【作者】 郭芳；

【导师】 杨向东；

【作者基本信息】 南华大学 ， 病理与病理生理学， 2005， 硕士

【摘要】 目的:经皮球囊冠状动脉腔内成形术(percutaneous transluminal coronaryangioplasty,PTCA)是冠心病治疗学上的一个重大进展,但PTCA术后3～6个月再狭窄(restenosis,RS)率较高,严重影响治疗效果。再狭窄的共同机制包括血管内皮细胞的损伤、平滑肌细胞(VSMC)的迁移、增殖、内膜增生、细胞外基质(ECM)沉积和血管重塑等过程。VSMC过度增殖是再狭窄形成的一个重要因素,而VSMC的凋亡是血管平衡细胞增殖的重要途径,增殖与凋亡稳态的失衡是影响再狭窄形成的重要因素。泛素-蛋白酶体途径(ubiquitin-proteasomepathway,UPP)广泛参与多种生理过程如细胞周期调控、DNA修复、细胞信号转导、细胞凋亡、蛋白的跨膜定位和细胞表面膜受体的内化等。越来越多的研究表明UPP与人类多种疾病的发生发展有关,最近的研究发现UPP可能参与动脉粥样硬化(atherosclerosis,AS)病变的发生发展。文献报道UPP抑制剂可以促进VSMC凋亡,调控UPP的表达可能是调控血管壁细胞凋亡的重要手段。 本实验选择UPP作为研究对象,主要是基于以下原因: (1)目前对UPP的研究主要集中在肿瘤领域,对UPP与心血管疾病关系的研究尚处于探索阶段。 (2)在前期动脉粥样硬化相关基因克隆的研究中,发现UPP相关基因可能是单核/巨噬细胞、内皮细胞氧化损伤和凋亡的相关基因,在ox-LDL处理的VGMC中亦有UPP的表达。 (3)蛋白酶体抑制剂MG132如果能够有效抑制血管狭窄,将为防治PTCA术后再狭窄提供新的手段,研究其作用分子机制将非常重要。 以UPP为靶标,研究血管壁细胞凋亡机制,探索平衡血管壁细胞增殖与凋亡新途径是防治球囊损伤后血管狭窄、重塑的重要策略,将为防治再狭窄提供新的方向。更多还原

