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

【摘要】： 前言 21世紀全球進入不可逆轉(zhuǎn)的老齡化社會,全球老齡化社會的到來使發(fā)達國家和發(fā)展中國家同樣面臨嚴重的人口社會問題。據(jù)中國老齡化工作委員會辦公室2006年12月23日公布《中國人口老齡化發(fā)展趨勢預測研究報告》:2005年底中國60歲以上人口近1.44億,占總?cè)丝诒壤?1%,2037年超過4億,我國老年人口正以年均3%的速度增長。為應對老齡化社會帶來的巨大人口健康問題,滿足我國社會經(jīng)濟實現(xiàn)長期可持續(xù)發(fā)展的重大需求,國家實施戰(zhàn)略計劃,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,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險的增加是增齡與疾病相互作用(age-diseaseinteractions)的結(jié)果;Baltimore衰老縱向研究證實年齡相關(guān)的動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,介導細胞內(nèi)活性氧生成增加可能是血管衰老的主要原因之一;活性氧在介導血管及內(nèi)皮細胞衰老中有重要作用。血管衰老機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立危險因子,增齡引起的血管改變可能成為治療和預防血管疾病的靶目標。 細胞凋亡學說是較為公認的衰老機理學說。其理論認為,衰老是細胞自發(fā)的、主動的過程,即生物從發(fā)育到衰老,在體預先即有時間安排,該時間安排稱為“生物鐘”,由基因程序所決定。由于啟動衰老的基因存在與“關(guān)閉了開關(guān)”基因(多效應定時基因)的存在,啟動和關(guān)閉衰老現(xiàn)象的發(fā)生,均按照時間程序進行,稱“程序性細胞死亡”(programmed cell death,PCD)或“細胞凋亡”(apoptosis)�？刂粕L發(fā)育的基因在各個時期均可開啟或關(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,為應對老齡化社會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需求。近年來,細胞凋亡與衰老關(guān)系的研究也受到重視,細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、老年癡呆和動脈粥樣硬化等都被認為與器官的老化有關(guān)。目前值得注意的是細胞凋亡引起血管衰老的機制尚不清楚,故我們設(shè)計如下的細胞實驗及動物實驗進行驗證凋亡調(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)生的機制。 方法 1.細胞實驗 (1)細胞分組:體外培養(yǎng)的人臍靜脈內(nèi)皮細胞株(HUVECs)分為對照組、AngⅡ組、Valsartan組,AngⅡ及Valsartan的終濃度均為10~(-6)mol/L (2)細胞衰老程度鑒定:通過β-gal染色、流式細胞術(shù)進行細胞周期分析鑒定細胞衰老程度 (3)通過AnnexinV-FICT標記的流式細胞術(shù)檢測凋亡早期事件的發(fā)生 (4)熒光顯微鏡和透射電子顯微鏡觀察觀察細胞形態(tài)學的變化 (5)RT-PCR分析各組細胞Bcl-2、Bax和Caspase-3的mRNA表達 (6)Western blot分析各組細胞Bcl-2、Bax和Caspase-3的蛋白表達 2.動物實驗 (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 (4)主動脈順應性測定 (5)主動脈內(nèi)皮細胞Bcl-2、Bax免疫組化染色檢測 (6)分別應用RT-PCR和Western bolt檢測各組大鼠Bcl-2、Bax和Caspase-3的mRNA及蛋白表達 結(jié)果 1.細胞實驗 經(jīng)AngⅡ刺激后,HUVECs的β-gal染色陽性細胞數(shù)增加,細胞向G_0-G_1期停滯,熒光顯微鏡可見明顯的細胞凋亡,透射電子顯微鏡可見細胞衰老的特征性改變和細胞核染色質(zhì)濃縮和凝聚。與AngⅡ誘導組相比,Valsartan組Bcl-2的mRNA及蛋白表達水平明顯增高,Bax和Caspase-3的mRNA及蛋白表達水平降低。 2.動物實驗 隨增齡,大鼠的主動脈管壁增厚,順應性下降,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;主動脈血管的順應性增高,其中彈性面積有顯著性差異;Bcl-2的mRNA及蛋白表達水平明顯增高,Bax的mRNA和Caspase-3及蛋白表達水平降低。 結(jié)論 1.AngⅡ可誘導體外培養(yǎng)的內(nèi)皮細胞老化,從而復制細胞衰老。 2.AngⅡ誘導的衰老HUVECs發(fā)生凋亡,提示細胞凋亡參與了AngⅡ誘導HUVECs衰老的過程;細胞凋亡是AngⅡ誘導的HUVECs老化的特征之一。 3.AngⅡ誘導血管內(nèi)皮細胞衰老的分子機制之一可能與Bcl-2、Bax mRNA及蛋白表達的失衡和Caspase-3的mRNA及蛋白表達水平降低有關(guān)。 4.Valsartan對血管內(nèi)皮細胞衰老有一定保護作用,為延緩細胞、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及蛋白表達的失衡和Caspase-3的mRNA及蛋白表達水平降低可能是血管衰老的重要分子機制之一。 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.
鏈夌爺絀舵寚鍑鴻綆＄粨鏋，

本文編號：1539024