AngⅡ-p22phox-活性氧通路介导血管衰老及siRNA靶向干预的实验研究
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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。
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, the
control 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 mRNA
and 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.
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。
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, the
control 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 mRNA
and 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.
引文
1. Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part Ⅰ: aging arteries: a"set up" for vascular disease. Circulation, 2003,107: 139—46.
2. Li H, Bai XJ, Chen XM. Progress on the study of mechanism and biomarkers of arterial and cardiac aging. Zhonghua Yi Xue Za Zhi, 2005,85:212—5.
3. Fozard JL, Vercryssen M, Reynolds SL, et al. Age differences and changes in reaction time: the Baltimore Longitudinal Study of Aging. J Gerontol, 1994,49: P179—89.
4. Costa PT Jr, McCrae RR. Psychological research in the Baltimore Longitudinal Study of Aging. Z Gerontol, 1993,26: 138—41.
5.白小涓,李虹,杨莹等.洛沙坦对心脏和动脉衰老保护作用的实验研究.中国老年学杂志,2005,25:171—173.
6.白小涓,李虹,杨军等.沈阳地区健康人群心血管预后中间终点指标评估.中华医学杂志,2005,85:1062.
7. Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part Ⅱ: the aging heart in health: links to heart disease. Circulation, 2003,107: 346—54.
8. Kingwell BA, Cameron JD, Dart AM. Large artery stiffness and baroreflex function. Circulation, 2002,105: e56.
9. Minamino T, Miyauchi H, Yoshida T, et al. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation, 2002,105: 1541—4.
10. van der Loo B, Labugger R, Skepper JN, et al. Enhanced peroxynitrite formation is associated with vascular aging. J Exp.Med, 2000,192:1731—44.
11. Asai K, Kudej RK, Shen YT, et al. Peripheral vascular endothelial dysfunction and apoptosis in old monkeys. Arterioscler Thromb Vasc Biol, 2000,20: 1493—9.
12. Belmin J, Corman B, Merval R, et al. Age - related changes in endothelial permeability and distribution volume of albumin in rat aorta. Am J Physiol, 1993,264: H679-85.
13. Grune T. Oxidative stress, aging and the proteasomal system. Biogerontology, 2000,1:31 - 40.
14 . Shringarpure R, Da vies KJ. Protein turnover by the proteasome in aging and disease. Free Radic Biol Med, 2002,32:1084 - 9.
15. Droge W. Oxidative stress and aging. Adv Exp Med Biol, 2003, 543:191-200.
16 . Munzel T, Keaney JF Jr. Are ACE inhibitors a "magic bullet" against oxidative stress?. Circulation, 2001,104:1571 - 4.
17. Li JM, Mullen AM, Yun S, et al. Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor - alpha. Circ Res, 2002,90:143-50.
18 . Tarpey MM, Fridovich I. Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxyni- trite. Circ Res, 2001,89.-224-36.
19 . Zhang C, Yang J, Jennings LK. Leukocyte-derived myeloper-oxidase amplifies high-glucose-induced endothelial dysfunction through interaction with high - glucose -stimulated, vascular non-leukocyte - derived reactive oxygen species. Diabetes, 2004,53:2950 - 9.
20 . Berry C, Hamilton CA, Brosnan MJ, et al. Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries. Circulation, 2000,101:2206 - 12.
21. Csiszar A, Ungvari Z, Edwards JG, et al. Aging-induced phe- notypic changes and oxidative stress impair coronary arteriolar function. Circ Res, 2002,90:1159-66.
22. Tham DM, Martin-McNulty B, Wang YX, et al. Angiotensin II injures the arterial wall causing increased aortic stiffening in apoli- poprotein E-deficient mice. Am J Physiol Regul Integr Comp Physiol, 2002,283: R1442—9.
23. Michel JB, Heudes D, Michel O, et al. Effect of chronic ANG Ⅰ—converting enzyme inhibition on aging processes. Ⅱ. Large arteries. Am J Physiol, 1994, 267: R124—35.
