本文關(guān)鍵詞：大鼠心梗模型中管家基因的選擇及ADAMTSs在心梗后心肌中的表達(dá)及機(jī)制　出處：《山東大學(xué)》2012年博士論文　論文類型：學(xué)位論文

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【摘要】：研究背景及目的 基因表達(dá)分析在許多生命科學(xué)研究領(lǐng)域里的重要性日益增加,其研究的深入將為探索疾病相關(guān)基因、了解基因表達(dá)調(diào)控、解析生命奧秘、從而最終為人類服務(wù)大有裨益。反轉(zhuǎn)錄實(shí)時(shí)熒光聚合酶鏈反應(yīng)是對(duì)特定信使RNA進(jìn)行定量研究的最靈敏方法。為了分析特定信使RNA含量的差別,要用內(nèi)部參照基因來進(jìn)行定量。管家基因有幾百種,目前,作為內(nèi)參使用最廣泛的管家基因是3磷酸甘油醛脫氫酶、β肌動(dòng)蛋白、18SrRNA和28SrRNA。教條地使用一種管家基因或盲目參照不同實(shí)驗(yàn)對(duì)象及條件所使用的管家基因作為內(nèi)參,一方面可能使基因表達(dá)的微小差異難以發(fā)現(xiàn),另一方面可能導(dǎo)致錯(cuò)誤甚至相反的結(jié)論。大鼠是一種常用制作心梗模型的實(shí)驗(yàn)動(dòng)物,對(duì)于冠心病相關(guān)基因的研究使用大鼠做動(dòng)物模型是許多實(shí)驗(yàn)室的首選。然而,心梗后大鼠內(nèi)參基因選擇問題還較少研究報(bào)道。 心肌梗死是威脅人類生命的重大疾病,心肌梗死動(dòng)物模型對(duì)于研究人類冠心病的發(fā)病機(jī)制、病理生理改變以及對(duì)治療方法的評(píng)估都具有重大意義。長期以來實(shí)驗(yàn)室研究是用結(jié)扎大鼠冠狀動(dòng)脈的方法來制備模型,但在實(shí)踐中,由于麻醉和人工呼吸的使用不當(dāng)、肺損傷以及左冠狀動(dòng)脈定位錯(cuò)誤等各種原因,造成動(dòng)物死亡或制模失敗。因此提高存活率及制模成功率是解決心肌梗死動(dòng)物模型的一個(gè)核心問題。我們對(duì)制作大鼠心梗模型的方法加以改進(jìn),明顯提高了大鼠的存活率,較好地解決了大鼠心梗模型的制備問題。 急性心梗是引起人類死亡與殘疾的主要疾病之一。心梗后心室重塑可導(dǎo)致心衰與死亡率的增加。在心室重塑過程中包括細(xì)胞外基質(zhì)(ECM)分子的集聚和降解的動(dòng)態(tài)變化。許多生物物質(zhì)包括蛋白酶,蛋白酶抑制劑及生長因子等對(duì)ECM的重建起作用。其中基質(zhì)金屬蛋白酶(MMPS)表達(dá)的增加與激活也牽涉其中。除基質(zhì)金屬蛋白酶外,還有一些蛋白酶家族可降解細(xì)胞外基質(zhì)。ADAMTS是一類分泌性金屬蛋白酶,其主要結(jié)構(gòu)包括解聚素,基質(zhì)金屬蛋白酶和血小板反應(yīng)素基序。在人類中其家族已發(fā)現(xiàn)19個(gè)成員,涉及裂解多種蛋白聚糖,膠原的代謝,抗血管新生,VWF多聚體的降解,以及胚胎器官發(fā)育,生殖等多種功能。其中某些成員可與細(xì)胞外基質(zhì)結(jié)合發(fā)揮作用。在頸動(dòng)脈粥樣硬化斑塊及冠脈不穩(wěn)定斑塊中的富巨噬細(xì)胞區(qū)域ADAMTS4與8的表達(dá)增加,在動(dòng)脈粥樣硬化發(fā)展過程中,ADAMTS4的表達(dá)上調(diào)。Versican是聚集蛋白聚糖家族成員之一,在組織中廣泛存在,參與傷口愈合及組織重塑過程。在冠脈結(jié)扎致心梗大鼠模型中其在心肌中表達(dá)并短暫升高,提示其參與了心梗后的炎癥反應(yīng)。ADAMTS4降解血管壁里的蛋白多聚糖versican,其作用位點(diǎn)在V1/V0versican的Glu1428-Ala1429。