母爱剥夺对缺血缺氧性脑损伤大鼠海马神经元凋亡的影响

发布时间：2018-09-19 15:08

【摘要】：目的研究母爱剥夺(maternal deprivation,MD)对缺血缺氧性脑损伤(Hypoxic-ischemic brain damage,HIBD)新生大鼠海马神经元凋亡水平的影响,并初步探讨MD对HIBD大鼠神经元凋亡调控的分子机制。材料与方法采用Rice法制备HIBD 7日龄SD大鼠模型,并同时设立假手术组。根据随机数字表法将HIBD大鼠随机分为对照组和MD组。对照组大鼠后期饲养按照标准饲养环境,MD组大鼠于HIBD造模后第2天开始,进行每日3小时的母子分离处理,直至出生后第21日龄为止。每组大鼠中,分别在14日龄、21日龄及28日龄中随机选取相等数量大鼠进行处死,收取相关脑组织标本。采用原位细胞凋亡检测法(Td T-mediated d UTP nick end labeling,TUNEL)检测各组大鼠海马神经元凋亡水平,采用免疫组化观察各组大鼠海马部位凋亡相关基因p53、Bcl-2及NF-κB表达情况。采用荧光实时定量聚合酶链式反应(RT-PCR)检测各组大鼠海马部位凋亡相关基因p53、Bcl-2及NF-κB的m RNA表达水平,采用Western blot检测各组大鼠海马部位中凋亡相关基因p53、Bcl-2、Bax及NF-κB的蛋白表达水平。结果1.TUNEL染色结果显示14日龄、21日龄及28日龄大鼠中,对照组与MD组海马神经元凋亡水平都高于假手术组,差异有统计学意义(P均0.05);21日龄及28日龄大鼠中,MD组大鼠海马神经元凋亡水平高于对照组,差异有统计学意义(P均0.05)。2.免疫组化结果显示:1)14日龄大鼠中:与假手术组相比,对照组与MD组海马部位p53、Bcl-2及NF-κB表达水平都升高,差异都有统计学意义(P均0.05);与对照组相比,MD组海马部位NF-κB表达水平升高,差异有统计学意义(P0.05),p53与Bcl-2表达水平无明显差异。2)21日龄大鼠中:与假手术组相比,对照组与MD组海马部位p53、Bcl-2及NF-κB表达水平都升高,差异都有统计学意义(P均0.05);与对照组相比,MD组海马部位NF-κB及p53表达水平升高,差异都有统计学意义(P均0.05),Bcl-2表达水平无明显差异。3)28日龄大鼠中:与假手术组相比,对照组NF-κB表达水平升高,差异有统计学意义(P0.05),p53和Bcl-2表达水平无明显差异;与假手术组或对照组相比,MD组海马区p53、NF-κB表达水平都升高,差异都有统计学意义(P均0.05),Bcl-2表达水平都显著降低,差异都有统计学意义(P0.05)。3.RT-PCR结果显示:1)14日龄大鼠中:与假手术组相比,对照组与MD组海马部位p53、Bcl-2及NF-κB m RNA表达水平都升高,差异都有统计学意义(P均0.05);与对照组相比,MD组海马部位p53、NF-κB m RNA表达水平升高,差异有统计学意义(P0.05),而Bcl-2 m RNA表达水平无明显变化。2)21日龄大鼠中:与假手术组相比,对照组与MD组海马部位p53、NF-κB m RNA表达水平升高,差异都有统计学意义(P均0.05),Bcl-2 m RNA表达水平无明显变化;与对照组相比,MD组海马部位p53、NF-κBm RNA表达水平升高,差异都有统计学意义(P均0.05),Bcl-2m RNA表达水平无明显差异。3)28日龄大鼠中:与假手术组相比,对照组与MD组大鼠海马部位p53、NF-κBm RNA表达水平升高,而Bcl-2 m RNA表达水平都降低,差异都有统计学意义(P均0.05);与对照组相比,MD组大鼠海马部位p53、NF-κB m RNA表达水平升高,Bcl-2m RNA表达水平降低,差异都有统计学意义(P均0.05)。4.Western blot结果显示:1)14日龄大鼠中:与假手术组相比,对照组与MD组海马部位p53、Bcl-2、Bax及NF-κB蛋白表达水平都升高,差异有统计学意义(P均0.05);与对照组相比,MD组海马部位p53与Bax蛋白表达水平升高,差异有统计学意义(P0.05),而Bcl-2与NF-κB蛋白表达水平无明显变化。2)21日龄大鼠中:与假手术组相比,对照组与MD组海马部位p53、Bax及NF-κB蛋白表达水平升高,差异有统计学意义(P均0.05),对照组Bcl-2表达水平升高,差异有统计学意义(P0.05),而MD组Bcl-2表达水平无明显变化;与对照组相比,MD组海马部位p53、Bax及NF-κB蛋白表达水平升高,差异都有统计学意义(P0.05),而Bcl-2蛋白表达水平降低,差异有统计学意义(P0.05)。3)28日龄大鼠中:与假手术组相比,对照组大鼠海马部位p53和NF-κB蛋白表达水平升高,差异有统计学意义(P0.05),Bcl-2及Bax蛋白表达水平无明显变化;与假手术组或对照相比,MD组大鼠海马部位p53、Bax及NF-κB蛋白表达水平都升高,差异有统计学意义(P均0.05),同时Bcl-2蛋白表达水平都降低,差异有统计学意义(P0.05)。4)在21日龄及28日龄大鼠中,MD组大鼠海马部位Bax与Bcl-2蛋白表达水平的比值都显著高于假手术组和对照组,差异都有统计学意义(P0.05)。结论MD干预致使HIBD大鼠海马神经元凋亡水平升高。细胞凋亡相关调控因子p53、Bcl-2、Bax及NF-κB参与MD对HIBD大鼠神经元凋亡的调控过程。
[Abstract]:Objective To study the effect of maternal deprivation (MD) on the apoptosis of hippocampal neurons in neonatal rats with hypoxic-ischemic brain damage (HIBD), and to explore the molecular mechanism of MD on the regulation of neuronal apoptosis in HIBD rats. The rats in the control group were fed according to the standard feeding environment. The rats in the MD group were separated from their mothers and children for 3 hours daily from the 2nd day after HIBD modeling until the 21st day after birth. At the age of 8 days, the same number of rats were randomly selected and sacrificed to collect the related brain tissue samples. The apoptosis level of hippocampal neurons in each group was detected by T D T-mediated D UTP nick end labeling (TUNEL), and the expression of apoptosis-related genes p53, Bcl-2 and NF-kappa B in the hippocampus was observed by immunohistochemistry. The expression levels of apoptosis-related genes p53, Bcl-2 and NF-kappa B in the hippocampus of rats in each group were detected by fluorescence real-time quantitative polymerase chain reaction (RT-PCR). The expression levels of apoptosis-related genes p53, Bcl-2, Bax and NF-kappa B in the hippocampus of rats in each group were detected by Western blot. Results 1. TUNEL staining showed that the expression levels of apoptosis-related genes p53, Bcl-2, Bax and NF-kappa B The apoptosis level of hippocampal neurons in the control group and the MD group was higher than that in the sham operation group (P 0.05). The apoptosis level of hippocampal neurons in the MD group was higher than that in the control group at 21 and 28 days of age (P 0.05). 