24.郭延松,吴宗贵,杨军柯等.三种大鼠动脉粥样硬化模型复制方法的比较.中国动脉硬化杂志,2003,11:465—469.
25.李文春,张红梅,孙万群等.不同段脐带静脉组织结构的定量分析及其临床意义.解剖学杂志,2004,27:79—81.
26. NEUMAN RE, LOGAN MA. The determination of collagen and elastin in tissues. J Biol Chem, 1950,186:549—56.
27. Didion SP, Ryan MJ, Baumbach GL, et al. Superoxide contributes to vascular dysfunction in mice that express human renin and angiotensinogen. Am J Physiol Heart Circ Physiol, 2002,283: H1569—76.
28. Didion SP, Ryan MJ, Didion LA, et al. Increased superoxide and vascular dysfunction in CuZnSOD—deficient mice. Cire Res, 2002, 91:938—44.
29. Lund DD, Faraci FM, Miller FJ Jr, et al. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation, 2000,101: 1027—33.
30.李建生,蒋士卿,姚培发等.老龄大鼠动脉壁形态与显微结构成份的定量分析.河南中医药学刊,1996,11:19—21.
31.李志华,甄志军,李剑.血管紧张素Ⅱ2型受体mRNA在大鼠心肌生长发育及老龄化中的表达变化.西安交通大学学报医学版,2003,24:446—447.
32.崔春梅.NAD(P)H氧化酶及其在高血压血管损害中的作用.国外医学·生理、病理科学与临床分册,2003,6:630—633.
33. Frank PG, Woodman SE, Park DS, et al. Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol, 2003,23: 1161—8.
34. Schachter F, Faure—Delanef L, Guenot F, et al. Genetic associations with human longevity at the APOE and ACE loci. Nat Genet,1994,6:29—32.
35. Hamet P, deBlois D. Endothelial and myocyte apoptosis—role of angiotensin Ⅱ. Can J Cardiol, 2001,17 Suppl A: 26A—8A.
36. Szmitko PE, Wang CH, Weisel RD, et al. Biomarkers of vascular disease linking inflammation to endothelial activation: Part Ⅱ. Circulation, 2003,108: 2041—8.
37. Taddei S, Virdis A, Mattei P, et al. Endothelium—dependent forearm vasodilation is reduced in normotensive subjects with familial history of hypertension. J Cardiovasc Pharmacol, 1992,20 Suppl 12:S193—5.
38. Challah M, Nadaud S, Philippe M, et al. Circulating and cellular markers of endothelial dysfunction with aging in rats. Am J Physiol,1997,273: H1941—8.
39.陈样新,傅国胜.血管紧张素Ⅱ及其受体与血管内皮功能的关系.临床心血管病杂志,2003,19:698—700.
40.从洪良,黄体钢,周丽娟等.NO、AngⅡ对大鼠主动脉内皮细胞功能及形态学影响的实验研究.高血压杂志,1999,7:374—377.
41. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A, 1995,92:9363—7.
42.张静,丁一,史学义等.不同类型标本的衰老相关β—半乳糖苷酶染色方法.河南医科大学学报,2000,35:334—335.
43. Serrano M, Lin AW, McCurrach ME, et al. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell, 1997, 88: 593—602.
44.姚爱玉,周建军,刘亚兵等.IL—1α体外诱导血管内皮细胞衰老及其机理初步探讨.自然科学进展,2003,13:1165—1169.
45. Krishnamurthy J, Torrice C, Ramsey MR, et al. Ink4a/Arf expression is a biomarker of aging. J Clin Invest, 2004,114:1299—307.
46.马国诏,陈生弟,陆国强等.白细胞介素一1α引起胶质瘤细胞U251中蛋白聚集体形成、活性氧含量和线粒体膜电位增高.中华神经外科杂志,2003,37:396—401.
47.和平,刘建平,景涛等.腺病毒介导AT2R基因转染表达抑制肾间 质纤维母细胞增值.中国现代医学杂志,2003,13:16—19.
