摘要
心脑血管疾病是发达国家人口死亡的主要原因,在我国的死亡率也呈逐年增高的倾向。而动脉粥性硬化(atherosclerosis, AS)性心血管疾病则被称为发达国家的“头号杀手”,它在发展中国家发病率也越来越高(Shimizu T,2008)。其中冠状动脉粥样硬化性心脏病(coronary atheroselerosis heart disease)简称冠心病(coronary heart disease, CHD)是一种常见疾病,已成为欧美国家最多见的心脏病种。1990年全球人口为53亿,死亡5000万,其中630万死于冠心病,占12.4%。1991年美国心脏协会公布,1988年冠心病死亡人数占总死亡的45.3%,约1/5的美国人在65岁前死于心血管病。作为亚洲国家,我国目前冠心病发病率和死亡率仍处于较低发国家的行列,但随着人们生活方式的改变,近年来有增高的趋势。在住院心脏病患者中,本病所占的比例也不断增加。而冠心病的基本病因为动脉粥样硬化(atherosderesis, As),而AS斑块的不稳定,引起局部血小板聚集,急性血栓形成,管腔急性闭塞是急性冠脉综合征(acute coronary syndrome, ACS)发生的共同机制。随着我国经济社会的发展,人民生活水平提高和饮食水平习惯改变,以及随之而来的人口的老龄化,动脉粥性硬化引发的疾病也逐渐成为我国主要死因(牟一鸣等,2001)。世界卫生组织预测,在世界范围内的传染性疾病病死率得到有效控制之后,到2020年心管管疾病将成为全世界第一死因。
动脉粥样硬化(atherosclerosis, AS)是心脑血管疾病的重要病理基础,防治AS是防治心脑血管病的根本措施。动脉粥样硬化(atherosclerosis, AS)是以富含脂质的斑块在大动脉壁聚集为特征的系统疾病,是心脑血管病的主要病理基础,它是一种多因素所致的动脉血管慢性疾病,集中表现为全身性血管内皮功能障碍(endothelialdysfunction)、血管内膜慢性炎症、纤维增生、管腔狭窄及血栓形成等。其主要临床表现是心肌梗塞、中风和外周血管疾病,常伴有高血压、高胆固醇血症或糖尿病等。AS病因病理复杂,目前尚未完全阐明,但可以确定的是AS是一类涉及到多种遗传因素和环境因素的复杂疾病,这些遗传因素和环境因素之间的相互作用最终导致斑块形成和出现随后的一系列临床事件。AS性血管疾病病因众多,其中不少与生活方式密切相关,包括吸烟、高糖高脂饮食、缺乏体力活动等习惯。从生物学角度来看,AS是一种长期慢性炎症疾病,与血脂紊乱密切相关,其发生发展是一个十分复杂的过程。AS性疾病临床事件主要为冠心病和中风。
因此,对AS发生机制的研究日益受到关注,血脂增高和紊乱是大多数AS发生的前提和基础,血管内皮功能障碍是AS发生的始发事件,多种因素相互作用,导致了AS的发生、发展。其中主要因素包括:血管内皮细胞功能障碍,血管平滑肌细胞增生,血小板和血液凝固状态异常,单核细胞、巨噬细胞浸润,自由基损伤,病毒及细菌感染,细胞凋亡,炎症和免疫反应,钙离子沉积,基因异常等。目前普遍认认同的发病机制是“损伤应激学说”(Ross R,1999)。该学说认为AS的过程包括以下几个步骤:内皮细胞(vascular endothelial cell, VEC)受各种刺激而损伤;单核细胞通过损伤处进入内膜,转变为巨噬细胞;巨噬细胞在与协同的T淋巴细胞分泌的细胞因子的作用下转变为巨噬细胞源性泡沫细胞;动脉中膜的血管平滑肌细胞迁入内膜,吞噬脂质形成平滑肌源性泡沫细胞,并增生迁移形成纤维帽;纤维帽被巨噬细胞分泌的基质金属蛋白酶和T淋巴细胞分泌的Y-干扰素削弱,使斑块破裂而移动,形成动脉粥样理化的栓子。
关于AS的形成,近年来免疫炎症学说越来越受到重视。研究认为AS是对血管内皮局部损伤的一种保护性炎症—纤维增殖性回应。如果损伤持续一段时间,这种回应则变得过度,最终成为疾病即斑块形成。在斑块的形成过程中脂质沉积是最重要的因素,也是损伤反应最早期的表现之一。伴随着脂质的沉积,氧化低密度脂蛋白胆固醇(oxLDL-C)的形成,循环中的白细胞和单核细胞被激活,并迁移到病变处,后者在oxLDL-C作用下变成活化的巨噬细胞,通过他们的清道夫受体,摄取oxLDL-C变成泡沫细胞,泡沫细胞的不断产生和堆积导致脂质条纹的形成。泡沫细胞死亡时则释放大量的胆固醇酷与血浆脂蛋白的沉积构成斑块下的脂质核心。炎症应答继续发展,T细胞活化,则引发纤维增殖反应最终形成纤维帽。在斑块形成的早期脂质核心小,纤维帽厚,斑块呈稳定状态,伴随着泡沫细胞的不断死亡和血浆脂类的沉积,斑块下的脂质核心不断增大,另一方面大量巨噬细胞浸润释放大量水解酶,尤其是金属蛋白酶系列,通过降解纤维帽以及抑制胶原纤维的生成使纤维帽逐渐变薄,从而使稳定斑块转为不稳定斑块,后者在内、外因素作用下,最终发生破裂导致急性冠脉事件发生。病理学家亦发现在斑块的破裂部位可见大量巨噬细胞浸润,炎性标记物表达增加。由此可见,斑块的不断进展与炎症反应的程度密切相关。斑块是否发生破裂与斑块内的炎症反应强度及巨噬细胞的浸润程度有密切的关系。动脉壁内脂纹或斑块形成是AS的特征性病变,而动脉内膜的巨噬细胞以及由中膜向内膜迁移的平滑肌细胞经由清道夫受体(scavenger receptor, SR)摄取氧化低密度脂蛋白(oxidizedLDL, oxLDL),从而形成巨噬/平滑肌细胞源性泡沫细胞,是AS特征性病变发生发展过程中的一个核心事件。
人们对衰老与长寿的研究逾近百年,但至今为止,控制人类寿命之迷仍未被揭开。尽管衰老本身不是一种疾病,但很多疾病与衰老相关,如肿瘤,老年痴呆(Alzheimer's disease),动脉硬化,代谢紊乱(metabolic disorders)等。心脏病和中风引起的死亡率随年龄呈指数增加,65-74岁死亡率超过40%,85岁及以上的死亡率将近60%。越来越多的研究发现,即使没有高血压,糖尿病及吸烟等各种危险因子存在,心血管系统随年龄增加亦趋于易发疾病和受损,这可能和衰老血管的细胞内稳态相关(Ungvari et al.,2010).在实验动物和人的身上都已证实,氧化损伤产生的活性氧(reactive oxygen species, ROS)激活NAD(P)H氧化酶及下调NO的活性,导致衰老进程的内皮细胞功能障碍,从而引发老年人冠状动脉疾病和中风。
热量限制(calorie restiction, CR)是指在提供生物体充分的营养成分如必需氨基酸、维生素等,保证生物体不发生营养不良的情况下,限制每日摄取的总热量。一般CR饮食是指减少动物随意进食量的30%—40%。热量限制功能以一科,被动的方式降低氧化和其他损伤,从而导致生命周期延长,可以调控从酵母到哺乳动物等生物体的生命周期。自1935年McCay等首次报道CR延长大鼠寿命以来,大量研究已表明CR是除遗传操作以外最强有力的延缓衰老的方法。热量限制不仅能维持许多年轻时的生理状态,还能延缓和预防一些与年龄相关疾病如心血管疾病、糖尿病、肿瘤的发生、发展。热量限制(caloric restriction, CR)是已经被证明具有抗衰老及延长寿限的作用(Russell JC2008; Fontana L,2009;),包括热量摄入限制和减少进食次数两种方式。CR对心血管系统具有多效保护作用,如保护血管(阻止动脉硬化进程),提高心肌耐受性及延缓心脏衰老(Shinmura K.2011),衰老进程中,转录因子NF-kB通过激活IL-1,IL-6,TNF-a等细胞因子介导机体长期慢性炎症反应,这种长期慢性炎症反应引发一系列老年相关疾病的关键。因此,有人提出可以通过抗氧化药物(如姜黄素(curcumin))来抑制依赖NF-kB信号的炎症反应,而延缓衰老(Sikora Eab et al,2010)。研究表明,CR可以降低炎症因子IL-6和leptin的表达(Reed JL,2010)。
本课题分为四个部分,首先依据已经发表的芯片数据结果进行生物信息学的分析和挖掘,具体为:来源于美国Miller SJ提交的GSE7281芯片数据集,Fischer344大鼠自然衰老,年龄分别为3,6,15,28个月,取大鼠动脉提取mRNA
进行全基因组芯片分析
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7281;和来源于美国Becker KG提交的GSE11845芯片数据集,选取数据集中正常对照(自由饮食),热量限制(间断饮食)及高脂(氢化椰子油,热量高于正常对照组的60%)三组样本数据,进行心脏,肝脏及肌肉相关基因分析。http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11845。挖掘动脉硬化与衰老相关基因。接下来建立Wistar大鼠模型,利用RT-PCR, ELISA等方法进行血清氧化低密度脂蛋白(OXLDL),C反应蛋白(C-reactive protein, CRP),血栓前体蛋白(TpP)等靶标的检测以及进行病理分析观察血管形态,对芯片数据挖掘的结果进行进一步的实验验证。在建立高糖饮食与热量限制的大鼠模型的基础上,进一步通过实时荧光定量PCR技术(Real-time quantitative Polymerase Chain Reaction, Real Time PCR)验证生物信息学结果并筛选得到31个同时响应热量限制与高脂高糖饮食的基因表达。通过建立HC诱导动脉硬化的细胞模型(细胞株EA.hy926),对高糖高胆固醇诱导EA.hy926细胞凋亡中对分子伴侣家族一员,HSP60蛋白的表达进行了初步的研究。
结果分述如下:
1.生物信息学挖掘动脉硬化与衰老相关基因。
通过年龄相关大鼠动脉芯片表达谱比较共获得166条探针(155个基因),其中有36个基因响应热量限制与高脂高糖饮食。其中Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt等参与糖代谢,在大鼠衰老的进程中呈下降趋势;而Gygl参与肝糖原合成,随年龄增加而持续上调。HK2, PFKP, Dlat, Pdp2, Acyl等是糖酵解与三羧酸循环的关键调控酶,Tkt, TECR与ACS15则是NADPH提供途径的关键酶。在大鼠衰老的进程中,糖代谢相关的酶表达下调,NADPH生成途径受阻,表明在个体发育成熟后,机体对能量的需求可能减少,NADPH下降可能将导致SIRT1的活性下降。有报道SIRT1对血管及在动脉硬化中具有保护作用,它在热量限制介导机体寿限的延长中起到非常重要调作用。因此,通过热量限制可能上调这些基因的表达,对机体的“燃料”进行充分燃烧,减少脂肪堆积,延长寿限。
