反转录实时荧光聚合酶链反应方法比较了四种管家基因在心梗大鼠心脏中的表达情况,采用GeNorm算法分析了哪些管家基因最适于心梗后大鼠基因表达研究。改进了大鼠心梗模型的制作方法,提高了手术成功率及大鼠存活率。探讨了ADAMTS4、ADAMTS8、versican、TIMP3在大鼠心梗后心脏中表达的动态变化、分布区域及可能机制,进一步认识急性心梗后局部ECM分子的变化及相互作用,为治疗提供新的理论依据。检测了IL-1β刺激内皮细胞后ADAMTSs的表达趋势。通过从冠状静脉窦取血检测了冠脉ADAMTS4及hs-CRP水平,并评估了PCI对ADAMTS4及hs-CRP水平的影响。本学位论文的主要研究内容及实验结果如下：
一、大鼠心梗模型中管家基因的选择
我们采用结扎冠脉前降支的方法制作心梗模型,结合实际工作,分析并挑选出使用广泛的4个标准管家基因：3磷酸甘油醛脱氢酶(GAPDH)、核糖体蛋白L13A(RPL13A)、β-肌动蛋白(ACTB)和结合区连接蛋白(ARBP),用反转录实时荧光聚合酶链反应方法比较了它们在心梗大鼠心脏中的表达情况,采用GeNorm算法分析哪些管家基因最适于心梗后大鼠基因表达的研究。结果示RPL13A、GAPDH、 ARBP及ACTB基因的M值分别为0.812、0.721、0.812和1.2,分析得出GAPDH及ARBP是在心梗模型中表达最稳定的基因。
二、大鼠心肌梗死模型制备的改进
选取健康雄性Wistar大鼠100只(体重230-270g),分为模型组(n=80)和假手术组(n=20)。3%戊巴比妥钠(40mg/kg)腹腔注射麻醉,气管切开,小动物呼吸机辅助呼吸,采用容量控制模式,潮气量3ml/100g,呼吸频率60-70次/min,吸呼比1：1。左侧胸部开胸,由第4肋间入胸,用乳突牵开器牵开肋骨,暴露心脏。在肺动脉圆锥和左心耳之间距主动脉根部约3mm处结扎左冠状动脉前降支。观察心电图变化,以肢体导联2个以上导联ST段抬高,或者肉眼观察结扎处下方大面积心肌表面变得苍白,周围瘀血征出现,室壁搏动减弱为结扎成功标准。假手术组大鼠只穿线不结扎。逐层关胸,撤除呼吸机,拔除气管插管。为防止气道狭窄及分泌物导致窒息,不缝合气管及颈部切口。术中至术后12h采用40W灯泡照射保暖,术后4h内注意观察气管切口处分泌物,及时用吸痰管清除。结果示心肌梗死模型制作的成功率为98.6%。模型组术后3周成活率为88.75%,假手术组存活率为100%。
通过对大鼠心梗模型制作方法的改进,提高了手术成功率及大鼠存活率。
三、大鼠心梗后心肌中ADAMTS4、ADAMTS8、versican、TIMP-3基因表达的动态变化
选择250-300g雄性Wistar大鼠。按前述方法制作心梗模型,设假手术组。分别于手术后0、3、6、12、24小时及3、7、14、21天(n=5)过量麻醉处死大鼠,迅速开胸摘取心脏,将结扎点下方梗死部位心肌分离切割,放入-70℃冰箱备用。用实时荧光定量RT-PCR方法分析梗死心肌中ADAMTS4、ADAMTS8、versican及TIMP-3mRNA表达,ELISA法检测(?)ADAMTS4的蛋白表达。结果示ADAMTS4表达在心梗后6及12小时明显升高(p<0.05),随后快速下降。而ADAMTS8则在心梗后6小时开始升高,至24小时达峰值,3天时仍持续在高水平(p<0.05),然后缓慢下降。心梗后versican mRNA水平显著增高,至3天时达峰值,且升高持续时间较长。TIMP3表达水平降低。ADAMTS4蛋白水平在心梗后6小时(p=0.026)、12小时(p=0.003)、24小时(p=0.002)及3天(p=0.009)显著增高。
ADAMTS4、ADAMTS8, versican及TIMP-3在心梗大鼠心脏中表达,且表现为不同的动态变化趋势。ADAMTS4、versican及TIMP-3的表达之间存在相互关系。
四、AAMTS4在梗死大鼠心脏血管内皮细胞及心肌细胞中表达
选择250-300g雄性Wistar大鼠制作心梗模型,设假手术组。手术后3天处死大鼠,取左心室制作冰冻切片。切片厚度为5um。免疫组织化学方法检测ADAMTS4、versican及aggrecan蛋白表达。对于阴性对照组,我们以等比稀释度用兔IgG处理切片。结果示ADAMTS4在梗死边缘区域心肌细胞中表达,在梗死区域及远离梗死区的正常心肌未见ADAMTS4表达。在梗死周围区域毛细血管内皮细胞ADAMTS4有弱表达,versican强表达。Aggrecan在心梗组及假手术组的心肌组织及血管内膜均未见表达。
ADAMTS4与versican共同表达于内皮细胞提示二者存在某种联系,其动态变化可能在心梗后基质重塑中起作用。
五、白介素1β刺激内皮细胞后ADAMTSs的表达
