| 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...
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