有氧运动对衰老大鼠海马ERK基因表达及其DNA甲基化水平的影响

发布时间：2018-04-25 11:12

本文选题：有氧运动 + 衰老　；参考：《成都体育学院》2017年硕士论文

【摘要】：目的:研究不同时期有氧运动干预对衰老大鼠海马ERK基因表达及其DNA甲基化的影响,探索有氧运动对海马ERK的影响及作用机制,探寻运动延缓脑衰老的最佳干预时期,为进一步揭示运动延缓脑衰老机制提供理论依据和实验资料。方法:3月龄成年雄性SPF级SD大鼠共75只。大鼠购进适应性喂养(1周)后,随机分成5组(n=15):对照组(C组)、衰老组(A组)、衰老前运动组(S1组)、衰老中运动组(S2组)、衰老后运动组(S3组)。除C组外,其余组均采用腹腔注射D-半乳糖方式建立亚急性衰老动物模型。S1、S2、S3组分别在衰老前、中、后进行中等负荷游泳运动。动物建模实验的最后一天断头法处死大鼠,迅速剥离大鼠海马,分别液氮保存、石蜡包埋以待用。动物建模过程中记录大鼠一般状态;尼氏染色观察大鼠海马神经元的形态结构变化;Western Blotting和Real-time PCR检测大鼠海马ERK基因和蛋白的表达;采用“飞行质谱法”检测大鼠海马ERK DNA甲基化水平。实验所得图像采集使用激光共聚焦扫描显微镜进行采集分析;实验所得数据使用SPSS20.0统计学软件进行处理和统计。结果:(1)大鼠一般状态:C组大鼠呈现正常状态;A组大鼠出现明显的衰老体征,表现为嗜睡、食欲不振、行动迟缓及活动减少、毛色枯黄脱落等现象;与C组比,S2组大鼠状态接近C组;与A组比,S2组大鼠状态较A组好;S1、S3组与A组相近;S1、S2、S3组比,S2组状态较好。(2)尼氏染色:C组大鼠海马的神经元形态结构正常;A组神经元细胞结构不完整,数量明显减少,部分神经元细胞核模糊不清,尼氏体着色变浅;与C组比,S2组接近C组;与A组比,S2组海马神经元排列相对有序,数量及细胞间距相对正常;S1、S3组与A组相似;S1、S2、S3组比,S2组大鼠较好,海马神经元的排列,数量,着色最好。(3)大鼠海马ERK蛋白及mRNA表达:海马ERK蛋白及mRNA表达趋势基本一致,C组表达最高,S2组次之,A组最低,且非常显著性低于其它各组(P0.01);与C组比,S2组接近C组,均非常显著性高于其它各组(P0.01);与A组比,S2组非常显著性高(P0.01),S1、S3组与A组相似,组间差异无统计学意义(P0.05);S1、S2、S3组比,S2组非常显著性高于其他两组(P0.01)。(4)大鼠海马ERK基因(基因序列全长:1509bp)甲基化检测:C组大鼠甲基化最低;A、S3组最高;S1组次之;与C组相比,S2组与C组相似,甲基化水平均相对较低,组间差异无统计学意义(P0.05);与A组比,S2组非常显著性低于A组(P0.01),S1、S3较A组均有不同程度的降低,组间差异无统计学意义(P0.05);与S1、S2、S3组比,S2组非常显著性高于其它两组(P0.01)。(5)大鼠海马ERK基因甲基化与蛋白和mRNA表达相关性分析:各组大鼠海马ERK基因甲基化与ERK蛋白和mRNA表达之间呈负相关(P0.05)。结论:(1)衰老使大鼠海马神经元的形态结构发生退行性改变,使ERK基因发生高甲基化,导致海马ERK蛋白和mRNA表达均出现下调,进而影响ERK信号通路的功能,使脑的功能下降。(2)有氧运动作为外源刺激因素之一,对基因表达具有一定的修饰作用。可以使ERK基因发生去甲基化,进而使该基因的蛋白和mRNA表达上调,修复海马神经元的形态结构,维持脑的功能。推测有氧运动使ERK基因发生去甲基化,是运动延缓脑衰老的可能机制之一。(3)在大鼠衰老的不同时期进行有氧运动干预,其效果不同。其中,在衰老过程中进行有氧运功干预效果最显著,说明有氧运动干预脑衰老应该在衰老过程中进行。
[Abstract]:Objective: To study the effect of aerobic exercise intervention on the expression of ERK gene and DNA methylation in hippocampus of aging rats, explore the effect of aerobic exercise on hippocampal ERK, explore the best intervention period to postpone brain senescence, and provide theoretical basis and experimental data to further reveal the mechanism of retarding the aging of brain aging. Method: 3 75 adult male SPF SD rats were randomly divided into 5 groups (n=15) after adaptive feeding (group C), aging group (group A), pre senescence group (group S1), aging exercise group (group S2) and aging group (S3 group). Except C group, the other groups were injected with D- galacose by intraperitoneal injection of D- galacose to establish subacute senescence animal models. Type.S1, S2, and S3 group were subjected to medium load swimming before and after aging respectively. The rats were killed in the last day of the animal modeling experiment, and the rat hippocampus was quickly stripped, the liquid nitrogen was preserved and paraffin embedded to be used. The animal modeling process was used to record the general state of the rats; Nissl staining observed the morphological changes of the hippocampus neurons in the rats. Western Blotting and Real-time PCR were used to detect the expression of ERK gene and protein in the hippocampus of rats; the level of ERK DNA methylation in hippocampus of rats was detected by "flight mass spectrometry". The experimental image collection was collected and analyzed by laser confocal scanning microscope, and the experimental data were processed and counted with SPSS20.0 statistics software. Fruit: (1) general state of rats: rats in group C showed normal state; group A rats showed