选取人脐静脉血管内皮细胞,以传代培养法培养细胞。分别以2.5ng/ml,5ng/ml,10ng/ml,20ng/ml浓度的IL-1β刺激内皮细胞,24h后收集细胞提取总RNA及蛋白。用10ng/ml IL-1β刺激内皮细胞,分别于刺激后2hh、6h、12h、24h、48h收集细胞提取总RNA及蛋白,设空白对照组,每组5个样本。RT-PCR测定ADAMTS1、4、8、9、15mRNA表达,Western-blot测定ADAMTS4蛋白表达。结果示IL-1β刺激内皮细胞后ADAMTS1、4、8、9、15mRNA表达均增高,但随时间变化趋势不同,ADAMTS4蛋白表达增高。
此结果提示ADAMTSs参与炎症反应,具体机制有待进一步阐明。
六、冠心病患者冠脉血中ADAMTS4水平的变化及经皮冠脉介入治疗对冠脉ADAMTS4水平的影响
根据冠脉造影结果,将81例患者分为对照组、简单病变组及复杂病变组,其中35例患者接受了PCI治疗。血液检测标本从冠状静脉窦获得。ADAMTS4及hs-CRP值分别通过ELISA及免疫浊度法检测。结果示复杂病变组ADAMTS4及hs-CRP值明显高于对照组及简单病变组(P<0.001)。所有研究对象及冠心病患者的ADAMTS4水平均与hs-CRP水平相关(r,=0.73, r2=0.763, P<0.01)。接受了PCI治疗的患者ADAMTS4值高于未接受PCI者(P<0.001), hs-CRP亦表现出相似结果(P=0.025)。
冠心病患者冠脉ADAMTS4及hs-CRP水平升高,且随病变复杂程度增加而升高。PCI后冠脉ADAMTS4及hs-CRP升高,其可能由PCI过程中球囊扩张或支架植入的机械挤压导致冠脉斑块破裂释放而出。
Background and objective
The importance of gene expression analysis in many areas of life science research is growing. Its in-depth study will benefit to explore disease-related genes, understand gene regulation, resolve the mysteries of life, and ultimately to service to humanity. Real-time fluorescence reverse transcription-polymerase chain reaction is the most sensitive method of studying specific messenger RNA quantitatively. To analyze the differences of the specific messenger RNA content, the internal reference gene is used to quantify. There are hundreds of housekeeping genes. At present, the most widely used internal reference housekeeping genes are GAPDH, β-actin,18SrRNA and28SrRNA. Using of one housekeeping gene singly or selection housekeeping genes blindly in accordance with different experimental subjects may make the small differences in gene expression difficult to find, on the other hand it may lead to errors or even opposite conclusions. Rat is a commonly used animal to be made myocardial infarction model. It is the first choice for many laboratories to use rats as animal model for coronary heart disease-related genes studies. However, there are fewer studies about the selection of reference genes in rats after myocardial infarction.
