OTUD 3敲除对神经干细胞中缝隙连接蛋白43表达的影响

EFFECT OF OTUD 3 KNOCKOUT ON THE EXPRESSION OF CONNEXIN 43 IN NEURAL STEM CELLS

目的探讨OTUD 3敲除对神经干细胞(NSCs)中缝隙连接蛋白43(Cx43)表达的影响。方法分离提取妊娠第14天时野生型(WT)和OTUD 3^(-/-)胚胎小鼠的脑皮质组织蛋白,同时进行体外培养WT-NSCs和OTUD 3^(-/-)-NSCs,并根据细胞状态传代,传代至第3代时,应用倒置显微镜观察第1、3天时OTUD 3^(-/-)-NSCs和WT-NSCs生长状态。用细胞免疫荧光染色检测WT-NSCs和OTUD 3^(-/-)-NSCs中Ki67、Nestin和β-tubulinⅢ的表达情况,应用Western blotting方法检测WT-NSCs和OTUD 3^(-/-)-NSCs中以及WT和OTUD 3^(-/-)胚胎小鼠脑皮质组织中OTUD3和Cx43表达变化情况。结果培养并传代至第3代的WT-NSCs和OTUD 3^(-/-)-NSCs生长状态良好,细胞免疫荧光染色显示Ki67、Nestin和β-tubulinⅢ染色阳性。与WT-NSCs相比,OTUD 3^(-/-)-NSCs中OTUD3和Cx43表达量显著下调(t=16.82、3.21,P<0.05);与WT胚胎小鼠相比,OTUD 3^(-/-)胚胎小鼠脑皮质组织中OTUD3和Cx43表达量显著下调(t=10.04、20.41,P<0.05)。结论OTUD 3敲除后,NSCs中Cx43表达量显著下调,Cx43可能在促进NSCs的增殖和神经发育过程中发挥调节作用。

Objective To investigate the effect of OTUD3 knockout on the expression of connexin 43(Cx43)in neural stem cells(NSCs).Methods Brain cortex tissue protein was isolated and extracted from wild-type(WT)and OTUD 3^(-/-)embryonic mice on day 14 of gestation,and WT-NSCs and OTUD 3^(-/-)-NSCs were cultured in vitro and subcultured according to cell status.After subculture to the third generation,an inverted microscope was used to observe the growth state of OTUD 3^(-/-)-NSCs and WT-NSCs on days 1 and 3.Immunofluorescent staining was used to measure the expression of Ki67,Nestin,andβ-tubulinⅢin WT-NSCs and OTUD 3^(-/-)-NSCs,and Western blotting was used to measure the changes in the expression of OTUD3 and Cx43 in WT-NSCs,OTUD 3^(-/-)-NSCs,and the cerebral cortex of WT and OTUD 3^(-/-)embryonic mice.Results WT-NSCs and OTUD 3^(-/-)-NSCs cultured and subcultured to the third generation showed a good growth status,and immunofluorescent staining showed positive staining of Ki67,Nestin,andβ-tubulinⅢ.Compared with WT-NSCs,OTUD 3^(-/-)-NSCs had significant reductions in the expression levels of OTUD3 and Cx43(t=16.82,3.21,P<0.05),and compared with WT embryonic mice,OTUD 3^(-/-)embryonic mice also had significant reductions in the expression levels of OTUD3 and Cx43 in the cerebral cortex(t=10.04,20.41,P<0.05).Conclusion The expression of Cx43 in NSCs is significantly downregulated after OTUD 3 knockout,and Cx43 may play a regulatory role in promoting the proliferation of NSCs and neurodevelopment.