【Abstract】 Aim Percutaneous transluminal coronary angioplasty, PTCA, is a great progress in the coronary heart disease therapeutics, but 3-6 months after PTCA, the rate of restenosis is quite high, this influences the result of treatment seriously. The common mechanisms of restenosis include these courses such as vein endothelial cells injury, intimal hyperplasia, the transplant and proliferation of vascular smooth muscle cells-VSMCs, extracellular matrix aggradation, vascular remodeling and so on. Recent study shows that the excessive hyperplasia of VSMCs is the key factor in determining the restenosis. Moreover, the apoptosis of VSMCs is the important approach of vascular cells proliferation balance. The unbalance of proliferation and apoptosis is a important factor that influences the formation of restenosis. The ubiquitin-proteasome pathway, UPP, takes part in a lot of physiological courses, such as cell cycle regulation, DNA repairing, cell signal transduction, cell apoptosis and the endocytosis of membrane receptor. Lots of experiments have demonstrated that UPP is involved in many human diseases. Latest researches indicate that the UPP may participate in the happening and developing of pathological change of atherosclerosis, AS. Documents report that UPP inhibitor can promote the apoptosis of VSMC. Regulating and controlling the expression of UPP may be the important means of regulating and controlling the apoptosis of vascular cell. We choose UPP as the object of the experiments, this is based upon the hereinafter reasons:(1)Presently, the studies on UPP are centralized in tumour field, the research of the relationship between UPP and cardiovascular disease is still in the procession of discovery.(2) The anterior researches of the clone of Atherosclerotic relevant genefound that UPP related genes may be the relevant gene of oxidation injury and apoptosis of monocyte and endothelial cell, and there are expressions of UPP on VSMCs dealing with ox-LDL. (3) If proteasome inhibitor MG132 can effectively inhibit vascular stenosis, it will offer new means for the prevention and cure of restenosis after PTCA, and the study of its molecular mechanism is very important. We take UPP as the drone, research the apoptosis mechanism of vascular cell, explore new means for the balance between proliferation and apoptosis of vascular cell. This is the important tactic about the prevention and cure of restenosis after balloon injury, and it will provide the prevention and cure of restenosis with a new direction.Methods:1. the part of animals experiments: using a balloon to injure the New Zealand white rabbit’s carotid and reproduce the model of vascular stenosis, then we put the proteasome inhibitor MG132 in the local of injuried vascular, observe the influence of proteasome inhibitor MG132 to vascular stenosis after balloon injury. Divide the 40 New Zealand white rabbits into four parts at random(the group of control, the group of hypercholesterol, the group of balloon injury+hypercholesterol, the group of balloon injury+MG132+hypercholesterol). And after feeding them respectively for 2 weeks, 4 weeks, 8 weeks, make pathology section, dye HE, check by pathology morphology. Then use the immunohistochemistry to observe the changes of Ubiquitin, p53, caspase3 protein.2. the part of cells experiments: The experiments have two parts, part 1 is to study the influence of proteasome inhibitor MG132 to the apoptosis of VSMC. The experimental groups are: ①control group; ②2μmol·L-1 MG132 treated for 24 and 48 hours; ③ 5μmol·L-1 MG132 treated for 24 and 48 hours ④ 10μmol·L-1 MG132 treated for 24 and 48 hours. Part 2 is to study the influence of proteasome inhibitor MG132 to the apoptosis of vein endothelial cell-VEC. The experimental groups are: ①control group; ②2μmol·L-1 MG132 treated for 24 hours; ③5μmol·L-1 MG132treated for 24 hours. The apoptotic cells were determined by DNA fragment analysis and flow cytometric analysis. Using RT-PCR to detect mRNA expression of Ub, El, E2, E3, 26s proteasome, caspase3, p53. The protein contents of caspase3 and p53 were analyzed by immunocytochemistry.Results: The result of pathology morphology checking shows that the carotid cavity of the group of balloon injury+hypercholesterol shrinks, the intimal thickens, and there are a lot of hyperplasia of foam cells and a large number of VSMCs. So, it has obvious stenosis. But there is no such kind of phenomenon in the group of balloon injury+MG132+hypercholesterol. The immunohistochemistry checks that the expression of ubiquitin protein enhanced in the carotid vessel wall of the group of balloon injury+hypercholesterol, the expression of p53 and caspase3 protein reduced. But in the group of balloon injury+MG132+hypercholesterol, there is an opposite condition. The apoptotic rate of VSMC presents increase after dealing by the proteasome inhibitor MG132 for 48 hours. The expression of UPP related genes in VSMC treated with MG132 were down-regulated especially in 48 hours, but the expression of mRNA of p53 and caspase3 were up-regulated. The protein contents of caspase3 and p53 were up-regulated by immunocytochemistry analysis. The apoptotic rate of VEC presents increase after dealing by the proteasome inhibitor MG132 for 24 hours. The expression of mRNA of caspase3 was up-regulated. The protein contents of caspase3 were up-regulated by immunocytochemistry analysis.Conclusion:1. Using the balloon to injure the New Zealand white rabbit’s carotid to reproduce the model of vascular stenosis, we observed that the vascular stenosis of carotid appeared. Putting the proteasome inhibitor MG132 in the local of injuried vascular could inhibit intimal hyperplasia, and inhibit vascular stenosis. Using MG132 interdict UPP, the expression of p53 and caspase3 were up-regulated.2. Proteasome inhibitor MG132 can induce the apoptosis of human umbilical artery VSMCs and human umbilical VECs.更多还原