【摘要】：背景：既往研究提示Bmi-1(B-cell specific moloney leukemia virus insertion site1)屬于多梳基因家族的轉(zhuǎn)錄抑制基因,可作為細(xì)胞周期、氧化應(yīng)激、DNA損傷修復(fù)和線粒體功能的共同調(diào)節(jié)靶分子,進(jìn)而在調(diào)控衰老進(jìn)程中起關(guān)鍵作用,但Bmi-1基因與腦衰老的相關(guān)性有待于進(jìn)一步明確。 目的：本學(xué)位論文旨在通過對三種不同的Bmi-1基因表達(dá)量的小鼠即Bmi-1基因全敲除、半劑量敲除和正常野生型小鼠的生理、生化及形態(tài)學(xué)等方面的表型進(jìn)行分析,來探討B(tài)mi-1基因與腦衰老的相關(guān)性,從而為研究靶向Bmi-1延緩衰老進(jìn)程提供科學(xué)依據(jù)。 方法： 1.第一部分應(yīng)用野生型C57BL1月齡、4月齡、5月齡、9月齡和20月齡的自然衰老小鼠,免疫組織化學(xué)染色和免疫印跡檢測Bmi-1和p53蛋白的表達(dá)變化。 2.第二部分采用2、4周齡的Bmi-1基因全敲除小鼠和野生型小鼠檢測腦羥自由基和丙二醛的水平以及MBP和GFAP的表達(dá)。 3.第三部分采用8月齡的Bmi-1基因半劑量敲除小鼠和野生型小鼠,運用Morris水迷宮檢測兩組小鼠學(xué)習(xí)記憶功能,流式細(xì)胞技術(shù)和生化試劑盒檢測活性氧簇以及氧化、抗氧化指標(biāo)的水平,免疫組織化學(xué)染色和電鏡檢測兩組小鼠病理組織學(xué)包括神經(jīng)元及膠質(zhì)細(xì)胞的改變,免疫印跡檢測Bmi-1相關(guān)蛋白的表達(dá)變化。 結(jié)果： 1.Bmi-1在成年之前隨著月齡的增長表達(dá)逐步上調(diào),并在4月齡的皮質(zhì)、齒狀回、小腦及嗅球的表達(dá)達(dá)到高峰,之后隨著衰老推進(jìn)表達(dá)逐步下調(diào)；而p53蛋白隨月齡增長而表達(dá)增高。 2.存在神經(jīng)退行性變性的4周齡Bmi-1基因敲除小鼠腦羥自由基和丙二醛水平都明顯高于野生型小鼠,但無明顯病理組織學(xué)損害的2周齡Bmi-1基因敲除小鼠腦僅羥自由基水平增高。 3.Bmi-1基因半劑量敲除小鼠在Morris水迷宮測試過程中學(xué)習(xí)記憶能力較野生型小鼠輕度降低,但無顯著性差異；活性氧簇、氧化應(yīng)激指標(biāo)丙二醛水平輕度上升,總超氧化物歧化酶和總抗氧化能力活性輕度下降,而羥自由基和還原型谷胱甘肽顯著增高。與野生型小鼠相比,Bmi-1基因半劑量敲除小鼠海馬錐體細(xì)胞變性或凋亡神經(jīng)元以及神經(jīng)元脂褐素沉積增多；突觸素免疫陽性產(chǎn)物、突觸囊泡以及線粒體的密度降低,并出現(xiàn)星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞的輕度活化。另外,Bmi-1下游基因p19、p27、p53在Bmi-1基因半劑量敲除小鼠腦中表達(dá)上調(diào),抗凋亡蛋白Bcl-2表達(dá)下調(diào)。 結(jié)論： 1.在生理性衰老進(jìn)程中存在Bmi-1表達(dá)下調(diào),p53表達(dá)逐步上調(diào)。 2.Bmi-1基因全敲除導(dǎo)致嚴(yán)重的氧化應(yīng)激損傷,是造成小鼠發(fā)生成熟前神經(jīng)退行性樣病變的主要機制。 3.成年Bmi-1半劑量敲除小鼠出現(xiàn)早期腦衰老表型,但存在一些代償機制如星形膠質(zhì)細(xì)胞的活化、抗氧化物如還原型谷胱甘肽合成增加部分糾正了因Bmi-1表達(dá)下調(diào)所致的腦氧化應(yīng)激損害。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R339.38

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