Myocardial infarction is a major disease to threat human life. Animal model of myocardial infarction is significant greatly for studying pathogenesis and pathophysiology of human coronary heart disease and evaluation of treatment methods. The method of coronary artery ligation is used to prepare the model ever since a long time ago. In practice, many reasons result in animal death or making model failure just as anesthesia, using respirator inadequately, lung injury and the left coronary artery positioning erroneously. Therefore, it is a core issue of improving survival and the success rate of making model to solve the question of animal model of myocardial infarction. We consummate the method of making rat model of myocardial infarction and improve the survival rate of rats significantly.
Acute myocardial infarction is a leading cause of death and disability in world. Ventricular remodeling after myocardial infarction can lead to heart failure and mortality. The dynamic change of extracellular matrix (ECM) accumulation and degradation is included in the process of ventricular remodeling. Many biological substances such as proteases, protease inhibitors and growth factors play a part in ECM reconstruction. The increased expression and activation of matrix metalloproteinases (MMPS) is also involved. In addition to matrix metalloproteinases, there are other protease families having the capacity to degrade the ECM. ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) is a group of secreted proteases that now incorporates19genes in humans, involving of cracking a variety of proteoglycans, collagen metabolism, anti-angiogenesis, VWF polymer degradation, the embryonic organ development, reproduction and other functions. Some of ADAMTS can bind to the ECM. ADAMTS4and8have been shown inflammatory regulated enzymes expressed in macrophage-rich areas of carotid atherosclerosis plaque and coronary instable plaque. ADAMTS4expression increases in the development of atherosclerosis process. Versican is a member of the aggrecan family, spread in the organization widely and participate in the process of wound healing and tissue remodeling. Versican expressed and incrased temporally in the myocardium of coronary artery ligation-induced myocardial infarction rat model, suggesting their involvement in the inflammatory response after myocardial infarction. ADAMTS4degrade versican of the vessel wall at V1/V0versican's Glu1428-Ala1429site. ADAMTS4can be blocked by the endogenous inhibitors TIMPs. ADAMTS4and ADAMTS5can be effectively suppressed by TIMP-3, while not be sensitive to TIMP-1,2and4.
In recent years the role of ADAMTS in inflammation and atherosclerosis is attentioned. Immunohistochemical analysis showed that ADAMTS1,4,5and8expressed in the human carotid artery plaque and coronary atherosclerotic plaque. ADAMTS4,5and8co-exist with macrophages and ADAMTS1co-exist with smooth muscle cells and endothelial cells. Versican is widely distributed in the organization and also present in the heart. Versican expression increased in the myocardial infarction heart and came from the mononuclear cells, suggesting their participation in the inflammatory response after myocardial infarction. Aggrecan is a cartilage-specific protein of chondroitin sulfate polysaccharide and is a substrate of ADAMTS. ADAMTS4and5showed strong activity in the degradation of aggrecan. It is not clear about the expression position of ADAMTS4in the heart after myocardial infarction. Whether versican express in the same area of ADAMTS4and whether aggrecan express in the heart after myocardial infarction are unclear. The shear stress up regulated the ADAMTS1mRNA expression in the human vein endothelial cells and cardiac microvascular endothelial cells. In the atherosclerotic plaque intima and proliferation/migration vascular smooth muscle cells, ADAMTS1mRNA expression increased. Stimulation macrophages with INF-γ, TNF-α and IL-1γ showed ADAMTS4,7,8,9expression increased, ADAMTS1and17expression decreased, while ADAMTS2,5,10is not affected after stimulation with INF-γ. ADAMTS4,7and8expressions increased, ADAMTS9slightly elevated after stimulation with TNF-α. ADAMTS4,7and8expressions were not affected, ADAMTS1and9increased in the early phase after stimulation with IL-1β. All of these indicate there are different expression and regulation mechanisms of ADAMTS family members. Previous observations suggest that (a) ADAMTS4plays an important role in the development of atherosclerosis and instability of atherosclerotic plaques;(b) ADAMTS4may serve as a marker of plaque destabilization and for predicting the severity of ACS. Percutaneous coronary intervention (PCI) procedure can be regarded as a model for mechanical induced plaque rupture. Thus studies on the inflammatory reaction during PCI could give information on mechanisms of plaque destabilization. Many clinical studies have evaluated the inflammatory response after PCI in patients with CAD. Since ADAMTS4has been proved to be inflammatory regulated enzymes and involved in the development of atherosclerosis and instability of atherosclerotic plaques, we speculated ADAMTS4level would elevate in the coronary circulation of patients with CAD and be influenced by PCI.
