【作者】 李虹；

【导师】 白小涓；

【作者基本信息】 中国医科大学 ， 内科学， 2006， 博士

【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。1999年中国已经进入老龄化社会,中国老龄人口以每年3%的速度递增,2020年中国老龄人口将达到3亿人。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。有研究指出老人心血管疾病风险的增加是增龄与疾病相互作用(age-disease interactions)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。为应对老龄化社会带来的巨大人口健康问题,血管衰老研究成为紧要的社会需求。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。 活性氧(ROS)学说是较为公认的衰老机理学说。自血管壁ROS发现以来,大量研究证实ROS是调节血管张力和结构的重要信号分子,在维持血管稳态、介导血管损伤中起关键性作用,NADH/NADPH氧化酶是血管壁ROS产生的主要来源,血管紧张素Ⅱ(AngⅡ)是NADH/NADPH氧化酶重要的刺激因素。活性氧产生和清除的异常与高血压病、糖尿病、缺血性心肌病等密切相关。已有研究显示,人内乳动脉和大鼠冠状动脉活性氧产量很低却同衰老密切相关,因而ROS在介导血管衰老中应居于重要的地位。究竟增龄引起血管的氧化应激及慢性炎症状态的机理是什么,是否有某种因素在血管衰老中起关键性的作用仍未明确,对此问题的解答必将引起衰老理论的突破,并找到预防延缓衰老的有效措施。近年来,血管紧张素系统的研究倍受关注,相关研究提示增龄相关的血管局部血管紧张素系统的改变在衰老血管重塑中有重要的作用,但是缺乏相关的系统研究。更多还原

