【摘要】：目的研究母愛剝奪(maternal deprivation,MD)對缺血缺氧性腦損傷(Hypoxic-ischemic brain damage,HIBD)新生大鼠海馬神經(jīng)元凋亡水平的影響,并初步探討MD對HIBD大鼠神經(jīng)元凋亡調(diào)控的分子機制。材料與方法采用Rice法制備HIBD 7日齡SD大鼠模型,并同時設立假手術組。根據(jù)隨機數(shù)字表法將HIBD大鼠隨機分為對照組和MD組。對照組大鼠后期飼養(yǎng)按照標準飼養(yǎng)環(huán)境,MD組大鼠于HIBD造模后第2天開始,進行每日3小時的母子分離處理,直至出生后第21日齡為止。每組大鼠中,分別在14日齡、21日齡及28日齡中隨機選取相等數(shù)量大鼠進行處死,收取相關腦組織標本。采用原位細胞凋亡檢測法(Td T-mediated d UTP nick end labeling,TUNEL)檢測各組大鼠海馬神經(jīng)元凋亡水平,采用免疫組化觀察各組大鼠海馬部位凋亡相關基因p53、Bcl-2及NF-κB表達情況。采用熒光實時定量聚合酶鏈式反應(RT-PCR)檢測各組大鼠海馬部位凋亡相關基因p53、Bcl-2及NF-κB的m RNA表達水平,采用Western blot檢測各組大鼠海馬部位中凋亡相關基因p53、Bcl-2、Bax及NF-κB的蛋白表達水平。結(jié)果1.TUNEL染色結(jié)果顯示14日齡、21日齡及28日齡大鼠中,對照組與MD組海馬神經(jīng)元凋亡水平都高于假手術組,差異有統(tǒng)計學意義(P均0.05);21日齡及28日齡大鼠中,MD組大鼠海馬神經(jīng)元凋亡水平高于對照組,差異有統(tǒng)計學意義(P均0.05)。2.免疫組化結(jié)果顯示:1)14日齡大鼠中:與假手術組相比,對照組與MD組海馬部位p53、Bcl-2及NF-κB表達水平都升高,差異都有統(tǒng)計學意義(P均0.05);與對照組相比,MD組海馬部位NF-κB表達水平升高,差異有統(tǒng)計學意義(P0.05),p53與Bcl-2表達水平無明顯差異。2)21日齡大鼠中:與假手術組相比,對照組與MD組海馬部位p53、Bcl-2及NF-κB表達水平都升高,差異都有統(tǒng)計學意義(P均0.05);與對照組相比,MD組海馬部位NF-κB及p53表達水平升高,差異都有統(tǒng)計學意義(P均0.05),Bcl-2表達水平無明顯差異。3)28日齡大鼠中:與假手術組相比,對照組NF-κB表達水平升高,差異有統(tǒng)計學意義(P0.05),p53和Bcl-2表達水平無明顯差異;與假手術組或?qū)φ战M相比,MD組海馬區(qū)p53、NF-κB表達水平都升高,差異都有統(tǒng)計學意義(P均0.05),Bcl-2表達水平都顯著降低,差異都有統(tǒng)計學意義(P0.05)。3.RT-PCR結(jié)果顯示:1)14日齡大鼠中:與假手術組相比,對照組與MD組海馬部位p53、Bcl-2及NF-κB m RNA表達水平都升高,差異都有統(tǒng)計學意義(P均0.05);與對照組相比,MD組海馬部位p53、NF-κB m RNA表達水平升高,差異有統(tǒng)計學意義(P0.05),而Bcl-2 m RNA表達水平無明顯變化。2)21日齡大鼠中:與假手術組相比,對照組與MD組海馬部位p53、NF-κB m RNA表達水平升高,差異都有統(tǒng)計學意義(P均0.05),Bcl-2 m RNA表達水平無明顯變化;與對照組相比,MD組海馬部位p53、NF-κBm RNA表達水平升高,差異都有統(tǒng)計學意義(P均0.05),Bcl-2m RNA表達水平無明顯差異。3)28日齡大鼠中:與假手術組相比,對照組與MD組大鼠海馬部位p53、NF-κBm RNA表達水平升高,而Bcl-2 m RNA表達水平都降低,差異都有統(tǒng)計學意義(P均0.05);與對照組相比,MD組大鼠海馬部位p53、NF-κB m RNA表達水平升高,Bcl-2m RNA表達水平降低,差異都有統(tǒng)計學意義(P均0.05)。4.Western blot結(jié)果顯示:1)14日齡大鼠中:與假手術組相比,對照組與MD組海馬部位p53、Bcl-2、Bax及NF-κB蛋白表達水平都升高,差異有統(tǒng)計學意義(P均0.05);與對照組相比,MD組海馬部位p53與Bax蛋白表達水平升高,差異有統(tǒng)計學意義(P0.05),而Bcl-2與NF-κB蛋白表達水平無明顯變化。2)21日齡大鼠中:與假手術組相比,對照組與MD組海馬部位p53、Bax及NF-κB蛋白表達水平升高,差異有統(tǒng)計學意義(P均0.05),對照組Bcl-2表達水平升高,差異有統(tǒng)計學意義(P0.05),而MD組Bcl-2表達水平無明顯變化;與對照組相比,MD組海馬部位p53、Bax及NF-κB蛋白表達水平升高,差異都有統(tǒng)計學意義(P0.05),而Bcl-2蛋白表達水平降低,差異有統(tǒng)計學意義(P0.05)。3)28日齡大鼠中:與假手術組相比,對照組大鼠海馬部位p53和NF-κB蛋白表達水平升高,差異有統(tǒng)計學意義(P0.05),Bcl-2及Bax蛋白表達水平無明顯變化;與假手術組或?qū)φ障啾?MD組大鼠海馬部位p53、Bax及NF-κB蛋白表達水平都升高,差異有統(tǒng)計學意義(P均0.05),同時Bcl-2蛋白表達水平都降低,差異有統(tǒng)計學意義(P0.05)。4)在21日齡及28日齡大鼠中,MD組大鼠海馬部位Bax與Bcl-2蛋白表達水平的比值都顯著高于假手術組和對照組,差異都有統(tǒng)計學意義(P0.05)。結(jié)論MD干預致使HIBD大鼠海馬神經(jīng)元凋亡水平升高。細胞凋亡相關調(diào)控因子p53、Bcl-2、Bax及NF-κB參與MD對HIBD大鼠神經(jīng)元凋亡的調(diào)控過程。
[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.
【學位授予單位】：成都醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R742

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