【作者】 张善锋；

【导师】 王明臣； 安玉会；

【作者基本信息】 郑州大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 背景和目的:衰老是人生长、成熟和退化3个阶段中退化期的表现。人衰老的形成经过漫长、持久而复杂的演变过程,有学者认为机体受到外环境(物理或化学因素)和内环境(生理和病理)的影响,产生过剩的自由基,它们极易对构成组织细胞的生物大分子(核酸、蛋白质、多糖、脂类)造成损伤性破坏。该损伤反应涉及到正常组织器官的形态结构和生理功能的完整性。当损伤程度超过修复能力导致组织器官丧失代偿能力时,则出现衰老或疾病,这就是目前医学界广泛研究的衰老自由基学说。 在一定时间内,连续给动物注射大剂量的D-半乳糖,使机体细胞内半乳糖浓度增高,在醛糖还原酶的催化下,还原成半乳糖醇,这种物质不能被细胞进一步代谢而堆积在细胞内,影响正常渗透压,导致细胞肿胀、功能障碍、代谢紊乳,同时产生大量的自由基,最终导致衰老的发生。半乳糖致衰老动物在体征和生化指标上与自然衰老动物毫无显著性差异,是较为理想的衰老模型。 叶黄素是一种天然植物提取物,属于类胡萝卜素。国外大量的流行病学证据表明,叶黄素在预防视黄斑退化、肿瘤的发生与发展、心血管疾病和增强机体免疫力等方面有着广泛的生物活性。 根据衰老的自由基代谢紊乱学说,本实验采用大鼠颈后部皮下注射D-半乳糖,建立亚急性衰老模型,同时系统的研究了不同剂量的叶黄素对衰老大鼠的干预作用。通过测定与衰老有关的行为学指标、免疫学指标及生化指标,探讨叶黄素对衰老大鼠的作用机理。同时,本实验利用鼠伤寒沙门菌组氨酸营养缺陷型回复突变试验(Ames试验)和体外中国仓鼠卵巢细胞CHO细胞染色体畸变试验进行研究,进一步检测了不同剂量的叶黄素抗突变作用及机理。我们的研究为探讨叶黄素抗衰老机理及叶黄素产品的开发提供了有价值的基础研究资料。更多还原