ADAMTS4能被內(nèi)源性的抑制劑TIMPs阻斷,ADAMTS4和ADAMTS5均能被TIMP-3有效抑制,而對(duì)TIMP-1,2,4卻基本上不敏感。 近年來ADAMTS在炎癥及動(dòng)脈粥樣硬化中的作用得到關(guān)注。免疫組化分析示ADAMTS1、4、5、8在人類頸動(dòng)脈病變及冠脈粥樣硬化斑塊處表達(dá),ADAMTS4、5、8與巨噬細(xì)胞共存而ADAMTS1與內(nèi)皮細(xì)胞及平滑肌細(xì)胞共存。Versican在組織中廣泛分布,亦存在于心臟中。versican在心梗后心臟中表達(dá)增高且其來源于滲出的單核細(xì)胞,提示其參與心梗后炎癥反應(yīng)。Aggrecan是一個(gè)軟骨特異性的硫酸軟骨素蛋白多聚糖,是ADAMTS的作用底物。ADAMTS4及5表現(xiàn)為較強(qiáng)的降解aggrecan活性。ADAMTS4在心梗后心臟中的具體表達(dá)部位,是否與versican在同一部位表達(dá)以及心梗后是否涉及aggrecan的表達(dá),目前尚不明確。 在剪切應(yīng)力作用下,人靜脈內(nèi)皮細(xì)胞及心臟微血管內(nèi)皮細(xì)胞的ADAMTS1mRNA表達(dá)上調(diào)。在動(dòng)脈粥樣硬化斑塊內(nèi)膜及增殖/遷移的主動(dòng)脈血管平滑肌細(xì)胞中,ADAMTS1的mRNA表達(dá)增高。應(yīng)用INF-γ、TNF-α及IL-1β刺激巨噬細(xì)胞發(fā)現(xiàn)INF-Y刺激后ADAMTS4、7、8、9表達(dá)增加,ADAMTS1及17表達(dá)下降,而ADAMTS2、5、10不受其影響,TNF-α刺激后ADAMTS4、7、8表達(dá)增加,ADAMTS9輕微升高,IL-1β刺激對(duì)ADAMTS4、7、8表達(dá)無影響,而ADAMTS1及9在早期升高,說明ADAMTS家族中不同成員表達(dá)及調(diào)控機(jī)制存在差異。 以往研究提示,在動(dòng)脈硬化發(fā)展及動(dòng)脈粥樣硬化斑塊不穩(wěn)定的過程中,ADAMTS4扮演著重要角色；ADAMTS4可以作為一種預(yù)示斑塊不穩(wěn)定及急性冠脈綜合征嚴(yán)重程度的指標(biāo)。經(jīng)皮冠脈介入治療(PCI)過程可以看作為機(jī)械擠壓誘導(dǎo)斑塊破裂的模型,因此研究PCI過程中的炎癥反應(yīng)或許可以為斑塊不穩(wěn)定機(jī)制提供線索。許多臨床研究報(bào)道了冠心病患者PCI后的炎癥反應(yīng)。既然ADAMTS4已被證明是炎癥調(diào)節(jié)酶且與動(dòng)脈硬化的發(fā)展及斑塊不穩(wěn)定相關(guān),我們猜測在冠心病患者冠脈循環(huán)中ADAMTS4水平可能升高且受PCI手術(shù)影響。 本研究用反轉(zhuǎn)錄實(shí)時(shí)熒光聚合酶鏈反應(yīng)方法比較了四種管家基因在心梗大鼠心臟中的表達(dá)情況,采用GeNorm算法分析了哪些管家基因最適于心梗后大鼠基因表達(dá)研究。改進(jìn)了大鼠心梗模型的制作方法,提高了手術(shù)成功率及大鼠存活率。探討了ADAMTS4、ADAMTS8、versican、TIMP3在大鼠心梗后心臟中表達(dá)的動(dòng)態(tài)變化、分布區(qū)域及可能機(jī)制,進(jìn)一步認(rèn)識(shí)急性心梗后局部ECM分子的變化及相互作用,為治療提供新的理論依據(jù)。檢測了IL-1β刺激內(nèi)皮細(xì)胞后ADAMTSs的表達(dá)趨勢(shì)。通過從冠狀靜脈竇取血檢測了冠脈ADAMTS4及hs-CRP水平,并評(píng)估了PCI對(duì)ADAMTS4及hs-CRP水平的影響。