母愛(ài)剝奪與慢性溫和應(yīng)激對(duì)大鼠抑郁樣行為的影響及紋狀體par-4基因表達(dá)的甲基化調(diào)控機(jī)制研究
發(fā)布時(shí)間：2018-08-18 14:25
【摘要】： 目的：研究母愛(ài)剝奪和慢性溫和應(yīng)激對(duì)大鼠抑郁樣行為、紋狀體前列腺凋亡反應(yīng)蛋白-4(prostate apoptosis response-4, par-4) mRNA及蛋白質(zhì)表達(dá)的影響,探索基因甲基化是否參與par-4基因表達(dá)的調(diào)控。 方法：SD大鼠按窩別隨機(jī)分為母愛(ài)剝奪組(n=17)、慢性應(yīng)激組(n=17,)、雙重應(yīng)激組(n=10)和對(duì)照組(n=17)。母愛(ài)剝奪組大鼠接受母愛(ài)剝奪應(yīng)激,慢性應(yīng)激大鼠組接受慢性溫和應(yīng)激,雙重應(yīng)激大鼠組接受母愛(ài)剝奪應(yīng)激和慢性溫和應(yīng)激,對(duì)照組大鼠不接受任何實(shí)驗(yàn)性處理。在大鼠13周齡時(shí)采用糖水偏愛(ài)實(shí)驗(yàn)、強(qiáng)迫游泳實(shí)驗(yàn)和曠場(chǎng)實(shí)驗(yàn)評(píng)定其焦慮、抑郁水平；采用實(shí)時(shí)熒光定量PCR (Real-timePCR)檢測(cè)紋狀體par-4及多巴胺D2受體(dopamine receptor D2, DRD2) mRNA表達(dá)水平；采用免疫印跡法(Western Blot)檢測(cè)紋狀體par-4蛋白質(zhì)表達(dá)水平；采用亞硫酸鹽法處理基因組DNA，，連接T載體后克隆測(cè)序檢測(cè)par-4基因啟動(dòng)子區(qū)DNA甲基化水平。 結(jié)果： 1.行為學(xué)實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)：與對(duì)照組相比,母愛(ài)剝奪組大鼠糖水偏愛(ài)率降低,漂浮時(shí)間延長(zhǎng),爬行總路程及直立次數(shù)減少(P0.05)；中央格比例及大便顆數(shù)與對(duì)照組相比無(wú)顯著差異(P0.05)。慢性應(yīng)激組與對(duì)照組相比糖水偏愛(ài)率降低,漂浮時(shí)間延長(zhǎng),直立次數(shù)減少(P0.05)；中央格比例、大便顆數(shù)及爬行總路程與對(duì)照組相比無(wú)顯著差異(P0.05)。雙重應(yīng)激組與對(duì)照組相比,漂浮時(shí)間延長(zhǎng),直立次數(shù)減少(P0.05)；糖水偏愛(ài)率、爬行總路程、中央格比例、大便顆數(shù)與對(duì)照組相比無(wú)顯著差異(P0.05)。 2.基因表達(dá)檢測(cè)結(jié)果發(fā)現(xiàn)：母愛(ài)剝奪組、慢性應(yīng)激組、雙重應(yīng)激組大鼠的紋狀體par-4及DRD2 mRNA表達(dá)水平相對(duì)于對(duì)照組均顯著降低(P0.05)。母愛(ài)剝奪組、慢性應(yīng)激組和雙重應(yīng)激組大鼠相對(duì)于對(duì)照組大鼠的par-4 mRNA表達(dá)量分別為0.53、0.33和0.61,相對(duì)于對(duì)照組的DRD2 mRNA表達(dá)量分別為0.31、0.33和0.54。且par-4mRNA表達(dá)水平與糖水偏愛(ài)率、漂浮時(shí)間、總路程、大便顆數(shù)、直立次數(shù)相關(guān)顯著(r值分別為0.408,0.537,0.504,0.478,0.464,P0.05)；與中央格比例相關(guān)不顯著(r=0.155,P0.05)。而DRD2mRNA表達(dá)水平與與漂浮時(shí)間、總路程、直立次數(shù)相關(guān)顯著(r值分別為0.390,0.389,0.400,P0.05)；與糖水比例、中央格比例及大便顆數(shù)相關(guān)不顯著(r值分別為0.133,0.070,0.094,P0.05)。四組大鼠紋狀體par-4蛋白質(zhì)表達(dá)水平差異顯著(F=14.597,P=0.000)，母愛(ài)剝奪組、慢性應(yīng)激組、雙重應(yīng)激組大鼠紋狀體par-4蛋白表達(dá)量相對(duì)于對(duì)照組大鼠顯著降低(P0.05),慢性應(yīng)激組和雙重應(yīng)激組par-4蛋白表達(dá)量低于母愛(ài)剝奪組(P0.05)。 3.克隆測(cè)序結(jié)果：四組大鼠的par-4基因啟動(dòng)子區(qū)總體甲基化水平組間差異不明顯(F=1.079,P=0.359)；但以CG位點(diǎn)來(lái)統(tǒng)計(jì)時(shí)發(fā)現(xiàn),有四個(gè)位點(diǎn)的甲基化水平組間差異顯著(F值分別為3.103,3.605,6.862,5.196,P0.05)；慢性應(yīng)激組有3個(gè)CG位點(diǎn)的甲基化水平高于對(duì)照組(P0.05),并有1個(gè)CG位點(diǎn)的甲基化水平低于對(duì)照組(P0.05)。 結(jié)論： 1.母愛(ài)剝奪、慢性溫和應(yīng)激和雙重應(yīng)激能使大鼠產(chǎn)生抑郁樣行為。 2.母愛(ài)剝奪、慢性溫和應(yīng)激及雙重應(yīng)激均能使大鼠紋狀體中par-4及DRD2 mRNA表達(dá)水平降低,降低程度與行為學(xué)指標(biāo)相關(guān)。母愛(ài)剝奪、慢性溫和應(yīng)激及雙重應(yīng)激均能使大鼠紋狀體par-4蛋白質(zhì)表達(dá)水平降低。 3.基因甲基化可能不是par-4基因表達(dá)下降的主要調(diào)控機(jī)制。
[Abstract]:AIM: To study the effects of maternal deprivation and chronic mild stress on depression-like behavior, expression of mRNA and protein of prostate apoptosis response-4 (par-4) in striatum of rats, and to explore whether gene methylation is involved in the regulation of Par-4 gene expression.
