蛋白酶体抑制剂MG132对脑室室管膜下区神经干细胞增殖潜能的影响

目的: 观察侧脑室注射蛋白酶体抑制剂MG132对脑室室管膜下区(SVZ)神经干细胞(NSCs)增殖能力的影响,了解蛋白酶体对NSCs增殖潜能的调控作用,从而进一步探讨蛋白酶体在帕金森病(PD)等老年性疾病神经元损伤中的可能作用机制。方法: 选90日龄健康BALB/c小鼠,左侧侧脑室注射10 μg MG132,于注射后3 d、7 d、14 d提取SVZ总蛋白,荧光酶标仪检测蛋白酶体活性。同时腹腔注射5-溴-2'-脱氧尿嘧啶核苷(BrdU),BrdU免疫荧光染色标记NSCs,动态观察蛋白酶体活性改变对NSCs增殖能力的影响。另设溶剂对照组左侧侧脑室注射等剂量DMSO,进行上述实验。结果: 侧脑室注射MG132 3 d、7 d后,SVZ蛋白酶体活性较DMSO对照组显著降低(P<0.05),同时SVZ BrdU⁺ NSCs也显著减少至21±4和22±3,与DMSO对照组相比差异显著(P<0.05)。随着MG132注射时间延长,14 d后SVZ蛋白酶体活性恢复至正常水平(P＞0.05),BrdU⁺ NSCs数量MG132组(82±4)与DMSO组(67±6)相比无显著差别(P＞0.05)。结论: 应用蛋白酶体可逆性抑制剂MG132短时期内能显著抑制SVZ蛋白酶体活性,并且能够降低NSCs增殖能力,提示蛋白酶体活性与NSCs增殖潜能密切相关。

Effects of proteasome inhibitor MG132 on proliferation of neural stem cells in subventricular zone

AIM: To investigate the role of proteasome in the degeneration of neuron in age-related diseases such as Parkinson disease (PD) by observing the effects of proteasome inhibitor MG132 on the proliferation of neural stem cells (NSCs) in the subventricular zone (SVZ).METHODS: Stereotaxic microinjection of proteasome inhibitor MG132 at a dose of 10 μg into the left lateral ventricle of 90-day-old mice was performed.The control mice were received the same volume of DMSO into the left lateral ventricle.Three days, 7 days and 14 days after injection, the total proteins isolat from SVZ were subject to proteasome activity assay using the fluorescence microplate reader.Meanwhile, the mice were administered with 5-bromo-2'-deoxyuridine (BrdU) by intraperitoneal injection.The numbers of BrdU-positive cells were counted to examine the effect of MG132 on the proliferation of NSCs.RESULTS: After microinjection of MG132 into the left lateral ventricle, the proteasome activity in SVZ was significantly decreased on day 3 and day 7 post-injection (P<0.05) as compared with the control animals.Furthermore, the numbers of BrdU⁺ cells in SVZ were significantly reduced to 21±4 on day 3 and to 22±3 on day 7 post-injection (P<0.05).After long periods of treatment with MG132, the proteasome activity in SVZ was restored to normal level on day 14 post-injection.At the same time, no statistical difference of BrdU⁺ cells between the mice treated with MG132 (82±4) and DMSO (67±6) was observed (P>0.05).CONCLUSION: Short-term injection of reversible proteasome inhibitor MG132 attenuates the proteasome activity in SVZ, which in turn inhibits the proliferation of NSCs, suggesting that the proteasome activity may be closely related to the proliferation potential of NSCs.