叶酸干预对染铝大鼠海马Aβ相关基因甲基化影响的研究
发布时间：2019-02-27 20:09
【摘要】：目的:研究叶酸干预对染铝大鼠Aβ沉积的相关基因(APP、PS1、BACE1)的甲基化影响。方法:健康清洁级雄性SD大鼠40只,按体重随机分成5组,对照组(生理盐水),染铝组(1.08mg/kg),叶酸干预低、中、高剂量组(0.7、1.5和3.4mg/kg),每组8只,各组大鼠腹腔注射相应剂量麦芽酚铝,同时叶酸干预组每日灌胃1ml/100g,连续60天。1.Morris水迷宫测定大鼠学习记忆能力,石墨炉原子吸收法测定大鼠脑铝含量;2.酶联免疫吸附法测定大鼠全基因组甲基化和海马Aβ的表达;3.甲基化特异性聚合酶链反应法测定大鼠海马组织APP、PS1、BACE1的甲基化,实时荧光定量聚合酶链法测定m RNA的表达,酶联免疫吸附法测定蛋白的表达。结果:1.Morris水迷宫结果显示:铝暴露可导致大鼠目标象限停留时间明显缩短和穿越原平台位置的次数明显减少,染铝组与对照组比较差异有统计学意义(P0.05);叶酸干预组可导致大鼠目标象限停留时间明显延长和穿越原平台位置的次数明显增多,与染铝组相比叶酸干预中、高剂量组差异有统计学意义(P0.05)。铝含量测定结果显示:铝暴露可导致大鼠脑铝含量增多,染铝组的大鼠的脑铝含量与对照组相比差异有统计学意义(P0.05),叶酸干预降低了大鼠的脑铝含量,与染铝组相比叶酸干预中、高剂量组差异有统计学意义(P0.05)。结果显示:叶酸改善大鼠学习记忆能力减少了铝在大鼠脑组织中的蓄积。2.大鼠全基因组甲基化测定结果显示:铝暴露可导致大鼠全基因组甲基化率显著降低,与对照组比较差异具有统计学意义(P0.05);而叶酸干预高剂量组全基因组甲基化率显著增加,与染铝组比较差异具有统计学意义(P0.05),与对照组比较差异无统计学意义(P0.05)。大鼠海马组织中Aβ蛋白检测结果显示:铝暴露可导致大鼠海马组织中Aβ蛋白表达显著增加,与对照组相比,染铝组的大鼠海马组织中Aβ蛋白差异有统计学意义(P0.05);而叶酸干预中、高剂量组全基因组甲基化率显著增加,与染铝组比较差异具有统计学意义(P0.05),与对照组比较差异无统计学意义(P0.05)。铝染毒能够使大鼠的全基因组甲基化率降低,同时全基因组的甲基化降低导致大鼠脑组织海马组织中Aβ蛋白表达增加,同时叶酸干预参与了基因修饰过程中的甲基化过程。3.MSP结果显示:对照组、叶酸干预组APP启动子甲基化条带均有表达,非甲基化条带没有表达,染铝组APP启动子甲基化条带未表达,非甲基化条带有表达。对照组、叶酸干预高剂量组BACE1启动子甲基化条带均有表达,非甲基化条带没有表达,染铝组、叶酸干预低和中剂量组BACE1启动子甲基化条带未表达,非甲基化条带有表达,结果表明,生理状态下APP、BACE1启动子是甲基化状态,染铝使得甲基化状态转化为非甲基化状态,叶酸干预改变麦芽酚铝导致大鼠海马组织APP、BACE1启动子的非甲基化,使之变成甲基化。对照组、叶酸干预剂量组PS1启动子甲基化条带未表达,非甲基化条带有表达,结果表明,生理状态下PS1启动子是非甲基化状态,染铝没有改变基因的非甲基化状态,叶酸干预没有改变PS1启动子的非甲基化状态。RT-PCR和ELISA法测定蛋白结果显示:铝暴露可导致大鼠APP、PS1、BACE1的m RNA和蛋白表达增加,叶酸干预改变了铝导致m RNA和蛋白表达增加。结论:1.亚慢性麦芽酚铝染毒损伤大鼠的认知功能,认知功能的改变与脑铝含量有关,叶酸可以改善染毒组的认知功能,改变了铝在脑组织中蓄积。2.叶酸干预能改变染铝引起的大鼠全基因组甲基化降低,主要表现为Aβ蛋白表达降低,叶酸干预大鼠全基因组甲基化的改变与大鼠学习记忆能力的改变相一致。提示叶酸干预铝毒性通过改变大鼠全基因组甲基化来降低Aβ蛋白表达。3.铝可以降低APP、BACE1的甲基化水平,增加APP、PS1和BACE1的m RNA表达,同时升高三个基因调控的蛋白表达水平,增加叶酸干预可以改善铝引起m RNA和蛋白质表达的变化。4.叶酸通过引起全基因组甲基化率升高和APP、BACE1的甲基化升高,导致APP的淀粉源性代谢途径减少,引起转录翻译水平的改变,从而降低海马组织中Aβ的蓄积,改善铝的神经毒性作用,最终改善大鼠的学习记忆能力和认知功能。
[Abstract]:Objective: To study the effects of folic acid on the methylation of associated gene (APP, PS1, BACE1) of A-type deposit in aluminum-stained rats. Methods:40 healthy and clean male SD rats were randomly divided into 5 groups, control group (normal saline), aluminum-infected group (1.08 mg/ kg) and folic acid in low, medium and high dose group (0.7, 1.5 and 3.4 mg/ kg). In that meantime,1 ml/100 g of the folic acid intervention group was given to the rats for 60 consecutive days.1. Morris water maze was used to measure the learning and memory ability of the rats, and the content of aluminum in the rat was determined by the graphite furnace atomic absorption method. The whole-genome methylation of rats and the expression of hippocampal A-antigen in rats were determined by enzyme-linked immunosorbent assay (ELISA). Methylation of APP, PS1 and BACE1 in the hippocampus of rats was determined by methylation-specific polymerase chain reaction, and the expression of m-RNA and the expression of protein were determined by real-time fluorescence quantitative polymerase chain reaction. Results:1. The results of the Morris water maze show that the exposure of aluminum can lead to a significant reduction in the residence time of the target quadrant of the rat and the number of the position of the original platform, and the difference between the group and the control group is statistically significant (P0.05). Folic acid intervention group can lead to a significant increase in the residence time of the target quadrant of the rat and the number of the position of the original platform, and the difference of the high-dose group in the folic acid intervention compared with that of the aluminum-infected group is statistically significant (P0.05). The results of the determination of aluminum content showed that the aluminum exposure could lead to an increase in the content of aluminum in the rat, and the content of aluminum in the rats exposed to the aluminum group was significantly different from that