本文關鍵詞：bFGF在煙草煙霧誘導大鼠血管平滑肌細胞增殖中的作用　出處：《中國醫(yī)科大學》2010年碩士論文　論文類型：學位論文

  更多相關文章： 煙草煙霧提取物 堿性成纖維細胞生長因子 血管平滑肌細胞 增殖

【摘要】： 前言 吸煙是動脈粥樣硬化的主要危險因素,對于人類在心血管方面的危害超過了吸煙導致的肺癌。我國是世界上煙草消耗最大的國家,國人60%男性和3%女性吸煙,吸煙者大約有3.5億人。吸煙對人類健康的危害是巨大的,2000年世界上有169萬人因吸煙死于心血管疾病,占全球心血管疾病死亡人數(shù)的11%,2002年有500萬人的死亡是由吸煙引起的,而我國每年有120萬人因吸煙導致死亡。 堿性成纖維細胞生長因子(basic Fibroblast Growth Factor,bFGF)是肝素結合生長因子家族成員之一,在體內(nèi)分布廣泛。bFGF具有多種生物學特性,促進細胞增殖是其主要作用之一。而血管平滑肌細胞增殖(vascular smooth muscle cell, VSMC)是動脈粥樣硬化、再狹窄和肺動脈高壓等多種心血管疾病的病理基礎。 本研究應用煙草煙霧提取物(cigarette smoke extract, CSE)刺激體外培養(yǎng)的大鼠VSMC,觀察其對大鼠VSMC增殖的影響并應用bFGF抗體中和bFGF生物功能,探討bFGF在CSE刺激VSMC增殖中的作用機制。 實驗方法 一、大鼠主動脈血管平滑肌細胞培養(yǎng) 細胞在37℃、5％CO2細胞培養(yǎng)箱中靜置培養(yǎng),每2天換液1次。當細胞長滿瓶底80%左右時,用0.25%胰蛋白酶消化傳代。取第3-8代的細胞,當細胞長至70%-80%融合時進行實驗。 二、實驗分組 1、用不同濃度(0,2.5%,5%,10%,20%)CSE刺激VSMC,采用四甲基偶氮唑鹽微量酶反應比色法(MTT法)觀察細胞增殖變化,免疫細胞化學法測定bFGF和增殖細胞核抗原(proliferating cell nuclear antigen, PCNA)蛋白的表達,同時用RT-PCR檢測bFGF mRNA表達。 2、用篩選出的最適CSE濃度處理VSMC不同時間(Oh,4h,8h,12h,24h)后,檢測bFGFmRNA, bFGF和PCNA蛋白的變化。 3、用bFGF抗體和最適濃度CSE干預VSMC 24h后檢測細胞增殖,bFGF和PCNA蛋白表達的變化。 三、實驗檢測方法 噻唑藍[3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide,MTT]比色法測定VSMC的增殖；用免疫細胞化學法測定bFGF, PCNA蛋白的表達；用RT-PCR檢測bFGF mRNA表達的變化。 實驗結果 1、與對照組相比,低濃度(2.5%)CSE組(P0.05)和中濃度(5%)CSE組增加明顯(P0.01),而高濃度(10%CSE)或過高濃度(20%CSE)組與對照組相比差異無顯著性(P0.05)。bFGFmRNA、蛋白和PCNA蛋白在對照組中有少量表達。與對照組相比,2.5%CSE組表達增加(P0.01),5%CSE組達到高峰。10%、20%CSE組仍高于對照組(P0.01)。 2、對照組(不加CSE組即Oh組)VSMC中有少量bFGF mRNA、蛋白和PCNA蛋白表達。5%CSE刺激4h后細胞內(nèi)bFGF mRNA、蛋白和PCNA蛋白表達增加(P0.01),bFGFmRNA于8h達高峰；bFGF和PCNA蛋白于12h達高峰。 3、bFGF抗體可顯著抑制5%CSE誘導的MTT吸光度(A值)和bFGF、PCNA蛋白表達增加。 結論 CSE可能是通過增加bFGF的表達促進大鼠VSMC的增殖。
[Abstract]:Foreword Smoking is the main risk factor of atherosclerosis, which is more harmful to human cardiovascular than lung cancer caused by smoking. China is the country with the largest consumption of tobacco in the world. 60% men and 3% women smoke in China, about 350 million smokers. Smoking is a great harm to human health. In 2000, 1.69 million people died of cardiovascular diseases because of smoking in the world. In 2002, 5 million deaths were caused by smoking. In China, there are 1.2 million deaths caused by smoking every year. Basic Fibroblast Growth FactorbFGFs are members of heparin binding growth factor family. BFGF is widely distributed in vivo and has many biological characteristics. The proliferation of vascular smooth muscle cells (VSMCs) and vascular smooth muscle cells (VSMC) is atherosclerosis. Restenosis and pulmonary hypertension are the pathological basis of many cardiovascular diseases. In this study, tobacco smoke extract (smoke extract) was used to stimulate rat VSMC in vitro. To investigate the effect of bFGF on the proliferation of VSMC in rats and to explore the mechanism of bFGF in VSMC proliferation stimulated by CSE, bFGF antibody was used to neutralize the biological function of bFGF. Experimental method 1. Cultured rat aortic vascular smooth muscle cells The cells were cultured in 37 鈩，

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