本文關(guān)鍵詞： 血管衰老 細(xì)胞凋亡 血管緊張素II Valsartan Bcl-2 Bax Caspase-3　出處：《中國(guó)醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

【摘要】： 前言 21世紀(jì)全球進(jìn)入不可逆轉(zhuǎn)的老齡化社會(huì),全球老齡化社會(huì)的到來使發(fā)達(dá)國(guó)家和發(fā)展中國(guó)家同樣面臨嚴(yán)重的人口社會(huì)問題。據(jù)中國(guó)老齡化工作委員會(huì)辦公室2006年12月23日公布《中國(guó)人口老齡化發(fā)展趨勢(shì)預(yù)測(cè)研究報(bào)告》:2005年底中國(guó)60歲以上人口近1.44億,占總?cè)丝诒壤?1%,2037年超過4億,我國(guó)老年人口正以年均3%的速度增長(zhǎng)。為應(yīng)對(duì)老齡化社會(huì)帶來的巨大人口健康問題,滿足我國(guó)社會(huì)經(jīng)濟(jì)實(shí)現(xiàn)長(zhǎng)期可持續(xù)發(fā)展的重大需求,國(guó)家實(shí)施戰(zhàn)略計(jì)劃,自2000年起設(shè)立重大衰老攻關(guān)課題(973課題)。而血管衰老研究成為緊要的社會(huì)需求,血管結(jié)構(gòu)、功能隨增齡發(fā)生的特征性重塑稱之為血管衰老。臨床研究證實(shí),隨增齡,許多心血管疾病發(fā)生率急劇上升,如高血壓病、冠心病、心功能不全、腦卒中等。有研究指出老年人心血管疾病風(fēng)險(xiǎn)的增加是增齡與疾病相互作用(age-diseaseinteractions)的結(jié)果;Baltimore衰老縱向研究證實(shí)年齡相關(guān)的動(dòng)脈特征性變化與增齡依賴的血管疾病的急劇增高密切相關(guān)。在我們前期“973”課題研究中血管衰老的機(jī)制研究取得如下成果:衰老血管有其特征性結(jié)構(gòu)和功能改變;隨增齡AngⅡ增高并從mRNA水平上調(diào)NADPH氧化酶p22phox表達(dá),介導(dǎo)細(xì)胞內(nèi)活性氧生成增加可能是血管衰老的主要原因之一;活性氧在介導(dǎo)血管及內(nèi)皮細(xì)胞衰老中有重要作用。血管衰老機(jī)理的研究是防治衰老血管重塑及相關(guān)疾病的基石。血管衰老是心血管疾病的獨(dú)立危險(xiǎn)因子,增齡引起的血管改變可能成為治療和預(yù)防血管疾病的靶目標(biāo)。 細(xì)胞凋亡學(xué)說是較為公認(rèn)的衰老機(jī)理學(xué)說。其理論認(rèn)為,衰老是細(xì)胞自發(fā)的、主動(dòng)的過程,即生物從發(fā)育到衰老,在體預(yù)先即有時(shí)間安排,該時(shí)間安排稱為“生物鐘”,由基因程序所決定。由于啟動(dòng)衰老的基因存在與“關(guān)閉了開關(guān)”基因(多效應(yīng)定時(shí)基因)的存在,啟動(dòng)和關(guān)閉衰老現(xiàn)象的發(fā)生,均按照時(shí)間程序進(jìn)行,稱“程序性細(xì)胞死亡”(programmed cell death,PCD)或“細(xì)胞凋亡”(apoptosis)�？刂粕L(zhǎng)發(fā)育的基因在各個(gè)時(shí)期均可開啟或關(guān)閉,有些在生命晚期發(fā)揮作用的基因可能控制衰老�；虬l(fā)生突變或基因功能喪失,是衰老主要原因,程序衰老理論能更好解釋衰老現(xiàn)象。 有研究指出血管結(jié)構(gòu)、功能隨增齡發(fā)生改變即血管衰老直接改變了各種心血管疾病發(fā)生的閾值和嚴(yán)重度,而血管衰老與損傷是造成人類死亡的主要原因之一。因此,為應(yīng)對(duì)老齡化社會(huì)帶來的巨大人口健康問題,血管衰老的研究將成為緊要的社會(huì)需求。近年來,細(xì)胞凋亡與衰老關(guān)系的研究也受到重視,細(xì)胞凋亡與衰老過程中組織器官功能的退化及衰老相關(guān)疾病的發(fā)生、發(fā)展密切相關(guān),如帕金森氏病、老年癡呆和動(dòng)脈粥樣硬化等都被認(rèn)為與器官的老化有關(guān)。目前值得注意的是細(xì)胞凋亡引起血管衰老的機(jī)制尚不清楚,故我們?cè)O(shè)計(jì)如下的細(xì)胞實(shí)驗(yàn)及動(dòng)物實(shí)驗(yàn)進(jìn)行驗(yàn)證凋亡調(diào)控基因與血管衰老相關(guān)性,并探討血管緊張素Ⅱ受體拮抗劑(AngiotensinⅡReceptor Blocker,ARB)纈沙坦(Valsartan)的干預(yù)作用,旨在從基因及蛋白水平,揭示血管衰老可能發(fā)生的機(jī)制。 方法 1.細(xì)胞實(shí)驗(yàn) (1)細(xì)胞分組:體外培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞株(HUVECs)分為對(duì)照組、AngⅡ組、Valsartan組,AngⅡ及Valsartan的終濃度均為10~(-6)mol/L (2)細(xì)胞衰老程度鑒定:通過β-gal染色、流式細(xì)胞術(shù)進(jìn)行細(xì)胞周期分析鑒定細(xì)胞衰老程度 (3)通過AnnexinV-FICT標(biāo)記的流式細(xì)胞術(shù)檢測(cè)凋亡早期事件的發(fā)生 (4)熒光顯微鏡和透射電子顯微鏡觀察觀察細(xì)胞形態(tài)學(xué)的變化 (5)RT-PCR分析各組細(xì)胞Bcl-2、Bax和Caspase-3的mRNA表達(dá) (6)Western blot分析各組細(xì)胞Bcl-2、Bax和Caspase-3的蛋白表達(dá) 2.