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

发布时间：2018-05-07 11:17

本文选题：神经干细胞 + S蛋白酶体β亚单位　；参考：《神经解剖学杂志》2017年05期

【摘要】：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。
[Abstract]:Aim: to optimize the protocol of proteasome subunit beta type-5 PSMB5-shRNA lentivirus infected neural stem cells (NSCS), observe the effect of down-regulation of PSMB5 expression on the proliferation and differentiation of NSCs, and explore the molecular mechanism of regulating NSCs potential. Methods: the PSMB5-shRNA lentivirus vector carrying the green fluorescent protein (GFP) gene was constructed, and the missense sequence control was used to infect the newborn mice with postnatal day 0 (P0). The positive rate of GFP was observed by inverted fluorescence microscope, the infection rate was calculated by RT-PCR, the silencing efficiency and proteasome activity of PSMB5 were detected by Western blot and fluorescence spectrophotometry. The number and diameter of neurospheres in control group and shRNA group were compared. The effect of PSMB5 gene silencing on the proliferative potential of NSCs was observed by CCK-8 assay. The effect of PSMB5 gene silencing on the differentiation of NSCs was observed by staining. Results the fluorescence expression of GFP reached its peak at 48 h after infection with NSCs and stable expression of GFP was observed after passage. The expression of PSMB5 mRNA and protein in PSMB5-shRNA group decreased by 66.49% 卤4.81% (P 0.001) and 33.1% 卤2.54%(P0.001).PSMB5-shRNA (43.4% 卤1.48%(P0.01).shRNA), respectively, when the complex MOI was 40 渭 g/ml. The positive rate of GFP was 92.5% 卤2.3%. The expression of PSMB5 mRNA and protein in PSMB5-shRNA group was lower than that in control group (66.49% 卤4.81%, P 0.001) and 33.1% 卤2.54%(P0.001).PSMB5-shRNA group (43.4% 卤1.48%(P0.01).shRNA group, respectively). The number of nerve balls was 126.5 卤8.4 significantly lower than that of the control group (163.5 卤9.5) P0.01G, and the average diameter of the neurosphere was 29.9 渭 m 卤2.6 渭 m significantly lower than that of the control group (42.9 渭 m 卤2.3 渭 m(P0.01).CCK-8). The results showed that the cell absorbance of the PSMB5-shRNA group was 0.36 卤0.04, and the positive rate of Tuj1 was 39.13% 卤8.14B in the PSMB5-shRNA group, which was significantly lower than that in the control group. Conclusion the silencing of PSMB5 gene can decrease the activity of NSCs proteasome and inhibit the proliferation and differentiation of NSCs in P0 phase mice.
【作者单位】：山西医科大学第一临床医学院;山西医科大学基础医学院解剖学教研室;
【基金】：国家级大学生创新创业训练项目(201510114005) 山西省自然科学基金(2015011132) 山西省归国留学人员基金项目(2014-033)
【分类号】：R329.2

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