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目的:探讨叔丁基对苯二酚(tBHQ)对成年小鼠神经干细胞(aNSCs)增殖分化能力的影响,寻找维持aNSCs活力的有效手段。方法:成年BALB/c小鼠腹腔注射tBHQ(每日16.7 mg/kg)7 d,收集室管膜下区(SVZ)的脑组织,应用荧光酶标仪定量检测其蛋白酶体的活性;同时小鼠腹腔注射5-溴脱氧尿嘧啶核苷(BrdU),Brd U免疫荧光染色检测SVZ内a NSCs的增殖能力。分离培养SVZ aNSCs,tBHQ(20μmol/L)作用7 d,分析比较tBHQ组与DMSO组a NSCs的蛋白酶体活性、BrdU阳性率以及形成神经球的数量和直径。应用1%的胎牛血清诱导a NSCs分化,早期神经元标记物β-Ⅲ微管蛋白(Tuj1)免疫荧光染色并比较tBHQ组与DMSO组a NSCs向神经元分化的能力。结果:小鼠腹腔注射tBHQ,其SVZ蛋白酶体活性较DMSO对照组上调12.9%±4.6%(P<0.05),并且BrdU标记实验显示tBHQ组阳性细胞数为31.3±6.3,与DMSO对照组(15.4±1.3)比较,aNSCs增殖能力显著提高(P<0.05)。体外实验结果也显示tBHQ组aNSCs蛋白酶体活性较DMSO对照组上升10.1%±0.8%(P<0.01)。tBHQ组Brd U阳性率(31.3%±3.2%)是DMSO对照组(20%±1.5%)的1.6倍(P<0.05)。tBHQ组神经球的数量和直径分别是DMSO对照组的1.7倍(P<0.05)和1.4倍(P<0.05),提示tBHQ促进aNSCs的自我更新。此外,tBHQ组Tuj1阳性率为26.5%±1.6%,较DMSO对照组18.6%±2.1%显著提高(P<0.05),提示tBHQ能够促进a NSCs向神经元方向分化。结论:tBHQ能够上调蛋白酶体活性,促进aNSCs的增殖与分化。
AIM: To investigate the effect of t-butyl hydroquinone (tBHQ) on the proliferation and differentiation of neural stem cells (aNSCs) in adult mice and to find an effective way to maintain the viability of aNSCs. Methods: Adult BALB / c mice were intraperitoneally injected with tBHQ (16.7 mg / kg per day) for 7 days to collect the brain tissue of the subventricular zone (SVZ). The activity of proteasome was quantified by fluorescence microplate reader. Rats were injected intraperitoneally with 5-bromodeoxyuridine (BrdU) and BrdU immunofluorescent staining to detect the proliferation of a NSCs in SVZ. The SVZ aNSCs were isolated and cultured for 7 days at tBHQ (20μmol / L). The proteasome activity, the positive rate of BrdU and the number and diameter of neurospheres in a group of tBHQ and DMSO were analyzed. NSCs were induced to differentiate into NSCs by 1% fetal bovine serum. Tuj1 immunofluorescent staining was used to detect neuronal differentiation of NSCs from tBHQ group and DMSO group. Results: Compared with DMSO control group, the activity of SVZ proteasome was up-regulated by tBHQ in mice by 12.9% ± 4.6% (P <0.05), and BrdU labeling assay showed that the number of positive cells in tBHQ group was 31.3 ± 6.3, ± 1.3), the proliferation of aNSCs significantly increased (P <0.05). The results of in vitro experiments also showed that the proteasome activity of aNSCs in tBHQ group increased by 10.1% ± 0.8% (P <0.01) compared with DMSO control group. The positive rate of BrdU in tBHQ group (31.3% ± 3.2%) was 1.6 times that in DMSO control group (20% ± 1.5%) (P <0.05). The numbers and diameters of neurospheres in the tBHQ group were 1.7-fold (P <0.05) and 1.4-fold (P <0.05) higher than those in the DMSO control group, respectively, suggesting that tBHQ promotes self-renewal of aNSCs. In addition, the positive rate of Tuj1 in tBHQ group was 26.5% ± 1.6%, which was significantly higher than that in DMSO control group (18.6% ± 2.1%, P <0.05), suggesting that tBHQ can promote the differentiation of a NSCs into neurons. Conclusion: tBHQ can upregulate proteasome activity and promote the proliferation and differentiation of aNSCs.