母爱剥夺与慢性温和应激对大鼠抑郁样行为的影响及纹状体par-4基因表达的甲基化调控机制研究
发布时间：2018-08-18 14:25
【摘要】： 目的：研究母爱剥夺和慢性温和应激对大鼠抑郁样行为、纹状体前列腺凋亡反应蛋白-4(prostate apoptosis response-4, par-4) mRNA及蛋白质表达的影响,探索基因甲基化是否参与par-4基因表达的调控。 方法：SD大鼠按窝别随机分为母爱剥夺组(n=17)、慢性应激组(n=17,)、双重应激组(n=10)和对照组(n=17)。母爱剥夺组大鼠接受母爱剥夺应激,慢性应激大鼠组接受慢性温和应激,双重应激大鼠组接受母爱剥夺应激和慢性温和应激,对照组大鼠不接受任何实验性处理。在大鼠13周龄时采用糖水偏爱实验、强迫游泳实验和旷场实验评定其焦虑、抑郁水平；采用实时荧光定量PCR (Real-timePCR)检测纹状体par-4及多巴胺D2受体(dopamine receptor D2, DRD2) mRNA表达水平；采用免疫印迹法(Western Blot)检测纹状体par-4蛋白质表达水平；采用亚硫酸盐法处理基因组DNA，，连接T载体后克隆测序检测par-4基因启动子区DNA甲基化水平。 结果： 1.行为学实验结果发现：与对照组相比,母爱剥夺组大鼠糖水偏爱率降低,漂浮时间延长,爬行总路程及直立次数减少(P0.05)；中央格比例及大便颗数与对照组相比无显著差异(P0.05)。慢性应激组与对照组相比糖水偏爱率降低,漂浮时间延长,直立次数减少(P0.05)；中央格比例、大便颗数及爬行总路程与对照组相比无显著差异(P0.05)。双重应激组与对照组相比,漂浮时间延长,直立次数减少(P0.05)；糖水偏爱率、爬行总路程、中央格比例、大便颗数与对照组相比无显著差异(P0.05)。 2.基因表达检测结果发现：母爱剥夺组、慢性应激组、双重应激组大鼠的纹状体par-4及DRD2 mRNA表达水平相对于对照组均显著降低(P0.05)。母爱剥夺组、慢性应激组和双重应激组大鼠相对于对照组大鼠的par-4 mRNA表达量分别为0.53、0.33和0.61,相对于对照组的DRD2 mRNA表达量分别为0.31、0.33和0.54。且par-4mRNA表达水平与糖水偏爱率、漂浮时间、总路程、大便颗数、直立次数相关显著(r值分别为0.408,0.537,0.504,0.478,0.464,P0.05)；与中央格比例相关不显著(r=0.155,P0.05)。而DRD2mRNA表达水平与与漂浮时间、总路程、直立次数相关显著(r值分别为0.390,0.389,0.400,P0.05)；与糖水比例、中央格比例及大便颗数相关不显著(r值分别为0.133,0.070,0.094,P0.05)。四组大鼠纹状体par-4蛋白质表达水平差异显著(F=14.597,P=0.000)，母爱剥夺组、慢性应激组、双重应激组大鼠纹状体par-4蛋白表达量相对于对照组大鼠显著降低(P0.05),慢性应激组和双重应激组par-4蛋白表达量低于母爱剥夺组(P0.05)。 3.克隆测序结果：四组大鼠的par-4基因启动子区总体甲基化水平组间差异不明显(F=1.079,P=0.359)；但以CG位点来统计时发现,有四个位点的甲基化水平组间差异显著(F值分别为3.103,3.605,6.862,5.196,P0.05)；慢性应激组有3个CG位点的甲基化水平高于对照组(P0.05),并有1个CG位点的甲基化水平低于对照组(P0.05)。 结论： 1.母爱剥夺、慢性温和应激和双重应激能使大鼠产生抑郁样行为。 2.母爱剥夺、慢性温和应激及双重应激均能使大鼠纹状体中par-4及DRD2 mRNA表达水平降低,降低程度与行为学指标相关。母爱剥夺、慢性温和应激及双重应激均能使大鼠纹状体par-4蛋白质表达水平降低。 3.基因甲基化可能不是par-4基因表达下降的主要调控机制。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R395.1
本文编号：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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R395.1
【参考文献】
相关期刊论文 前2条
1 朱熊兆;彭素芳;马秀玲;李婷;;母爱剥夺对成年大鼠情绪及多巴胺转运蛋白基因表达的影响[J];中南大学学报(医学版);2010年01期
2 王玮文;邵枫;刘美;林文娟;;早期应激对抑郁相关行为及神经内分泌反应的长期影响[J];心理科学进展;2006年06期
本文编号：2189749
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