In this study, we compared four housekeeping genes'expression in hearts of myocardial infarction rats with real-time fluorescence reverse transcription-polymerase chain reaction method and analyzed which is the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm algorithms. We consummate the method of making rat model of myocardial infarction and improve the operation success rate and survival rate of rats. The dynamic expression changes and distribution area of ADAMTS4, ADAMTS8, versican, TIMP3and the possible mechanisms were studied. The changes and interactions of local ECM molecules after acute myocardial infarction were further understanded and a new theoretical basis for treatment was provided. ADAMTSs expression trend in the endothelial cells after stimulation withlL-1β were detected. We detected coronary ADAMTS4and high-sensitivity C-reactive levels using coronary sinus sampling and assessed the effect of PCI on ADAMTS4and hs-CRP levels. The main research contents and results are as follows:
1. Selection of housekeeping genes in rat model of myocardial infarction We made myocardial infarction models using the anterior descending coronary artery ligation method,combined with practical work, analyzed and selected the four widely used standard housekeeping genes:
glyceraldehydes-3-phosphate dehydrogenase (GAPDH), ribosomal protein L13A (RPL13A), beta-actin (ACTB) and acidic ribosomal phosphoprotein PO (ARBP). We compared their expression in the rat heart after myocardial infarction using real-time fluorescent reverse transcription polymerase chain reaction, analyzed which is the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm algorithms. The results showed the M values of RPL13A, GAPDH, ARBP and ACTB gene were0.812,0.721,0.812and1.2respectively. The results suggest that GAPDH and ARBP are the most stable genes for the rat myocardial infarction model.
2. Improvement of the rat myocardial infarction model
One hundred male Wistar rats (weight230-270g) were divided into model group (n=80) and sham-operatered group (n=20). The rats were anaesthetised with sodium pentobarbital (40mg/kg, intraperitoneally), operated tracheostomy, then ventilated with an automatic breathing apparatus using the volume control mode(tidal volume,3ml/100g, respiratory rate,60-70cycles/min, respiratory ratio,1:1). An left anterior thoracotomy was performed at the fourth intercostal space and rib was retracted with the mastoid retractor for exposing the heart. The left anterior descending coronary artery was ligated approximately3mm from its origin with the use of6-0silk to induce MI. The induction of MI was confirmed by a cardiac surface color change from reddish to a pale color and by ST-segment elevation documented by continuous electrocardiographic monitoring or left ventricular motion weakened. Sham-operated rats underwent similar surgery but without coronary artery ligation. Then closed thoracic cavity layer by layer, removed ventilator and tracheal intubation. To prevent the airway stegnosis and secretions lead to suffocation, the trachea and the neck incision were not closed. Warmed the rats with40W light bulb12h after surgery, observed the airway secretions4h after surgery and removed with suction tube in time. The results showed the success rate of making myocardial infarction model was98.6%, the survival rate of model group3weeks after surgery was88.75%and survival rate of sham-operated group was100%. We consummate the method of making rat model of myocardial infarction and improve the operation success rate and survival rate of rats.
