Bmi-1基因与脑衰老的相关性研究
发布时间：2018-09-05 12:07
【摘要】：背景：既往研究提示Bmi-1(B-cell specific moloney leukemia virus insertion site1)属于多梳基因家族的转录抑制基因,可作为细胞周期、氧化应激、DNA损伤修复和线粒体功能的共同调节靶分子,进而在调控衰老进程中起关键作用,但Bmi-1基因与脑衰老的相关性有待于进一步明确。 目的：本学位论文旨在通过对三种不同的Bmi-1基因表达量的小鼠即Bmi-1基因全敲除、半剂量敲除和正常野生型小鼠的生理、生化及形态学等方面的表型进行分析,来探讨Bmi-1基因与脑衰老的相关性,从而为研究靶向Bmi-1延缓衰老进程提供科学依据。 方法： 1.第一部分应用野生型C57BL1月龄、4月龄、5月龄、9月龄和20月龄的自然衰老小鼠,免疫组织化学染色和免疫印迹检测Bmi-1和p53蛋白的表达变化。 2.第二部分采用2、4周龄的Bmi-1基因全敲除小鼠和野生型小鼠检测脑羟自由基和丙二醛的水平以及MBP和GFAP的表达。 3.第三部分采用8月龄的Bmi-1基因半剂量敲除小鼠和野生型小鼠,运用Morris水迷宫检测两组小鼠学习记忆功能,流式细胞技术和生化试剂盒检测活性氧簇以及氧化、抗氧化指标的水平,免疫组织化学染色和电镜检测两组小鼠病理组织学包括神经元及胶质细胞的改变,免疫印迹检测Bmi-1相关蛋白的表达变化。 结果： 1.Bmi-1在成年之前随着月龄的增长表达逐步上调,并在4月龄的皮质、齿状回、小脑及嗅球的表达达到高峰,之后随着衰老推进表达逐步下调；而p53蛋白随月龄增长而表达增高。 2.存在神经退行性变性的4周龄Bmi-1基因敲除小鼠脑羟自由基和丙二醛水平都明显高于野生型小鼠,但无明显病理组织学损害的2周龄Bmi-1基因敲除小鼠脑仅羟自由基水平增高。 3.Bmi-1基因半剂量敲除小鼠在Morris水迷宫测试过程中学习记忆能力较野生型小鼠轻度降低,但无显著性差异；活性氧簇、氧化应激指标丙二醛水平轻度上升,总超氧化物歧化酶和总抗氧化能力活性轻度下降,而羟自由基和还原型谷胱甘肽显著增高。与野生型小鼠相比,Bmi-1基因半剂量敲除小鼠海马锥体细胞变性或凋亡神经元以及神经元脂褐素沉积增多；突触素免疫阳性产物、突触囊泡以及线粒体的密度降低,并出现星形胶质细胞和小胶质细胞的轻度活化。另外,Bmi-1下游基因p19、p27、p53在Bmi-1基因半剂量敲除小鼠脑中表达上调,抗凋亡蛋白Bcl-2表达下调。 结论： 1.在生理性衰老进程中存在Bmi-1表达下调,p53表达逐步上调。 2.Bmi-1基因全敲除导致严重的氧化应激损伤,是造成小鼠发生成熟前神经退行性样病变的主要机制。 3.成年Bmi-1半剂量敲除小鼠出现早期脑衰老表型,但存在一些代偿机制如星形胶质细胞的活化、抗氧化物如还原型谷胱甘肽合成增加部分纠正了因Bmi-1表达下调所致的脑氧化应激损害。
[Abstract]:Background: previous studies have suggested that Bmi-1 (B-cell specific moloney leukemia virus insertion site1) is a transcriptional suppressor gene of multicomb gene family, which can be used as a target molecule for cell cycle, oxidative stress damage repair and mitochondrial function regulation. Then it plays a key role in the regulation of aging process, but the correlation between Bmi-1 gene and brain aging needs to be further clarified. Objective: to analyze the physiological, biochemical and morphological phenotypes of three kinds of mice with different Bmi-1 gene expression levels, that is, full knockout of Bmi-1 gene, half dose knockout and normal wild type mice. To explore the correlation between Bmi-1 gene and brain aging, so as to provide scientific basis for the study of targeted Bmi-1 to delay the aging process. Methods: 1. In the first part, the expression of Bmi-1 and p53 protein was detected by immunohistochemical staining and Western blotting in natural aging mice aged 9 and 20 months, 4 months old and 5 months old of wild type C57BL1. 2. In the second part, the levels of hydroxyl radical and malondialdehyde (MDA) and the expression of MBP and GFAP in brain of Bmi-1 gene knockout mice and wild type mice were detected. In the third part, Bmi-1 gene half-dose knockout mice and wild-type mice were used to detect learning and memory function by Morris water maze, reactive oxygen species cluster and oxidation were detected by flow cytometry and biochemical kit. The expression of Bmi-1 related protein was detected by immunoblotting assay and immunohistochemical staining and electron microscope examination, including the changes of neuron and glial cells in the two groups. Results: the expression of 1.Bmi-1 increased gradually with the increase of age before adulthood, and reached the peak in cortex, dentate gyrus, cerebellum and olfactory bulb at 4 months of age, and then down-regulated gradually with the development of aging. The expression of p53 protein increased with age. 2. 2. The levels of hydroxyl radical and malondialdehyde in 4-week-old Bmi-1 knockout mice with neurodegenerative degeneration were significantly higher than those in wild-type mice. However, the level of only hydroxyl radical in the brain of 2-week-old Bmi-1 knockout mice without obvious histopathological damage was increased. The learning and memory ability of 3.Bmi-1 half-dose knockout mice was slightly lower than that of wild-type mice during the Morris water maze test. But there was no significant difference. The level of MDA increased slightly, total superoxide dismutase (SOD) and total antioxidant activity decreased slightly, but hydroxyl radical and reduced glutathione increased significantly. Compared with wild-type mice, the denaturation or apoptosis of hippocampal pyramidal cells and the deposition of lipofuscin in hippocampal pyramidal cells of half-dose knockout mice were increased, and the density of synaptophysin immunoreactive products, synaptic vesicles and mitochondria were decreased. There was mild activation of astrocytes and microglia. In addition, the expression of p19p27 p553 in the brain of half-dose knockout mice with Bmi-1 gene was up-regulated, and the expression of anti-apoptotic protein Bcl-2 was down-regulated. Conclusion: 1. During physiologic senescence, down-regulation of Bmi-1 expression and up-regulation of p53 expression were found, and 2.Bmi-1 gene knockout caused severe oxidative stress injury, which was the main mechanism of premature neurodegenerative lesion in mice. 3. Half dose knockout mice of adult Bmi-1 have early aging phenotype, but there are some compensatory mechanisms such as astrocyte activation. Increased synthesis of antioxidants such as reduced glutathione partially corrected brain oxidative stress damage caused by down-regulation of Bmi-1 expression.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R339.38
