20S蛋白酶体对SVZ神经干细胞增殖潜能的调控作用
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(P<0.05)。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(P<0.05),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(P>0.05)。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(P<0.05)；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(P<0.05),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。
Objective:To reveal the effect of the activity of proteasome on proliferation potential in neural stem cell, observe the expression of 20S proteasome of neural stem cells in SVZ and the activity change of 20S proteasome in the aging process, to provide a new breakthrough to delay the aging process of the body
Methods:Mice were divided into five groups which are embryonic 14 days (E14)、postnatal day (P0)、Postnatal 30 days (P30)、Postnatal 60 days (P60)、Postnatal 540 days (P540) 1.to observe the expression of nestin and 20S proteasome of neural stem cells in SVZ through immunofluorescence.2.to extract proteins in SVZ from the E14、P0、P30、P60、P540 mouse, then semi-quantitative analysis the expression change of 20s proteasome at different ages with Western Blot method.3.to extract the proteins in SVZ from the E14、P0、P30、P60、P540mouse, to detect the activity of 20S proteasome of different ages in mice by fluorimetric method.4. To inject proteasome inhibitor MG132 into lateral ventricle of the adult mouse, then take the intraperitoneal injection of Brdu after 3days、7 days, to detect proteasome inhibitors on the proliferative potential of NSCs by immunofluorescence staining BrdU+.
Results:1.20S proteasome positive cells can be seen in both cytoplasm and the nucleus, round, distributed along the lateral ventricle wall, and the neural stem cell marker nestin also expressed in the same cells which suggest 20S proteasome may have a regulatory role in NSCs.2. The western-blot results show that the expression of 20S proteasome gradually decreased with age. The expression of 20S proteasome from embryonic mouse is highest, compared to aged mouse, it has a statistically significant difference (p< 0.05).3.Proteasome activity of the test results also show the activity of 20S proteasome is on the decrease with age, in which the activity of 20S proteasome from E14、P0、P30 mice were significantly higher than which from the P540 mice (p<0.05), but the activity of 20S proteasome from P60 mice and P540 mice has no significant difference(p>0.05).4. MG132 intervention results showed that after injection of MG132 3days and 7 days, The number of BrdU+ cells are decreased in SVZ. After injection of MG132 3days, The number of BrdU+ cells are decreased (2±0.1), compared with the DMSO group (22±3), it has a statistically significant difference (p<0.05). After injection of MG132 7days, The number of BrdU+ cells are decreased (3±1), compared with the DMSO group (8±3), it has a statistically significant difference (p<0.05). Which indicate the decreased activity of 20Sproteasome led to the decrease of proliferative potential in NSCs.
Conclusion:(1)the expression and activity of 20S proteasome gradually reducedand in SVZ NSCs with age; which suggest the decreased activity of 20S proteasome may result in the senescence of NSCs and the aging of the body.
(2) Proteasome inhibitors can significantly inhibit the proliferation potential of NSCs, indicating that aging of the body may make proteasome dysfunction which induce to the depletion of NSCs library and aging-related diseasesby.
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(P<0.05)。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(P<0.05),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(P>0.05)。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(P<0.05)；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(P<0.05),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。
Objective:To reveal the effect of the activity of proteasome on proliferation potential in neural stem cell, observe the expression of 20S proteasome of neural stem cells in SVZ and the activity change of 20S proteasome in the aging process, to provide a new breakthrough to delay the aging process of the body
Methods:Mice were divided into five groups which are embryonic 14 days (E14)、postnatal day (P0)、Postnatal 30 days (P30)、Postnatal 60 days (P60)、Postnatal 540 days (P540) 1.to observe the expression of nestin and 20S proteasome of neural stem cells in SVZ through immunofluorescence.2.to extract proteins in SVZ from the E14、P0、P30、P60、P540 mouse, then semi-quantitative analysis the expression change of 20s proteasome at different ages with Western Blot method.3.to extract the proteins in SVZ from the E14、P0、P30、P60、P540mouse, to detect the activity of 20S proteasome of different ages in mice by fluorimetric method.4. To inject proteasome inhibitor MG132 into lateral ventricle of the adult mouse, then take the intraperitoneal injection of Brdu after 3days、7 days, to detect proteasome inhibitors on the proliferative potential of NSCs by immunofluorescence staining BrdU+.
Results:1.20S proteasome positive cells can be seen in both cytoplasm and the nucleus, round, distributed along the lateral ventricle wall, and the neural stem cell marker nestin also expressed in the same cells which suggest 20S proteasome may have a regulatory role in NSCs.2. The western-blot results show that the expression of 20S proteasome gradually decreased with age. The expression of 20S proteasome from embryonic mouse is highest, compared to aged mouse, it has a statistically significant difference (p< 0.05).3.Proteasome activity of the test results also show the activity of 20S proteasome is on the decrease with age, in which the activity of 20S proteasome from E14、P0、P30 mice were significantly higher than which from the P540 mice (p<0.05), but the activity of 20S proteasome from P60 mice and P540 mice has no significant difference(p>0.05).4. MG132 intervention results showed that after injection of MG132 3days and 7 days, The number of BrdU+ cells are decreased in SVZ. After injection of MG132 3days, The number of BrdU+ cells are decreased (2±0.1), compared with the DMSO group (22±3), it has a statistically significant difference (p<0.05). After injection of MG132 7days, The number of BrdU+ cells are decreased (3±1), compared with the DMSO group (8±3), it has a statistically significant difference (p<0.05). Which indicate the decreased activity of 20Sproteasome led to the decrease of proliferative potential in NSCs.
Conclusion:(1)the expression and activity of 20S proteasome gradually reducedand in SVZ NSCs with age; which suggest the decreased activity of 20S proteasome may result in the senescence of NSCs and the aging of the body.
(2) Proteasome inhibitors can significantly inhibit the proliferation potential of NSCs, indicating that aging of the body may make proteasome dysfunction which induce to the depletion of NSCs library and aging-related diseasesby.
引文
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