Studies On The Effect Of Notch1 Signal Pathway On The Proliferation Of Cerebrospinal Fluid-contacting Neurons

Objective:to explore whether Notch1 signaling pathways regulating erebrospinal fluid-contacting neurons?CSF-c Ns?proliferation.Methods:1.The peripheral nerve tissue of the upper central canal of the cervical spinal cord of C57BL/6 mice was extracted within 24 hours after birth,and the CSF-c Ns were sorted and purified by flow cytometry.2.The spheroidization of CSF-c Ns cultured in vitro was observed and passaged continuously.Immunofluorescence was used to detect the expression of neural stem cell markers in neurospheres.3.EDU detection kit,CCK-8detection kit and immunofluorescence labeled proliferation marker Ki67 were used to detect the proliferation ability of the third generation of neurospheres formed by CSF-c Ns.4.Western Blot was used to detect the expression of Notch1 signal pathway in CSF-c Ns.5.The CSF-c Ns suspended in vitro were divided into 4 groups.Group A:control group:serum-free nerve culture medium;Group B:DMSO group:serum-free nerve culture medium+0.05%DMSO;Group C:DAPT group:serum-free nerve culture medium+50 umol/L DAPT?0.05%DMSO configuration?;Group D:Jagged-1group:serum-free nerve culture medium+5 umol/L Jagged-1.The proliferation ability of neurospheres formed by CSF-c Ns in 4 groups was detected by EDU detection kit,CCK-8 detection kit and immunofluorescence labeled proliferation marker Ki67.Results:1.After flow sorting,the purity of CSF-c Ns was?94.5±2.03?%,and the survival rate was?93.82±2.58?%.2.Immunofluorescence showed that PKD2L1,a marker of CSF-c Ns,was highly expressed in neurospheres and co-expressed with Nestin,a marker of neural stem cells,and Ki67,a marker related to the proliferation of Sox2,cells.The positive rate of EDU staining was?42.32±1.80?%.There was no significant difference in OD value between 96h and 120h after CCK-8 staining?P=0.44?,but there was significant difference in OD value among other groups?P<0.05?.It is suggested that the neurospheres formed by CSF-c Ns have the characteristics of neural stem cells and good proliferation ability.3.The protein extracted from the neurospheres sample was detected by Western Blot,and the expression of Notch1,NICD and Hes1 protein was detected,which indicated that there was Notch1 signal pathway in the CSF-c Ns.4.After the addition of Jagged-1 to the neurospheres formed by CSF-c Ns,it was found that the expression of Notch1,NICD and Hes1 protein in the control group was higher than that in the control group(P_Notch1<0.01),indicating that Jagged-1 could activate Notch1 signal pathway.At 72h,96h and 120h after CCK-8 detection,the OD value of cells in Jagged-1 group was higher than that in control group(P_72h=0.03,P_96h=0.02,P_120h=0.01),and immunofluorescence showed that the A.U.of Ki67 protein in Jagged-1 group was higher than that in control group?P<0.01?;EDU detection showed that the positive cell rate in Jagged-1 group was higher than that in control group?P<0.01?.It was suggested that Jagged-1 can enhance the proliferation of CSF-c Ns.5.After the addition of DAPT to the neurospheres formed by CSF-c Ns,it was found that the expression of Notch1,NICD and Hes1 protein in the control group was lower than that in the control group(P_Notch1<0.01),indicating that DAPT could inhibit Notch1signal pathway.At 72h,96h and 120h after CCK-8 detection,the OD value of cells in DAPT group was lower than that in control group(P_72h<0.01;P_96h<0.01;P_120h<0.01),and immunofluorescence showed that the A.U.of Ki67 protein in Jagged-1 group was lower than that in control group?P<0.01?;EDU detection showed that the positive cell rate in DAPT group was lower than that in control group?P<0.01?.It was suggested that DAPT can inhibit the proliferation of CSF-c Ns.Conclusion:1.CSF-c Ns have the potential of neural stem cells in vitro.2.Notch1 protein is expressed in CSF-c Ns.3.Activating Notch1 signal pathway can enhance the proliferation ability of CSF-c Ns,and silencing Notch1 signal pathway can reduce the proliferation ability of CSF-c Ns.