本文编号：2224219
[Abstract]:Background: previous studies have suggested that Bmi-1 (B-cell specific moloney leukemia virus insertion site1) is a transcriptional suppressor gene of multicomb gene family, which can be used as a target molecule for cell cycle, oxidative stress damage repair and mitochondrial function regulation. Then it plays a key role in the regulation of aging process, but the correlation between Bmi-1 gene and brain aging needs to be further clarified. Objective: to analyze the physiological, biochemical and morphological phenotypes of three kinds of mice with different Bmi-1 gene expression levels, that is, full knockout of Bmi-1 gene, half dose knockout and normal wild type mice. To explore the correlation between Bmi-1 gene and brain aging, so as to provide scientific basis for the study of targeted Bmi-1 to delay the aging process. Methods: 1. In the first part, the expression of Bmi-1 and p53 protein was detected by immunohistochemical staining and Western blotting in natural aging mice aged 9 and 20 months, 4 months old and 5 months old of wild type C57BL1. 2. In the second part, the levels of hydroxyl radical and malondialdehyde (MDA) and the expression of MBP and GFAP in brain of Bmi-1 gene knockout mice and wild type mice were detected. In the third part, Bmi-1 gene half-dose knockout mice and wild-type mice were used to detect learning and memory function by Morris water maze, reactive oxygen species cluster and oxidation were detected by flow cytometry and biochemical kit. The expression of Bmi-1 related protein was detected by immunoblotting assay and immunohistochemical staining and electron microscope examination, including the changes of neuron and glial cells in the two groups. Results: the expression of 1.Bmi-1 increased gradually with the increase of age before adulthood, and reached the peak in cortex, dentate gyrus, cerebellum and olfactory bulb at 4 months of age, and then down-regulated gradually with the development of aging. The expression of p53 protein increased with age. 2. 2. The levels of hydroxyl radical and malondialdehyde in 4-week-old Bmi-1 knockout mice with neurodegenerative degeneration were significantly higher than those in wild-type mice. However, the level of only hydroxyl radical in the brain of 2-week-old Bmi-1 knockout mice without obvious histopathological damage was increased. The learning and memory ability of 3.Bmi-1 half-dose knockout mice was slightly lower than that of wild-type mice during the Morris water maze test. But there was no significant difference. The level of MDA increased slightly, total superoxide dismutase (SOD) and total antioxidant activity decreased slightly, but hydroxyl radical and reduced glutathione increased significantly. Compared with wild-type mice, the denaturation or apoptosis of hippocampal pyramidal cells and the deposition of lipofuscin in hippocampal pyramidal cells of half-dose knockout mice were increased, and the density of synaptophysin immunoreactive products, synaptic vesicles and mitochondria were decreased. There was mild activation of astrocytes and microglia. In addition, the expression of p19p27 p553 in the brain of half-dose knockout mice with Bmi-1 gene was up-regulated, and the expression of anti-apoptotic protein Bcl-2 was down-regulated. Conclusion: 1. During physiologic senescence, down-regulation of Bmi-1 expression and up-regulation of p53 expression were found, and 2.Bmi-1 gene knockout caused severe oxidative stress injury, which was the main mechanism of premature neurodegenerative lesion in mice. 3. Half dose knockout mice of adult Bmi-1 have early aging phenotype, but there are some compensatory mechanisms such as astrocyte activation. Increased synthesis of antioxidants such as reduced glutathione partially corrected brain oxidative stress damage caused by down-regulation of Bmi-1 expression.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R339.38
【参考文献】
相关期刊论文 前1条
1 宗峰,丁国宪,程蕴琳;H_2O_2对人淋巴细胞8-羟基-2'-脱氧鸟苷三磷酸酶表达及端粒长度的影响[J];南京医科大学学报(自然科学版);2005年04期
，本文编号：2224219
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