论文标题:心肌老化与非酶糖基化的相关研究
Study in the Relationship between the Myocardial Aging and Glycation in the Aged Rat
论文作者
论文导师 程蕴琳,论文学位 博士,论文专业 老年医学
论文单位 南京医科大学,点击次数 114,论文页数 126页File Size7883K
2007-04-01论文网 http://www.lw23.com/lunwen_41227687/
cardiac aging;; nonenzymatic glycosylation;; D-galactose;; Oxidative stress;; mtDNA;; ALT-711;; EGB761
目的机体衰老是老年人所面临的共同病理、生理发展过程，如何延缓衰老，是目前老年医学热门研究课题，而只有明确其机制，才能有的放矢地去干预和治疗。研究衰老、老年病的发病机制，需要可靠的衰老模型，应用自然衰老模型成本高，周期长，目前国内外根据衰老学说已研制出较多的衰老动物模型，我国学者根据非酶糖基化交联学说研制出D-半乳糖衰老动物模型，但此模型有关心血管系统在组织、细胞及分子水平缺少系统性研究，特别与自然衰老之间的可比性试验资料较少，因此本试验分别从心脏整体结构及心功能水平，电镜下心肌细胞及超微结构的变化、心肌组织内非酶糖基化终末期产物、抗氧化物质的含量，及线粒体DNA的缺失率，观察2组的相关性，为制作D-半乳糖模型提供老化依据，同时探讨心肌衰老发生发展的途径。 非酶糖基化与心肌老化有明显相关性，它可以通过AGEs的形成，触发氧化应激，从而导致线粒体DNA突变，引起心肌结构及功能的改变，目前有什么途径能阻止其产生和发展，减少或逆转对心肌的损伤呢?已成为研究的热门话题，但都处于动物实验及临床试验阶段。在国外研制出AGEs交联蛋白裂解剂，4，5-Dimethyl-3-(2-oxo-Phenylethyl)-Thiazolium Chloride (ALT-711)，它可以使已经被AGEs修饰的蛋白裂解，恢复蛋白的结构及功能，本试验经过ALT-711对自然衰老及D-半乳糖模型衰老的干预治疗，观察其心脏结构及功能的改变，及组织AGEs和抗氧化能力的变化，mtDNA缺失的情况，为心脏衰老的治疗寻找试验依据。 中医中药在延年益寿抗衰老方面已有悠久历史，银杏叶是目前国内外广泛关注和热门的中药，银杏叶提取物(EGB)具有多种药用价值，已被国内外认可，许多西方国家大量进口银杏叶，进行提取制成口服或注射液出口，国内银杏叶产量大，已被广泛用于治疗心脑血管疾病，但银杏叶对心肌老化的作用，目前报导较少。本研究旨在探讨银杏叶对自然衰老和D-半乳糖衰老模型心肌的干预治疗作用，观察电镜下心肌超微结构、心脏二维超声结构及功能、心肌内组织水平AGEs的水平，抗氧化物质SOD、GSH-Px的活性，及氧化产物MDA的变化，以ALT-711为对照组，观察银杏叶对心肌的抗衰老作用，为中药抗心肌老化，提供依据。 方法分组：1．SD健康成年雄性大鼠20只(180-220g)，分为D-半乳糖老化模型组、对照组(每组各10只)。建立D-半乳糖老化模型：每天予D-半乳糖50mg／kg／d腹部皮下注射，对照组每天给予同等容量的生理盐水皮下注射；另设自然老化组10只(20-22月龄)。2．ALT-711干预治疗组：在2种衰老模型各10只，均给予ALT-711以10mg／kg．day灌胃。2．银杏叶提取液干预治疗组：2种衰老模型各10只，均给予EGB100mg／kg．day口服，连续观察16周。 于实验第16周在10％戊巴比妥(1ml／100g)静脉麻醉后常规探查心脏结构及血流并测定心功能。取心肌组织电镜下观察心肌的超微结构，测定心肌内AGEs含量，抗氧化指标SOD、GSH-Px、MDA及线粒体DNA的缺失率。 结果1、自然衰老和半乳糖组分别与正常组比较，电镜下结构有相似的结构紊乱，包括肌丝断裂，润盘模糊，线粒体肿胀，心脏二维超声结构提示左心室心肌肥厚，重量增加，心脏功能减退，2组衰老组心肌内AGEs含量增加(P＜0.05)，抗氧化指标SOD、GSH-Px减少(P＜0.05)，氧化代谢产物MDA增加(P＜0.05)，线粒体DNA的缺失率增加(P＜0.05)，2组衰老组之间无显著性差别。 2、2组衰老组经ALT-711干预治疗后，电镜心脏超微结构有改善，包括肌丝排列整齐，线粒体致密，无水肿，润盘清晰和连续，心脏二维超声提示2组衰老组心肌的重塑均有改善，包括室间隔厚度减轻，左室重量减轻，心脏的舒张功能提高(p＜0.05)，心肌内AGEs含量减少(P＜0.05)，抗氧化指标SOD、GSH-Px增加(P＜0.05)，氧化代谢产物MDA减少(P＜0.05)，线粒体DNA的缺失率减少(P＜0.05)。 3、2组衰老组经EGB干预治疗后，心脏超微结构有改善，包括肌丝排列整齐，线粒体致密，无水肿，润盘清晰和连续，心脏二维超声提示2组衰老组心肌的重塑均有改善，包括室间隔厚度减轻，左室重量减轻，心脏的舒张功能提高(p＜0.05)，心肌肉AGEs含量减少(P＜0.05)，抗氧化指标SOD、GSH-Px增加(P＜0.05)，氧化代谢产物MDA减少，线粒体DNA的缺失率减少(P＜0.05)。 结论1、自然衰老和半乳糖衰老模型具有形似的形态学改变，具有可比性，同时衰老和非酶糖基化及氧化应激相关，导致线粒体缺失，最终心脏功能减退和心肌重塑。 2、ALT-711能够逆转心肌的结构的破坏，降低心肌内糖基化终末期产物的含量，改善抗氧化功能，减少线粒体的损伤。 3、EGB能够和ALT-711同样起到改善心肌老化的作用，能够逆转心肌的结构的破坏，降低心肌内糖基化终末期产物的含量，改善抗氧化功能，减少线粒体的损伤。