of the control group (P0.05). The difference of high-dose group was statistically significant (P0.05). The results showed that the ability of folic acid to improve the learning and memory of rats decreased the accumulation of aluminum in the brain of rats. The results of the whole genome methylation of the rats showed that the exposure of aluminum can lead to a significant decrease in the whole genome methylation rate of the rat, and the difference of the whole genome methylation of the rats with the control group is statistically significant (P0.05); and the whole genome methylation rate of the high-dose group of the folic acid intervention group is obviously increased, The difference between the two groups was statistically significant (P0.05), and the difference between the control group and the control group was not statistically significant (P0.05). The results of the detection of A-type protein in the hippocampus of the rats showed that the expression of the A-protein in the hippocampus of the rats increased significantly, compared with that in the control group (P0.05); and in the folic acid intervention, The methylation rate of the whole genome in the high-dose group was significantly increased, and the difference between the group and the treated group was statistically significant (P0.05), and the difference between the control group and the control group was not significant (P0.05). Aluminum exposure can reduce the whole genome methylation rate of the rat, while the methylation of the whole genome leads to an increase in the expression of the A-protein in the hippocampus of the rat brain tissue, while the folic acid intervention is involved in the methylation process in the gene modification process. The methylation bands of APP promoter in the folic acid intervention group were both expressed, the non-methylated bands were not expressed, and the methylation bands of APP promoter in the staining group were not expressed and the non-methylated strips were expressed. in that control group, the methylation band of the BACE1 promoter in the high-dose group of the folic acid intervention group is express, the non-methylated band is not express, and the methylated band of the BACE1 promoter in the low-and medium-dose group is not expressed, and the non-methylated strip has the expression, and the result shows that the APP in the physiological state The BACE1 promoter is a methylated state, and the stained aluminum causes the methylation state to be converted into a non-methylated state, and the folic acid intervention changes the non-methylation of the APP and BACE1 promoter in the hippocampus of the rat, so that the methylation state is changed into a methylation state. In the control group, the methylation of PS1 promoter in the folic acid intervention dose group was not expressed, and the non-methylated strip was expressed. The results showed that the PS1 promoter in the physiological state was non-methylated and that the staining of aluminum did not change the non-methylated state of the gene. Folic acid intervention did not change the non-methylated state of the PS1 promoter. The results showed that the expression of mRNA and protein of APP, PS1 and BACE1 in rats increased by RT-PCR and ELISA. Conclusion:1. The cognitive function and cognitive function of subchronic maltol were related to the content of the brain and aluminum, and the folic acid could improve the cognitive function of the exposed group and change the accumulation of aluminum in the brain tissue. Folic acid intervention can change the whole-genome methylation of rats, which is mainly manifested as the decrease of the expression of A-protein, and the change of the whole-genome methylation of the folic acid in the rats is consistent with the change of the learning and memory ability of the rats. It