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

发布时间：2017-12-30 22:17

本文关键词：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　出处：《山东大学》2012年博士论文　论文类型：学位论文

【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(p0.05),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(p0.05),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(P0.001)。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, P0.01)。接受了PCI治疗的患者ADAMTS4值高于未接受PCI者(P0.001), 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。
[Abstract]:Background and purpose of research
Gene expression analysis is important in many fields of life science research is increasing, further research will explore the disease related genes, to understand the regulation of gene expression, analysis of the mysteries of life and ultimately serve mankind. Be of great advantage of reverse transcription real-time polymerase chain reaction is the most sensitive method for quantitative study of specific messenger RNA in order to analyze. Specific messenger RNA content difference, to quantitatively by internal reference genes. There are hundreds of housekeeping genes as reference, at present, is the most widely used housekeeping gene glyceraldehyde 3 phosphate dehydrogenase, beta actin, 18SrRNA and 28SrRNA. use a dogma of housekeeping genes or blindly refer to housekeeping genes used by different experimental objects and conditions as a reference, one hand may make small differences in gene expression are difficult to find, on the other hand may lead to erroneous or even opposite conclusions The rat is a common experimental animal model of myocardial infarction for production, research on genes associated with coronary heart disease using the rat animal model is preferred in many laboratories. However, the problem is less reported rat reference genes after myocardial infarction.
Myocardial infarction is a major disease that threatens human life, animal model of myocardial infarction in the pathogenesis of human disease, is of great significance to the pathophysiological changes and to evaluate the treatment methods. Long laboratory research is to prepare the model with the method of coronary artery ligation in rats, but in practice, due to improper use of narcotic and artificial respiration, lung injury and left coronary artery positioning errors and other reasons, resulting in the death of the animal or mold failure. Therefore a key problem to improve survival rate and the success rate of model is to solve the animal model of myocardial infarction. We made the rat model of myocardial infarction methods improved, significantly improved the survival rate rats, effectively solves the problem of preparing rat model of myocardial infarction.
Acute myocardial infarction is one of the main diseases causing human death and disability. After myocardial infarction ventricular remodeling and heart failure can lead to increased mortality. In the ventricular remodeling process including extracellular matrix (ECM) dynamic changes of molecular aggregation and degradation. Many biological substances including protease, protease inhibitors and reconstruction of growth factors on ECM which play a role. The matrix metalloproteinase (MMPS) expression with increased activation is also involved. In addition to matrix metalloproteinases, and some proteases can degrade the extracellular matrix.ADAMTS is a secretory protein metal enzyme, which is mainly composed of a disintegrin and metalloproteinase gene sequence and platelet response. The family has been found 19 members in humans, involving various cracking proteoglycan, collagen metabolism, anti angiogenesis, degradation of VWF oligomers, and embryonic organ development, a variety of reproduction etc. Function. Some members with extracellular matrix binding. Play a role in carotid atherosclerotic plaque and unstable coronary plaque in ADAMTS4 macrophage rich regions and 8 increased expression in the development of atherosclerosis in the process, the upregulation of the expression of.Versican ADAMTS4 is one of the members of the family aggregation of proteoglycans, widely exists in the organization, and participate in wound healing the tissue remodeling process. In the model by coronary artery ligation in rats with myocardial infarction in the myocardial expression and transient increase, the inflammatory reaction of.ADAMTS4 degradation of its participation in the vascular wall after myocardial infarction in aggrecan versican, site of action can be endogenous inhibitor TIMPs in V1/V0versican Glu1428-Ala1429.ADAMTS4, ADAMTS4 and ADAMTS5 can be TIMP-3 effective suppression of TIMP-1,2,4 is basically not sensitive.