3. The dynamic expression of ADAMTS4, ADAMTS8, versican and TIMP-3genes in rat myocardium after myocardial infarction
Chose250-300g male Wistar rats and made myocardial infarction model by the method mentioned above. pats were sacrificed anesthesia overdose at3h,6h,12h,24h,3days,7days,14days, or21days (n=5) after surgery and removed the heart rapidly. The infarcted myocardium below the ligature points were separated and put into the-70℃refrigerator. The ADAMTS4, ADAMTS8, versican and TIMP-3mRNA expression were analyzed by real-time fluorescence quantitative RT-PCR. ADAMTS4protein expression was analyzed by ELISA assay. The results showed ADAMTS4mRNA expression was significantly increased in the infarcted hearts6h and12h after MI compared with the level in sham-operated hearts (p<0.05), then decreased rapidly. While the relative level of ADAMTS8increased at6h, peaked at24h, remained high at3days(p<0.05),then decreased gradually. After myocardial infarction versican mRNA levels were significantly increased, peaked at3days and remain high for a long time. TIMP3expression levels decreased. ADAMTS4protein levels was significantly increased at6hours (p=0.026),12hours (p=0.003),24hours (p=0.002) and3days (p=0.009) after myocardial infarction.
ADAMTS4, ADAMTS8, versican, and TIMP-3mRNA expressed in rat heart after myocardial infarction and presented different dynamic trends. There are interrelationship among the expression of ADAMTS4, versican, and TIMP-3.
4. ADAMTS4expressed in endothelial cells and myocardial cells of the rat infarcted heart
Male Wistar rats (250-300g) were chosed to make myocardial infarction model. Rats were killed3days after surgery and the left ventricle were taken to produce frozen section with slice thickness5um. ADAMTS4, versican and aggrecan protein expression were detected by immunohistochemistry method. For the negative control group, we use the same dilution of rabbit IgG to handle slices. The results showed ADAMTS4expressed in myocardial cells at marginal zone of the infarcted area and did not express at normal myocardium and area away from infracted myocardium. At marginal zone of the infarcted area ADAMTS4expressed weakly and versican had strong expression in capillary endothelial cells. Aggrecan showed no expression in the cardiac tissue and vascular endothelium of MI group and sham-operation group. ADAMTS4and versican expression co-existed in endothelial cells hinted a link between them. The dynamic change may play a role in matrix remodeling after myocardial infarction.
5. The expression of ADAMTSs in endothelial cells stimulated with interleukin-1β
Selected human umbilical vein endothelial cells to subculture. Stimulated endothelial cells with2.5ng/ml,5ng/ml,10ng/ml,20ng/ml IL-1β respectively (n=5) and collected cells to extract total RNA and protein24hours after stimulation. Stimulated endothelial cells with10ng/ml IL-1β and collected cells to extract total RNA and protein respectively2h,6h,12h,24h,48h (n=5) after stimulation. Blank control group were set up. ADAMTS1,4,8,9,15mRNA expression were analyzed by RT-PCR and protein expression by Western-blot. The results showed after stimulation with IL-1β ADAMTS1,4,8,9,15mRNA expression were all increased, but the trends with time was different, ADAMTS4protein expression increased.
The results suggest that ADAMTSs involved in inflammation, the specific mechanism remains to be further clarified.
6. The change of coronary level of ADAMTS4and effect of percutaneous coronary intervention on ADAMTS4in patients with coronary artery disease
The81subjects were classified according to their coronary angiographic findings into control, simple, and complex groups. PCI was performed in35patients. Blood samples were taken from the coronary sinus. ADAMTS4and hs-CRP levels were measured by ELISA and turbidometry respectively. Results:ADAMTS4and hs-CRP showed higher level in complex group than in control and simple group (P<0.001). The ADAMTS4level showed a positive correlation with the hs-CRP level whether from all patients or from patients with CAD (r1=0.73, r2=0.763, P<0.01). The ADAMTS4value was higher in patients who underwent PCI than in those who did not (P<0.001), and the same was found regarding CRP (P=0.025).
Coronary ADAMTS4and hs-CRP levels elevated in patients with CAD and increased with the complexity of the lesions. The marked increase of ADAMTS4and hs-CRP after PCI in patients with CAD can be attributed to their release from the coronary atheroma secondary to the direct mechanical effect applied on the atheroma itself by balloon inflation and stent deployment.
引文
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