Background The aging of the organisms involves the commonpathologic and physiologic processes which the elderly people are facingwith. How to defer the aging process is the hot topic in the field ofgerontology. To identify the mechanism of senility would certainly helpto prevent and treat for the old peoplperson. Objective To investigate the mechanisms of aging and senile disease, wewould demand the reliable model of aging. The natural aging model ishigher price and need long time. In the present, the artificial animalmodel of aging was developed by the international scholar. TheD-galactose aging model was established, according to the glycosylationand cross-link theory in our country. The study in the cardiovascularaging of D-gal model is seldom. Few materials showed the comparabilitybetween the D-gal and natura old animal model. In this experiment, thevariation in the cardiac structure and function was to be observed. Theultrastructure of myocardial cell, the contents of AGEs, SOD, GSH-Px,MDA and mtDNA deletion were observed. We try to approach thepathway of aging and provide the confidence of D-gal model rat. The glycosylation is higher relative with the cardiac aging, whichtriggers the oxidative stress and induces the mtDNA mutation by theAGEs formation. What would prevent and hamper the AGEs formationand improve the cardiac structure and function was the hot subject,However, the study keeps in the animal test and clinical experiment stage.The crosslink breaker,3-phenacyl-4,5-dimethylthiazolium chloride(ALT-711), improves arterial and ventricular function in older rhesusmonkeys and vascular compliance in humans. In this experiment, weobserved the changes in the cardiac structure and function and thecontents of AGEs, SOD, GSH-Px and MDA through the ALT-711administration. There were more experience to lengthen life in the traditionalmedicine. The ginkgo leaf has multiple effects and is ratified by theinternational countries. Many west countries import the ginkgo leaf andextract the oral administration or injection, then to export it. There ismuch ginkgo leaf in our country. The ginkgo leaf is extensively appliedby brain and cardiovascular patients, but seldom to be researched in themyocardial aging. In this test, we investigated the effects of ExtractGingko biloba (EGB761) to the heart aging in the rat, including naturaland D-gal aging. We observed the changes in the cardiac structure andfunction and the contents of AGEs, SOD, GSH-Px and MDA through theEGB-761 administration. Method 1. The rats were divided into young group, D-gal aging modelgroup and natural aging group, the aging model(n=10,weight200～250mg) was made by injecting D-galactose,50mg/Kg.day, and atthe same time, natural aging group group(n=10,weight 500～600mg) wasgiven the same volume NS injecting. 2. The ALT-711 administrationgroup was given ALT-711 10mg/Kg.day by intragastric administrationfor 16 weeks. 