48. Gaur RK. RNA interference: a potential therapeutic tool for silencing splice isoforms linked to human diseases. Biotechniques, 2006,Apr: Suppl: 15—22.
49. Scherr M, Morgan MA, Eder M. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem, 2003,10:245—56.
50. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double—stranded RNA in Caenorhabditis elegans. Nature, 1998,391: 806—11.
51. Sheen FM, Levis RW. Transposition of the LINE—like retrotransposon TART to Drosophila chromosome termini. Proc Natl Acad Sci U S A, 1994,91: 12510—4.
52. Timmons L, Fire A. Specific interference by ingested dsRNA. Nature, 1998,395: 854.
53.吕辉,贺智敏.RNA干扰技术的演进及其在基因功能和基因治疗研究中的应用.中南大学学报(医学版),2005,50:1—12.
54. Bargmann CI. High—throughput reverse genetics: RNAi screens in Caenorhabditis elegans. Genome Biol, 2001, 2: 1005.
55. Zhang JQ, Ma YX, Wang DW et al. Selective knockdown of Angiotensin Ⅱ receptor subtype la in rat vascular smooth muscle cells by RNA interference. Zhonghua Xin Xue Guan Bing Za Zhi, 2006,34: 54—59.
56. Miyano—Kurosaki N, Takaku H. Gene silencing of virus replication by RNA interference. Handb Exp Pharmacol, 2006,173: 151—71.
57. Leirdal M, Sioud M. Gene silencing in mammalian cells by preformed small RNA duplexes. Biochem Biophys Res Commun, 2002,295:744—8.
58. Heidenreich O, Krauter J, Riehle H, et al. AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8: 21)—positive leukemic cells. Blood, 2003,101: 3157—63.
59. Ingelbrecht I, Van Houdt H, Van Montagu M, et al. Posttran- scriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc Natl Acad Sci U S A, 1994,91:10502-6.
60. Dzitoyeva S, Dimitrijevic N, Manev H. Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system. Mol Psychiatry, 2001, 6:665-70.
61. Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science, 2001,293:834 - 8.
62. Holen T, Amarzguioui M, Wiiger MT, et al. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res, 2002,30:1757 - 66.
63. Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 2001,107:297-307.
64. Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Onco- gene, 2002,21:5716-24.
65. Wall NR, Shi Y. Small RNA: can RNA interference be exploited for therapy. Lancet, 2003,362:1401 - 3.
66. Caplen NJ. RNAi as a gene therapy approach. Expert Opin Biol Ther, 2003,3:575-86.
67 . Yang D, Buchholz F, Huang Z et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci U S A, 2002, 99:9942-7.
68. Mittal V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet, 2004,5:355-65.
69. Ui-Tei K, Naito Y, Takahashi F et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res, 2004,32:936 - 48.
70. Reynolds A, Leake D, Boese Q et al. Rational siRNA design for RNA interference. Nat Biotechnol, 2004,22:326-30.
71. Zhang DX, Pan B, Liu B, et al. Experimental strategies of the application of RNAi technique in mammalian cells. Yi Chuan, 2005,27: 839-44.
72. Hu XX. RNA interference (RNAi) as novel approach for gene silencing-review. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2005,13:1141 -4.
73 . Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21 - nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001,411:494-8.
74. Hiscott A, McLellan DS. Graft -versus-host disease in alloge- neic bone marrow transplantation;the role of monoclonal antibodies in prevention and treatment. Br J Biomed Sci, 2000,57:163 -9.
75. Caplen NJ, Parrish S, Imani F, et al. Specific inhibition of gene expression by small double - stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A, 2001,98:9742 - 7.
76. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22 - nucleotide RNAs. Genes Dev, 2001,15:188 - 200.
77. Chen L, Zheng XX. Inhibition of CD40 expression on CA46 cell lines by hairpin siRNA eukaryotic expression vectors. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005,21:163-6.
