本文關(guān)鍵詞：bFGF在煙草煙霧誘導(dǎo)大鼠血管平滑肌細(xì)胞增殖中的作用　出處：《中國(guó)醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 煙草煙霧提取物 堿性成纖維細(xì)胞生長(zhǎng)因子 血管平滑肌細(xì)胞 增殖

【摘要】： 前言 吸煙是動(dòng)脈粥樣硬化的主要危險(xiǎn)因素,對(duì)于人類在心血管方面的危害超過(guò)了吸煙導(dǎo)致的肺癌。我國(guó)是世界上煙草消耗最大的國(guó)家,國(guó)人60%男性和3%女性吸煙,吸煙者大約有3.5億人。吸煙對(duì)人類健康的危害是巨大的,2000年世界上有169萬(wàn)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(wàn)人的死亡是由吸煙引起的,而我國(guó)每年有120萬(wàn)人因吸煙導(dǎo)致死亡。 堿性成纖維細(xì)胞生長(zhǎng)因子(basic Fibroblast Growth Factor,bFGF)是肝素結(jié)合生長(zhǎng)因子家族成員之一,在體內(nèi)分布廣泛。bFGF具有多種生物學(xué)特性,促進(jìn)細(xì)胞增殖是其主要作用之一。而血管平滑肌細(xì)胞增殖(vascular smooth muscle cell, VSMC)是動(dòng)脈粥樣硬化、再狹窄和肺動(dòng)脈高壓等多種心血管疾病的病理基礎(chǔ)。 本研究應(yīng)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(duì)大鼠VSMC增殖的影響并應(yīng)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(jī)制。 實(shí)驗(yàn)方法 一、大鼠主動(dòng)脈血管平滑肌細(xì)胞培養(yǎng) 細(xì)胞在37℃、5％CO2細(xì)胞培養(yǎng)箱中靜置培養(yǎng),每2天換液1次。當(dāng)細(xì)胞長(zhǎng)滿瓶底80%左右時(shí),用0.25%胰蛋白酶消化傳代。取第3-8代的細(xì)胞,當(dāng)細(xì)胞長(zhǎng)至70%-80%融合時(shí)進(jìn)行實(shí)驗(yàn)。 二、實(shí)驗(yàn)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(yīng)比色法(MTT法)觀察細(xì)胞增殖變化,免疫細(xì)胞化學(xué)法測(cè)定bFGF和增殖細(xì)胞核抗原(proliferating cell nuclear antigen, PCNA)蛋白的表達(dá),同時(shí)用RT-PCR檢測(cè)bFGF mRNA表達(dá)。 2、用篩選出的最適CSE濃度處理VSMC不同時(shí)間(Oh,4h,8h,12h,24h)后,檢測(cè)bFGFmRNA, bFGF和PCNA蛋白的變化。 3、用bFGF抗體和最適濃度CSE干預(yù)VSMC 24h后檢測(cè)細(xì)胞增殖,bFGF和PCNA蛋白表達(dá)的變化。 三、實(shí)驗(yàn)檢測(cè)方法 噻唑藍(lán)[3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide,MTT]比色法測(cè)定VSMC的增殖；用免疫細(xì)胞化學(xué)法測(cè)定bFGF, PCNA蛋白的表達(dá)；用RT-PCR檢測(cè)bFGF mRNA表達(dá)的變化。 實(shí)驗(yàn)結(jié)果 1、與對(duì)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(guò)高濃度(20%CSE)組與對(duì)照組相比差異無(wú)顯著性(P0.05)。bFGFmRNA、蛋白和PCNA蛋白在對(duì)照組中有少量表達(dá)。與對(duì)照組相比,2.5%CSE組表達(dá)增加(P0.01),5%CSE組達(dá)到高峰。10%、20%CSE組仍高于對(duì)照組(P0.01)。 2、對(duì)照組(不加CSE組即Oh組)VSMC中有少量bFGF mRNA、蛋白和PCNA蛋白表達(dá)。5%CSE刺激4h后細(xì)胞內(nèi)bFGF mRNA、蛋白和PCNA蛋白表達(dá)增加(P0.01),bFGFmRNA于8h達(dá)高峰；bFGF和PCNA蛋白于12h達(dá)高峰。 3、bFGF抗體可顯著抑制5%CSE誘導(dǎo)的MTT吸光度(A值)和bFGF、PCNA蛋白表達(dá)增加。 結(jié)論 CSE可能是通過(guò)增加bFGF的表達(dá)促進(jìn)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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