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降脂及抗脂质氧化药物的筛选
谢亚利
学位类型硕士
导师李维琪
2004-06-10
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业微生物学
关键词降脂 肝癌细胞hepg2 载脂蛋白ai 抗脂质氧化 氧化低密度脂蛋白 巨噬细胞
摘要据流行病学调查,心血管疾病随着人们生活水平的提高和生活方式的改 变,已经成为发达国家及部分发展中国家人民的主要杀手。心血管疾病主要病因是高血清总胆固醇,所以近年来人们都把降脂药的研 究和开发重点放到降低血清总胆固醇特别是低密度脂蛋白一胆固醇(LDL-C) 水平上,这些药能显著降低血清总胆固醇,防止及降低动脉粥样硬化和冠心病 发生,发展和死亡,对于预防和治疗心血管疾病功不可没。但它们通常只能减 少内源性胆固醇合成及降低家族性高胆固醇血症引起的冠心病的发展或恶化, 对于治疗外源性胆固醇增多引起的动脉粥样硬化,对于消除已进入巨噬细胞内 的胆固醇和抑制由此导致的动脉粥样硬化形成,使动脉粥样硬化血管回复正 常,则作用不大。近年来人们普遍注意到提高血浆高密度脂蛋白-胆固醇(HDL-C)的含量可以有效改善这些问题。高密度脂蛋白一胆固醇(HDL-C) 的功能主要是胆固醇逆转运:把血液、外周组织中的胆固醇运送到肝脏去处理, 从而维持胆固醇水平恒定。提高血浆高密度脂蛋白一胆固醇(L-C)的含量 最重要是要提高载脂蛋白AI水平。另外大量资料证明,动脉粥样硬化的发生、发展与低密度脂蛋白氧化修饰 有关。最近的研究还发现,除从AS病变中能分离出的LDL有部分发生了氧化 修饰外,血管病变中通常含有抗氧化型LDL的抗体;血循环中存在的可与氧 化型LDL反应的抗体,其滴度与AS严重程度密切相关。抗脂质氧化药物可以 保护巨噬细胞免受脂质过氧化损伤和防止低密度脂蛋白氧化所导致的泡沫样 变,并能防止和减轻动脉粥样硬化形成。本论文根据以上降脂和脂质氧化机理,建立新一代降脂药和抗脂质氧化药 物筛选的方法,为中药现代化服务。本论文主要目的:建立载脂蛋白AI作为靶标,用MW-ELISA进行检测的 新一代降脂药筛选方法以及基于巨噬细胞吞噬氧化低密度脂蛋白的抗脂质氧 化药物的筛选方法。本论文方法:通过离体培养的人肝癌细胞HepG2(该细胞体外仍保持ApoAI 以及其他载脂蛋白的分泌能力),建立微波一酶联免疫吸附法(MW-ELISA),分析几种传统药物提取物对HePG2细胞分泌APOAI的影响,旨在建立起一种 高通量筛选降脂药的方法;为建立抗脂质氧化药筛选模型,首先分离提取人血 中低密度脂蛋白一胆固醇,利用Cu+2氧化修饰低密度脂蛋白一胆固醇,产生过氧 化脂质,建立小鼠腹腔巨噬细胞吞噬氧化低密度脂蛋白过氧化脂质的实验模型, 检测细胞分泌脂质过氧化活性中间物丙二醛(MDA)的水平和该系统超氧化 物歧化酶(SOD)活力,通过药物干预前后,丙二醛水平和超氧化物歧化酶活 力变化,筛选具有抗脂质氧化潜力的药物。 在验证两种筛选模型的可行性时,选用已经在传统验方或动物实验模型上 有确切降脂或抗脂质氧化作用的多种中药材(泽泻、虎杖,郁金,山碴、马齿 芡、绞股蓝)提取物,希望既能考证两种筛选模型的可行性,又可进一步验证 这些药物及其有效成分的临床作用。本论文的结论:实验结果证明泽泻、绞股蓝、山植、郁金的提取物在我们 的降脂药筛选模型上具有相对明显的提高APoAI表达的作用,而绞股蓝、马 齿觉,虎杖、山植等的提取物在抗脂质氧化模型上确实具有明显的抗氧化作用, 实验发现不同的调脂药物(liporegulators)在我们的降脂和抗脂质氧化模型上 有不同的作用机制,本课题建立的方法可以作为降脂药和抗脂质氧化药物筛选的一种简单方法。
其他摘要According to epidemiological research result. following living standard promotion and changed living style.cardiovascular disease has become the citizen's murderer in developed and some developing country. The major cause of cardiovascular disease is high serum total cholesterol level,so people put emphasis of the development of lipid-lowering medicine on lowering serum total cholesterol especially low density lipoprotein-cholesterol(LDL-C) in recent years,these drags can evidently lower serum total cholesterol,prevent and reduce the roduction,development and death caused by atheroscelerosis and coronary heart disease,they are greatly contributing to prevent and treat cardiovascular disease.But they only function on reduction of endogenous cholesterol synthesis and coronary heart disease depravation caused by familial hypercholesterolemia.To remedy atheroscelerosis caused by exogenous cholesterol elevation,remove cholesterol intruded into macrophages and prevent atheroscelerosis developing, recovery the blood vessel from atheroscelerosis,All of those medicines show little function.In recent years people popularly notice that elevating high density lipoprotein-cholesterol(HDL-C) level in plasma can improve the status which low density lipoprotein-cholesterol comes up against.High density lipoprotein-cholesterol play a major function as transporting cholesterol from blood and perpheral tissues to liver to dispose,to keep the total cholesterol at a certain level.In elevating high density lipoprotein-cholesterol(HDL-C) level in plasma, elevating the level of apolipoprotein AI plays the most important role. In additions great deal of data have proved that atheroscelerosis formation and developing correlate with low density lipoprotein oxidization.Recent research also illustrates,not only we can