In recent years, the role of ADAMTS in inflammation and atherosclerosis in entions. Immunohistochemical analysis showed that the expression of ADAMTS1,4,5,8 in human carotid artery lesions and coronary atherosclerotic plaques, ADAMTS4,5,8 and macrophage coexistence ADAMTS1 and endothelial cells and smooth muscle cells of the coexistence of.Versican are widely distributed in tissues, also exists in the heart of.Versican expression in mononuclear cells increased and the it comes from the exudate in the heart after myocardial infarction, inflammatory reaction in.Aggrecan after myocardial infarction is a cartilage specific chondroitin sulfate proteoglycan, is a substrate of.ADAMTS4 ADAMTS and 5 showed strong.ADAMTS4 degradation activity of aggrecan after myocardial infarction in the heart specific expression sites, and in the same versican site expression after myocardial infarction and is involved in the expression of aggrecan is unclear.
The effect of shear stress, up-regulated expression of human umbilical vein endothelial cells and cardiac microvascular endothelial cells. ADAMTS1mRNA in atherosclerotic plaque intima and proliferation / migration of vascular smooth muscle cells, the ADAMTS1 expression of mRNA. Application of INF- gamma, TNF- alpha and IL-1 beta stimulated macrophages was found after INF-Y stimulation increased ADAMTS4,7,8,9 expression, decreased the expression of ADAMTS1 and 17 ADAMTS2,5,10, and is not affected by it, TNF- stimulated the expression of ADAMTS4,7,8 increased after ADAMTS9, slightly elevated, IL-1 stimulation had no effect on the expression of ADAMTS4,7,8, ADAMTS1 and 9 increased in the early stage, indicating the existence of different expression and regulation mechanism of ADAMTS family members in difference.
Previous studies suggest that in the process of atherosclerosis and development of atherosclerotic plaque instability, ADAMTS4 plays an important role; ADAMTS4 can be used as a sign of plaque instability and acute coronary syndrome severity. Percutaneous coronary intervention (PCI) process can be viewed as a mechanical extrusion induced plaque rupture model, so the research the inflammatory reaction in the process of PCI may provide clues for the mechanism of plaque instability. Many clinical studies reported the inflammatory reaction in patients with coronary heart disease after PCI. Since ADAMTS4 has been shown to be the development and regulation of enzyme and plaque inflammation and atherosclerosis is not stable, we guess at the level of ADAMTS4 in patients with coronary heart disease in the coronary circulation may be increased and the effect of PCI surgery.
This study used reverse transcription real-time polymerase chain reaction method to compare the expression of four housekeeping genes in the heart of myocardial infarction in rats, using GeNorm algorithm to analyze what the most suitable housekeeping gene expression of gene in rats after myocardial infarction. The improved rat model of myocardial infarction, improve the success rate of surgery and rats. The survival rate. The effects of ADAMTS4, ADAMTS8, versican, dynamic changes of the expression of TIMP3 in rat heart after myocardial infarction in the distribution area and the possible mechanism, to further understand the changes after acute myocardial infarction and local ECM molecular interaction, provide a new theoretical basis for the treatment. To detect the expression trend of ADAMTSs IL-1 beta stimulated endothelial cells after. The blood taken from the coronary sinus to detect coronary ADAMTS4 and hs-CRP levels, and to evaluate the effect of PCI on ADAMTS4 and hs-CRP. In this thesis, the main research contents and experimental results such as Under the:
The selection of housekeeper genes in rat myocardial infarction model
We used the anterior descending coronary artery ligation method to produce myocardial infarction, combined with the actual work, we analyze and choose the widely used 4 criteria: housekeeping gene glyceraldehyde 3 phosphate dehydrogenase (GAPDH), ribosomal protein L13A (RPL13A), beta actin (ACTB) and binding domain connected protein (ARBP), their expression in the heart of myocardial infarction in rats was studied by reverse transcription polymerase chain reaction method, using GeNorm algorithm for analysis of gene expression in rats after myocardial infarction which is most suitable for the housekeeping gene. The results showed that RPL13A, GAPDH, ARBP and ACTB gene M were 0.812,0.721,0.812 and 1.2, GAPDH analysis and ARBP is the most stable gene expression in myocardial infarction model.
Two, improvement of rat model of myocardial infarction