78. Hannon GJ. RNA interference. Nature, 2002,418:244 -51.
2. Li H, Bai XJ, Chen XM. Progress on the study of mechanism and biomarkers of arterial and cardiac aging. Zhonghua Yi Xue Za Zhi, 2005,85:212—5.
3. Fozard JL, Vercryssen M, Reynolds SL, et al. Age differences and changes in reaction time: the Baltimore Longitudinal Study of Aging. J Gerontol, 1994,49: P179—89.
4. Costa PT Jr, McCrae RR. Psychological research in the Baltimore Longitudinal Study of Aging. Z Gerontol, 1993,26: 138—41.
5.白小涓,李虹,杨莹等.洛沙坦对心脏和动脉衰老保护作用的实验研究.中国老年学杂志,2005,25:171—173.
6.白小涓,李虹,杨军等.沈阳地区健康人群心血管预后中间终点指标评估.中华医学杂志,2005,85:1062.
7. Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part Ⅱ: the aging heart in health: links to heart disease. Circulation, 2003,107: 346—54.
8. Kingwell BA, Cameron JD, Dart AM. Large artery stiffness and baroreflex function. Circulation, 2002,105: e56.
9. Minamino T, Miyauchi H, Yoshida T, et al. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation, 2002,105: 1541—4.
10. van der Loo B, Labugger R, Skepper JN, et al. Enhanced peroxynitrite formation is associated with vascular aging. J Exp.Med, 2000,192:1731—44.
11. Asai K, Kudej RK, Shen YT, et al. Peripheral vascular endothelial dysfunction and apoptosis in old monkeys. Arterioscler Thromb Vasc Biol, 2000,20: 1493—9.
12. Belmin J, Corman B, Merval R, et al. Age - related changes in endothelial permeability and distribution volume of albumin in rat aorta. Am J Physiol, 1993,264: H679-85.
13. Grune T. Oxidative stress, aging and the proteasomal system. Biogerontology, 2000,1:31 - 40.
14 . Shringarpure R, Da vies KJ. Protein turnover by the proteasome in aging and disease. Free Radic Biol Med, 2002,32:1084 - 9.
15. Droge W. Oxidative stress and aging. Adv Exp Med Biol, 2003, 543:191-200.
16 . Munzel T, Keaney JF Jr. Are ACE inhibitors a "magic bullet" against oxidative stress?. Circulation, 2001,104:1571 - 4.
17. Li JM, Mullen AM, Yun S, et al. Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor - alpha. Circ Res, 2002,90:143-50.
18 . Tarpey MM, Fridovich I. Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxyni- trite. Circ Res, 2001,89.-224-36.
19 . Zhang C, Yang J, Jennings LK. Leukocyte-derived myeloper-oxidase amplifies high-glucose-induced endothelial dysfunction through interaction with high - glucose -stimulated, vascular non-leukocyte - derived reactive oxygen species. Diabetes, 2004,53:2950 - 9.
20 . Berry C, Hamilton CA, Brosnan MJ, et al. Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries. Circulation, 2000,101:2206 - 12.
21. Csiszar A, Ungvari Z, Edwards JG, et al. Aging-induced phe- notypic changes and oxidative stress impair coronary arteriolar function. Circ Res, 2002,90:1159-66.
22. Tham DM, Martin-McNulty B, Wang YX, et al. Angiotensin II injures the arterial wall causing increased aortic stiffening in apoli- poprotein E-deficient mice. Am J Physiol Regul Integr Comp Physiol, 2002,283: R1442—9.
23. Michel JB, Heudes D, Michel O, et al. Effect of chronic ANG Ⅰ—converting enzyme inhibition on aging processes. Ⅱ. Large arteries. Am J Physiol, 1994, 267: R124—35.
24.郭延松,吴宗贵,杨军柯等.三种大鼠动脉粥样硬化模型复制方法的比较.中国动脉硬化杂志,2003,11:465—469.