isolate partly oxidized low density lipoprotein from atheroscelerosis pathological change position, also we often find the antibody of oxidized-LDL, moreover the concentration of the antibody relates with the atheroscelerosis degree, antioxidants can prevent macrophages from injury by lipid oxidization and being induced to be foam cells by low density lipoprotein oxidization,then prevent and reduce atheroscelerosis. According the mechanism of lowering-lipid and lipid oxidization above.this project is to establish a method of screening new generation of lipid-lowering medicine and antioxidant.to serve for traditional Chinese medicine modernization. Major objectives of this project: to establish a new method for screening lipid-lowering medicine,which comprises aplipoprotein AI as target and microwave-enzyme-linked immunosorbent assay as detect ing measure,also screening lipid antioxidant method based on the mechanism of macrophages licking up oxidized-LDL was established in the project. Methods of this project: to establish screening lipid-lowering medicine method, human hepatic carcinoma cells(HepG2 cells) were cultured in vitro,which still remains the ability of aplipoprotein AI and other apolipoproteins secretion in vitro, MW-ELISA then was established for detecting.the extracts of several Chinese traditional drugs were added in cultured HepG2 to stimulate aplipoprotein AI secretion, through above we hope to obtain an approach to high throughput screening lipid-lowering medicine.To establish screening lipid antioxiditant model,first low density lipoprotein-cholesterol was isolated from human plasma,then used Cu+2 to oxidize low density lipoprotein-cholesterol.produced lipid peroxide,established mouse peritoneal macrophages licking up oxidized-LDL model,detected macrophages secreting lipid peroxide active intermediate-MDA level and SOD activity,through the MDA level and SOD activity changing when there were drugs extract intervention or not,we may screen the medicine which have anti-oxidative potential. when validating the feasibility of two above screening model,we selected several herbital plants (Alisma orientalis, Giant Rnotweed P. E., Curcuminoids, Crataegus pinnatifida, Portulaca oleracea, Gynostemma P. E.) , those have been proved to have some effect in lipid -lowering or lipid antioxidation in Chinese traditional medicine prescription and animal model,we hope it not only textually research feasibility of two above screening model,but also validate those drugs as well as extracts have clinical effect in more. Conclusions of this project: the result indicates that the extracts from Alisma orientalis, Gynostemma P. E., Crataegus pinnatifida ,Curcuminoids evidently have abilitity to evelating apolipoprotein AI level in our lipid-lowering model,and the extracts from Gynostemma P. E., Portulaca oleracea, Giant Knotweed P.E., Crataegus pinnatifida evidently have abilitity to antioxidation,According to the results,we find those different liporegulators have different function mechanism in the established lipid-lowering and lipid antioxidation model, the established methods can serve as simple lipid-lowering medicine and lipid antioxidation screening.
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4456
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
推荐引用方式
GB/T 7714
谢亚利. 降脂及抗脂质氧化药物的筛选[D]. 北京. 中国科学院研究生院,2004.
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