20S蛋白酶体对SVZ神经干细胞增殖潜能的调控作用
发布时间：2019-01-01 12:51
【摘要】： 目的：观察小鼠室管膜下区(Subventricular zone,SVZ)神经干细胞(Neural Stem Cells, NSCs)20S蛋白酶体的表达,以及机体老化进程中SVZ 20S蛋白酶体的活性变化,旨在揭示蛋白酶体活性改变对NSCs增殖潜能的影响,为延缓机体衰老进程提供新的突破方向。 方法：将小鼠分为胚胎(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组。 1.应用20S蛋白酶体/nestin(神经干细胞标志物)免疫荧光双标法观察SVZ NSCs 20S蛋白酶体的表达。2.提取E14、P0、P30、P60、P540小鼠SVZ蛋白,通过Western Blot半定量分析不同年龄阶段小鼠20S蛋白酶体的表达变化。3.提取E14、P0、P30、P60、P540小鼠SVZ蛋白,应用荧光分光光度法检测不同年龄阶段小鼠20S蛋白酶体的活性。4.将1μl MG132(30mg/kg)注入成年小鼠侧脑室,分别在3d、7d后腹腔注射BrdU (5mg/kg),通过免疫荧光染色观察BrdU阳性细胞数,检测蛋白酶体抑制剂对NSCs增殖潜能的影响。 结果：1.20S蛋白酶体免疫阳性细胞沿侧脑室侧壁分布,阳性细胞呈圆形,胞浆与胞核内均可见阳性表达,并且20S蛋白酶体阳性表达与NSCs标志物nestin共存,提示20S蛋白酶体可能对NSCs具有调控作用。2. Western Blot结果表明随年龄增加20S蛋白酶体表达水平逐渐降低,其中胚胎小鼠SVZ 20S蛋白酶体表达量最高(1.22±0.1),老年小鼠20S蛋白酶体表达量最低(0.6±0.05),二者相比差异有统计学意义(P0.05)。3.蛋白酶体活性检测结果表明随着年龄增加蛋白酶体活性出现递减趋势,其中胚胎、新生、幼年小鼠20S蛋白酶体活性显著高于老年小鼠(P0.05),但成年小鼠与老年小鼠20S蛋白酶体活性差异无统计学意义(P0.05)。4.MG132干预实验结果显示注入MG132 3d、7d后脑室SVZBrdU阳性细胞减少,统计分析结果显示注入MG1323d后BrdU阳性细胞数减少(2±0.1),与溶剂对照DMSO组(22±3)相比差异有统计学意义(P0.05)；注入MG132 7d后BrdU阳性细胞数减少(3±1),与溶剂对照DMSO组(8±3)相比差异有统计学意义(P0.05),提示蛋白酶体活性降低可导致NSCs增殖潜能下降。 结论：1..NSCs表达20S蛋白酶体,且随年龄增加SVZ NSCs 20S蛋白酶体表达量及活性逐步降低,提示20S蛋白酶体活性降低可能是导致NSCs衰老进而使机体老化的关键因素。 2.应用蛋白酶体抑制剂能够显著抑制NSCs的增殖潜能,提示蛋白酶体功能异常导致NSCs库耗竭可能是衰老相关疾病发生的重要原因。
[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 卤0.1), and the expression of 20s proteasome in aged mice was the lowest (0.6 卤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 卤0.1) after MG1323d injection, which was significantly different from that of the solvent control group (22 卤3) (P0.05). The number of BrdU positive cells decreased (3 卤1) after 7 days of MG132 injection, which was significantly different from that of the solvent control DMSO group (8 卤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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2397595
[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 卤0.1), and the expression of 20s proteasome in aged mice was the lowest (0.6 卤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 卤0.1) after MG1323d injection, which was significantly different from that of the solvent control group (22 卤3) (P0.05). The number of BrdU positive cells decreased (3 卤1) after 7 days of MG132 injection, which was significantly different from that of the solvent control DMSO group (8 卤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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 朱茜;20S蛋白酶体参与调控神经干细胞衰老进程[D];山西医科大学;2012年
，本文编号：2397595
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