生物信息学的分析表明,随着年龄的增长,大鼠动脉血管的细胞粘附因子Vcam1, Icam1, Postn, Fn1, Sdc1, Mfge8, Spp1, Jup, Tln1上调表达,表明内皮细胞随年龄增长功能下降,容易诱发动脉硬化。与血管发育相关的Agt, Colla1, Gja5随年龄增长而下调,而参于血管调节与重塑Bgn, Acvrl1, Fgfr1, Box等则上升。这些基因可能参与高血压的发生,推测热量限制可能抑制Bgn, Acvrll的表达。此外,参与免疫的HSPD1与抗氧化的Pex19, Cyld等基因随年龄呈下降趋势,热量限制显著提高他们的表达。随着年龄的增长,动脉硬化的易感基因表达上调,表明患动脉硬化的风险增加;而热量限制可以下调这些基因的表达,推测其可能预防或延缓动脉硬化的发生。
2.高糖高脂诱导大鼠动脉硬化模型建立以及生物信息学结果的实验验证。
将44只6月和40只11月大的SPF级的Wistar雄性大鼠进行随机分组,FC(自由饮食组)给予基本饲料和普通算来水喂养;CR(热量限制组)每天定时给予基本饲料100-150g/3只老鼠,此根据老鼠一天能刚吃完饲料重量而定;HC(高糖高脂组),饲料组成为:基本饲料+15%猪油+3%胆固醇+5%精制糖。各组大鼠进行2个月和4个月喂食后,心脏取血后,通过血清OXLDL和血浆TpP含量检测,以及血液中CRP的mRNA检测表明,本实验通过高糖高脂喂养的大鼠更有诱发动脉硬化的可能性。并且随年龄增长,长期高糖高脂饮食罹患动脉硬化的可能性更大,而热量限制饮食即可减低此风险。
在建立高糖饮食与热量限制的大鼠模型的基础上,通过Q-PCR检测生物信息学筛选得到31个同时响应热量限制与高脂高糖饮食的基因表达。结果表明,热量限制可以上调成年大鼠动脉中Aldh5al (F=5.301, P=0.022), Ldha (F=2.215, P=0.016), Hk2(F=3.556, P=0.061), Pfkp (F=3.045, P=0.085), Slc2a4(F=4.006, P=0.046), Dlat (F=3.389, P=0.068), Tkt(F=0.177,P=0.008), Gygl (F=5.831,p=0.017)等参与糖代谢相关的基因,而高糖高脂的饮食则起到相反的作用。同时,热量限制还可以有效抑制Icam1(F=3.739. P=0.045)和Tln1(1.546, P=0.050)的表达,但对Vcam1(F=0.177, P=0.267)和Fn1(F=1.495, P=0.102)作用不明显。Zou Y等报道热量限制可以有效抑制不同年龄大鼠l cam1和Vcam1的表达,但热量限制的时间持续13和31个月。本研究是在成年鼠后进行热量限制4个月进行检测,可能由于处理时间偏短,从而影响了结果。此外,热量限制还可以有效抑制Bgn(F=13.189, P=0.001), Acvrll(F=2.306, P=0.142)的表达,而上调HSPD1(F=3.191, P=0.075), Pexl9(2.474, P=0.126), Cyld(F=1.888, P=0.194)等基因的表达。高糖饮食则相反。
3.高糖高胆固醇诱导EA.hy926细胞凋亡中对HSPD1基因表达的影响
随胆固醇浓度的增加,EA.hy926细胞活性下降,呈浓度依赖性。0.8mM胆固醇+5mM葡萄糖共培养12h的细胞活性与正常对照组(5mM葡萄糖+0mM胆固醇)无显著差异(P>0.056),而0.8mM胆固醇+25mM葡萄糖共培养12h的细胞活性与正常对照组(25mM葡萄糖+0mM胆固醇)具显著性差异(F=579.023,P=0.001)。5mM或25mM葡萄糖+0.8mM胆固醇处理EA.hy926细胞不同时间后,细胞活性呈时间依赖性下调,48h后细胞大部分变圆,脱落(F=79.758,P<0.001)。流式检测表明此时,与低糖低脂处理的细胞比较,高糖高胆固醇处理的细胞大部分发生凋亡(F=127.068,P<0.001),HSPD1基因表达显著上调(F=6.959,P=0.006)。
通过上述三个部分的实验,我们得出以下结论:
1.随着年龄的增长,动脉硬化的易感基因表达上调,表明患动脉硬化的风险增加。
2.热量限制可以下调这些基因的表达,推测其可能预防或延缓动脉硬化的发生。
3.高糖高脂喂养的大鼠,长期的HC饮食可使血液中的OXLDL升高;血浆中TpP水平显著高于FC组;而CR组显著低于FC组;从动脉血管横切图看,HC组的血管内皮显著增厚,并不同程度的形成泡沫细胞。表明HC组大鼠动脉已有不同程度动脉硬化的病变,更有诱发动脉硬化的可能性。并且随年龄增长,长期高糖高脂饮食罹患动脉硬化的可能性更大,而热量限制饮食即可减低此风险。
4.热量限制可以上调成年大鼠动脉中Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gygl等参与糖代谢相关的基因,而高糖高脂的饮食则起到相反的作用。同时,热量限制还可以有效抑制Icaml, Tlnl, Bgn, Acvrll, Fgfrl, Bax的表达,而上调HSPD1, Pex19, Cyld等基因的表达。高糖饮食则相反。即并且随着年龄的增长,动脉硬化的易感基因表达上调,患动脉硬化的风险增加;而热量限制可以下调这些基因的表达,从而预防或延缓动脉硬化的发生。相反,高糖高脂饮食则起相反的作用,加快发病及病变的进程。
5.持续激活的HSP60通过激活人血管内皮细胞表达TNF-a,E-选择素,ICAM1,IL6等细胞因子,促进自身炎症发应从而诱发AS。无论是高糖,高糖高脂,或是低糖高脂处理EA.hy926内皮细胞,都可以诱导HSPD1的nRNA表达,但高糖高脂诱导表达的效果最显著。因此,适当控制饮食摄入糖与脂含量,可预防或延缓AS的发生
Cardiovascular disease is the predominant cause of death in developed countries population, mortality also showed a tendency to increase year by year in China. Atherosclerosis (AS) cardiovascular disease is called the number one killer in developed countries, and the incidence in developing countries is also high (Shimizu, T,2008). Coronary atherosclerotic heart disease referred to as coronary heart disease (CHD) is a common disease, has become a most common kinds of heart disease in the United States and Europe. In1990, The global population is5.3billion, and the death of50million, of which6.3million died of coronary heart disease, accounting for12.4%.1991American Heart Association announced that deaths of coronary heart disease account for45.3%to total deaths in1988, and about1/5of Americans die of cardiovascular disease before the age of65. As an Asian country, coronary heart disease morbidity and mortality of China is in the ranks of lower-prone countries, but with the changing life styles, it increased in recent years. In hospitalized patients with heart disease, the proportion of the disease are also increasing. The basic pathogenesis of coronary heart disease is atherosclerosis (As). The common mechanism are AS plaque instability, caused by local platelet aggregation and acute thrombosis, acute occlusion of the lumen, and acute coronary syndrome (ACS). With China's economic and social development and improved living standards and dietary level of habit change, and the consequent aging of the population, atherosclerosis sclerosis diseases gradually become China's leading causes of death (Mu Yi-ming,2001). The World Health Organization predicted that upto2020after worldwide infectious disease case fatality rate has been effectively controlled, Heart tube disease will become the world's first cause of death.
