發(fā)布時(shí)間：2018-03-16 05:11

  本文選題：心肌梗死　切入點(diǎn)：載脂蛋白A-I結(jié)合蛋白　出處：《第四軍醫(yī)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：背景隨著社會(huì)人口老齡化和人民生活水平提高,冠心病已經(jīng)成為危害人類健康的主要疾病之一。對(duì)于缺血性心臟病的治療,最重要的是恢復(fù)心肌血液灌注,解除心肌組織缺血、缺氧和營(yíng)養(yǎng)物質(zhì)供應(yīng)不足的情況。再生的血管可以很大程度上改善心肌的供血,從而緩解疾病的進(jìn)展。心肌缺血缺氧后如何促進(jìn)血管再生、改善微循環(huán)的研究越來越受到重視,這已經(jīng)成為缺血性心臟病,尤其是糖尿病性心肌病治療學(xué)研究的重點(diǎn)方向之一。近期研究發(fā)現(xiàn),周圍組織分泌的載脂蛋白A-I結(jié)合蛋白(Apo A-1 Binding Protein,AIBP)可以抑制血管形成。那么從理論上推斷,阻斷AIBP活性可以刺激血管再生長(zhǎng),但目前AIBP在心肌微血管內(nèi)皮細(xì)胞(Cardiac Microvascular Endothelial Cells,CMECs)以及心肌組織中的作用尚無報(bào)道。為此我們研究阻斷心肌血管AIBP活性是否可以刺激心肌缺血后心臟微血管生成和新生,從根本上解決缺血性心臟病和許多終末期心力衰歇患者的心肌微循環(huán)障礙問題,從而改善心肌細(xì)胞生存的微環(huán)境和營(yíng)養(yǎng)能量物質(zhì)的供給,為尋找缺血性心臟病的潛在新治療靶點(diǎn)奠定一定的理論基礎(chǔ)。目的1.探討大鼠CMECs體外分離、培養(yǎng)及鑒定的方法和慢病毒轉(zhuǎn)染、篩選及阻斷大鼠AIBP基因的表達(dá)情況。2.觀察阻斷AIBP基因的表達(dá)對(duì)大鼠CMECs血管再生功能的影響。3.動(dòng)物實(shí)驗(yàn)研究阻斷心肌血管AIBP基因表達(dá)對(duì)大鼠心梗后冠脈微血管再生的影響。方法1.大鼠CMECs體外分離培養(yǎng)和鑒定以及慢病毒轉(zhuǎn)染篩選采用混合酶消化法和差速貼壁法分離SD大鼠CMECs,并采用乙酰低密度脂蛋白(Dil-Ac-LDL)吞噬檢測(cè)法進(jìn)行鑒定,取P2-P3 CMECs進(jìn)行慢病毒介導(dǎo)的AIBP sh RNA轉(zhuǎn)染,阻斷CMECs AIBP基因表達(dá)。采用RT-PCR和Western Blot檢測(cè)正常及轉(zhuǎn)染后的CMECs AIBP基因和蛋白的表達(dá)水平,篩選出有效阻斷AIBP表達(dá)的基因序列。2.阻斷AIBP基因表達(dá)對(duì)CMECs血管再生能力的研究取P2-P3 CMECs進(jìn)行阻斷AIBP基因表達(dá)轉(zhuǎn)染,與Control組和L-NC組進(jìn)行比較,采用Westren Blot檢測(cè)細(xì)胞內(nèi)VEGFR2和p AKT蛋白的表達(dá)、Alama Blue比色法檢測(cè)細(xì)胞增殖能力、Transwell小室遷移實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力、Matrigel基質(zhì)膠管樣結(jié)構(gòu)形成實(shí)驗(yàn)檢測(cè)細(xì)胞血管再生能力。3.動(dòng)物實(shí)驗(yàn)觀察阻斷大鼠心肌血管AIBP的表達(dá)對(duì)心梗后微血管再生的影響建立大鼠心梗模型并于心梗處注射慢病毒,采用Western Blot檢測(cè)正常及轉(zhuǎn)染后的心肌中AIBP蛋白的表達(dá)水平、免疫組化檢測(cè)CD31的表達(dá)水平、Masson三色法檢測(cè)心肌纖維化程度和心臟超聲檢測(cè)心臟功能。結(jié)果1.成功分離、鑒定大鼠CMECs,培養(yǎng)5 d-6 d時(shí)成熟的CMECs相互融合并緊密連接,呈多邊形“鋪路石樣”改變,對(duì)Ac-LDL的吞噬功能正常。Western Blot檢測(cè)結(jié)果與RT-PCR相互印證,成功篩選出有效阻斷AIBP表達(dá)的基因序列。2.體外實(shí)驗(yàn)結(jié)果表明,阻斷AIBP表達(dá)后上調(diào)CMECs VEGFR2的表達(dá)與AKT磷酸化水平�？商岣逤MECs的增殖能力、細(xì)胞遷移能力以及成管能力(P0.01)。3.動(dòng)物實(shí)驗(yàn)結(jié)果表明,阻斷AIBP表達(dá)后心梗區(qū)域心肌微血管再生數(shù)顯著增多,心梗后心肌纖維化程度顯著下降、顯著改善心梗后大鼠左室心功能(P0.01)。結(jié)論阻斷心肌血管AIBP基因的表達(dá)可以顯著促進(jìn)心肌微血管再生。體外實(shí)驗(yàn)中阻斷大鼠CMECs AIBP基因的表達(dá)可以促進(jìn)大鼠心肌微血管再生能力;體內(nèi)動(dòng)物實(shí)驗(yàn)中,阻斷大鼠心肌血管AIBP基因的表達(dá)同樣可以顯著促進(jìn)心梗后冠脈微血管再生,改善心功能。因此,阻斷心肌血管AIBP基因的表達(dá)可以成為治療缺血性心臟病的一個(gè)潛在治療靶點(diǎn),為臨床促心肌微血管再生治療提供了堅(jiān)實(shí)的理論基礎(chǔ)。
[Abstract]:With the social background of the aging population and the improvement of people's living standard, coronary heart disease has become one of the major diseases that endanger human health. For the treatment of ischemic heart disease, the most important is to restore the myocardial blood perfusion, relieve myocardial tissue ischemia, hypoxia and nutrient supply shortage. The regeneration of blood vessels can improve myocardial largely the blood supply, so as to relieve the progression of the disease. How to promote angiogenesis after myocardial ischemia, improve microcirculation research more and more attention, which has become the ischemic heart disease, especially one of the key research direction of the treatment of diabetes cardiomyopathy. A recent study found that tissue surrounding the secretion of apolipoprotein A-I (Apo binding protein A-1 Binding Protein, AIBP) can inhibit angiogenesis. So theoretically inferred that blocking AIBP activity can stimulate blood vessel growth, but the current AIBP In cardiac microvascular endothelial cells (Cardiac Microvascular Endothelial Cells, CMECs) and the role in myocardial tissue has not been reported. So we study whether blocking myocardial vascular AIBP activity after myocardial ischemia can stimulate cardiac angiogenesis and myocardial microcirculation of newborn, no ischemic heart disease and many end-stage heart failure patients fundamentally the solution, thereby improving myocardial cell