25.李文春,张红梅,孙万群等.不同段脐带静脉组织结构的定量分析及其临床意义.解剖学杂志,2004,27:79—81.
26. NEUMAN RE, LOGAN MA. The determination of collagen and elastin in tissues. J Biol Chem, 1950,186:549—56.
27. Didion SP, Ryan MJ, Baumbach GL, et al. Superoxide contributes to vascular dysfunction in mice that express human renin and angiotensinogen. Am J Physiol Heart Circ Physiol, 2002,283: H1569—76.
28. Didion SP, Ryan MJ, Didion LA, et al. Increased superoxide and vascular dysfunction in CuZnSOD—deficient mice. Cire Res, 2002, 91:938—44.
29. Lund DD, Faraci FM, Miller FJ Jr, et al. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation, 2000,101: 1027—33.
30.李建生,蒋士卿,姚培发等.老龄大鼠动脉壁形态与显微结构成份的定量分析.河南中医药学刊,1996,11:19—21.
31.李志华,甄志军,李剑.血管紧张素Ⅱ2型受体mRNA在大鼠心肌生长发育及老龄化中的表达变化.西安交通大学学报医学版,2003,24:446—447.
32.崔春梅.NAD(P)H氧化酶及其在高血压血管损害中的作用.国外医学·生理、病理科学与临床分册,2003,6:630—633.
33. Frank PG, Woodman SE, Park DS, et al. Caveolin, caveolae, and endothelial cell function. Arterioscler Thromb Vasc Biol, 2003,23: 1161—8.
34. Schachter F, Faure—Delanef L, Guenot F, et al. Genetic associations with human longevity at the APOE and ACE loci. Nat Genet,1994,6:29—32.
35. Hamet P, deBlois D. Endothelial and myocyte apoptosis—role of angiotensin Ⅱ. Can J Cardiol, 2001,17 Suppl A: 26A—8A.
36. Szmitko PE, Wang CH, Weisel RD, et al. Biomarkers of vascular disease linking inflammation to endothelial activation: Part Ⅱ. Circulation, 2003,108: 2041—8.
37. Taddei S, Virdis A, Mattei P, et al. Endothelium—dependent forearm vasodilation is reduced in normotensive subjects with familial history of hypertension. J Cardiovasc Pharmacol, 1992,20 Suppl 12:S193—5.
38. Challah M, Nadaud S, Philippe M, et al. Circulating and cellular markers of endothelial dysfunction with aging in rats. Am J Physiol,1997,273: H1941—8.
39.陈样新,傅国胜.血管紧张素Ⅱ及其受体与血管内皮功能的关系.临床心血管病杂志,2003,19:698—700.
40.从洪良,黄体钢,周丽娟等.NO、AngⅡ对大鼠主动脉内皮细胞功能及形态学影响的实验研究.高血压杂志,1999,7:374—377.
41. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A, 1995,92:9363—7.
42.张静,丁一,史学义等.不同类型标本的衰老相关β—半乳糖苷酶染色方法.河南医科大学学报,2000,35:334—335.
43. Serrano M, Lin AW, McCurrach ME, et al. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell, 1997, 88: 593—602.
44.姚爱玉,周建军,刘亚兵等.IL—1α体外诱导血管内皮细胞衰老及其机理初步探讨.自然科学进展,2003,13:1165—1169.
45. Krishnamurthy J, Torrice C, Ramsey MR, et al. Ink4a/Arf expression is a biomarker of aging. J Clin Invest, 2004,114:1299—307.
46.马国诏,陈生弟,陆国强等.白细胞介素一1α引起胶质瘤细胞U251中蛋白聚集体形成、活性氧含量和线粒体膜电位增高.中华神经外科杂志,2003,37:396—401.
47.和平,刘建平,景涛等.腺病毒介导AT2R基因转染表达抑制肾间 质纤维母细胞增值.中国现代医学杂志,2003,13:16—19.