Atherosclerosis (As) is an important pathological basis of cardiovascular and cerebrovascular diseases, prevention AS is a fundamental measure of prevention and treatment of cardiovascular diseases. Atherosclerosis (AS) is characterized by a lipid-rich plaques in the aorta wall gathering system disease, and is the major pathological basis of cardiovascular and cerebrovascular disease.it is a multi-factor chronic arterial disease, the concentrated expression of systemic vascular endothelial function obstacles (endothelialdysfunction,), intimal chronic inflammation, fibrosis, stenosis and thrombosis formation. The main clinical manifestations of myocardial infarction, stroke and peripheral vascular disease, often accompanied by hypertension, hypercholesterolemia or diabetes. Because of the complexity of the etiology and pathology, AS has not yet been fully elucidated, but it is certain that AS is related to a variety of genetic and environmental factors of complex diseases, the interaction between these genetic factors and environmental factors ultimately lead to plaque formation and the emergence of series of subsequent clinical events. Many AS vascular disease etiology, which is closely related to many lifestyles, including smoking, high fat diet, lack of physical activity habits. From the biological perspective, AS is a chronic inflammatory disease, and dyslipidemia is closely related to its development and it is a very complex process. The clinical events of AS disease are coronary heart disease and stroke.
Therefore, the AS mechanism have become an increasing concern, elevated blood lipids and disorder are the premise and basis of most of the AS. Endothelial dysfunction is the originating events of AS; and a variety of factors interact, result in the occurrence and development of AS. The main factors include:vascular endothelial cell dysfunction, vascular smooth muscle cells, platelets and blood coagulation state of exception, monocytes, macrophage infiltration, free radical damage, viral and bacterial infections, apoptosis, inflammation and immune response, calcium deposition, and genetic abnormalities. The pathogenesis is now generally recognized as "injury stress theory (Ross, R,1999). The process involves in AS containing the following steps:a variety of stimuli and injury of endothelial cells (vascular of endothelial cell, VEC); monocytes transmit through the lesion into the intima and tranform into macrophages; macrophages together with the role of T lymphocytes secrete cytokines transmit into macrophage-derived foam cells; medial vascular smooth muscle cells move into the intima, engulfed lipid to form foam cell then proliferation and migration to form a fibrous cap; fiber cap weakened by matrix metalloproteinases and y-dry detoxification secreted by macrophage and T lymphocyte, the formation of atherosclerosis physics and chemistry of the emboli.
As to the formation of AS, There are more and more attention paid to the immune and inflammatory theory in recent years. Studies suggest that AS is a protective inflammation-fiber proliferative response to the local damage of the vascular endothelial. If the injury sustained period of time, this response becomes excessive, and eventually become diseases that plaque formation. Lipid deposition in the plaque formation process is the most important factor in the damage response and is also one of the earliest performance. Along with lipid deposition, the formation of oxidized low-density lipoprotein cholesterol (by oxLDL-C), circulating leukocytes and monocytes are activated and migrate to the lesion, which become activated by oxLDL-C under macrophages through their scavenger receptor uptake by oxLDL-C into a foam cell generation and accumulation of foam cells lead to the formation of lipid stripes. The release of the deposition of cholesterol cool plasma lipoproteins from the foam cell death constitute the plaque lipid core. Inflammatory response to the continued development of T cell activation, triggered fiber proliferative response ultimately the formation of the fibrous cap. Early lipid core plaque formation in small, fibrous cap thickness, plaque steady state, along with the continued death of foam cells and the deposition of plasma lipids, lipid core plaque growing on the other hand a large number of infiltration of macrophages to release large amounts of hydrolytic enzymes, especially metalloproteinases series, gradually thinning the fibrous cap by degrading the fibrous cap, as well as inhibit the formation of collagen fibers, so that stable plaques transmit into unstable plaques, and the latter influenced by the inner and outside factors, and the final breakdown lead to acute coronary events. The pathologist also found a large number of macrophage infiltration, and the increased expression of inflammatory markers in the plaque rupture site. Thus, the continuous progress of the plaque and the extent of the inflammatory response are closely related. The rupture of the plaque is closely related to the intensity of inflammation and macrophage infiltration in plaque. The formation of the fatty streak or plaque within the arterial wall is the characteristic lesions of AS. However, Macrophages as well as central and membrane migration to the intima of the arterial intima smooth muscle cells via the scavenger receptor (scavenger receptor, SR) uptaking of oxidized low-density lipoprotein (oxidizedLDL, oxLDL), and thus the formation of macrophage/smooth muscle cells derived foam cells, is a central event in the development of AS process.
Over the past century, people have done some research on aging and longevity but so far, no news has been announced. Although aging itself is not a disease but many diseases associated with aging such as cancer, Alzheimer's (Alzheimer's disease), atherosclerosis, metabolic disorders (metabolic disorders). Heart disease and stroke mortality with age exponential increase in mortality at age65-74more than40percent, nearly60%of the mortality rate of85years. Growing body of research found that, even in the absence of other risk factors as hypertension, diabetes and smoking, cardiovascular system increases with age have become more prone to disease and damage, this may homeostasis and aging vascular cells related (Ungvari et al,2010). In experimental animals and human body have confirmed that the oxidative damage generated by reactive oxygen species reactive oxygen species (ROS) in activation of NAD (P) H oxidase and lowered the activity of NO, leading to endothelial cell dysfunction of the aging process, causing the elderly coronary artery disease and stroke.
Calorie restriction (calorie restiction, CR) is to provide the organism full of nutrients such as essential amino acids, vitamins, etc., to ensure that organisms do not occur malnutrition, limiting the daily intake of total calories. General CR diet is reduced by30%-40%of the animal free food intake. The function of caloric restriction in a passive way to reduce oxidation and other damage, leading to prolonged life cycle, can regulate the life cycle from yeast to mammals and other organisms. Since1935McCay, first reported in the CR to prolong rats life, a large number of studies have shown that CR is the most powerful anti-aging methods In addition to genetic manipulation. Calorie restriction not only to maintain the physiological state of many a young man, but also delay and prevent the age-related diseases such as cardiovascular disease, diabetes, tumor occurrence and development. Calorie restriction (caloric restriction, CR) has been shown to have anti-aging and extend the life of the role (Russell JC2008; Fontana, L,2009), including calorie intake limit and reduce the consumption of the number of two ways. Multi-effect protective effect of CR on the cardiovascular system, such as protection of blood vessels (to prevent atherosclerosis process) to improve myocardial tolerance and delay cardiac aging (Shinmura K,2011). In the aging process, the activation of IL-1, IL-6and TNF-a and other cytokine by the transcription factor of NF-kB, mediated body chronic inflammation, which lead to the key of a series of aging-related diseases. Therefore, it was suggested by antioxidant drugs (such as curcumin (curcumin)) to suppress the inflammatory response dependent on NF-kB signaling and aging (Sikora, the Eab et al.,2010). It is shown that the CR can reduce the inflammatory cytokine IL-6and leptin expression (Reed, JL,2010).
This topic is divided into four parts, Firstly, based on published microarray data results, we do bioinformatics analysis and data mining, as the following:derived from the GSE7281microarray data sets submitted by the United States Miller, the SJ, Fischer344rats of natural aging, aged3,6,15,28months, the artery mRNA was extracted from rat to do whole genome microarray analysis. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7281, Submitted from the United States Becker, KG the GSE11845chip data set, select the data set in normal controls (free diet), calorie restriction (discontinuous diet) and fat (hydrogenated coconut oil, heat higher than60%of the normal control group) of three groups of samplesdata, heart, liver and muscle-related gene analysis. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11845.
