大鼠血管平滑肌细胞蛋白组学的研究以及尼古丁对血管平滑肌细胞蛋白和基因表达影响的研究

【作者】 武卫东；

【导师】 赵荣瑞； 乔中东；

【作者基本信息】 山西医科大学 ， 生理学， 2005， 博士

【摘要】 背景和目的近年来,随着人类基因组计划的顺利进行和完成,生物医学科学的研究进入了后基因组时代。在这个时代,生命科学的主要研究对象是功能基因组学,包括结构基因组研究和蛋白质组研究等。蛋白质组学研究目前是生物医学研究领域热点课题之一,已经广泛的应用于基础医学和临床医学,并取得了较大的成绩。目前,蛋白质组学在临床医学研究中主要应用于各种肿瘤细胞的研究和心血管疾病的研究。近年来,心血管疾病的蛋白质组学研究主要集中在1. 心血管疾病蛋白差异表达的研究: 1992年,Baker 等人利用2DE 分离了1 500 多种人心肌蛋白,并对其中12个蛋白进行了鉴定。Corbett 及Weekes J 两个研究小组在对人及其动物模型牛的扩张性心肌病的研究中,发现与正常心肌组织相比,有100 多种蛋白质的表达是下调。2. 心血管疾病生物标志物的研究: Fromm 用蛋白质组学的研究手段着手监测MI发生后TnI的降解;You SA 等将蛋白质组的方法运用于寻找冠状动脉粥样硬化发病的生物标志物的研究,发现冠状动脉病患者的铁蛋白轻链的表达量显著增加。血管平滑肌细胞的增殖和凋亡在动脉粥样硬化、血管重构等病理状态的形成和发展过程中起着重要的作用。研究血管平滑肌细胞在生理和病理状态下的形态学、基因和蛋白表达的变化对研究动脉粥样硬化及其相关性疾病如:心血管疾病、中风等的发病机理有着不可替代的作用。目前,国外有学者报道了新生小鼠和成年小鼠血管平滑肌细胞的蛋白组学研究;Taurin S 等用蛋白组学的研究方法研究了对照组和喹巴因处理组平滑肌细胞的疏水和亲水蛋白,并鉴定了差异表达的蛋白。尼古丁是体内重要的致病因子,可以通过破坏血管内皮细胞的功能、调节脂质代谢、直接或间接的刺激血管平滑肌细胞增殖等方面来促进动脉粥样硬化及其相关性疾病如:心血管疾病、脑血管疾病、间歇性跛行以及肿瘤的形成和发展。但是其致病的分子生物学机制尚不是十分明了。因此:为了对尼古丁致病机理有一个全面的了解,我们在实验中应用蛋白组学的更多还原

【Abstract】 Objective Recently, proteomic analysis technology has been used extensively in biological innovative researches and has become one of the most essential technologies in functional genomics. The proteomics is the study of various protein properties (expression level, post-translation modification, etc) at the whole cell level to obtain a global, integrated view of disease processes, physiological and biochemical processes of cells and regulatory networks at the protein level. Proteomic technology has also been applied in studying cell growth and regulation, stimulus response, protein function, etc. By using proteomic technology, it has become possible to analyze simultaneously hundreds or even thousands of protein. Epidemiological studies have shown that nicotine, the major component of cigarette smoking, is a dominant risk factor of cardiovascular disease. Although the underlying mechanisms are not completely understood, there are substantial evidences indicating that the primary role of cigarette smoking in cardiovascular disease is to promote the formation and development of atherosclerosis. Nicotine could promote atherosclerosis formation via its following effects: ①initiating changes in the lipid metabolism, resulting in decreased high-density lipoprotein cholesterol, increased low-density lipoproteins, and higher levels of oxidized LDL, an atherogenetic lipid; ②causing injury to the vascular endothelium by direct cytotoxicity and impairment of nitrogen monoxide (NO) bioactivity, leading to endothelial cell dysfunction and initiating the pathogenesis of atherosclerosis, which is the primary vascular lesion of cardiovascular disease); ③modulating the phenotype of SMCs and stimulating secretion of growth factors, and hence activating the medial smooth muscle cells migration into the intima to engender proliferation and producing extracellular matrix protein, which is a key event in the vascular response to injury. However, atherosclerosis is a multifactorial disease and the underlying mechanisms available are still controversial). Therefore, more basic information of nicotine-related SMCs pathophysiogical changes are yet to be ascertained. In this study, we used the high-resolution 2-D electrophoresis technology, PMF and MS/MS to analyze the possible protein expression changes of nicotine-stimulated SMCs. At the same time, using RealtimeRT-PCR methods studied the changes of all those encode genes. The aim of this study is to provide some evidences for further understanding of the molecular mechanisms of nicotine-related diseases such as atherosclerosis, cardiovascular diseases, and cancer, etc. Material and methods 1. Cell culture: Healthy male Wistar rats weighting 300-350g were afforded. Aortic smooth muscle cells (SMCs) were isolated from medial explants of the thoracic aorta, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 150mmol/L N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 100U/ml penicillin, and 100μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Cells passaged 3-10 were used in our experiment. 2. The cytoactive effect of nicotine on SMCs: treating SMCs 24h with 0.1μM, 1.0μM, 10μM, 100μM or without nicotine to study the cell activity by two methods: MTT and LDH methods. 3. Two-dimensional gel electrophoresis: after SMCs treated with 10μM 24h or without nicotine, the SMCs were lysised by lysis solution, and the cell total protein were acquired. Then, the total proteins were subjected to two-dimensional gel electrophoresis. 4. Gel analysis: When the SDS-PAGE was finished, the Proteins were stained in a solution containing Coomassie brilliant blue R-250 (0.1% W/V), methanol (45%W/V) and acetic acid (10%W/V) for 4 hours. Destaining was carried out in the same solution without Coomassie brilliant blue. Gels were dried, exposed, scanned using an ImageScanner II (Amersham Biosciences) andanalyzed by calculation of volumes and density of the spots with the ImageMaster 2D Platinum software更多还原