葉酸干預(yù)對染鋁大鼠海馬Aβ相關(guān)基因甲基化影響的研究
發(fā)布時(shí)間：2019-02-27 20:09
【摘要】：目的:研究葉酸干預(yù)對染鋁大鼠Aβ沉積的相關(guān)基因(APP、PS1、BACE1)的甲基化影響。方法:健康清潔級雄性SD大鼠40只,按體重隨機(jī)分成5組,對照組(生理鹽水),染鋁組(1.08mg/kg),葉酸干預(yù)低、中、高劑量組(0.7、1.5和3.4mg/kg),每組8只,各組大鼠腹腔注射相應(yīng)劑量麥芽酚鋁,同時(shí)葉酸干預(yù)組每日灌胃1ml/100g,連續(xù)60天。1.Morris水迷宮測定大鼠學(xué)習(xí)記憶能力,石墨爐原子吸收法測定大鼠腦鋁含量;2.酶聯(lián)免疫吸附法測定大鼠全基因組甲基化和海馬Aβ的表達(dá);3.甲基化特異性聚合酶鏈反應(yīng)法測定大鼠海馬組織APP、PS1、BACE1的甲基化,實(shí)時(shí)熒光定量聚合酶鏈法測定m RNA的表達(dá),酶聯(lián)免疫吸附法測定蛋白的表達(dá)。結(jié)果:1.Morris水迷宮結(jié)果顯示:鋁暴露可導(dǎo)致大鼠目標(biāo)象限停留時(shí)間明顯縮短和穿越原平臺(tái)位置的次數(shù)明顯減少,染鋁組與對照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05);葉酸干預(yù)組可導(dǎo)致大鼠目標(biāo)象限停留時(shí)間明顯延長和穿越原平臺(tái)位置的次數(shù)明顯增多,與染鋁組相比葉酸干預(yù)中、高劑量組差異有統(tǒng)計(jì)學(xué)意義(P0.05)。鋁含量測定結(jié)果顯示:鋁暴露可導(dǎo)致大鼠腦鋁含量增多,染鋁組的大鼠的腦鋁含量與對照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05),葉酸干預(yù)降低了大鼠的腦鋁含量,與染鋁組相比葉酸干預(yù)中、高劑量組差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)果顯示:葉酸改善大鼠學(xué)習(xí)記憶能力減少了鋁在大鼠腦組織中的蓄積。2.大鼠全基因組甲基化測定結(jié)果顯示:鋁暴露可導(dǎo)致大鼠全基因組甲基化率顯著降低,與對照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05);而葉酸干預(yù)高劑量組全基因組甲基化率顯著增加,與染鋁組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05),與對照組比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。大鼠海馬組織中Aβ蛋白檢測結(jié)果顯示:鋁暴露可導(dǎo)致大鼠海馬組織中Aβ蛋白表達(dá)顯著增加,與對照組相比,染鋁組的大鼠海馬組織中Aβ蛋白差異有統(tǒng)計(jì)學(xué)意義(P0.05);而葉酸干預(yù)中、高劑量組全基因組甲基化率顯著增加,與染鋁組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05),與對照組比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。鋁染毒能夠使大鼠的全基因組甲基化率降低,同時(shí)全基因組的甲基化降低導(dǎo)致大鼠腦組織海馬組織中Aβ蛋白表達(dá)增加,同時(shí)葉酸干預(yù)參與了基因修飾過程中的甲基化過程。3.MSP結(jié)果顯示:對照組、葉酸干預(yù)組APP啟動(dòng)子甲基化條帶均有表達(dá),非甲基化條帶沒有表達(dá),染鋁組APP啟動(dòng)子甲基化條帶未表達(dá),非甲基化條帶有表達(dá)。對照組、葉酸干預(yù)高劑量組BACE1啟動(dòng)子甲基化條帶均有表達(dá),非甲基化條帶沒有表達(dá),染鋁組、葉酸干預(yù)低和中劑量組BACE1啟動(dòng)子甲基化條帶未表達(dá),非甲基化條帶有表達(dá),結(jié)果表明,生理狀態(tài)下APP、BACE1啟動(dòng)子是甲基化狀態(tài),染鋁使得甲基化狀態(tài)轉(zhuǎn)化為非甲基化狀態(tài),葉酸干預(yù)改變麥芽酚鋁導(dǎo)致大鼠海馬組織APP、BACE1啟動(dòng)子的非甲基化,使之變成甲基化。對照組、葉酸干預(yù)劑量組PS1啟動(dòng)子甲基化條帶未表達(dá),非甲基化條帶有表達(dá),結(jié)果表明,生理狀態(tài)下PS1啟動(dòng)子是非甲基化狀態(tài),染鋁沒有改變基因的非甲基化狀態(tài),葉酸干預(yù)沒有改變PS1啟動(dòng)子的非甲基化狀態(tài)。RT-PCR和ELISA法測定蛋白結(jié)果顯示:鋁暴露可導(dǎo)致大鼠APP、PS1、BACE1的m RNA和蛋白表達(dá)增加,葉酸干預(yù)改變了鋁導(dǎo)致m RNA和蛋白表達(dá)增加。結(jié)論:1.亞慢性麥芽酚鋁染毒損傷大鼠的認(rèn)知功能,認(rèn)知功能的改變與腦鋁含量有關(guān),葉酸可以改善染毒組的認(rèn)知功能,改變了鋁在腦組織中蓄積。2.葉酸干預(yù)能改變?nèi)句X引起的大鼠全基因組甲基化降低,主要表現(xiàn)為Aβ蛋白表達(dá)降低,葉酸干預(yù)大鼠全基因組甲基化的改變與大鼠學(xué)習(xí)記憶能力的改變相一致。提示葉酸干預(yù)鋁毒性通過改變大鼠全基因組甲基化來降低Aβ蛋白表達(dá)。3.鋁可以降低APP、BACE1的甲基化水平,增加APP、PS1和BACE1的m RNA表達(dá),同時(shí)升高三個(gè)基因調(diào)控的蛋白表達(dá)水平,增加葉酸干預(yù)可以改善鋁引起m RNA和蛋白質(zhì)表達(dá)的變化。4.葉酸通過引起全基因組甲基化率升高和APP、BACE1的甲基化升高,導(dǎo)致APP的淀粉源性代謝途徑減少,引起轉(zhuǎn)錄翻譯水平的改變,從而降低海馬組織中Aβ的蓄積,改善鋁的神經(jīng)毒性作用,最終改善大鼠的學(xué)習(xí)記憶能力和認(rèn)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R114
本文編號(hào)：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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R114
【引證文獻(xiàn)】
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