100 healthy male Wistar rats (weight 230-270g) were selected, and divided into model group (n=80) and sham operation group (n=20.3%) sodium pentobarbital (40mg/kg) intraperitoneal injection of anesthesia, tracheotomy, small animal ventilator assisted breathing, using the volume control mode, tidal volume 3ml/100g, the respiratory rate of 60-70 /min, breath 1:1. on the left chest chest, from the fourth intercostal space, with mastoid retractor ribs, expose the heart. Between the pulmonary artery and the left atrial appendage from the aortic root cone about 3mm ligation of the left anterior descending coronary artery. To observe the changes of electrocardiogram, limb lead more than 2 ST elevation, or below the eye ligation of large area myocardial surface pale, blood stasis syndrome appeared around the wall, beating weakened into a successful ligation standard. Sham group rats without coronary artery ligation. By closed chest, removal of the ventilator, gas extraction tube to prevent airway stenosis. Secretions cause suffocation, trachea and neck incision without suture. During the operation to 12h after operation using 40W bulb to keep warm, observe the incision of trachea secretions within 4h after operation, timely use of sputum suction tube removed. Results showed the success ratio of the model for myocardial infarction 98.6%. group 3 weeks after operation. The survival rate was 88.75%. The sham operation group the survival rate was 100%.
By improving the method of making the rat myocardial infarction model, the success rate of the operation and the survival rate of rats were improved.
Three, dynamic changes of ADAMTS4, ADAMTS8, versican and TIMP-3 gene expression in myocardium after myocardial infarction in rats
250-300g male Wistar rats. Myocardial infarction model produced by the aforementioned method, respectively. The sham operation group at 0,3,6,12,24 hours after surgery and 3,7,14,21 days (n=5) rats were killed by overdose anesthesia, thoracotomy rapid removal of the heart, cardiac infarction below the ligation point separation cut into -70 C refrigerator. Analysis of ADAMTS4, myocardial infarction in ADAMTS8 using real-time fluorescence quantitative RT-PCR method, the expression of versican and TIMP-3mRNA, ELISA assay (?) the expression of ADAMTS4 protein. Results showed that the expression of ADAMTS4 in myocardial infarction after 6 hours and 12 hours were significantly increased (P0.05), followed by rapid decline. While ADAMTS8 after myocardial infarction increased at 6 hour, 24 hours to reach peak, 3 the day continued at a high level (P0.05), and then decreased slowly. The level of mRNA versican increased significantly after myocardial infarction, and increased to 3 at the peak, a longer duration of.TIMP3 expression level of.ADAMTS4 protein levels were reduced in myocardial infarction The following 6 hours (p=0.026), 12 hours (p=0.003), 24 hours (p=0.002) and 3 days (p=0.009) were significantly higher.
ADAMTS4, ADAMTS8, versican and TIMP-3 were expressed in the heart of myocardial infarction rats, and showed different dynamic trends. There were correlations between.ADAMTS4, versican and TIMP-3 expression.
Four, AAMTS4 expression in the cardiac vascular endothelial cells and cardiac myocytes of infarcted rats
250-300g male Wistar rats model of myocardial infarction, the sham operation group. Rats were sacrificed 3 days after surgery, the left ventricle made frozen sections. The slice thickness of 5um. for ADAMTS4 was detected by immunohistochemistry, the expression of versican and aggrecan protein. The negative control group, we compared the dilution of rabbit IgG slices. The results showed that the expression of ADAMTS4 in the infarct border zone of myocardial cells, the expression in the infarction region and far from the infarct area of normal myocardium. No ADAMTS4 weak expression in the peri infarct area of capillary endothelial cells ADAMTS4, versican strong expression of.Aggrecan in myocardial infarction group and sham operation group of myocardial tissue and the vascular endothelium showed no expression.
The expression of ADAMTS4 and versican in endothelial cells suggests that there are some connections between the two, and the dynamic changes may play a role in the remodeling of the matrix after the myocardial infarction.
Five, the expression of ADAMTSs after interleukin 1 beta stimulation of endothelial cells
Selection of human umbilical vein endothelial cells, the passage cultured cells. In 2.5ng/ml, 5ng/ml, 10ng/ml, 20ng/ml concentration of IL-1 beta stimulated endothelial cells, 24h cells were collected to extract total RNA and protein. 10ng/ml IL-1 beta stimulated endothelial cells were stimulated following 2hh, 6h, 12h, 24h, 48h collection the cell extract total RNA and protein, a blank control group, each group of 5 samples of.RT-PCR ADAMTS1,4,8,9,15mRNA expression was detected by Western-blot assay, the expression of ADAMTS4 protein. The results showed that IL-1 beta stimulated endothelial cells after the expression of ADAMTS1,4,8,9,15mRNA was increased, but the different trends over time, the expression of ADAMTS4 protein increased.
The results suggest that ADAMTSs is involved in the inflammatory response, and the specific mechanisms need to be further elucidated.
Six, changes in the level of ADAMTS4 in coronary artery blood and percutaneous coronary intervention in patients with coronary heart disease

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R341

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