動(dòng)物實(shí)驗(yàn) (1)分組:Wistar健康大鼠60只分成3組:青年組(3-4月齡,n=20只);18-20月齡大鼠隨機(jī)分為兩組:衰老組(n=20只)、Valsartan組(n=20只),衰老組正常飲水,Valsartan組(n=20只,飲用水予以Valsartan 30mg.kg~(-1).d~(-1)),相同條件下喂養(yǎng)至22-24月齡 (2)石蠟切片制備及HE染色,Masson染色 (3)血漿MOD、SOD檢測(cè) (4)主動(dòng)脈順應(yīng)性測(cè)定 (5)主動(dòng)脈內(nèi)皮細(xì)胞Bcl-2、Bax免疫組化染色檢測(cè) (6)分別應(yīng)用RT-PCR和Western bolt檢測(cè)各組大鼠Bcl-2、Bax和Caspase-3的mRNA及蛋白表達(dá) 結(jié)果 1.細(xì)胞實(shí)驗(yàn) 經(jīng)AngⅡ刺激后,HUVECs的β-gal染色陽性細(xì)胞數(shù)增加,細(xì)胞向G_0-G_1期停滯,熒光顯微鏡可見明顯的細(xì)胞凋亡,透射電子顯微鏡可見細(xì)胞衰老的特征性改變和細(xì)胞核染色質(zhì)濃縮和凝聚。與AngⅡ誘導(dǎo)組相比,Valsartan組Bcl-2的mRNA及蛋白表達(dá)水平明顯增高,Bax和Caspase-3的mRNA及蛋白表達(dá)水平降低。 2.動(dòng)物實(shí)驗(yàn) 隨增齡,大鼠的主動(dòng)脈管壁增厚,順應(yīng)性下降,內(nèi)皮功能受損,與衰老組相比,Valsartan組血漿丙二醛濃度明顯降低,超氧化物歧化酶濃度明顯升高;主動(dòng)脈血管的順應(yīng)性增高,其中彈性面積有顯著性差異;Bcl-2的mRNA及蛋白表達(dá)水平明顯增高,Bax的mRNA和Caspase-3及蛋白表達(dá)水平降低。 結(jié)論 1.AngⅡ可誘導(dǎo)體外培養(yǎng)的內(nèi)皮細(xì)胞老化,從而復(fù)制細(xì)胞衰老。 2.AngⅡ誘導(dǎo)的衰老HUVECs發(fā)生凋亡,提示細(xì)胞凋亡參與了AngⅡ誘導(dǎo)HUVECs衰老的過程;細(xì)胞凋亡是AngⅡ誘導(dǎo)的HUVECs老化的特征之一。 3.AngⅡ誘導(dǎo)血管內(nèi)皮細(xì)胞衰老的分子機(jī)制之一可能與Bcl-2、Bax mRNA及蛋白表達(dá)的失衡和Caspase-3的mRNA及蛋白表達(dá)水平降低有關(guān)。 4.Valsartan對(duì)血管內(nèi)皮細(xì)胞衰老有一定保護(hù)作用,為延緩細(xì)胞、血管衰老開辟新途徑。 5.血管衰老有其特征性生理改變,Bcl-2、Bax的mRNA及蛋白表達(dá)的失衡和Caspase-3的mRNA及蛋白表達(dá)水平降低可能是血管衰老的重要分子機(jī)制之一。 6.Valsartan有一定的改善血管衰老的作用。
[Abstract]:Preface
The aging society in twenty-first Century the world entered an aging society, with the arrival of the global population is also facing a serious social problem in developed countries and developing countries. According to the China Aging Working Committee Office announced in December 23, 2006 "development trend of aging population China Prediction Research Report": by the end of 2005 people over the age of 60 China nearly 144 million, the proportion of the total population of 11% in 2037, more than 400 million, China's elderly population is growing at an annual rate of 3%. The huge population health problems to deal with the aging of society, to meet the demand of social economy in our country to achieve a major long-term sustainable development, the national implementation of the strategic plan, since 2000 the establishment of major Aging Project (973 