2. Immunohistochemical results showed that: 1) the apoptosis level of hippocampal neurons in the 14-day-old rats was higher than that in the sham operation group (P 0.05). Compared with the operation group, the expression levels of p53, Bcl-2 and NF-kappa B in the hippocampus of the control group and the MD group were significantly higher (P 0.05); Compared with the control group, the expression level of NF-kappa B in the hippocampus of the MD group was significantly higher (P 0.05), and the expression levels of p53 and Bcl-2 were not significantly different. The expression levels of p53, Bcl-2 and NF-kappa B in the hippocampus of the MD group and the control group were significantly higher than those of the MD group (P 0.05). The expression levels of NF-kappa B and p53 in the hippocampus of the MD group were significantly higher than those of the control group (P 0.05). There was no significant difference in the expression level of Bcl-2 between the control group and the sham operation group. The expression level of kappa B increased, the difference was statistically significant (P 0.05), the expression levels of p53 and Bcl-2 were not significantly different; compared with sham operation group or control group, the expression levels of p53 and NF-kappa B in the hippocampus of MD group were increased, the difference was statistically significant (P 0.05), the expression level of Bcl-2 was significantly decreased, the difference was statistically significant (P 0.05). 1) In 14-day-old rats, the expression levels of p53, Bcl-2 and NF-kappa B m RNA in the hippocampus of control group and MD group were significantly higher than those of sham-operation group (P 0.05); compared with the control group, the expression levels of p53 and NF-kappa B m RNA in the hippocampus of MD group were significantly higher than those of sham-operation group (P 0.05), but the expression level of Bcl-2 m RNA was not significantly changed (P 0.05). Hua.2) 21-day-old rats: Compared with sham operation group, the expression levels of p53 and NF-kappa B m RNA in the hippocampus of control group and MD group were significantly higher than those of sham operation group (P 0.05). The expression levels of Bcl-2 m RNA in the hippocampus of MD group had no significant change (P 0.05). Compared with the control group, the expression levels of p53 and NF-kappa B m RNA in the hippocampus of MD group were significantly higher (P 0.05). There was no significant difference in the expression of 2m RNA. 3) In 28-day-old rats, compared with sham-operated rats, the expression of p53 and NF-kappa B m RNA in the hippocampus of control group and MD group increased, while the expression of Bcl-2m RNA decreased (all P 0.05); compared with the control group, the expression of p53, NF-kappa B m RNA in the hippocampus of MD group increased, and Bcl-kappa B m RNA in the hippocampus increased. Western blot results showed that: 1) Compared with sham-operated group, the expression levels of p53, Bcl-2, Bax and NF-kappa B protein in hippocampus of control group and MD group were higher than those of sham-operated group, and the difference was statistically significant (P 0.05); compared with control group, the expression levels of p53 and Bax protein in hippocampus of MD group were significantly higher (P 0.05). The expression levels of p53, Bax and NF-kappa B in the hippocampus of the control group and the MD group were higher than those of the sham operation group (P 0.05). The expression levels of Bcl-2 and NF-kappa B in the control group were higher than those in the sham operation group (P 0.05). The expression of Bcl-2 in the hippocampus of MD group was significantly higher than that of the control group (P 0.05), while the expression of Bcl-2 protein was significantly lower (P 0.05). The expression levels of p53, Bax and NF-kappa B protein in hippocampus of MD rats were higher than those of sham operation group and control group, and the difference was statistically significant (P 0.05). The expression levels of Bcl-2 protein were lower than those of sham operation group and control group. The expression of Bax and Bcl-2 protein in hippocampus of MD group was significantly higher than that of sham-operated group and control group (P 0.05). Conclusion MD intervention can increase the level of apoptosis of hippocampal neurons in HIBD rats. Bax and NF- kappa B are involved in the regulation of MD on neuronal apoptosis in HIBD rats.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742

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