Preliminary Study On The Expression And Function Of Salt-induced Kinase 1 In Neural Stem Cells

Background:Brain function and plasticity depend on the complex structure of neural circuits builting up during development.Neurogenesis in the mammalian brain is a highly precise and finely regulated process,which relies on a complex and precise regulatory network involving genetic,environmental,biochemical and physical factors.The proliferation and differentiation of neural stem cells（NSCs）is a key link in the process of neurogenesis,so it is of great significance to search for the regulatory mechanisms of the proliferation and differentiation of NSCs for the study of brain function and neurological disorders.In recent years,studies have reported that AMP-activated protein kinase（AMPK）family plays an important regulatory role in neurogenesis and affects the proliferation and differentiation of NSCs.Thus,the salt-inducible kinase 1（Sik1）of this family was originally found in the adrenal glands of rats on a high Salt diet,and its current research is limited to renal tissue,macrophages and tumor cells,and there has not been any relevant literature on the association between Sik1 and NSCs.Objective:This study focused on the spatial distribution of the expression of Sik1 in the brain of mouse,especially the expression of Sik1 in NSCs to further explore the regulatory role of Sik1 in the process of proliferation and differentiation of NSCs.Methods:1.To explore the expression pattern of Sik1 in the nervous system:1)The m RNA was extracted from the organs of seven-day-old C57BL/6mice and reverse transcribed into c DNA for RT-PCR;2)The m RNA of olfactory bulb,cortex,hippocampus,striatum,thalamus,midbrain and cerebellum was extracted from the brain tissues of adult C57BL/6 mice.And the m RNA was reverse transcripted for RT-PCR;3)The tissue of mice at different developmental stages（E14,E16,E18,P0,P3,P7,P14,1M,2M）was extracted and the m RNA was extracted.After reversing,RT-PCR and Real-time PCR were used for study;4)The co-labeling of Sik1,GFAP,SOX2,Nestin,MCM2and Brd U in NSCs cultured in vitro and adherent cells were observed by fluorescence multi-target method.2.The proliferation of NSCs cultured in vitro from Sik1 knock out mice:1)The diameters of the neurospheres were measured;2)Brd U was exogenously added into the medium of NSCs cultured in vitro in the control group and the knocking out of Sik1 group.After a period of time,the positive rate of Brd U was compared.3.The differentiation of NSCs from Sik1knock out mice in vitro:By inducing differentiation of cultured NSCs in vitro,the ratio of GFAP⁺/Hoechst⁺and Tuj1⁺/Hoechst⁺in NSCs of control group and the knocking out of Sik1 gene group was compared.4.The proliferation of NSCs in the DG region of Sik1 knock out adult mice:Sik1^+/+and Sik1^-/-mice were intraperitoneally injected with Brd U every 2 hours for 7 times and were sacrificed 2 hours after the last injection.The frozen sections were dehydrated and fixed for immunofluorescence staining,and then the number of Brd U and Ki67 positive cells was compared after knocking out of Sik1 gene.Results:1.The expression pattern of Sik1 in the nervous system:Sik1 is richly expressed in brain tissues,including olfactory bulb,hippocampus,striatum,thalamus and midbrain;The expression of Sik1 gene was significantly higher in embryonic stage than in adult stage,and reached the peak at P0,and then showed a downward trend until 2M;Sik1 was expressed in GFAP,Sox2and Nestin positive NSCs,as well as in MCM2 and Brd U positive NSCs.2.The proliferation of NSCs cultured in vitro from Sik1 knock out mice:When the number of experimental cases was 5,Statistics showed that the diameters of the neurospheres in the control group was 117.7±5.25μm,and that of the knocking out of Sik1 gene group was 116.1±3.75μm;When the number of samples was 6,the Brd U positive rate of the control group was 55.00±2.18%,and that of the knocking out of Sik1 gene group was 51.67±4.04%.3.The differentiation of NSCs from Sik1 knock out mice in vitro:When the number of experimental cases was 4,the GFAP⁺/Hoechst⁺ratio of control group was 35.11±6.74%,and that of the knocking out of Sik1 gene group was 28.32±3.91%;The Tuj1⁺/Hoechst⁺ratio of control group was 2.480±0.53%,and that of the knocking out of Sik1 gene group was1.97±0.60%.4.The proliferation of NSCs in the DG region of Sik1 knock out adult mice:When the number of experimental cases was 3,the number of Brd U positive cells in control group and the knocking out of Sik1 gene group was 245.7±26.10 and 179.7±28.72,respectively.The number of Ki67 positive cells in control group and the knocking out of Sik1 gene group was 315.7±15.59 and 266.7±15.19,respectively.Conclusion:Sik1 was richly expressed in the mouse brain;Sik1 was highly expressed in embryonic period and expressed in NSCs;After knocking out of Sik1 gene,there was no significant difference in the proliferation ability of cultured NSCs in vitro,and the differentiation ability of NSCs showed a downward trend.And the proliferation ability of NSCs in the hippocampal DG region of adult mice showed a downward trend,suggesting that Sik1 may paly a potential regulatory role in the proliferation and differentiation of NSCs.