【摘要】： 目的：觀察小鼠室管膜下區(qū)(Subventricular zone,SVZ)神經(jīng)干細(xì)胞(Neural Stem Cells, NSCs)20S蛋白酶體的表達(dá),以及機(jī)體老化進(jìn)程中SVZ 20S蛋白酶體的活性變化,旨在揭示蛋白酶體活性改變對NSCs增殖潛能的影響,為延緩機(jī)體衰老進(jìn)程提供新的突破方向。 方法：將小鼠分為胚胎(Embryonic 14 days, E14)、新生(Postnatal day, P0)、幼年(Postnatal 30 days, P30)、成年(Postnatal 60 days, P60)、老年(Postnatal 540 days, P540)5組。 1.應(yīng)用20S蛋白酶體/nestin(神經(jīng)干細(xì)胞標(biāo)志物)免疫熒光雙標(biāo)法觀察SVZ NSCs 20S蛋白酶體的表達(dá)。2.提取E14、P0、P30、P60、P540小鼠SVZ蛋白,通過Western Blot半定量分析不同年齡階段小鼠20S蛋白酶體的表達(dá)變化。3.提取E14、P0、P30、P60、P540小鼠SVZ蛋白,應(yīng)用熒光分光光度法檢測不同年齡階段小鼠20S蛋白酶體的活性。4.將1μl MG132(30mg/kg)注入成年小鼠側(cè)腦室,分別在3d、7d后腹腔注射BrdU (5mg/kg),通過免疫熒光染色觀察BrdU陽性細(xì)胞數(shù),檢測蛋白酶體抑制劑對NSCs增殖潛能的影響。 結(jié)果：1.20S蛋白酶體免疫陽性細(xì)胞沿側(cè)腦室側(cè)壁分布,陽性細(xì)胞呈圓形,胞漿與胞核內(nèi)均可見陽性表達(dá),并且20S蛋白酶體陽性表達(dá)與NSCs標(biāo)志物nestin共存,提示20S蛋白酶體可能對NSCs具有調(diào)控作用。2. Western Blot結(jié)果表明隨年齡增加20S蛋白酶體表達(dá)水平逐漸降低,其中胚胎小鼠SVZ 20S蛋白酶體表達(dá)量最高(1.22±0.1),老年小鼠20S蛋白酶體表達(dá)量最低(0.6±0.05),二者相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3.蛋白酶體活性檢測結(jié)果表明隨著年齡增加蛋白酶體活性出現(xiàn)遞減趨勢,其中胚胎、新生、幼年小鼠20S蛋白酶體活性顯著高于老年小鼠(P0.05),但成年小鼠與老年小鼠20S蛋白酶體活性差異無統(tǒng)計(jì)學(xué)意義(P0.05)。4.MG132干預(yù)實(shí)驗(yàn)結(jié)果顯示注入MG132 3d、7d后腦室SVZBrdU陽性細(xì)胞減少,統(tǒng)計(jì)分析結(jié)果顯示注入MG1323d后BrdU陽性細(xì)胞數(shù)減少(2±0.1),與溶劑對照DMSO組(22±3)相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)；注入MG132 7d后BrdU陽性細(xì)胞數(shù)減少(3±1),與溶劑對照DMSO組(8±3)相比差異有統(tǒng)計(jì)學(xué)意義(P0.05),提示蛋白酶體活性降低可導(dǎo)致NSCs增殖潛能下降。 結(jié)論：1..NSCs表達(dá)20S蛋白酶體,且隨年齡增加SVZ NSCs 20S蛋白酶體表達(dá)量及活性逐步降低,提示20S蛋白酶體活性降低可能是導(dǎo)致NSCs衰老進(jìn)而使機(jī)體老化的關(guān)鍵因素。 2.應(yīng)用蛋白酶體抑制劑能夠顯著抑制NSCs的增殖潛能,提示蛋白酶體功能異常導(dǎo)致NSCs庫耗竭可能是衰老相關(guān)疾病發(fā)生的重要原因。
[Abstract]:Aim: to observe the expression of (Neural Stem Cells, NSCs) 20s proteasome in mouse subependymal area (Subventricular zone,SVZ) neural stem cells and the activity of SVZ 20s proteasome during aging. The aim of this study was to reveal the effect of proteasome activity change on the proliferative potential of NSCs and to provide a new breakthrough direction for delaying the aging process of the body. Methods: the mice were divided into five groups: Embryonic 14 days, E14, (Postnatal day, P0, Postnatal 30 days, P30, Postnatal 60 days, P60, Postnatal 540 days, P540. 1. The expression of 20 S proteasome / nestin (neural stem cell marker) was detected by immunofluorescence double labeling method. 2. The SVZ protein was extracted from E14 P0P0 P30 P60 P540 mice and the changes of 20s proteasome expression in mice at different ages were semi-quantitatively analyzed by Western Blot. 3. The SVZ protein was extracted from E14 P0P0 P30 P60 P540 mice and the activity of 20s proteasome in mice at different ages was detected by fluorescence spectrophotometry. 4. 1 渭 l MG132 (30mg/kg) was injected into the lateral ventricle of adult mice, and BrdU (5mg/kg) was injected intraperitoneally after 3 days. The number of BrdU positive cells was observed by immunofluorescence staining, and the effect of proteasome inhibitor on the proliferation of NSCs was detected. Results: 1.The immunoreactive cells of 20s proteasome were distributed along the lateral wall of lateral ventricle, the positive cells were round, the positive expression of 20s proteasome was observed in cytoplasm and nucleus, and the positive expression of 20s proteasome was coexisted with NSCs marker nestin. These results suggest that the 20s proteasome may have a regulatory effect on NSCs. The results of Western Blot showed that the expression level of 20s proteasome decreased with age. The expression of 20s proteasome in embryonic mice was the highest (1.22 鹵0.1), and the expression of 20s proteasome in aged mice was the lowest (0.6 鹵0. 05). The difference was statistically significant (P0.05). The results of proteasome activity test showed that the proteasome activity decreased with the increase of age. The 20s proteasome activity of embryonic, newborn and juvenile mice was significantly higher than that of old mice (P0.05). However, there was no significant difference in the activity of 20s proteasome between adult mice and aged mice (P0.05). The results of 4.MG132 intervention showed that the number of SVZBrdU positive cells in the ventricle decreased 7 days after MG132 injection. The results of statistical analysis showed that the number of BrdU positive cells decreased (2 鹵0.1) after MG1323d injection, which was significantly different from that of the solvent control group (22 鹵3) (P0.05). The number of BrdU positive cells decreased (3 鹵1) after 7 days of MG132 injection, which was significantly different from that of the solvent control DMSO group (8 鹵3) (P0.05), which suggested that the decrease of proteasome activity could lead to the decrease of NSCs proliferation potential. Conclusion: 1..NSCs expresses 20s proteasome, and the expression and activity of SVZ NSCs 20s proteasome decrease gradually with age, which suggests that the decrease of 20s proteasome activity may be the key factor leading to NSCs aging and body aging. 2. The application of proteasome inhibitor can significantly inhibit the proliferation potential of NSCs, suggesting that the depletion of NSCs library caused by abnormal proteasome function may be an important cause of aging related diseases.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 朱茜;20S蛋白酶體參與調(diào)控神經(jīng)干細(xì)胞衰老進(jìn)程[D];山西醫(yī)科大學(xué);2012年

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本文編號：2397595