阻断心肌血管AIBP基因表达对心肌微血管再生作用的研究

发布时间：2018-03-16 05:11

本文选题：心肌梗死　切入点：载脂蛋白A-I结合蛋白　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景随着社会人口老龄化和人民生活水平提高,冠心病已经成为危害人类健康的主要疾病之一。对于缺血性心脏病的治疗,最重要的是恢复心肌血液灌注,解除心肌组织缺血、缺氧和营养物质供应不足的情况。再生的血管可以很大程度上改善心肌的供血,从而缓解疾病的进展。心肌缺血缺氧后如何促进血管再生、改善微循环的研究越来越受到重视,这已经成为缺血性心脏病,尤其是糖尿病性心肌病治疗学研究的重点方向之一。近期研究发现,周围组织分泌的载脂蛋白A-I结合蛋白(Apo A-1 Binding Protein,AIBP)可以抑制血管形成。那么从理论上推断,阻断AIBP活性可以刺激血管再生长,但目前AIBP在心肌微血管内皮细胞(Cardiac Microvascular Endothelial Cells,CMECs)以及心肌组织中的作用尚无报道。为此我们研究阻断心肌血管AIBP活性是否可以刺激心肌缺血后心脏微血管生成和新生,从根本上解决缺血性心脏病和许多终末期心力衰歇患者的心肌微循环障碍问题,从而改善心肌细胞生存的微环境和营养能量物质的供给,为寻找缺血性心脏病的潜在新治疗靶点奠定一定的理论基础。目的1.探讨大鼠CMECs体外分离、培养及鉴定的方法和慢病毒转染、筛选及阻断大鼠AIBP基因的表达情况。2.观察阻断AIBP基因的表达对大鼠CMECs血管再生功能的影响。3.动物实验研究阻断心肌血管AIBP基因表达对大鼠心梗后冠脉微血管再生的影响。方法1.大鼠CMECs体外分离培养和鉴定以及慢病毒转染筛选采用混合酶消化法和差速贴壁法分离SD大鼠CMECs,并采用乙酰低密度脂蛋白(Dil-Ac-LDL)吞噬检测法进行鉴定,取P2-P3 CMECs进行慢病毒介导的AIBP sh RNA转染,阻断CMECs AIBP基因表达。采用RT-PCR和Western Blot检测正常及转染后的CMECs AIBP基因和蛋白的表达水平,筛选出有效阻断AIBP表达的基因序列。2.阻断AIBP基因表达对CMECs血管再生能力的研究取P2-P3 CMECs进行阻断AIBP基因表达转染,与Control组和L-NC组进行比较,采用Westren Blot检测细胞内VEGFR2和p AKT蛋白的表达、Alama Blue比色法检测细胞增殖能力、Transwell小室迁移实验检测细胞迁移能力、Matrigel基质胶管样结构形成实验检测细胞血管再生能力。3.动物实验观察阻断大鼠心肌血管AIBP的表达对心梗后微血管再生的影响建立大鼠心梗模型并于心梗处注射慢病毒,采用Western Blot检测正常及转染后的心肌中AIBP蛋白的表达水平、免疫组化检测CD31的表达水平、Masson三色法检测心肌纤维化程度和心脏超声检测心脏功能。结果1.成功分离、鉴定大鼠CMECs,培养5 d-6 d时成熟的CMECs相互融合并紧密连接,呈多边形“铺路石样”改变,对Ac-LDL的吞噬功能正常。Western Blot检测结果与RT-PCR相互印证,成功筛选出有效阻断AIBP表达的基因序列。2.体外实验结果表明,阻断AIBP表达后上调CMECs VEGFR2的表达与AKT磷酸化水平。可提高CMECs的增殖能力、细胞迁移能力以及成管能力(P0.01)。3.动物实验结果表明,阻断AIBP表达后心梗区域心肌微血管再生数显著增多,心梗后心肌纤维化程度显著下降、显著改善心梗后大鼠左室心功能(P0.01)。结论阻断心肌血管AIBP基因的表达可以显著促进心肌微血管再生。体外实验中阻断大鼠CMECs AIBP基因的表达可以促进大鼠心肌微血管再生能力;体内动物实验中,阻断大鼠心肌血管AIBP基因的表达同样可以显著促进心梗后冠脉微血管再生,改善心功能。因此,阻断心肌血管AIBP基因的表达可以成为治疗缺血性心脏病的一个潜在治疗靶点,为临床促心肌微血管再生治疗提供了坚实的理论基础。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.2

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