Mining of atherosclerosis and aging-related genes. Then set up Wistar rat model, Using the RT-PCR, ELISA methods, we do serum oxidized low-density lipoprotein (of OXLDL), C-reactive protein (C-of reactive protein, CRP), thrombus precursor protein (TpP) targets detection and pathological analysis of the observed vascular morphology, and further experimental validation of the results of the microarray data mining. High carbohydrate diet and calorie restriction in a rat model based on the real-time fluorescence quantitative PCR (Real-time quantitative Polymerase Chain Reaction and Real Time PCR), to verify the results of bioinformatics and filter31 while responding to caloric restriction gene expression and high-fat high-sugar diet. EA.hy926cell apoptosis through the establishment of the HC induced cell model of atherosclerosis (cell line of EA. Hy926), high glucose and high cholesterol induced a preliminary study of a molecular chaperone family of HSP60protein expression. The results are as follows:
1. Bioinformatics mining of atherosclerosis and aging-related genes.
The total of166probes (155genes) were obtained from the Age-related rat arterial microarray, of which36genes respond to calorie restriction and high-fat high-sugar diet. The Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, etc. involved in glucose metabolism, a downward trend in the aging process in rats; Gyg1involved in glycogen synthesis, increased with age and continued upward. HK2, PFKP, Dlat, Pdp2, Acyl, etc. are a key regulatory enzyme of glycolysis and the citric acid cycle, Tkt, TECR, and ACS15are the key enzyme of the NADPH pathway. Along with the aging process in rats, the glucose metabolism enzyme expression and NADPH formation routes blocked, suggesting that individual mature, the body may reduce the energy demand of NADPH, the decline may lead to decreased activity of SIRT1, It is reported that SIRT1has a protective effect to blood vessels and atherosclerosis, and it play a very important role in mediating the body the life extension by calorie restriction. Therefore, caloric restriction may increase the expression of these genes, the body's "fuel" burning, reduce fat accumulation, and to extend the lifespans.
Bioinformatics analysis showed that the increase with age, rat arterial vascular cell adhesion molecule Vcam1, Icaml, Postn, the Fn1, Sdcl, Mfge8, Sppl, Jup, Tln1upregulated, indicating that the decline of endothelial cells increased with agelikely to cause atherosclerosis. Agt, Collal, Gja5which related to the vascular development, will decrease, and Bgn, Acvrll, Fgfrl, Bax which are involved in vascular regulation and remodeling will rose. These genes may be involved in the occurrence of hypertension, suggesting that caloric restriction may inhibit the expression of Bgn, Acvrl1. In addition, the HSPD1involved in the immune and antioxidant genes such as Pex19, Cyld declined with aging, calorie restriction significantly increased their expression. With aging, atherosclerosis susceptibility genes were up-regulated, indicating that the increase in the risk of suffering from arteriosclerosis; calorie restriction lowered the expression of these genes, suggesting that it may prevent or delay atherosclerosis.
2. High glucose and fat induced atherosclerosis Rat model and experimental verification of bioinformatics result.
44, six months and40, eleven months SPF male Wistar rats were randomized to FC (free diet group) to give basic feed and count runoff feeding; CR (calorie restriction group) time every day to give the basic feed100~150g/3mice, according to the mouse day had just eaten the feed weight; HC (high-sugar high-fat group), feed composition:basic feed+15%lard and3%cholesterol,5%refined sugar. Rats in each group after2months and4months feeding, cardiac blood was collected by the serum OXLDL and the plasma TpP content testing, and blood CRP mRNA detection showed that this experiment in rats fed by high glucose and fat more the possibility of induced atherosclerosis. And with aging, they are more likely to suffer from arteriosclerosis in long-term high-fat diet, however, calorie restriction diet can reduce this risk.
Q-PCR was done to verificated the bioinformatics results, Based on the high-sugar diet and calorie restriction rat model, screened31gene expression in response to calorie restriction and high-fat high-sugar diet. The results show that calorie restriction can increase the adult rat arterial Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gygl and other genes involved in glucose metabolism, high-sugar high-fat diet have the opposite effect. The same time, calorie restriction can effectively inhibit the expression of Icam1and Tln1, but not obvious effect on Vcaml and Fnl gene. Zou, Y reported that caloric restriction can effectively inhibit the expression of different ages the rat Icam1and Vcam1, but the heat restrictions are thirteen months and thirty-one months. In this study, the adult mice were calorie restriction for four months for testing, maybe due to the shorter processing time, thus affecting the results. In addition, calorie restriction can effectively inhibit the expression of Bgn, Acvrl1, Fgfr1, Bax, and activate HSPD1, Pex19, Cyld gene expression. The high carbohydrate diet is the opposite.
3. High glucose and high cholesterol induce EA.hy926cell apoptosis and have effect on HSPD1gene expression.
With increasing cholesterol concentration of EA. Hy926cell activity dropped to a concentration dependent manner. But the cell activity has no significantly different between the0.8mM cholesterol+5mM and glucose co-cultured for12h and normal control group (5mM glucose+0mM cholesterol)(P>0.05); There is significant difference between the0.8mM cholesterol+25mM and glucose co-cultured12h and normal control group (25mM glucose+0mM and cholesterol)(P<0.01).5mM or25mM glucose+0.8mM cholesterol dealing with EA.hy926cells at different times, cell activity was time-dependent decrease, most of the cells rounded off after48h. Flow cytometry showed that the majority ofcells treated with high sugar and high cholesterol treated cells undergo apoptosis (50%) contrast to low-sugar low-fat treatment, and HSPD1gene expression was significantly upregulated (F=6.959, P=0.006).
Through above three-Part test, we draw the following conclusions:
1. With aging, atherosclerosis susceptibility genes upregulated, indicating that the risk of an increased risk of atherosclerosis.
2. Calorie restriction can downregulate the expression of those genes, suggesting that it may prevent or delay atherosclerosis occurrence.
3. High sugar and high fat fed rats, the long-term HC diet can increase blood OXLDL; the plasma TpP level significantly higher than the FC group; CR group were significantly lower than the FC group; from the arterial cross-section diagram, HC group of vascular endothelial significantly is thicker, and varying degrees of formation of foam cells. So that, the HC rats artery has varying degrees of atherosclerosis lesions, the possibility of more-induced atherosclerosis. And with aging, It is more likely to suffer from arteriosclerosis in long-term high-fat diet, however, calorie restriction diet can reduce this risk.
4. Caloric restriction can increase the Aldh5al, Ldha, Hk2, Pfkp, Slc2a4, Dlat, Tkt, Gyg1and other genes involved in glucose metabolism in the adult rat arteries, high-sugar and high-fat diet have the opposite effect. The same time, calorie restriction can effectively inhibit the expression of Icam1, Tlnl, Bgn, Acvrll, Fgfr1, Bax, and to upregulate HSPD1, Pex19, Cyld gene expression. The high carbohydrate diet is the opposite. And with aging, atherosclerosis susceptibility gene expression increased, which raised risk of atherosclerosis; calorie restriction lowered the expression of these genes, thus preventing or delaying the occurrence of atherosclerosis. In contrast, high-fat diet is counterproductive to speed up the process of onset and lesions.
5. The sustained activation of HSP60activate the expression of TNF-a, E-selectin factors, ICAM1, IL6, cytokines by the human vascular endothelial cells to promote the inflammation and induce the AS. Although high-sugar, high sugar and fat or low sugar and high fat treatment on lipid EA.hy926endothelial cells can induce HSPD1mRNA expression, but high glucose and fat-induced expression of the most significant effect. Therefore, an appropriate control of dietary intake of sugar and fat content, can prevent or delay the occurrence of AS.
引文
[1]牟一鸣,傅蓉,王秋娟,吴玉林。炎症相关细胞在动脉粥样硬化发生发展过程中的作用[j]。药学与临床研究,2011,19(2)151-155。
[2]Mayerl C, Lukasser M, Sedivy R etal. Atherosclerosis research from past to present--on the track of two pathologists with opposing views, Carl von Rokitansky and Rudolf Virchow. Virchows Arch,2006,Jul,449 (1):96-103.
[3]FalkE.Pathogenesisofatheroselerosis.J Am Coll Cardlol,2006,47:C7-12.
[4]AcharSA, KunduS, NorerossWA.Diagnosisofacutecoronarysyndrome.Am FamPhysieian,2005,72:119-26.
[5]LargeGA.Contemporary management of acute coronary syndrome. Postgrad Med J,2005,81:217-22.
[6]PrasadA, MathewV, HolmesDRJr, etal. Current management of Non-ST-segment-elevation acute coronary syndrome:reconciling the results of randomized controlled trials. Eur HeartJ,2003,24:1544-53.
