凋亡调控基因与血管衰老相关性的实验研究
发布时间：2018-02-26 17:28
本文关键词： 血管衰老 细胞凋亡 血管紧张素II Valsartan Bcl-2 Bax Caspase-3 出处：《中国医科大学》2008年博士论文 论文类型：学位论文
【摘要】： 前言 21世纪全球进入不可逆转的老龄化社会,全球老龄化社会的到来使发达国家和发展中国家同样面临严重的人口社会问题。据中国老龄化工作委员会办公室2006年12月23日公布《中国人口老龄化发展趋势预测研究报告》:2005年底中国60岁以上人口近1.44亿,占总人口比例11%,2037年超过4亿,我国老年人口正以年均3%的速度增长。为应对老龄化社会带来的巨大人口健康问题,满足我国社会经济实现长期可持续发展的重大需求,国家实施战略计划,自2000年起设立重大衰老攻关课题(973课题)。而血管衰老研究成为紧要的社会需求,血管结构、功能随增龄发生的特征性重塑称之为血管衰老。临床研究证实,随增龄,许多心血管疾病发生率急剧上升,如高血压病、冠心病、心功能不全、脑卒中等。有研究指出老年人心血管疾病风险的增加是增龄与疾病相互作用(age-diseaseinteractions)的结果;Baltimore衰老纵向研究证实年龄相关的动脉特征性变化与增龄依赖的血管疾病的急剧增高密切相关。在我们前期“973”课题研究中血管衰老的机制研究取得如下成果:衰老血管有其特征性结构和功能改变;随增龄AngⅡ增高并从mRNA水平上调NADPH氧化酶p22phox表达,介导细胞内活性氧生成增加可能是血管衰老的主要原因之一;活性氧在介导血管及内皮细胞衰老中有重要作用。血管衰老机理的研究是防治衰老血管重塑及相关疾病的基石。血管衰老是心血管疾病的独立危险因子,增龄引起的血管改变可能成为治疗和预防血管疾病的靶目标。 细胞凋亡学说是较为公认的衰老机理学说。其理论认为,衰老是细胞自发的、主动的过程,即生物从发育到衰老,在体预先即有时间安排,该时间安排称为“生物钟”,由基因程序所决定。由于启动衰老的基因存在与“关闭了开关”基因(多效应定时基因)的存在,启动和关闭衰老现象的发生,均按照时间程序进行,称“程序性细胞死亡”(programmed cell death,PCD)或“细胞凋亡”(apoptosis)。控制生长发育的基因在各个时期均可开启或关闭,有些在生命晚期发挥作用的基因可能控制衰老。基因发生突变或基因功能丧失,是衰老主要原因,程序衰老理论能更好解释衰老现象。 有研究指出血管结构、功能随增龄发生改变即血管衰老直接改变了各种心血管疾病发生的阈值和严重度,而血管衰老与损伤是造成人类死亡的主要原因之一。因此,为应对老龄化社会带来的巨大人口健康问题,血管衰老的研究将成为紧要的社会需求。近年来,细胞凋亡与衰老关系的研究也受到重视,细胞凋亡与衰老过程中组织器官功能的退化及衰老相关疾病的发生、发展密切相关,如帕金森氏病、老年痴呆和动脉粥样硬化等都被认为与器官的老化有关。目前值得注意的是细胞凋亡引起血管衰老的机制尚不清楚,故我们设计如下的细胞实验及动物实验进行验证凋亡调控基因与血管衰老相关性,并探讨血管紧张素Ⅱ受体拮抗剂(AngiotensinⅡReceptor Blocker,ARB)缬沙坦(Valsartan)的干预作用,旨在从基因及蛋白水平,揭示血管衰老可能发生的机制。 方法 1.细胞实验 (1)细胞分组:体外培养的人脐静脉内皮细胞株(HUVECs)分为对照组、AngⅡ组、Valsartan组,AngⅡ及Valsartan的终浓度均为10~(-6)mol/L (2)细胞衰老程度鉴定:通过β-gal染色、流式细胞术进行细胞周期分析鉴定细胞衰老程度 (3)通过AnnexinV-FICT标记的流式细胞术检测凋亡早期事件的发生 (4)荧光显微镜和透射电子显微镜观察观察细胞形态学的变化 (5)RT-PCR分析各组细胞Bcl-2、Bax和Caspase-3的mRNA表达 (6)Western blot分析各组细胞Bcl-2、Bax和Caspase-3的蛋白表达 2.动物实验 (1)分组:Wistar健康大鼠60只分成3组:青年组(3-4月龄,n=20只);18-20月龄大鼠随机分为两组:衰老组(n=20只)、Valsartan组(n=20只),衰老组正常饮水,Valsartan组(n=20只,饮用水予以Valsartan 30mg.kg~(-1).d~(-1)),相同条件下喂养至22-24月龄 (2)石蜡切片制备及HE染色,Masson染色 (3)血浆MOD、SOD检测 (4)主动脉顺应性测定 (5)主动脉内皮细胞Bcl-2、Bax免疫组化染色检测 (6)分别应用RT-PCR和Western bolt检测各组大鼠Bcl-2、Bax和Caspase-3的mRNA及蛋白表达 结果 1.细胞实验 经AngⅡ刺激后,HUVECs的β-gal染色阳性细胞数增加,细胞向G_0-G_1期停滞,荧光显微镜可见明显的细胞凋亡,透射电子显微镜可见细胞衰老的特征性改变和细胞核染色质浓缩和凝聚。与AngⅡ诱导组相比,Valsartan组Bcl-2的mRNA及蛋白表达水平明显增高,Bax和Caspase-3的mRNA及蛋白表达水平降低。 2.动物实验 随增龄,大鼠的主动脉管壁增厚,顺应性下降,内皮功能受损,与衰老组相比,Valsartan组血浆丙二醛浓度明显降低,超氧化物歧化酶浓度明显升高;主动脉血管的顺应性增高,其中弹性面积有显著性差异;Bcl-2的mRNA及蛋白表达水平明显增高,Bax的mRNA和Caspase-3及蛋白表达水平降低。 结论 1.AngⅡ可诱导体外培养的内皮细胞老化,从而复制细胞衰老。 2.AngⅡ诱导的衰老HUVECs发生凋亡,提示细胞凋亡参与了AngⅡ诱导HUVECs衰老的过程;细胞凋亡是AngⅡ诱导的HUVECs老化的特征之一。 3.AngⅡ诱导血管内皮细胞衰老的分子机制之一可能与Bcl-2、Bax mRNA及蛋白表达的失衡和Caspase-3的mRNA及蛋白表达水平降低有关。 4.Valsartan对血管内皮细胞衰老有一定保护作用,为延缓细胞、血管衰老开辟新途径。 5.血管衰老有其特征性生理改变,Bcl-2、Bax的mRNA及蛋白表达的失衡和Caspase-3的mRNA及蛋白表达水平降低可能是血管衰老的重要分子机制之一。 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.
鏈夌爺绌舵寚鍑鸿绠＄粨鏋，
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