【Abstract】 Background and Purpose:Aging is a manifestation of degeneration stage during the three stages of a life cycle-growth, maturing and degeneration. Aging is a long, lasting and complex developing process. Some scholars believe that the body produces extra free radical influenced by internal environment (physical or chemical factors) and external environment (physiological or pathological factors). The extra free radical is likely to destroy macromolecules (nucleic acid, protein, sugar and fat) of tissue cells. The damage reaction circulates badly and extends layer by layer. The wholeness of tissue structure and physiological function is also destroyed. Illness or aging occurs when destroy beyond repairing results in compensation of tissue disappering.This is free radical theory studied widely in present medicinal community.In a certain period of time, lager dose D-galatose is injected into rats’bodies, which enables the density of D-galatose inside the cells to increase.D-galatose catalyzed by aldose reducase is deoxygenized to galactitol. Galactitol can not be metabolized, so it accumulates in cell and influences osmotic pressure and results in cell bulging, function obstacle and metabolism disturbance. Meanwhile, it produces a plenty of free radical and leads to aging finally. Aging rats caused by D-galatose are similar to natural aging rat in biochemical behavior, which is better aging model.Lutein is a kind of natural plant extract, which belongs to carorenes. Plenty of foreign epidemiological studies showed that lutein has extensive biological activities in preventing macular degeneration, tumor, cardiovascular diseases and enhancingbody immunity. In china, lutein is used only in forage industry; its biological activity has not been regarded highly by food industry and medicine industry.According to free radical aging theory, rats were injected with D- galatose to make a subacute aging model, on the basis of which the ant-aging effect of various doses of lutein was studied. In order to study the mechanism of anti-aging of lutein, we determined the behavior, biochemistry indices and immunity indices relating to aging. To study the mechanism of antimutation of lutein, we detected the mutation and antimutation of lutein with Salmonella typhimurium strain mutagenicity test (Ames test) and chromosome aberration test of Chinese hamster ovary cell. Our research provides valuable basic research information for studying the mechanism of antiaging of lutein and exploiting lutein product. Methods:To study anti-aging of lutein in animal level, subcuitis of the rats’ necks injected with D-galatose 125mg/kg.bw for 6 weeks to make subacute aging model, on the basis of which the ant-aging effect of lutein on aging model was studied. During the experiment, behaviors (locomotor activity, aspect, hair) were observed at any time, the value of rat weight was acquired once a week, After 42 days’ stomach rearing, the ability of learning and memory were tested by Y-maze. Then the rats of each group were killed. Blood, liver, brain, spleen, thymus gland, lens, retina were collected quickly in ice box. Spleen and thymus gland values were calculated, the activity of SOD, CAT and GSH-Px in serum, liver and brain tissues were detected. The content of MDA and GSH in serum, liver and brain were also measured. The activity of SOD and The content of MDA in lens and retina were determined.To study antimutation and mutation of lutein in prokaryotic cell level, we detected the mutation and antimutation of lutein with Salmonella typhimurium strain mutagenicity test (Ames test). In order to clarify the mechanism of antimutation of lutein, four different adding drug ways were used: A,incubating mutagen and lutein together for 30min, then add bacteria; B,incubating lutein and bacteria together for 30min, then add mutagen; C incubating mutagen and bacteria together for 30min, then add lutein;D, incubating lutein, mutagen and bacterial together for 30min.In eukaryotic cell level, chromosome aberration test of Chinese hamster ovary cell was done to detect the mutation and antimutation of lutein. Results:1. The characters of rats’aspect. The rats in normal group are good at activity, response and diet.Two weeks after first injecting, the rats of aging group and solvent group compared with the other rats were scorcher in hair, slower in action, less in diet and cachexia, aggravation with time. Positive group and lutein groups of different dose epilated from the 4th week and alleviated from the 6th week, their behavior and aspect are similar to normal group.2. Viscera value test. Compared with normal group, spleen and thymus gland values of aging group were significantly decreased (P<0.05); There were no significant different between aging group and solvent group (P>0.05); Compared with solvent group, the values of lutein group were increased (P<0.05), the values of lutein group were similar to vitamin E group(P>0.05).3. Y-maze test. In learning and memory test, the result of aging group was obviously decreased than that of normal group(P<0.05),there was no obviously difference between solvent group and positive group(P>0.05), the test result of different dose of lutein group was obviously increased than that of solvent group(P<0.05), there were no significant differences between positive group and moderate dose lutein group(P>0.05).4. The activities of antioxidant enzymes(SOD, GSH-Px, CAT and GSH) and the levels of MDA in serum, brain and liver in aging group were significantly different from those of normal group(P<0.05).There were significant differences among solvent group, aging group and lutein group(P>0.05). The activities of free radical related enzymes(SOD> GSH-Px^ CAT and GSH) in different tissues were decreased during senile phase. As a product of free radical metabolism, the quantity of MDA was increased with age.5. To compare aging group with normal group. The activities of SOD in lens and retina were obviously decreased and the quantities of MDA were increased (P<0.05), just the same as solvent group and lutein groups, vitamin E group and lutein groups.There were no significant differences between solvent group and aging group (P>0.05).6. Ames test. In mutation test, Lutein groups have no more 2 times than negative group and there were significant differences compared with positive group(P<0.05), In antimutation test, comparing with positive group, Lutein groups can decrease the colonies in TA98 and TA100 strains. In different adding drag way test, way A, B, C, D have no significant differences(P<0.05).7. Chromosome aberration test of Chinese hamster ovary cell (CHO) cell. In mutation test, the number of aberrations in lutein groups is lower than 5%, and there are significant differences with positive group (P<0.05). In antimutation test, lutein groups have significant differences with positive group (P<0.05).Conclusions:1. The rats injected with D-galatose could result in decrease of the learning and memory ability and changes of aging-related indices including SOD> MDA, GSH-Px^ CAI\ GSH . The aging animal model could be established by this method.2. Lutein we used could increase viscera value of the rats with D-galatose and improve their immunity.3. Lutein we used could improve learning and memory ability of the rats with D-galatose.4. Lutein we used could eliminate superfluous free radical, thus it has anti-oxidation.5. Lutein we used could significantly improve the anti-oxidation ability in lens and retina in the rats with D-galatose.6. Lutein has no mutation but antimutation.7. The mechanism of antimutation of lutein is not only desmutation to mutagen but renovation to mutation cell.更多还原