[7]YamaguehiT, NakamuraM, MitsuoK, etal.Percutaneous coronary intervention in acute coronary syndrome.Inten Med,2000,39:337-8.
[8]ShenCX, ChenHZ, GeJB.The role of inflanllnatory stress in acute coronary syndrome. Chin Med J(Engl),2004,117:133-9.
[9]Greeh ED, Ramsdale DR.Acute coronary syndromerST segment elevation Myocardial Infaretion. BMJ,2003,326:1379-81.
[10]Panteghini M. Acute coronary syndrome:biochemical strategies in the troponin era. Chest,2002,122:1428-35.
[11]KodamaK, AsakuraM, UedaY, etal.The role of plaque rupture in the development of acute coronary syndrome evaluated by the coronary angioseope. Intern Med,2000,39:333-5.
[12]马文丽,郑文岭.DNA芯片技术的方法与应用[M]。广州:广东科技出版社,2002:7-5359-3065-4。
[13]Bjoorkholm, B.etal.Comparison of genetic divergence and fitness between twosubelones of Helicobacter pylori.Infect.Immun.69,7832-7838,2001.
[14]黄修献,李利华,吴新华.血管内皮细胞与动脉粥样硬化。医学综述,2010,16(18):2724-6.
[15]Ross R. Atherosclerosis-an inflammatory disease。N Engl J Med,1999,340(2):115-26.
[16]陈雁,杨晓晶,白海涛.血管内皮损伤与修复的研究进展。医学综述,2010,16(17):2573-6.
[17]Chiu JJ, Usami S, Chien S. Vascular endothelial responses to altered shear stress: pathologic implications for atherosclerosis. Ann Med,2009,41(1):19-28.
[18]Wong WT, Ng CH, Tsang SY, et al. Relative contribution of individual oxidized components in ox-LDL to inhibition on endothelium-dependent relaxation in rat aorta. Nutr Metab Cardiovase Dis,2009,21(3):158-64.
[19]Matsumoto Y, Adams V, Walther C, et al. Reduced number and function of endothelial progenitor cells in patients with aortic valve stenosis:a novel concept for valvular endothelial cell repair. Eur Heart J,2009,30(3):346-55.
[20]Ishikado A, Nishio Y, Yamane K, et al. Soy phosphatidylcholine inhibited TLR4-mediated MCP-1 expression in vascular cells. Atherosclerosis,2009,205(2): 404-12.
[21]张韶辉,高东升.巨噬细胞移动抑制因子在动脉粥样硬化中的研究进展。国际内科学杂志,2009,36(9):524-7.
[22]Chen L, Yang G, Zhang X, et al. Induction of MIF expression by oxidized LDL via activation of NF-kappa B in vascular smooth muscle cells. Atherosclerosis, 2009,207(2):428-33.
[23]Seizer P, Schonberger T, Schott M, et al. EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and MCSF during foam cell formation [J]. Atherosclerosis,2010,209(1):51-7.
[24]Kim HR, Lee DM, Lee SH, et al. Chlorogenic acid suppresses pulmonary eosinophilia, IgE production, and Th2-type cytokine production in an ovalbumin-induced allergic asthma:activation of STAT-6 and JNK is inhibited by chlorogenic acid. Int Immunopharmacol,2010,10(10):1242-8.
[25]Cheng Y, Zhang J, Hou W, et al. Immunoregulatory effects of sinomenine on the T-bet/GATA-3 ratio and Th1/Th2 cytokine balance in the treatment of mesangial proliferative nephritis. Int Immunopharmacol,2009,9(7-8):894-9.
[26]Xinqiang S, Fei L, Nan L, et al. Therapeutic efficacy of experimental rheumatoid arthritis with low-dose methotrexate by increasing partially CD4+CD25+Treg cells and inducing Thl to Th2 shift in both cells and cytokines.Biomed Pharmacother,2010,64(7):463-71.
[27]Von Vietinghoff S, Ley K. Interleukin 17 in vascular inflammation [J]. Cytokine Growth Factor Rev,2010,21 (6):463-9.
[28]Mohamed R, Bermudez-Fajardo A, Oviedo-Orta E. Th17 cells induction accelerated atherosclerosis development in ApoE(-/-) mice. Atherosclerosis, 2010,213(1):e10.
[29]Xie JJ, Wang J, Tang TT, et al. The Thl7/Treg functional imbalance during atherogenesis in ApoE(-/-) mice. Cytokine,2010,49(2):185-93.
[30]Clarke MC, Littlewood TD, Figg N, et al. Chronic apoptosis of vascular smooth muscle cells accelerates atherosclerosis and promotes calcification and medial degeneration. Circ Res,2008,102(12):1529-38.
[31]李磊,戴敏.动脉粥样硬化血管内皮分泌功能失调与平滑肌细胞增殖。中国药理学通报,2010,26(2):155-8.
[1]牟一鸣,傅蓉,王秋娟,吴玉林。炎症相关细胞在动脉粥样硬化发生发展过程中的作用[j]。药学与临床研究,2011,19(2)1 51-155。
[2]黄修献,李利华,吴新华.血管内皮细胞与动脉粥样硬化[J].医学综述2010,16(18):2724-6.
[3]Ross R. Atherosclerosis -an inflammatory disease [J].N Engl J Med,1999,340(2):115-26.
[4]陈雁,杨晓晶,白海涛.血管内皮损伤与修复的研究进展[J].医学综述,2010,16(17):2573-6.
[5]张韶辉,高东升.巨噬细胞移动抑制因子在动脉粥样硬化中的研究进展[J].国际内科学杂志,2009,36(9):524-7.
[6]李磊,戴敏.动脉粥样硬化血管内皮分泌功能失调与平滑肌细胞增殖[J].中国药理学通报,2010,26(2):155-8.
[7]Wong WT, Ng CH, Tsang SY, et al. Relative contribution of individual oxidized components in ox-LDL to inhibition on endothelium -dependent relaxation in rat aorta [J]. Nutr Metab Cardiovase Dis,2009,21(3):158-64.
[8]朱宁.氧化修饰低密度脂蛋白与动脉粥样硬化.国外医学(生理、病理科学与临床分册),1994,14:157-158.
[9]胡云,徐伟,潘佳佳,等.急性时相血清淀粉样蛋白A与2型糖尿病颈动脉粥样硬化的关系.临床内科杂志,2008,25(10):684-686.
[10]付丽,汪俊军,强红娟,等.糖尿病患者血浆氧化、丙二醛修饰低密度脂蛋白及其自身抗体水平.检验医学,2008,23(1):69-71.
[11]Mallamaci F, Zoccali C, Cuzzola F, et al. Adiponectin in essential hypertension. J Nephrol,2002,15:507-511.
[12]华洁,牛国中,李保华. 急性脑血管患者血清C反应蛋白测定的临床义。中华急诊医学杂志,2003,12(7):483-484.
[13]安有芬,唐仁满.定量CRP检测的临床价值。西藏医药杂志,2002,23(3):48-49。
[14]刘运双. CRP与心血管事件。 国外医学·临床生物化学与检验学分册,2003,24(3):142-143。
[15]Gambino R. C-reactive protein-undervalued, underutilizde. Clin Chem,1997, 43 (11):2017.
[16]Dodson PM, Shine B. Retinal vein occlusion:C-reactive protein and arterialhypertension. Acta Ophthal Mologica,1984,64:123-130.
[17]Fichtlscherer S, Ronsenberer G, Dirk H et al. Elevated C-reactive Proteinlevels and impaired endothelial vasoreactivity in patients with coronaryartery disease. Circulation,2000,102:1000-1006.
[18]吴强。不同期高血压病患者血浆一氧化氮和内皮素及循环内皮细胞计数的变化。临床荟萃,1999,14(8):341。
[19]Bhagat K, Vallance P.Inflammatory cytokines impair-endothe liwm dependentdilation in human veins in vivo. Circulation,1997,96:3042-3047.
[20]Hayaishr Okano R, Yamsaki Y, Katakami N, et al. Elevated C-reactive protein associates with early-stage carotid atherosclerosis in young subjectswith type ldiabetes. Diabetes Care,2002,25(8):1432-1438.
[21]叶平,王节,尚延忠,等. C-反应蛋白与动脉粥样硬化形成有关。中国动脉硬化杂志,2001,9(2):146-148.
[22]但三利,邹晓霞.血清C-反应蛋白与高血压病心功能改变的关系及临床意义。第三军医大学学报,2005,27(5):1604-1606.
[23]高文静,郝冰,吴寿岭. C-反应蛋白与原发性高血压的关系研究。中国综合临床,2005,21(3):209-211.
[24]吕萍,李自成,常青。高血压病患者C-反应蛋白变化的临床意义。第二军医大学学报,2005,26(9):1034-1036.