Methods: SD rats were randomly divided into three groups: maternal deprivation group (n = 17), chronic stress group (n = 17), double stress group (n = 10) and control group (n = 17). At the age of 13 weeks, the levels of anxiety and depression were assessed by sugar preference test, forced swimming test and open field test, and the expression of Par-4 and dopamine D2 receptor (DRD2) mRNA in striatum were detected by real-time fluorescence quantitative PCR (Real-time PCR). The expression level of Par-4 protein in striatum was detected by Blot assay. Genomic DNA was treated by sulfite method. DNA methylation in promoter region of Par-4 gene was detected by cloning and sequencing after connecting T vector.
Result:
1. The results of behavioral experiment showed that compared with the control group, the rate of glucose preference decreased, the floating time prolonged, the total crawling distance and the number of erection decreased (P 0.05); there was no significant difference in the proportion of central grid and the number of stools between the two groups (P 0.05). Compared with the control group, the rate of glucose preference and the floating time decreased in the chronic stress group. Compared with the control group, the floating time was prolonged and the number of upright stools was decreased (P 0.05); the sugarwater preference rate, the total crawling distance, the proportion of central grid, the number of stools and the total crawling distance had no significant difference (P 0.05). .05).
2. The expression of Par-4 and DRD2 mRNA in the striatum of rats in maternal deprivation group, chronic stress group and double stress group was significantly lower than that in control group (P 0.05). The expression of Par-4 mRNA in maternal deprivation group, chronic stress group and double stress group was 0.53, 0.33 and 0.61 respectively. The expression levels of Par-4 mRNA were significantly correlated with the sugar preference rate, floating time, total distance, stool size and erection times (r values were 0.408, 0.537, 0.504, 0.478, 0.464, P 0.05), respectively, and were not significantly correlated with the central grid ratio (r = 0.155, P 0.05). Floating time, total distance and erection times were significantly correlated (r values were 0.390, 0.389, 0.400, P 0.05), but not significantly correlated with the ratio of sugar to water, the ratio of central grid and the number of stools (r values were 0.133, 0.070, 0.094, P 0.05), respectively. Compared with the control group, the expression of Par-4 protein in the striatum of the double stress group was significantly decreased (P 0.05). The expression of Par-4 protein in the chronic stress group and the double stress group was lower than that in the maternal deprivation group (P 0.05).
3. Cloning and sequencing results: There was no significant difference in the overall methylation level of Par-4 gene promoter region among the four groups (F = 1.079, P = 0.359); however, there were significant differences in the methylation level among the four groups (F values were 3.103, 3.605, 6.862, 5.196, P 0.05), and methylated water at three CG sites in chronic stress group. The level of methylation in the 1 CG loci was lower than that in the control group (P0.05), and the level of methylation in the control group was lower than that in the control group (P0.05).