本學(xué)位論文的主要研究內(nèi)容及實(shí)驗(yàn)結(jié)果如下： 一、大鼠心梗模型中管家基因的選擇 我們采用結(jié)扎冠脈前降支的方法制作心梗模型,結(jié)合實(shí)際工作,分析并挑選出使用廣泛的4個(gè)標(biāo)準(zhǔn)管家基因：3磷酸甘油醛脫氫酶(GAPDH)、核糖體蛋白L13A(RPL13A)、β-肌動(dòng)蛋白(ACTB)和結(jié)合區(qū)連接蛋白(ARBP),用反轉(zhuǎn)錄實(shí)時(shí)熒光聚合酶鏈反應(yīng)方法比較了它們?cè)谛墓４笫笮呐K中的表達(dá)情況,采用GeNorm算法分析哪些管家基因最適于心梗后大鼠基因表達(dá)的研究。結(jié)果示RPL13A、GAPDH、 ARBP及ACTB基因的M值分別為0.812、0.721、0.812和1.2,分析得出GAPDH及ARBP是在心梗模型中表達(dá)最穩(wěn)定的基因。 二、大鼠心肌梗死模型制備的改進(jìn) 選取健康雄性Wistar大鼠100只(體重230-270g),分為模型組(n=80)和假手術(shù)組(n=20)。3%戊巴比妥鈉(40mg/kg)腹腔注射麻醉,氣管切開,小動(dòng)物呼吸機(jī)輔助呼吸,采用容量控制模式,潮氣量3ml/100g,呼吸頻率60-70次/min,吸呼比1：1。左側(cè)胸部開胸,由第4肋間入胸,用乳突牽開器牽開肋骨,暴露心臟。在肺動(dòng)脈圓錐和左心耳之間距主動(dòng)脈根部約3mm處結(jié)扎左冠狀動(dòng)脈前降支。觀察心電圖變化,以肢體導(dǎo)聯(lián)2個(gè)以上導(dǎo)聯(lián)ST段抬高,或者肉眼觀察結(jié)扎處下方大面積心肌表面變得蒼白,周圍瘀血征出現(xiàn),室壁搏動(dòng)減弱為結(jié)扎成功標(biāo)準(zhǔn)。假手術(shù)組大鼠只穿線不結(jié)扎。逐層關(guān)胸,撤除呼吸機(jī),拔除氣管插管。為防止氣道狹窄及分泌物導(dǎo)致窒息,不縫合氣管及頸部切口。術(shù)中至術(shù)后12h采用40W燈泡照射保暖,術(shù)后4h內(nèi)注意觀察氣管切口處分泌物,及時(shí)用吸痰管清除。結(jié)果示心肌梗死模型制作的成功率為98.6%。模型組術(shù)后3周成活率為88.75%,假手術(shù)組存活率為100%。 通過對(duì)大鼠心梗模型制作方法的改進(jìn),提高了手術(shù)成功率及大鼠存活率。 三、大鼠心梗后心肌中ADAMTS4、ADAMTS8、versican、TIMP-3基因表達(dá)的動(dòng)態(tài)變化 選擇250-300g雄性Wistar大鼠。按前述方法制作心梗模型,設(shè)假手術(shù)組。分別于手術(shù)后0、3、6、12、24小時(shí)及3、7、14、21天(n=5)過量麻醉處死大鼠,迅速開胸摘取心臟,將結(jié)扎點(diǎn)下方梗死部位心肌分離切割,放入-70℃冰箱備用。用實(shí)時(shí)熒光定量RT-PCR方法分析梗死心肌中ADAMTS4、ADAMTS8、versican及TIMP-3mRNA表達(dá),ELISA法檢測(?)ADAMTS4的蛋白表達(dá)。結(jié)果示ADAMTS4表達(dá)在心梗后6及12小時(shí)明顯升高(p0.05),隨后快速下降。而ADAMTS8則在心梗后6小時(shí)開始升高,至24小時(shí)達(dá)峰值,3天時(shí)仍持續(xù)在高水平(p0.05),然后緩慢下降。心梗后versican mRNA水平顯著增高,至3天時(shí)達(dá)峰值,且升高持續(xù)時(shí)間較長。TIMP3表達(dá)水平降低。ADAMTS4蛋白水平在心梗后6小時(shí)(p=0.026)、12小時(shí)(p=0.003)、24小時(shí)(p=0.002)及3天(p=0.009)顯著增高。 ADAMTS4、ADAMTS8, versican及TIMP-3在心梗大鼠心臟中表達(dá),且表現(xiàn)為不同的動(dòng)態(tài)變化趨勢(shì)。