[25]吴寿岭,高竞生,郝冰等. 血清高敏C反应蛋白浓度与高血压病的相关性研究。中华心血管病杂志,2003,31(12):917-920。
[26]孙芹敏,韩放,孙国华,等。原发性高血压并高尿酸血症与C-反应蛋白的关系探讨。大连医科大学学报,2004,26(4):290-292。
[27]Bautista LE, Lopez-Jaramillo P, Vera LM, et al. Is C-reactive protein an Independent risk factor for essential hypertension?. J Hypertens,2001,19(5): 857-861.
[28]Bautista LE, Vera LM, Arenas IA, et al. Independent association between inflammatory markers (C-reactive protein, interleukin-6, and TNF-a) and essential hypertension.J Human Hypertens,2005,19:149-154.
[29]Ferandez-Real JM, Vayreda M, Richart C, et al. Circulating interleukin 6 levels, blood pressure, and insulin sensitivity in apparently healthymen and women. J Clin Endocrinol Metab,2001,86(3):1154-1159.
[30]Sung KC, Suh JY, Kim BS, et al. High sensitivity C-reactive protein as an independent risk factor for essentival hypertension.Am J Hpertens,2003,16(6): 429-433.
[31]Fichtlscherer S, Rosenberger G, Walter DH, et al. Elevated Creactive protein levels and impaired endothelial vasoreactivity in patients with coronary artery disease. Circulation,2000,102(9):1000-6.
[32]Yasmin, CarmelM,McEniery, et al. C-Reactive Protein Is Associated With Arterial Stiffness in Apparently Healthy Individuals. Arterioscler Thromb Vase Biol,2004,24(5):969-74.
[33]Perticone F, Maio R, Sciacqua A, et al. Endothelial dysfunction and CRP are risk factors for diabetes in essential hypertension. Diabetes,2007,56 (10): 2425-32.
[34]Dotsenko O, Chaturvedi N, Thom SA, et al. Platelet and leukocyte activation, atherosclerosis and inflammation in european and south asian men. J Thromb Haemost,2007,5(10):2036-42.
[35]Matoh T, Miyazaki M, Une H, et al. Does elevated high-sensitivity serum C-reactive protein associate with hypertension in non-obese Japanese males. Clin Exp Hypertens,2007,29(6):395-401.
[36]Shankar A, Li J, Nieto FJ, et al. Association between C-reactive protein level and peripheral arterial disease among US adults with cardiovascular disease, diabetes, or hypertension. Am Heart J,2007,154(3):495-501.
[37]Naya M, Tsukamoto T, Morita K, et al. Plasma interleukin-6, CRP and tumor necrosis factor-alpha can p redict coronary endothelial dysfunction in hypertensive patients. Hypertens Res,2007,30(6):541-8.
[38]Sesso HD, Wang L, Buring JE, et al. Comparison of interleukin-6 and C-reactive protein for the risk of developing hypertension in women. Hypertension,2007,49(3):304-10.
[39]Carv illeDG, D im itr ijev icN, W alshM, et a.1 Thrombus precur sor pro te in(T pP):M arker o f thrombosis ear ly in the pathogenesis o fmyoca rd ial infa rction. ClinChem,1996,42(9):1537-1541.
[40]Ha rker LA, M arn KG, Fuster V, et a.l T hrombosis in cardiovascu lar d isorders.Philadephia:WB Saunders,1992,1-16.
[41]laCapra S, A rkelYS, Ku DH. The use o f thrombus precursor protein, D-dimer,prothromb in fragment 1.2, and thromb in antithromb in in the exc lusion o f prox-Imal deep ve in thrombosis and pulmonary embolism.B lood Coagul F ibr inolysis,2000,11(4):371-377.
[1]Nathan C. Points of control in inflammation. Nature 2002;420:846-852.
[2]牟一鸣,傅蓉,王秋娟,吴玉林。炎症相关细胞在动脉粥样硬化发生发展过程中的作用。药学与临床研究,2011,19(2)151-155。
[3]黄修献,李利华,吴新华.血管内皮细胞与动脉粥样硬化。医学综述2010,16(18):2724-6.
[4]陈雁,杨晓晶,白海涛.血管内皮损伤与修复的研究进展。医学综述,2010,16(17):2573-6.
[5]张韶辉,高东升.巨噬细胞移动抑制因子在动脉粥样硬化中的研究进展。国际内科学杂志,2009,36(9):524-7.
[6]李磊,戴敏.动脉粥样硬化血管内皮分泌功能失调与平滑肌细胞增殖。中国药理学通报,2010,26(2):155-8.
[7]CINES DB, POLLAK ES, BUCK CA, et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood,1998,91(10):3527-3561.
[8JMENDES RIBEIRO AC, BRUNINI TM, ELLORY JC, et al.Abnormalities in L-arginine transport and nitric oxide biosynthesis in chronic renal and heart failure. Cardiovasc Res,2001,49 (4):697-712.
[9]温绍君,刘洁琳.血管内皮细胞功能研究概况。生殖医学杂志,2005,1(5)310-313.
[10]刘宏,孙刚.血管内皮功能与动脉弹性的关系.医学综述,2005,11(9):819-821.
[11]Ross R, Glomset JA. Atherosclerosis and arterial smooth muscle cell: proliferation of smooth muscle is a key event in the genesis of the lesion of atherosclerosis. Science,1973,180 (93):1332-1339.
[12]王雪梅.超声评价动脉硬化兔血管内皮舒张功能的实验研究.中国药物与临床杂志,2007,7(2):112-114.
[13]Callow AD. Endothelial dysfunction in atherosclerosis. Vascul Phamacol,2002, 38(5):257-258.
[14]Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenes is.Nature,1992; 359(6 398):843-845
[15]陈喜容,朱惠斌.益气活血方对自由基介导肿瘤坏死因子。湖南中医学院学报,1998,18(3):13-15
[16]李维根,李昭波,高云秋,等.运动对自发性高血压大鼠心肌血管内皮生长因子及其基因表达的影响。中国运动医学杂志,1999,18(2):115-116
[17]Duvall WL. Endothelial dys function and antioxidants.Mt Sinai J Med 2005;72(2):71-80
[18]Li D, Liu L, Chen H, et al. LOX-1, an oxidized LDL endothelial receptor, induces CD40/CD40L s ignaling in human coronary artery endothelial cells.Arterioscler Thromb Vase Biol 2003;23(5):816-821
[19]Suwaidi JA, Hamasaki S, Higano ST, et al. Long-term follow-up of patients with mild coronary artery disease and endothelial dys function. Circulation 2000;101(9):948-954
[20]Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dys function on adverse long-term outcome of coronary heart disease.Circulation 2000; 101 (16):1899-1906
[21]Halcox J,Sachenke W,Zalos G,et al. Prognos ticvalue of coronary vascular endothelialdys function.Circulation 2001;104(25):3091-3096
[22]Modena MG, Bonetti L, Coppi F, et al. Prognos tic role of reversible endothelialdys function inhypertensive postmenopausal women.J Am Coll Cardiol 2002;40(3):505-510
[23]M andalK, JahangiriM, Xu Q. Autoimmunity to heat shock proteins in atherosclerosis.Autoimmun Rev,2004,3 (2):31-37.
[24]Erkkila L, La itinen K, H aasio K, et al. H eat shock protein 60 auto immunity and earlylipid lesions in cholesterol-fed C57BL/6J mice during Chlamydia pneumoniae infection. Atherosclerosis,2004,177(2):321-328.
[25]Wick G, KnoflachM, Xu Q. Autoimmune and inflamma torymechanisms in atherosc lerosis. Annu Rev Immunol,2004,22:361-403.
[26]George J, Shoenfeld Y, AfekA, et al. Enhanced fatty streak formation in C57BL /6J mice by immunization with heat shock prote in65. A rteriosc lerThromb VascBiol,1999,19(3):505-510.
[27]M anda 1 K, Jahang iriM, Xu Q. Autoimmunity to heat shock proteins in atherosclerosis. Autoimmune Rev,2004,3(2):31-37.
[28]Erkkila L, Laitinen K, Haasio K,et al. Heat shock protein 60 auto immunity and earlylipid lesions in cholesterol-fed C57BL/6J m ice during Chlamydia pneumoniae infection. Atherosclerosis,2004,177(2):321-328.
[29]Bobryshev YV, Load RSA. Mapping of vascular dendritic cells in atherosclerotic arteries suggests their involvement in local immune in flammatory reactions. C ard iovasc Res,1998,37(3):799-810.
[30]Bodet tiT J, JacobsonE, W an C, et a.l Molecular evidence to support the expansion of the hostrange of Chlamydophila pneumoniae to includ e reptiles aswell as humans, horses, koalas and amphibians. J Syst Appl Microbiol 2002, 25(1):146-152.
