靶向PSMB5基因的shRNA慢病毒载体对神经干细胞增殖和分化潜能的影响

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%(P0.001)和33.1%±2.54%(P0.001)。PSMB5-shRNA组蛋白酶体活性较对照组下降43.4%±1.48%(P0.01)。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(P0.01),神经球的平均直径为29.9μm±2.6μm显著低于对照组42.9μm±2.3μm(P0.01)。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(P0.001),PSMB5-shRNA组Tuj1+阳性率为39.13%±8.14%,较对照组显著降低(P0.01)。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。

Effects of shRNA lentiviral vectors targeting PSMB5 on the proliferation and differentiation of neural stem cells

Objective:To optimize the project of 20S proteasome subunit beta type-5 (PSMB5)-shRNA gene infection by lentiviral vectors on neural stem cells (NSCs),then to observe the effect of PSMB5 on proliferation and differentiation of NSCs,and to investigate the molecular mechanism regulating the NSCs potential.Methods:We constructed PSMBS-shRNA lentiviral vectors carrying the green fluorescent protein (GFP) gene to infect the NSCs derived from the new born mouse (postnatal day 0).A missense sequence control was also set.The positive rate of GFP was observed by inverted fluorescence microscope,and the transfection rate was calculated.The expressions of PSMB5 mRNA and protein were analysed by Real-time PCR and Western Blot.The 20S proteasomal activities were analysed through the 20S assay Kit.The proliferation ability of NSCs was analysed through the number and diameter change of neural spheres and CCK-8 assay.The differentiation ability of NSCs was analysed by the Tuj1 + staining.Results:The GFP-positive cells appeared at 24 hour after transfection,got fastigium about 48 hours after transfection,and expressed stably after cell pas-sage.Under the condition of MOI =40,polybrene =3 μg/ml,the GFP+ cells could reach (92.5％ ±2.3％) at 48 h after transfection.Compared with the control group,the mRNA and protein level of PSMB5 in PSMBS-shRNA group were decreased by (66.49％ ± 4.81％) (P ＜ 0.001) and (33.1％ ± 2.54％) (P ＜ 0.001) respectively than control group.The proteasomal activities in PSMB5-shRNA group was decreased by (43.4％ ± 1.48％) (P ＜ 0.01) than control group.Meanwhile the proliferation ability of the NSCs transfected with PSMB5-shRNA was decreasing.The number of neural spheres in PSMB5-shRNA group (126.5 ± 8.4) was significantly lower than that in the control group (163.5 ± 9.5,P ＜ 0.01)and the diameter (29.9 μm ± 2.6 μm) was lower than control group (42.9 μm ±-2.3 μm,P ＜ 0.01).The CCK-8 results showed that the absorbance value of PSMBS-shRNA group (0.36 ± 0.04) was lower than that in con trol group (0.59 ±0.03,P ＜0.001),The relative level of Tujl + cells in PSMB5-shRNA group (39.13％ ± 8.14％)was lower than the control(P ＜ 0.01),These indicated that the proliferation and differentiation capacity of the NSCs in PSMB5-shRNA group was decreasing.Conclusion:The efficient PSMB5 silencing on P0 NSCs in vitro could decrease the proteasomal activity and restrain the proliferation and differentiation capacity.