survival and nutritional micro environment of energy supply, and lay a theoretical foundation for a potential new therapeutic target for ischemic heart disease. Objective to explore 1. rat CMECs in vitro culture and identification methods, and lentiviral transfection, screening and blocking AIBP gene expression in rat.2. observation of blocking the expression of AIBP gene on vascular regeneration function of CMECs rat animal experiment study on effect of.3. blocking myocardial vascular expression of AIBP gene on Effect of coronary microvascular regeneration in rats after myocardial infarction. CMECs in vitro culture and identification of 1. rats and the lentivirus was screened by mixed enzyme digestion and differential centrifugation separation of CMECs SD rats, and the acetylated low density lipoprotein (Dil-Ac-LDL) detection method of phagocytosis were identified from P2-P3 CMECs AIBP sh RNA transfected with lentivirus mediated expression of CMECs, blocking AIBP gene. The expression level of AIBP gene by CMECs and RT-PCR and Western protein in normal and Blot detection after transfection, screening effective blocking.2. gene sequence of AIBP expression inhibition of AIBP gene expression on CMECs vascular regeneration ability of P2-P3 CMECs inhibition of AIBP gene expression transfection, compared with Control group and L-NC group, the expression of Westren Blot and P VEGFR2 detected AKT protein, Alama Blue assay was used to detect cell proliferation, Transwell small room to move Shift test cell migration, formation of the.3. animal experiment to observe the expression of AIBP in rat myocardial vascular blocking effect on angiogenesis after myocardial infarction to establish the rat model of myocardial infarction and myocardial infarction at injection of lentivirus vascular test cell regeneration Matrigel matrix tube like structure, the expression level of AIBP protein in normal and Western Blot detection after transfection in the myocardium, the expression level of immunohistochemical detection of CD31, cardiac function detection of myocardial fibrosis and cardiac ultrasound Masson trichrome method. Results 1. successful separation, identification of CMECs rats, cultured for 5 D-6 D mature CMECs fusion and closely connected, polygonal cobblestone like change, on the phagocytic function Ac-LDL Blot and RT-PCR.Western normal test results confirm each other, successfully screened effectively blocking the expression of AIBP.2. gene sequence in vitro experiment showed that blocking AIBP The expression of CMECs was upregulated after VEGFR2 expression and phosphorylation of AKT. CMECs can improve the ability of proliferation, cell migration and tube formation ability of (P0.01).3. animal experiment results show that blocking the expression of AIBP after myocardial infarction in regional myocardial microvascular regeneration were significantly increased after myocardial infarction, fibrosis degree decreased significantly, significantly improved left ventricular the rat heart function after myocardial infarction (P0.01). Conclusion the expression of AIBP gene in myocardial vascular occlusion can significantly promote myocardial angiogenesis. In vitro blocking CMECs gene expression in AIBP rats can promote rat myocardial microvascular regeneration; animal experiment in vivo, blocking the gene expression of myocardial vessels in AIBP rats can also be significant promote coronary microvascular regeneration after myocardial infarction and improve heart function. Therefore, blocking the expression of myocardial vascular AIBP gene could be a potential therapeutic target for the treatment of ischemic heart disease. It provides a solid theoretical basis for the treatment of myocardium microvascular regeneration.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.2

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