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目的:探讨p53对神经干细胞(neural stem cells,NSCs)增殖能力的影响,寻求维持NSCs活力的有效途径。方法:分离出生后0 d(postnatal day 0,P0)和生后90 d(postnatal day 90,P90)小鼠前脑室管膜下区(subventricular zone,SVZ)组织,培养NSCs。比较P0和P90 NSCs增殖能力并通过q PCR和Western Blot检测衰老相关分子p53和p21在新生和成年NSCs的表达变化。应用p53抑制剂PFT-α(20μmol/L)连续作用于P90 NSCs 72 h,通过Brd U掺入实验和CCK-8实验,分析抑制p53对NSCs增殖能力的影响。结果:随年龄增加,NSCs的增殖能力降低,P90组神经球的数量和直径分别是P0组的22.9%±1.2%(P<0.01)和63.5%±3.7%(P<0.05)。P90NSCs p53和p21 mRNA表达水平分别较P0 NSCs显著增高了1.4±0.05和1.2±0.04(P<0.01)。Western Blot结果证明P90 NSCs p53蛋白的表达水平比P0 NSCs增加了1.2±0.01倍(P<0.05)。经p53抑制剂PFT-α连续处理P90 NSCs 72 h后,CCK-8结果显示PFT-α组吸光度值1.1±0.02高于DMSO组0.8±0.03(P<0.05),Brd U掺入实验也显示PFT-α组Brd U阳性细胞率为43.3%±4.0%显著高于DMSO组24.8%±3.1%(P<0.05)。结论:p53信号通路激活可能是导致成年小鼠NSCs增殖能力下降的重要因素,应用p53抑制剂PFT-α能够增加成年NSCs的增殖能力。
Objective: To investigate the effect of p53 on the proliferation of neural stem cells (NSCs) and to find an effective way to maintain the viability of NSCs. Methods: NSCs were isolated from postnatal day 0 (P0) and postnatal day 90 (P90) subventricular zone (SVZ) in mice. The proliferation of P0 and P90 NSCs was compared and the expression of the aging related molecules p53 and p21 in neonatal and adult NSCs was detected by q PCR and Western Blot. The effect of p53 on proliferation of NSCs was analyzed by BrdU incorporation assay and CCK-8 assay using PFT-α (20μmol / L) as a p53 inhibitor for 72 h. Results: With the increase of age, the proliferation of NSCs decreased. The numbers and diameters of neurospheres in P90 group were 22.9% ± 1.2% (P <0.01) and 63.5% ± 3.7% (P <0.05) respectively in P0 group. P90NSCs p53 and p21 mRNA levels were significantly higher than P0 NSCs 1.4 ± 0.05 and 1.2 ± 0.04 (P <0.01). The results of Western Blot showed that the expression of p53 protein in P90 NSCs was increased by 1.2 ± 0.01 times than that of P0 NSCs (P <0.05). CCK-8 results showed that the absorbance value of PFT-α group was 1.1 ± 0.02 higher than that of DMSO group (P <0.05) (P <0.05) after treated with PFT-α for 72 h. BrdU incorporation assay also showed that PFT The positive rate of Brd U positive cells in -α group was 43.3% ± 4.0%, significantly higher than that in DMSO group 24.8% ± 3.1% (P <0.05). CONCLUSION: Activation of p53 signaling pathway may be an important factor leading to the decreased proliferation of adult mouse NSCs. Application of p53 inhibitor PFT-α can increase the proliferation of adult NSCs.