Notch1 signal pathway promotes the proliferation of cerebrospinal fluid-contacting neurons in vitro

English Keywords:Cerebrospinal fluid-contacting neurons  Neural stem cells  Notch1 signal pathway  Proliferation  Rat

English Abstract:

  【Abstract】 Objectives: To explore whether Notch1 signaling pathways regulate the proliferation of cerebrospinal fluid-contacting neurons(CSF-cNs). 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-cNs were sorted and purified by fluorescence-activated cell sorter (FACS). CSF-cNs were cultured in suspension and subcultured. (2)The CSF-cNs was passaged to the second generation, and the CSF-cNs' optical density(OD) value was detected by CCK-8 at 0h, 24h, 48h, 72h, 96h, and 120h. After 5 days of culture, the co-expression of CSF-cNs specific marker polycystic kidney disease-2-like-1(PKD2L1) and neural stem cell markers(Nestin and Sox2), and proliferation marker(Ki67) was detected by immunofluorescence. (3)The third generation CSF-cNs suspended in vitro were divided into 4 groups. Control group: serum-free nerve culture medium; Dimethyl sulfoxide(DMSO) group: serum-free nerve culture medium + 0.05%DMSO; (3, 5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester(DAPT) group: serum-free nerve culture medium + 50umol/L DAPT(0.05% DMSO configuration); Jagged-1 group: serum-free nerve culture medium + 5umol/L Jagged-1. The OD value of CSF-cNs was detected by CCK-8 at 0h, 24h, 48h, 72h, 96h, and 120h. After 5 days of culture, the expression of PKD2L1, Notch1, NICD, Hes1, and βactin proteins was detected by Western Blot. The fluorescence intensity arbitrary unit(A.U.) value of Ki67 protein, a marker of cell proliferation, was detected by immunofluorescence. Results: (1)After FACS, the purity of CSF-CNS was (94.5±2.03)%, and the survival rate was (93.64±2.35)%. CSF-cNs can be continuously passaged down and form the neurospheres. (2)The OD value of the second-generation CSF-cNs was statistically different at 0h, 24h, 48h, 72h, and 96h(P<0.05), but there was no significant difference between 96h and 120h(P=0.44). PKD2L1 can be co-expressed with Nestin, Sox2 or Ki67 in CSF-cNs. (3)There was no statistical difference in the expression of PKD2L1 protein among different groups(P=0.27). In Jagged-1 group, the expression of Notch1, NICD and Hes1 proteins was higher than control group(P<0.01). At 72h, 96h and 120h, the OD value was significantly higher than control group(P72h=0.03, P96h=0.02, P120h=0.01). The A.U. value of Ki67 protein was significantly higher than control group(P<0.01). In DAPT group, the expression of Notch1, NICD and Hes1 proteins was lower than control group(P<0.01). At 72h, 96h and 120h, the OD value was significantly lower than control group(P<0.01). The A.U. value of Ki67 protein was significantly lower than control group(P<0.01). There was no statistical difference in all indexes between the DMSO group and the control group(P>0.05). Conclusions: Activating Notch1 signal pathway can enhance the proliferation ability of CSF-cNs, while silencing Notch1 signal pathway can reduce the proliferation ability of CSF-cNs.