48. Gaur RK. RNA interference: a potential therapeutic tool for silencing splice isoforms linked to human diseases. Biotechniques, 2006,Apr: Suppl: 15—22.
49. Scherr M, Morgan MA, Eder M. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem, 2003,10:245—56.
50. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double—stranded RNA in Caenorhabditis elegans. Nature, 1998,391: 806—11.
51. Sheen FM, Levis RW. Transposition of the LINE—like retrotransposon TART to Drosophila chromosome termini. Proc Natl Acad Sci U S A, 1994,91: 12510—4.
52. Timmons L, Fire A. Specific interference by ingested dsRNA. Nature, 1998,395: 854.
53.吕辉,贺智敏.RNA干扰技术的演进及其在基因功能和基因治疗研究中的应用.中南大学学报(医学版),2005,50:1—12.
54. Bargmann CI. High—throughput reverse genetics: RNAi screens in Caenorhabditis elegans. Genome Biol, 2001, 2: 1005.
55. Zhang JQ, Ma YX, Wang DW et al. Selective knockdown of Angiotensin Ⅱ receptor subtype la in rat vascular smooth muscle cells by RNA interference. Zhonghua Xin Xue Guan Bing Za Zhi, 2006,34: 54—59.
56. Miyano—Kurosaki N, Takaku H. Gene silencing of virus replication by RNA interference. Handb Exp Pharmacol, 2006,173: 151—71.
57. Leirdal M, Sioud M. Gene silencing in mammalian cells by preformed small RNA duplexes. Biochem Biophys Res Commun, 2002,295:744—8.
58. Heidenreich O, Krauter J, Riehle H, et al. AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8: 21)—positive leukemic cells. Blood, 2003,101: 3157—63.
59. Ingelbrecht I, Van Houdt H, Van Montagu M, et al. Posttran- scriptional silencing of reporter transgenes in tobacco correlates with DNA methylation. Proc Natl Acad Sci U S A, 1994,91:10502-6.
60. Dzitoyeva S, Dimitrijevic N, Manev H. Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system. Mol Psychiatry, 2001, 6:665-70.
61. Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science, 2001,293:834 - 8.
62. Holen T, Amarzguioui M, Wiiger MT, et al. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res, 2002,30:1757 - 66.
63. Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 2001,107:297-307.
64. Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Onco- gene, 2002,21:5716-24.
65. Wall NR, Shi Y. Small RNA: can RNA interference be exploited for therapy. Lancet, 2003,362:1401 - 3.
66. Caplen NJ. RNAi as a gene therapy approach. Expert Opin Biol Ther, 2003,3:575-86.
67 . Yang D, Buchholz F, Huang Z et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci U S A, 2002, 99:9942-7.
68. Mittal V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet, 2004,5:355-65.
69. Ui-Tei K, Naito Y, Takahashi F et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res, 2004,32:936 - 48.
70. Reynolds A, Leake D, Boese Q et al. Rational siRNA design for RNA interference. Nat Biotechnol, 2004,22:326-30.
71. Zhang DX, Pan B, Liu B, et al. Experimental strategies of the application of RNAi technique in mammalian cells. Yi Chuan, 2005,27: 839-44.
72. Hu XX. RNA interference (RNAi) as novel approach for gene silencing-review. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2005,13:1141 -4.
73 . Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21 - nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001,411:494-8.
74. Hiscott A, McLellan DS. Graft -versus-host disease in alloge- neic bone marrow transplantation;the role of monoclonal antibodies in prevention and treatment. Br J Biomed Sci, 2000,57:163 -9.
75. Caplen NJ, Parrish S, Imani F, et al. Specific inhibition of gene expression by small double - stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A, 2001,98:9742 - 7.
76. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22 - nucleotide RNAs. Genes Dev, 2001,15:188 - 200.
77. Chen L, Zheng XX. Inhibition of CD40 expression on CA46 cell lines by hairpin siRNA eukaryotic expression vectors. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005,21:163-6.
78. Hannon GJ. RNA interference. Nature, 2002,418:244 -51.