project). The research vessel aging has become a critical social demand, vascular structure, characteristic function remodeling with aging occurs is called vascular aging. Clinical studies Actually, with the increase of age, the incidence of a sharp rise in many cardiovascular diseases, such as hypertension, coronary heart disease, heart failure, stroke and so on. There is increasing research pointed out that the risk of cardiovascular diseases in the elderly is aging and disease interactions (age-diseaseinteractions) results; longitudinal study confirmed the changes of arterial characteristics associated with increasing age age dependent vascular disease Baltimore aging is closely related to a sharp increase in the mechanism research achievements are as follows. Our previous "vessel 973" research in aging: vascular aging has its specific structure and function change; with the increase of age and increased expression of Ang II in the level of mRNA and upregulation of NADPH oxidase p22phox, mediated by reactive oxygen species in the formation is one of the major causes of vascular aging; active oxygen has an important role in mediating vascular and endothelial cell senescence. The research mechanism of vascular aging is the prevention and treatment of aging Vascular aging is the cornerstone of related diseases. Vascular aging is an independent risk factor of cardiovascular disease. Vascular changes caused by aging may become a target for treatment and prevention of vascular diseases.
The cell apoptosis theory about aging is widely accepted. The theory holds that aging is a cell autonomous, active process, biological from development to aging, in the body that pre schedule, the schedule is called "biological clock", decided by the program. The gene start aging gene and "switched" gene (multi effect timing gene) the existence of startup and shutdown aging phenomenon, according to the time of the procedure, called "programmed cell death" (programmed cell, death, PCD) or "apoptosis" (apoptosis). Control the growth and development of genes in each period can be opened or closed some genes may play a role, to control aging in later life. Gene mutation or loss of gene function is the main cause of aging, aging process theory can better explain the phenomenon of aging.
鏈夌爺絀舵寚鍑?guó)櫋�綆＄粨鏋，

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