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目的:优化20S蛋白酶体β5亚单位(proteasome subunit beta type-5,PSMB5)-shRNA慢病毒感染神经干细胞(neural stem cells,NSCs)的方案,观察PSMB5表达下调对NSCs增殖和分化能力的影响,探讨调控NSCs潜能的分子机制。方法:构建携带绿色荧光蛋白(green fluorescent protein,GFP)基因的PSMB5-shRNA慢病毒载体,并设错义序列对照,感染新生小鼠(postnatal day 0,P0)NSCs。倒置荧光显微镜观察GFP阳性率,计算感染率,RT-PCR、免疫印迹和荧光分光光度法检测PSMB5沉默效率和蛋白酶体活性。比较对照组和shRNA组神经球的数量和直径,利用CCK-8实验观察PSMB5基因沉默对NSCs增殖潜能的影响。Tuj1染色观察PSMB5基因沉默对NSCs分化能力的影响。结果:PSMB5-shRNA慢病毒感染NSCs 24 h后可见GFP荧光表达,48 h达峰值,传代后可见GFP稳定表达。其感染复数MOI为40,polybrene 3μg/ml时,感染48 h GFP阳性率可达92.5%±2.3%;PSMB5-shRNA组PSMB5 mRNA和蛋白表达水平分别较对照组降低66.49%±4.81%(P<0.001)和33.1%±2.54%(P<0.001)。PSMB5-shRNA组蛋白酶体活性较对照组下降43.4%±1.48%(P<0.01)。shRNA组NSCs的增殖能力降低,神经球数量为126.5±8.4显著低于对照组163.5±9.5(P<0.01),神经球的平均直径为29.9μm±2.6μm显著低于对照组42.9μm±2.3μm(P<0.01)。CCK-8结果表明PSMB5-shRNA组细胞吸光度值0.36±0.04,显著低于对照组0.59±0.03(P<0.001),PSMB5-shRNA组Tuj1+阳性率为39.13%±8.14%,较对照组显著降低(P<0.01)。结论:PSMB5基因沉默可降低NSCs蛋白酶体活性抑制P0期小鼠NSCs增殖分化能力。
OBJECTIVE: To optimize the 20S proteasome subunit beta type-5 (PSMB5) -shRNA lentivirus infection of neural stem cells (NSCs) and to observe the effect of down-regulation of PSMB5 on the proliferation and differentiation of NSCs Molecular Mechanism of Regulating the Potential of NSCs. Methods: PSMB5-shRNA lentiviral vector carrying green fluorescent protein (GFP) gene was constructed and missense sequence control was used to infect postnatal day 0 (P0) NSCs. The positive rate of GFP was observed by inverted fluorescence microscope, the infection rate was calculated, and the silencing efficiency and proteasome activity of PSMB5 were detected by RT-PCR, immunoblot and fluorescence spectrophotometry. The numbers and diameters of neurospheres in control and shRNA groups were compared. The effects of PSMB5 silencing on the proliferation of NSCs were observed by CCK-8 assay. The Effect of PSMB5 Gene Silencing on the Differentiation of NSCs by Tuj1 Staining. Results: GFP expression was observed in 24 h after PSMB5-shRNA lentivirus infection, reaching a peak at 48 h. After transfection, stable expression of GFP was observed. The infection rate of MOI was 40 and polybrene 3μg / ml, the positive rate of GFP was up to 92.5% ± 2.3% 48 h after infection, and the expression of PSMB5 mRNA and protein in PSMB5-shRNA group decreased by 66.49% ± 4.81% (P < 0.001) and 33.1% ± 2.54% (P <0.001). PSMB5-shRNA group proteasome activity than the control group decreased 43.4% ± 1.48% (P <0.01). The proliferation of NSCs in shRNA group was significantly lower than that in control group (126.5 ± 8.4, 163.5 ± 9.5, P <0.01). The mean diameter of neurospheres was 29.9μm ± 2.6μm, which was significantly lower than that of the control group (42.9μm ± 2.3μm) (P <0.01). The results of CCK-8 showed that the cell absorbance of PSMB5-shRNA group was 0.36 ± 0.04, significantly lower than that of control group (0.59 ± 0.03) (P <0.001). The positive rate of Tuj1 + in PSMB5-shRNA group was 39.13% ± 8.14% P <0.01). Conclusion: Silencing of PSMB5 can decrease the proteasome activity of NSCs and inhibit the proliferation and differentiation of NSCs in P0 phase.