[31]da Costa CP, K irschn ing C J, Busch D, et al. Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydiapneum on iae. EurJ Immunol 2002,32:2460-2470.
[32]K ikuta LC, Pu olakka inen M, Kuo CC, et a.l Isolation and sequence an alysis of the Chlamydia pneumoniae GroE operonl.In fection and Imm unity 1991, 59(12):4665-4669.
[33]O ch iaiY, Fukush iH, Yan C,et al. Comparative analysis of the putative aminoacid sequences of chlamydia 1 heat shock protein 60 and Escherich in coli GroEL. JV etM ed Sci 2000,62(9):941-945.
[34]WICK G, KNOFLACH M, XU Q. Autoimmune and inflammatory mechanisms in atherosclerosis. Annu Rev Immunol,2004,22:361-403.
[35]BURIAN K, KIS Z, VIROK D, et al. Independent and joint effects of antibodies to human heat-shock protein 60 and Chlamydia pneumoniae infection in the development of coronary atherosclerosis. Circulation,2001,103(11):1503-1508.
[36]KLEINDIENST R, XU Q, WILLEIT J, et al. Immunology of atherosclerosis. Demonstration of heat shock protein 60 expression and T lymphocytes bearing alpha/beta or gamma/delta receptor in human atherosclerotic lesions. Am J Pathol,1993,142(6):1927-1937.
[37]XUQ, SCHETT G, PERSCHINKA H, et al. Serum soluble heat shock protein 60 is elevated in subjects with atherosclerosis in a general population. Circulation,2000,102(1):14-20.
[38]POCKLEY A G, WU R, LEMNE C, et al. Circulating heat shock protein 60 is associated with early cardiovascular disease.Hypertension,2000,36(2):303-307.
[39]CHEN W, SYLDATH U, BELLMANN K, et al. Human 60-kDa heatshock protein:a danger signal to the innate immune system. J Immunol,1999,162 (6):3212-3219.
[40]HANSSON G K, LIBBY P, SCHONBECK U, et al. Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res,2002,91 (4):281-291.
[41]TSAN M F, GAO B. Cytokine function of heat shock proteins. Am J Physiol Cell Physiol,2004,286 (4):C739-744.
[42]ERKKILA L, LAITINEN K, HAASIO K, et al. Heat shock protein 60 autoimmunity and early lipid lesions in cholesterol-fed C57BL/6JBom mice during Chlamydia pneumoniae infection. Atherosclerosis,2004,177 (2): 321-328.
[43]HUITTINEN T, LEINONEN M, TENKANEN L, et al. Autoimmunity to human heat shock protein 60, Chlamydia pneumoniae infection, and inflammation in predicting coronary risk. Arterioscler Thromb Vase Biol,2002,22 (3):431-437.
[44]ZHU J, KATZ R J, QUYYUMI A A, et al. Association of serum antibodies to heat-shock protein 65 with coronary calcification levels:suggestion of pathogen-triggered autoimmunity in early atherosclerosis. Circulation,2004,109 (1):36-41.
[45]Nomenclature and criteria for diagnosis of ischemic heart disease. Report of the Joint International Society and Federation of Cardiology/World Health Organization task force on standardization of clinical nomenclature. Circulation, 1979,59 (3):607-609.
[46]ZHU J, QUYYUMI A A, WU H, et al. Increased serum levels of heat shock protein 70 are associated with low risk of coronary artery disease. Arterioscler Thromb Vasc Biol,2003,23 (6):1055-1059.
[47]PROHASZKA Z, DUBA J, LAKOS G, et al. Antibodies against human heat-shock protein (hsp) 60 and mycobacterial hsp65 differ in their antigen specificity and complement-activating ability. Int Immunol,1999,11 (9): 1363-1370.
[48]POCKLEY A G, DE FAIRE U, KIESSLING R, et al. Circulating heat shock protein and heat shock protein antibody levels in established hypertension. J Hypertens,2002,20 (9):1815-1820.
[49]XU Q, WILLEIT J, MAROSI M, et al. Association of serum antibodies to heat-shock protein 65 with carotid atherosclerosis. Lancet,1993,341 (8840): 255-259.
[50]H aranaga S, Ikejim a H, Yam aguch i H, et a.l Analys is of Chlamydia pneumon iae growth in cells by reverse transcription-PCR targeted to bacterial gene transcripts. Clin and Diagn Lab Immun 2002,9(2) 313319.
[1]Fontana L, Meyer TE, Klein S, Holloszy JO. Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans. Proc Natl Acad Sci U S A.2004,101:6659-6663.
[2]Masoro EJ.Caloric restriction and aging:an update.J.Exp Gerontol,2000; 35(3):299-305
[3]Weindruch R, Walford RL. Dietary restriction in mice beginning at one year of age:effect on life-span and spontaneous cancer incidence. Science,1982; 215:14152-1418
[4]范林傅发源李曦铭限制热量摄入对超重人群长寿、代谢适应性和氧化应激生物学标记物的影响中华医学杂志2006;86[21]:1511
[5]HeilbronnLK,dejongeL,FriSardMI,etal for the pennington CALERIE Team.effect of 6-months calorie ristriction on biomarkers of longevity,metabolic adaptation,and oxidative stress in overweight individuals:a randomized controlled trial.JAMA 2006; 295:1539-1548
[6]Oberley L W. Superoxide Dismutase. M. C RC Press,1982
[7]Kawaquchi M,Furuya H,Patel P M. Neuroprotective effectsof anesthetic agents.J Anesth,2005; 19(2):150-156
[8]周元红 钟守琳 自由基与人体健康中外健康文摘2007;4[16]:629-630
[9]Harman D. Aging:A theory based on free radical and radical and radiation chemistry. J Geron,1956; 11(3):298-300
[10]Nystrom T. Thefree-radical hypothesis of aging goesprokaryotic J Cell Mol Life Sci,2003; 60(7):1333-1341
[11]Droge W. Oxidative stress and aging. J.Adv Exp Med Biol,2003; 543:191-200
[12]Masoro EJ. Caloric restriction and aging:an update. Exp Gerontol,2000; 35:299-305
[13]Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science, 1996; 273:59-63
[14]Ramsey JJ, Harper ME, Weindruch R. Restriction of energyintake, energy expenditure, and aging. Free Radic BioMed,2000; 29:946-968
[15]Tavernarakis N, Driscoll M. Caloric restriction andlifespan:a role for protein turnover Mech Ageing Dev,2002; 123:215-229
[16]Boonefoy,M,Drai,J,Kostka,T.Anti oxidantsto slow ageing,facts and perpectives.press Med,2002; 31(25):1174-1184.
[17]Lopez-Torres M, Gredilla R, Sanz A, et al. Influence of aging and long-term caloric restrictiononoxygen radical generation and oxidative DNA damage in rat liver mitochondria. J.Free Radic Biol Med,2002; 32:882-889.
[18]Mockett RJ, Bayne A-CV, KwongL K, et al. Ectopic expressionof catalase in Drosophila mitochondria increases stress resistance but not longevity. Free Radic Biol Med,2003; 34:207-217.
[19]Orr W C, Sohal R S. Doesoverexpressionof Cu, Zn-SOD extend life span inDrosophila melanogaster. J.Exp Gerontol,2003; 38:227-230.
[20]Melov S, Coskun P, Patel M, et al. Mitochondrial disease in superoxide dismutase mutant miceJ. Proc Natl Acad Sci USA,1999; 96:846-851.
[21]Lopez-Torres M, Gredilla R, Sanz A, et al. Influence of aging and long-term caloric restriction on oxygen radical generation and oxidative DNA damage in rat liver mitochondria. J.Free Radic Biol Med,2002; 32:882-889.
[22]杜历生限食延寿机理及其实践广西中医学院学报2003;6[4]:74-75
[23]VlassaraHBrownleeMCeramiASpecific macrophage recept or activity for advanced glycosylationand products inversely correlated with insulinlevelsinvivo.Diabetes,1988: 37:456-461
[24]程胜求膳食限制对糖化最用的影响第二军医大学学报1995,16(4)354-354.
[25]陈澍蔡如森余爱琴热量限制对大鼠非酶糖化蛋白产物的影响ChinJGeriatr 1997;16[6]: 374-374
[26]Stokkan KA et al,Brain Res,1991; 545:66-72.