is suggested that the effect of folic acid on aluminum toxicity can reduce the expression of A-protein by changing the whole genome methylation of the rat. Aluminum can reduce the methylation level of APP and BACE1, increase the expression of m-RNA of APP, PS1 and BACE1, and increase the level of protein expression regulated by three genes. The increase of the methylation rate of the whole genome and the increase of the methylation of the APP and BACE1 resulted in a decrease in the starch-derived metabolic pathway of the APP, resulting in a change in the level of transcription translation, thereby reducing the accumulation of the A-gene in the hippocampus tissue and improving the neurotoxic effect of aluminum, And finally, the learning and memory ability and the cognitive function of the rat are improved.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114
本文编号：2431540
[Abstract]:Objective: To study the effects of folic acid on the methylation of associated gene (APP, PS1, BACE1) of A-type deposit in aluminum-stained rats. Methods:40 healthy and clean male SD rats were randomly divided into 5 groups, control group (normal saline), aluminum-infected group (1.08 mg/ kg) and folic acid in low, medium and high dose group (0.7, 1.5 and 3.4 mg/ kg). In that meantime,1 ml/100 g of the folic acid intervention group was given to the rats for 60 consecutive days.1. Morris water maze was used to measure the learning and memory ability of the rats, and the content of aluminum in the rat was determined by the graphite furnace atomic absorption method. The whole-genome methylation of rats and the expression of hippocampal A-antigen in rats were determined by enzyme-linked immunosorbent assay (ELISA). Methylation of APP, PS1 and BACE1 in the hippocampus of rats was determined by methylation-specific polymerase chain reaction, and the expression of m-RNA and the expression of protein were determined by real-time fluorescence quantitative polymerase chain reaction. Results:1. The results of the Morris water maze show that the exposure of aluminum can lead to a significant reduction in the residence time of the target quadrant of the rat and the number of the position of the original platform, and the difference between the group and the control group is statistically significant (P0.05). Folic acid intervention group can lead to a significant increase in the residence time of the target quadrant of the rat and the number of the position of the original platform, and the difference of the high-dose group in the folic acid intervention compared with that of the aluminum-infected group is statistically significant (P0.05). The results of the determination of aluminum content showed that the aluminum exposure could lead to an increase in the content of aluminum in the rat, and the content of aluminum in the rats exposed to the aluminum group was significantly different from that of the control group (P0.05). The difference of high-dose group was statistically significant (P0.05). The results showed that the ability of folic acid to improve the learning and memory of rats decreased the accumulation of aluminum in the brain of rats. The results of the whole genome methylation of the rats showed that the exposure of aluminum can lead to a significant decrease in the whole genome methylation rate of the rat, and the difference of the whole genome methylation of the rats with the control group is statistically significant (P0.05); and the whole genome methylation rate of the high-dose group of the folic acid intervention group is obviously increased, The difference between the two groups was statistically significant (P0.05), and the difference between the control group and the control group was not statistically significant (P0.05). The results of the detection of A-type protein in the hippocampus