Nrf2靶向siRNA对小鼠神经干细胞增殖和分化能力的影响

The effects of Nrf2 targeting siRNA on proliferation and differentiation of mouse neural stem cells

目的:探讨核因子E2相关因子2(Nrf2)对小鼠神经干细胞(NSCs)增殖和分化能力的影响。方法:分离新生小鼠海马组织,培养NSCs,采用电穿孔法将Nrf2 siRNA序列转染至NSCs,分别用real time RT-PCR和Western Blot检测敲减效率;运用BrdU掺入实验、Tuj1免疫荧光染色和CCK-8法检测Nrf2靶向siRNA对NSCs的增殖、分化能力和细胞活力的影响。通过脑立体定位技术,在成年小鼠海马部位注射Nrf2抑制剂Brusatol,应用Western Blot检测Nrf2的蛋白表达水平;通过Ki67、DCX免疫荧光染色检测下调Nrf2对海马神经发生的影响。利用活性氧簇(ROS)水平测定法和real time RT-PCR分别检测活性氧水平和氧化应激相关通路超氧化物歧化酶(SOD1和SOD2)的表达。结果:Nrf2的siRNA序列转染至NSCs,real time RT-PCR结果显示siRNA的敲减效率为(54.56±7.05)%(P<0.01),Western Blot结果显示siRNA的敲减效率为(39.50±7.90)%(P<0.01);与NC-siRNA组相比,Nrf2-siRNA组的BrdU阳性率、细胞活力均下降(P<0.01),1%胎牛血清诱导分化后,Nrf2-siRNA组Tuj1阳性率下降(P<0.01);同时Nrf2-siRNA组SOD1和SOD2的mRNA水平均较NC-siRNA组下降(P<0.05),而ROS水平较NC-siRNA组升高(P<0.05)。脑内注射Nrf2抑制剂Brusatol后,与Control组相比,Brusatol组的海马组织Nrf2蛋白表达水平降低(P<0.01);Brusatol组Ki67阳性、DCX阳性细胞数量均减少(P<0.05和P<0.01);此外Brusatol组SOD1 mRNA和SOD2水平均较Control组下降(P<0.05),而ROS水平较Control组升高(P<0.05)。结论:Nrf2基因敲减导致NSCs氧化应激水平升高,进而抑制了NSCs增殖和分化能力。

Objective:To research the impact of nuclear factor E2-related factor 2(Nrf2)on the proliferation and differentiation capability of mouse neural stem cells(NSCs).Methods:NSCs were dissociated from the hippocampus dentate gyrus of the mice at postnatal days 0.Nrf2 siRNA sequences were transfected into NSCs by electroporation,and the knockdown efficiency was detected by real time RT-PCR and Western Blot,respectively;BrdU incorporation assay,Tuj1 immunofluorescence staining and CCK-8 assay were used to detect the effects of Nrf2 targeting siRNA on the proliferation,differentiation ability and cell viability of NSCs.Adult mice were stereotaxically injected with the Nrf2 inhibitor Brusatol into the hippocampus,the protein expression level of Nrf2 was detected by Western Blot,and the effect of down-regulation of Nrf2 on hippocampal neurogenesis was examined by Ki67 and DCX immunofluorescence staining.The effects of down-regulation of Nrf2 on reactive oxygen levels and the superoxide dismutase(SOD1 and SOD2)of oxidative stress-related pathways were examined using DCFH-DA and real time RT-PCR.Results:NSCs were targeted to electrotransfect siRNA sequences of Nrf2.Real time RT-PCR results showed that the knockdown efficiency of siRNA was(54.56±7.05)%(P<0.01),Western Blot results showed that the knockdown efficiency of siRNA was(39.50±7.90)%(P<0.01).The BrdU positive rate and cell viability of the Nrf2-siRNA group were decreased compared with the NC-siRNA group(P<0.01);After differentiation was induced by 1%fetal bovine serum,Tuj1 positive rate was decreased in Nrf2-siRNA group compared with NC-siRNA group(P<0.01);Meanwhile,the mRNA levels of both SOD1 and SOD2 in the Nrf2-siRNA group decreased compared with the NC-siRNA group(P<0.05),while ROS levels increased compared with the NC-siRNA group(P<0.05).After intracerebral injection of Nrf2 inhibitor Brusatol,Nrf2 protein expression levels in hippocampal tissue were lower than in the control group(P<0.01).Compared with control group,the number of Ki67+and DCX+cells were decreased in Brusatol group(P<0.05 and P<0.01);In addition,the mRNA levels of both SOD1 and SOD2 decreased in the Brusatol group compared with the control group(P<0.05),while ROS levels increased compared with the control group(P<0.05).Conclusion:Nrf2 gene knockdown resulted in increased levels of oxidative stress in NSCs,which in turn inhibited NSCs proliferation and differentiation ability.