3. The EGB administration group was given EGB10mg/Kg.day by intragastric administration for 16 weeks. Themyocardial histopathological changes under electron microscope, thecontents of AGEs and oxidative mark in the cardiac tissue were measured.The mitochondrial DNA (mtDNA) deletion level in cardiac cell wasexamined using polymerase chain reaction (PCR). Results 1. D-gal and natural aging heart gained more LV weight andLA than young control group significantly (P＜0.05). The electronmicroscope showed morghology damage, including the myofilamentbreaking, dropsy mitochondria and unclear moist disc in the 2 aginggroups than the control group. The consentration of SOD and GSH-Pxwere significantly lower in the D-gal and natural aging group than that inthe control group (P＜0.05). The AGEs, MDA level in the cardiac tissuewas higher than the control group significantly (P＜0.05). The rate ofmtDNA deletion significantly increased in the 2 aging group than in thecontrol group (P＜0.05). 2. The electron microscope showed morghology improveing, including the myofilament in order, normal mitochondriaand clear moist disc in the ALT-711administration group than in theaging control group. The cardiac structure and function improved in theALT-711 administration group than in the aging control group. Theconsentration of SOD and GSH-Px were significantly higher in theALT-711 treatment group than in the aging control group (P＜0.05). TheAGEs, MDA level in the cardiac tissue was lower than in the controlgroup significantly (P＜0.05). The rate of mtDNA deletion significantlyincreased in the ALT-711 administration group than in the aging controlgroup (P＜0.05). 3. In the EGB-761 administration group, the electronmicroscope showed morghology improveing, including the myofilamentin order, normal mitochondria and clear moist disc than in the agingcontrol group. The cardiac structure and function improved in the EGBadministration group than in the aging control group. The consentrationof SOD and GSH-Px were significantly higher in the EGBadministration group than in the control group (P＜0.05) and had nodifferent with the ALT-711 group. The AGEs and MDA level in thecardiac tissue was lower than in the control aging group significantly(P＜0.05) and had no different with the ALT-711 group. The rate ofmtDNA deletion significantly decreased in the EGB administration groupthan in the control aging group (P＜0.05) and has no different with theALT-711 group. Conelusion 1. There was comparability between the D-gal and naturalaging models, which included the changes of morghology and correlatedwith nonenzymatic glycosylation and oxidative stress. The mtDNA wasinduced and results in the myocard remodel, which decrease the heartfunction. 2. ALT-711 had some anti-aging effect by inhibiting NEG, reducing thefree radical and decreasing the mtDNA deletion. ALT-711 can improvethe cardiac structure and function. 3, EGB had anti-aging effect by inhibiting NEG, reducing the free radicaland decreasing the mtDNA deletion.EGB can improve the cardiacstructure and function and had similar role with ALT-711.
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