Conclusion:
1. maternal deprivation, chronic mild stress and dual stress can induce depression like behavior in rats.
2. Deprivation of maternal love, chronic mild stress and dual stress can reduce the expression of Par-4 and DRD2 mRNA in rat striatum, and the degree of reduction is related to behavioral indicators. Deprivation of maternal love, chronic mild stress and dual stress can reduce the expression of Par-4 protein in rat striatum.
3. gene methylation may not be the main regulatory mechanism of Par-4 gene expression decline.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R395.1
本文編號(hào)：2189749
[Abstract]:AIM: To study the effects of maternal deprivation and chronic mild stress on depression-like behavior, expression of mRNA and protein of prostate apoptosis response-4 (par-4) in striatum of rats, and to explore whether gene methylation is involved in the regulation of Par-4 gene expression.
Methods: SD rats were randomly divided into three groups: maternal deprivation group (n = 17), chronic stress group (n = 17), double stress group (n = 10) and control group (n = 17). At the age of 13 weeks, the levels of anxiety and depression were assessed by sugar preference test, forced swimming test and open field test, and the expression of Par-4 and dopamine D2 receptor (DRD2) mRNA in striatum were detected by real-time fluorescence quantitative PCR (Real-time PCR). The expression level of Par-4 protein in striatum was detected by Blot assay. Genomic DNA was treated by sulfite method. DNA methylation in promoter region of Par-4 gene was detected by cloning and sequencing after connecting T vector.
Result:
1. The results of behavioral experiment showed that compared with the control group, the rate of glucose preference decreased, the floating time prolonged, the total crawling distance and the number of erection decreased (P 0.05); there was no significant difference in the proportion of central grid and the number of stools between the two groups (P 0.05). Compared with the control group, the rate of glucose preference and the floating time decreased in the chronic stress group. Compared with the control group, the floating time was prolonged and the number of upright stools was decreased (P 0.05); the sugarwater preference rate, the total crawling distance, the proportion of central grid, the number of stools and the total crawling distance had no significant difference (P 0.05). .05).
2. The expression of Par-4 and DRD2 mRNA in the striatum of rats in maternal deprivation group, chronic stress group and double stress group was significantly lower than that in control group (P 0.05). The expression of Par-4 mRNA in maternal deprivation group, chronic stress group and double stress group was 0.53, 0.33 and 0.61 respectively. The expression levels of Par-4 mRNA were significantly correlated with the sugar preference rate, floating time, total distance, stool size and erection times (r values were 0.408, 0.537, 0.504, 0.478, 0.464, P 0.05), respectively, and were not significantly correlated with the central grid ratio (r = 0.155, P 0.05). Floating time, total distance and erection times were significantly correlated (r values were 0.390, 0.389, 0.400, P 0.05), but not significantly correlated with the ratio of sugar to water, the ratio of central grid and the number of stools (r values were 0.133, 0.070, 0.094, P 0.05), respectively. Compared with the control group, the expression of Par-4 protein in the striatum of the double stress group was significantly decreased (P 0.05). The expression of Par-4 protein in the chronic stress group and the double stress group was lower than that in the maternal deprivation group (P 0.05).
3. Cloning and sequencing results: There was no significant difference in the overall methylation level of Par-4 gene promoter region among the four groups (F = 1.079, P = 0.359); however, there were significant differences in the methylation level among the four groups (F values were 3.103, 3.605, 6.862, 5.196, P 0.05), and methylated water at three CG sites in chronic stress group. The level of methylation in the 1 CG loci was lower than that in the control group (P0.05), and the level of methylation in the control group was lower than that in the control group (P0.05).
Conclusion:
1. maternal deprivation, chronic mild stress and dual stress can induce depression like behavior in rats.
2. Deprivation of maternal love, chronic mild stress and dual stress can reduce the expression of Par-4 and DRD2 mRNA in rat striatum, and the degree of reduction is related to behavioral indicators. Deprivation of maternal love, chronic mild stress and dual stress can reduce the expression of Par-4 protein in rat striatum.
3. gene methylation may not be the main regulatory mechanism of Par-4 gene expression decline.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R395.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 朱熊兆;彭素芳;馬秀玲;李婷;;母愛(ài)剝奪對(duì)成年大鼠情緒及多巴胺轉(zhuǎn)運(yùn)蛋白基因表達(dá)的影響[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年01期
2 王瑋文;邵楓;劉美;林文娟;;早期應(yīng)激對(duì)抑郁相關(guān)行為及神經(jīng)內(nèi)分泌反應(yīng)的長(zhǎng)期影響[J];心理科學(xué)進(jìn)展;2006年06期
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