【Abstract】 ObjectiveIn 1999, China has entered an aged society. The aged population in China will increase by 3 percent per year and will reach 300 million in 2020. Structural and functional changes of blood vessel with advancing age are namely vascular aging. Clinical study indicated with advancing age, the incidence of many cardiovascular diseases such as hypertension, coronary heart disease, heart dysfunction and stroke increased rapidly. Some studies also indicated that the increased risk of cardiovascular diseases in aged population is the result of age disease interactions. The Baltimore longitudinal aging study found the age—related structural and functional changes in blood vessel were closely correlated with age—depended cardiovascular diseases. Therefore, the researches on vascular aging have been the impelling social requirement with aged society coming rapidly. The study of vascular aging, mechanism is the footstone for prevention and treatment of vascular remodeling and the related diseases. Vascular aging is the independent risk factor for cardiovascular diseases and the aging related structural and functional changes in blood vessel may be the latent target for prevention and treatment of cardiovascular diseases.The reactive oxygen species theory about aging is widely accepted. In recent years, reactive oxygen species(ROS) existing in vascular wall were discovered, related research indicated NADH/NADPH oxidase was the primary source of ROS in all kinds of vessel cells, p22phox is the essential subunit of NADH/NADPH oxidase activity, angiotensin II stimula-ted ROS production through NADH/NADPH oxidase, and possibly correlated with vascular remodification of vascular diseases, for example, it was showed NADH/NADPH oxidase contributed to the development of Ang II —dependent hypertension. As a result, ROS contributed much to the vascular aging regulation. But what is the primary mechanism for oxidation stress and chronic inflammation induced by aged organism is still unknown. To answer this question successfully will bring breakthrough for aging study and throw much light on the aging interventions. Nowadays, many studies have focused on angiotensin system, and many results showed that the local angiotensin system contribute much to the aging vascular remodeling. Unfortunately, the above theory need to be further researched.Based on above results, the following hypothesis is formed that Ang II up —regulate the expression of p22phox subunit of NADH/NADPH oxidase,so endothelial cells overproduce ROS, therefore endothelial dysfunction sets up and maintains the vascular aging process.Methods1. Animal experiment1. 1 Wistar rats were divided into three groups, the young group(3~ month old),the old control group(22~24 month old), valsartan group x treated with valsartan 30mg ? kg"1 ? d"1 for 5 months from 17 — 18 month of age).1. 2 Aorta artery structure and function were analysed.1. 3 Angiotensin H in plasma and aorta were detected.1. 4 ROS level of aortic tissues was investigated.1. 5 RT—PCR and Western blot were used to analyze the mRNA and protein expression of NADH/NADPH oxidase p22phox, angiotensin type 1 and 2 receptor (AT,R, AT2R).2. Cell experiment2.1 The cultured HUVECs in vitro were divided into 3 groups, thecontrol group, AngH group (stimulated with 10 6mol/l Angfl for 48h), Angll plus valsartan group(valsartan was added to cells lh before 10~6 mol/1 Angll ).2. 2 P—gal stain,cellcycle analysis, pi6 exprssion were used to identify cell aging status.2. 3 ROS and NO level in cells and medium were examined.2. 4 RT — PCR and Western blot were used to analysis the mRNA and protein expression of p22phox, angiotensin type 1 and 2 receptor (AT,R,AT2R).3. siRNA experiment3. 1 Design 3 kinds of siRNA and synthesis siRNA by Silencer? siR-NA Construction Kit (Ambion). Using siRNA to transfect HUVECs cultured in vitro and select the most powerful and most suitable transfec-tion concentration and time.3. 2 HUVECs were divided into 3 groups: Control group, Ang H group, siRNA group, Ang U + siRNA group.3. 3 (3—gal stain,cellcycle analysis and pi6 exprssion was used to i-dentify cell aging status.3. 4 ROS and NO level in cells and medium were examined.3. 5 RT —PCR and Western blot were used to analysis the mRNA and protein expression of p22phox, angiotensin type 1 and 2 receptor (AT,R,ATzR).Results1. Animal experiment1. 1 With advancing age, aorta artery structure and function changed, namely, aging aorta artery remodeled.1. 2 Angiontensin II and ROS level of aortic tissues increased.1. 3 mRNA and protein expression of p22phox and AT2R increased", but ATiR expression attenuated in aging aortic tissues.1.4 Valsartan treatment reduced ROS level, downregulated mRNAand protein expression of p22phox in aging aorta arteries, alleviated aorta structural and functional remodeling.2. Cell experiment2. 1 Ang II stimulation enhanced the positive cell number of |3 — gal stained HUVEC, depressed cell proliferation, and increased the protein expression of pi6, at the same time, stimulated cells producted less NO and more ROS.2. 2 The mRNA and protein expression of p22phox and AT2R in cells stimulated by AngII increased, the expression of AT]R decreased.2. 3 Valsartan treatment alleviated aging associated changes, decreased ROS production and increased NO production, and downregulated the mRNA and protein expression of p22phox.3. siRNA experiment3. 1 Among the 3 kinds of siRNA, siRNA—1 was the most powerful at silencing p22phox mRNA expression and was selected.3. 2 Silencing effect was the most powerful at 24h and 36h, and 50nM/L was the most suitable concentration.3. 3 Compared with Ang II group, the mRNA and protein expression of p22phox and AT2R in cells dereased, the expression of AT\R increased in Ang II + siRNA group.3. 4 Comparede with Ang H group, ROS level decreased and NO level increased in Ang II + siRNA group.3. 5 More cells stopped in Go —Gi stage in Ang II + siRNA group than that in Ang H group.Conclusion1. Age related vascular remodeling changes came forth in rats belonging to age group and the animal model was constructed successfully.2. AngiontensinII and ROS level of aorta increased, the mRNA and protein expression of p22phox and AT2R increased, but AT^R expression attenuated in aging aortic tissues.3. Valsartan treatment reduced ROS level, downregulated the mRNA and protein expression of p22phox in aging aorta arteries, alleviated aorta structural and functional changes.4. siRNA — 1 selected in the experiment silenced the expression of p22phox effectively, maybe siRNA construted in vitro is a readily and reliable techniques for silencing genes.5. NADH/NADPH oxidase is the primary source of ROS in endothe-lial cells, p22phox is the essential subunit of NADH/NADPH oxidase activity. After silencing the mRNA expression of p22phox by siRNA, ROS productivity decreased sharply in cells. RNAi will be the new and promising technology for prevention and treatment of cardiovascular diseases.6. AT2 R expression increased sharply in aged endothelial cells induced by Ang II and after the mRNA expression of p22phox silenced by siRNA, AT2R expression decreased markedly, which indicated the increasing expression of AT2R was an protective and regulatory mechanism for cell anti—aging process.7. Ang II inducing endothelial cells senescence is possibly important factor for vascular aging. Ang H —p22phox—ROS is the main pathway for endothelial cell senescence. Valsartan contributes some protective effects to the vascular aging by blocking ATiR.更多还原