obvious aging signs, showing somnolence, loss of appetite, slow movement and decrease of activity, and shedding and shedding of hair yellow and so on. Compared with group C, the state of group S2 was close to that of C group; compared with group A, the state of S2 group was better than that of A group; S1, S3 group and A group; S1 (2) Nissl staining: the hippocampal neurons in group C rats were normal, the neuron cell structure in group A was incomplete, the number of neurons was obviously reduced, the nucleus of some neurons were blurred and the coloring of Nissl body became shallow; compared with group C, the S2 group was close to the C group; compared with the A group, the hippocampal neurons in the group S2 were relatively orderly, the quantity and space distance were relatively normal. S1, S3 group was similar to group A; S1, S2, S3 group was better than group S2, and the arrangement, quantity and coloring of hippocampal neurons were best. (3) the expression of ERK protein and mRNA in hippocampus of rats: hippocampal ERK protein and mRNA expression trend was basically the same, C group was the highest, the group was the lowest, and very significant lower than that of other groups. Significantly higher than the other groups (P0.01), compared with group A, group S2 was very significant (P0.01), S1, S3 group was similar to A group, and there was no significant difference between groups (P0.05); S1, S2, S3 group ratio, S2 group was significantly higher than the other two groups. (4) methylation test of rat hippocampus: the lowest methylation of rats A, S3 group was the highest and S1 group. Compared with group C, S2 group was similar to C group, and the level of methylation was relatively low, and there was no significant difference between groups (P0.05). Compared with group A, the S2 group was significantly lower than the A group (P0.01), S1, and the group had no statistical significance. In other two groups (P0.01). (5) the correlation analysis of methylation of ERK gene in hippocampus of rats and expression of protein and mRNA expression: the methylation of ERK gene in hippocampus of rats was negatively correlated with ERK protein and mRNA expression (P0.05). Conclusion: (1) senescence resulted in degenerative changes in morphological structure of hippocampal neurons in rats, resulting in the hypermethylation of ERK gene and leading to the sea. The expression of ERK protein and mRNA decreased, thus affecting the function of ERK signaling pathway and reducing the function of the brain. (2) aerobic exercise, as one of the exogenous stimuli, has a certain modification effect on gene expression. It can cause the ERK gene to demethylation and then up the protein and mRNA expression of the base and repair the hippocampal neurons. Form structure, maintain the function of brain. It is suggested that aerobic exercise can demethmethylation of ERK gene, which is one of the possible mechanisms to delay brain aging. (3) the effect of aerobic exercise is different in the different period of aging of rats. The effect of aerobic exercise during aging is the most significant, indicating that aerobic exercise intervention in brain senescence. It should be carried out during the aging process.

【学位授予单位】：成都体育学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：G804.7

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