[27]ChenHL,MostoslavskyR,Developmental defects and P53 hyperacetylation in sir2 homolog deficient mice.proc Natl Acad sci USA,2003; 100:10794-10799
[28]Barzilai N, Gabriely I. The role of fat depletion in the biological benefits of caloric restriction. J Nutr,2001; 131:903S-906S
[29]金军华张铁梅童坦君热量限制延缓衰老作用机制的研究进展中华老年医学杂志2004;23[1]:66-68
[30]李国林印大中限食延缓衰老最新研究进展国外医学老年医学分册2008;29[1]:1-3
[31]Panowski SH.Wollf S Aguilaniu H et al PHA-4/Foxa mediates diet-restriction-induced longevity of C elegans Nature. 2007;477(7144):550-555.
[32]Bishop NA Guarente L.Two neurons mediate diet-restriction-induced longevity in C elegans Nature.2007;477(7144):545-549.
[33]Trayhurn P,Beattie JH.physiological role of adipose tisue:white adipose tissue as an endocrine and secetory organ. J.PROC NutrSoc,2001; 60:329-339
[34]Nora Kloting.Matthias Bluher.Extended longevity and insulin signaling in adipose tissue. J.Experimental Gerontology,2005; 40:878-883
[35]Das SK, Gilhooly CH, Golden JK, Pittas AG, Fuss PJ, Cheatham RA, Tyler S, Tsay M, McCrory MA, Lichtenstein AH, Dallal GE, Dutta C, Bhapkar MV, Delany JP, Saltzman E, Roberts SB. Long-term effects of 2 energy-restricted diets differing in glycemic load on dietary adherence, body composition, and metabolism in CALERIE:a 1-y randomized controlled trial. Am J Clin Nutr.2007,85:1023-1030.
[36]McCay CM, Crowell MF, Maynard LA. The effect of retarded growth upon the length of life span and ultimate body size, J. Nutr.1935,10:63-79.
[37]Weindruch R, Walford RL, Fligiel S, Guthrie D. The retardation of aging in mice by dietary restriction:longevity, cancer, immunity and lifetime energy intake. J Nutr,1986,116(4):641-654.
[38]Lin SJ, Defossez PA, Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science,2000, 289(5487):2126-2128.
[39]Ingram DK, Anson RM, de Cabo R, Mamczarz J, Zhu M, Mattison J, Lane MA, Roth GS. Development of calorie restriction mimetics as a prolongevity strategy, Ann. N Y Acad. Sci,2004,1019:412-423.
[40]Kaeberlein M, McVey M, Guarente L. The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev,1999,13:2570-2580.
[41]Lin SJ, Kaeberlein M, Andalis AA, Sturtz LA, Defossez PA, Culotta VC, Fink GR, Guarente L. Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature,2002,418(6895):344-348.
[42]Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature,2003,425(6954):191-196.
[43]Michan S, Sinclair D. Sirtuins in mammals:insights into their biological function. Biochem J,2007,404:1-13.
[44]Tannenbaum A. The initiation and growth of tumors. Introduction. Effects of underfeeding. Am J Cancer,1940,38:335-350.
[45]Kritchevsky D. Caloric restriction and experimental carcinogenesis. Hybrid Hybridomics,2002,21:147-151.
[46]Verdery RB, Ingram DK, Roth GS, Lane MA. Caloric restriction increases HDL2 levels in rhesus monkeys (Macaca mulatta). Am J Physiol,1997,273: E714-E719.
[47]Edwards IJ, Rudel LL, Terry JG, Kemnitz JW, Weindruch R, Zaccaro DJ, Cefalu WT. Caloric restriction lowers plasma lipoprotein (a) in male but not female rhesus monkeys.Exp Gerontol,2001,36:1413-1418.
[48]Jung SH, Park HS, Kim KS, Choi WH, Ahn CW, Kim BT, Kim SM, Lee SY, Ahn SM, Kim YK, Kim HJ, Kim DJ, Lee KW. Effect of weight loss on some serum cytokines in human obesity:increase in IL-10 after weight loss. J Nutr Biochem,2008,19(6):371-375.
[49]Pereira MA, Swain J, Goldfine AB, Rifai N, Ludwig DS. Effects of a low-glycemic load diet on resting energy expenditure and heart disease risk factors during weight loss. JAMA,2004,292:2482-2490.
[50]Walford RL, Harris SB, Gunion MW. The calorically restricted low-fat nutrient-dense diet in Biosphere 2 significantly lowers blood glucose, total leukocyte count, cholesterol, and blood pressure in humans. Proc Natl Acad Sci U S A.1992,89:11533-11537.
[51]Lefevre M, Redman LM, Heilbronn LK, Smith JV, Martin CK, Rood JC, Greenway FL, Williamson DA, Smith SR, Ravussin E; Pennington CALERIE team. Caloric restriction alone and with exercise improves CVD risk in healthy non-obese individuals.Atherosclerosis,2009,203(1):206-213.
[52]Adler A, Messina E, Sherman B, Wang Z, Huang H, Linke A, Hintze TH. NAD (P) Hoxidase-generated superoxide anion accounts for reduced control of myocardial O2consumption by NO in old Fischer 344 rats. Am J Physiol Heart Circ Physiol,2003,285:H1015-H1022.
[53]Raitakari M, Ilvonen T, Ahotupa M, Lehtimaki T, Harmoinen A, Suominen P, Elo J, Hartiala J, Raitakari OT. Weight reduction with very-lowcaloric diet and endothelial function in overweight adults:role of plasma glucose. Arterioscler Thromb Vase Biol,2004,24:124-128.
[54]Sasaki S, Higashi Y, Nakagawa K, Kimura M, Noma K, Hara K, Matsuura H, Goto C, Oshima T, Chayama K. A low-calorie diet improves endothelium-dependent vasodilation in obese patients with essential hypertension. Am J Hypertens,2002,15:302-309.
[55]Mattagajasingh I, Kim CS, Naqvi A, Yamamori T, Hoffman TA, Jung SB, DeRicco J, Kasuno K, Irani K. SIRT1 promotes endotheliumdependent vascular relaxation by activating endothelial nitric oxide synthase. Proc Natl Acad Sci U S A,2007,104:14855-14860.
[56]Ungvari ZI, Orosz Z, Labinskyy N, Rivera A, Xiangmin Z, Smith KE, Csiszar A. Increased mitochondrial H2O2 production promotes endothelial NF-kB activation in aged rat arteries. Am J Physiol Heart Circ Physiol,2007,293: H37-H47.
[57]Murdoch CE, Zhang M, Cave AC, et al. NADPH oxidase-dependent redox signaling in cardiac hypertrophy, remodeling and failure. Cardiovasc Res,2006, 71 (2):208-215.
[58]Crow MT, Mani K, Nam YJ, et al. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res,2004,95 (10):957-970.
[59]Kujoth GC, Hiona A, Pugh TD, et all Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging Science,2005,309 (5733):481-484.
[60]Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science, 1996,273:59-63.
[61]Guo Z, Mitchell-Raymundo F, Yang H, Ikeno Y, Nelson J, Diaz V, Richardson A, Reddick R. Dietary restriction reduces atherosclerosis and oxidative stress in the aorta of apolipoprotein E-deficient mice. Mech Ageing Dev,2002,123: 1121-1131.
[62]Cefalu WT, Wang ZQ, Bell-Farrow AD, Collins J, Morgan T, Wagner JD. Caloric restriction and cardiovascular aging in cynomolgus monkeys (Macaca fascicularis):metabolic, physiologic, and atherosclerotic measures from a 4-year intervention trial. J Gerontol A Biol Sci Med Sci,2004,59:1007-1014.
[63]Walford RL, Mock D, Verdery R, MacCallum T. Calorie restriction in biosphere 2:alterations in physiologic, hematologic, hormonal, and biochemical parameters in humans restricted for a 2-year period.J Gerontol A Biol Sci Med Sci,2002,57(6):B211-224.
[64]Alcendor RR, Kirshenbaum LA, Imai S, Vatner SF, Sadoshima J. Silent information regulator 2alpha, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes. Circ Res,2004,95(10):971-980.
[65]Pillai, JB, Isbatan A, Imai S and Gupta MP. Poly (ADP-ribose) polymerase-1-dependent cardiac myocyte cell death during heart failure is mediated by NAD+ depletion and reduced Sir2a deacetylase activity. J. Biol. Chem,2005,280:43121-43130.
[66]Zhou B, Wu L-J, Li L-H, Tashiro S, Onodera S, Uchiumi F, Ikejima T. Silibinin protects against isoproterenol-induced rat cardiac myocyte injury through mitochondrial pathway after up-regulation of SIRT1. J Pharmacol Sci,2006, 102:387-395.
[67]Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, Kessler B, et al. Calorie Restriction Promotes Mammalian Cell Survival by Inducing the SIRT1 Deacetylase.Science,2004,305(5682):390-392.
[68]Clarke MC, Littlewood TD, Figg N, et al. Chronic apoptosis of vascular smooth muscle cells accelerates atherosclerosis and promotes calcification and medial degeneration. Circ Res,2008,102(12):1529-38.