of the rats showed that the expression of the A-protein in the hippocampus of the rats increased significantly, compared with that in the control group (P0.05); and in the folic acid intervention, The methylation rate of the whole genome in the high-dose group was significantly increased, and the difference between the group and the treated group was statistically significant (P0.05), and the difference between the control group and the control group was not significant (P0.05). Aluminum exposure can reduce the whole genome methylation rate of the rat, while the methylation of the whole genome leads to an increase in the expression of the A-protein in the hippocampus of the rat brain tissue, while the folic acid intervention is involved in the methylation process in the gene modification process. The methylation bands of APP promoter in the folic acid intervention group were both expressed, the non-methylated bands were not expressed, and the methylation bands of APP promoter in the staining group were not expressed and the non-methylated strips were expressed. in that control group, the methylation band of the BACE1 promoter in the high-dose group of the folic acid intervention group is express, the non-methylated band is not express, and the methylated band of the BACE1 promoter in the low-and medium-dose group is not expressed, and the non-methylated strip has the expression, and the result shows that the APP in the physiological state The BACE1 promoter is a methylated state, and the stained aluminum causes the methylation state to be converted into a non-methylated state, and the folic acid intervention changes the non-methylation of the APP and BACE1 promoter in the hippocampus of the rat, so that the methylation state is changed into a methylation state. In the control group, the methylation of PS1 promoter in the folic acid intervention dose group was not expressed, and the non-methylated strip was expressed. The results showed that the PS1 promoter in the physiological state was non-methylated and that the staining of aluminum did not change the non-methylated state of the gene. Folic acid intervention did not change the non-methylated state of the PS1 promoter. The results showed that the expression of mRNA and protein of APP, PS1 and BACE1 in rats increased by RT-PCR and ELISA. Conclusion:1. The cognitive function and cognitive function of subchronic maltol were related to the content of the brain and aluminum, and the folic acid could improve the cognitive function of the exposed group and change the accumulation of aluminum in the brain tissue. Folic acid intervention can change the whole-genome methylation of rats, which is mainly manifested as the decrease of the expression of A-protein, and the change of the whole-genome methylation of the folic acid in the rats is consistent with the change of the learning and memory ability of the rats. It is suggested that the effect of folic acid on aluminum toxicity can reduce the expression of A-protein by changing the whole genome methylation of the rat. Aluminum can reduce the methylation level of APP and BACE1, increase the expression of m-RNA of APP, PS1 and BACE1, and increase the level of protein expression regulated by three genes. The increase of the methylation rate of the whole genome and the increase of the methylation of the APP and BACE1 resulted in a decrease in the starch-derived metabolic pathway of the APP, resulting in a change in the level of transcription translation, thereby reducing the accumulation of the A-gene in the hippocampus tissue and improving the neurotoxic effect of aluminum, And finally, the learning and memory ability and the cognitive function of the rat are improved.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114
【引证文献】
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