ADAMTS4、versican及TIMP-3的表達(dá)之間存在相互關(guān)系。 四、AAMTS4在梗死大鼠心臟血管內(nèi)皮細(xì)胞及心肌細(xì)胞中表達(dá) 選擇250-300g雄性Wistar大鼠制作心梗模型,設(shè)假手術(shù)組。手術(shù)后3天處死大鼠,取左心室制作冰凍切片。切片厚度為5um。免疫組織化學(xué)方法檢測ADAMTS4、versican及aggrecan蛋白表達(dá)。對(duì)于陰性對(duì)照組,我們以等比稀釋度用兔IgG處理切片。結(jié)果示ADAMTS4在梗死邊緣區(qū)域心肌細(xì)胞中表達(dá),在梗死區(qū)域及遠(yuǎn)離梗死區(qū)的正常心肌未見ADAMTS4表達(dá)。在梗死周圍區(qū)域毛細(xì)血管內(nèi)皮細(xì)胞ADAMTS4有弱表達(dá),versican強(qiáng)表達(dá)。Aggrecan在心梗組及假手術(shù)組的心肌組織及血管內(nèi)膜均未見表達(dá)。 ADAMTS4與versican共同表達(dá)于內(nèi)皮細(xì)胞提示二者存在某種聯(lián)系,其動(dòng)態(tài)變化可能在心梗后基質(zhì)重塑中起作用。 五、白介素1β刺激內(nèi)皮細(xì)胞后ADAMTSs的表達(dá) 選取人臍靜脈血管內(nèi)皮細(xì)胞,以傳代培養(yǎng)法培養(yǎng)細(xì)胞。分別以2.5ng/ml,5ng/ml,10ng/ml,20ng/ml濃度的IL-1β刺激內(nèi)皮細(xì)胞,24h后收集細(xì)胞提取總RNA及蛋白。用10ng/ml IL-1β刺激內(nèi)皮細(xì)胞,分別于刺激后2hh、6h、12h、24h、48h收集細(xì)胞提取總RNA及蛋白,設(shè)空白對(duì)照組,每組5個(gè)樣本。RT-PCR測定ADAMTS1、4、8、9、15mRNA表達(dá),Western-blot測定ADAMTS4蛋白表達(dá)。結(jié)果示IL-1β刺激內(nèi)皮細(xì)胞后ADAMTS1、4、8、9、15mRNA表達(dá)均增高,但隨時(shí)間變化趨勢(shì)不同,ADAMTS4蛋白表達(dá)增高。 此結(jié)果提示ADAMTSs參與炎癥反應(yīng),具體機(jī)制有待進(jìn)一步闡明。 六、冠心病患者冠脈血中ADAMTS4水平的變化及經(jīng)皮冠脈介入治療對(duì)冠脈ADAMTS4水平的影響 根據(jù)冠脈造影結(jié)果,將81例患者分為對(duì)照組、簡單病變組及復(fù)雜病變組,其中35例患者接受了PCI治療。血液檢測標(biāo)本從冠狀靜脈竇獲得。ADAMTS4及hs-CRP值分別通過ELISA及免疫濁度法檢測。結(jié)果示復(fù)雜病變組ADAMTS4及hs-CRP值明顯高于對(duì)照組及簡單病變組(P0.001)。所有研究對(duì)象及冠心病患者的ADAMTS4水平均與hs-CRP水平相關(guān)(r,=0.73, r2=0.763, P0.01)。接受了PCI治療的患者ADAMTS4值高于未接受PCI者(P0.001), hs-CRP亦表現(xiàn)出相似結(jié)果(P=0.025)。 冠心病患者冠脈ADAMTS4及hs-CRP水平升高,且隨病變復(fù)雜程度增加而升高。PCI后冠脈ADAMTS4及hs-CRP升高,其可能由PCI過程中球囊擴(kuò)張或支架植入的機(jī)械擠壓導(dǎo)致冠脈斑塊破裂釋放而出。
[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

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R341

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