Study On The Proliferation And Differentiation Of Neural Stem Cells Induced By IGFBP-2

Objective:To investigate the role of IGFBP-2 in the proliferation and differentiation of neural stem cells and its possible mechanism.Methods:1.Identification the expression of IGFBP-2 between olfactory ensheathing cells-conditioned medium(OCM)and ACss conditioned medium(ACM):OECs were isolated and cultured from the olfactory bulbs of 1-day-old postnatal mouse to prepare serum-free condition medium of OECs.ACss were isolated and cultured from the cortex of brain of 1-day-old postnatal mouse to prepare serum-free condition medium of ACss.SDS-PAGE electrophoresis and protein mass spectrometry were used to analyze the expression of IGFBP-2 between ACM and OCM.Confirm the results by using Western Blot and Elisa kit.2.Exogenous IGFBP-2 promotes NSCs proliferation:the third generation C17.2,when cell growth to 80%fusion use,according to different concentration of IGFBP-2 3 concentration group and 1 control group,the concentration groups were added to a final concentration of 125 ng/ml,250 ng/ml,500 ng/ml IGFBP-2,the blank control group equal amount of PBS.after 5 min to detect the expression of protein kinase 1/2 and protein kinase 1/2 activation;cell growth was observed and counted after 5 d under the microscope,and cells were collected for Western Blot detection of proliferating cell nuclear antigen(Ki67,PCNA)expression situation,determine the change of the cell cycle by flow cytometry and calculate the concentration group the proliferation coefficient,MTT detection of cell growth curve.3.Exogenous IGFBP-2 affects the diff-erentiation in NSCs:The third generation C17.2,when cell growth to 80%fusion standby,this experiment was divided into 2 groups:induced by DMEM/F12 culture medium group;the OCM induction medium group.Each group within the subdivision of 3 concentration group and 1 control group,the concentration group respectively with a final concentration of 125 ng/ml,250 ng/ml,500 ng/ml IGFBP-2,the blank control group equal amount of PBS.induced after 5 d cells were collected for detection of Western Blot cells(Nestin),nestin glial fibrillary acidic protein(GFAP)and beta tubulin Ⅲ(TUJ-1)expression,immunofluorescence staining GFAP,TUJ-1 positive cells were counted by flow cytometry analysis,GFAP,TUJ-1 positive cells.Results:1.Analysis of the expression of IGFBP-2 OCM and ACM Western Blot OCM:the expression of IGFBP-2 protein in high expression than ACM.Electrophoresis and mass spectrometry analysis showed that OCM IGFBP-2 is about 2.3 times that of ACM,ELISA test results showed that the concentration of IGFBP-2 was 188+48.84 ng/ml in OCM,ACM concentration was 49.25+21.14 ng/ml;2.Effect of IGFBP-2 on proliferation of NSCs:with the increase of IGFBP-2 concentration,the number of C17.2 cells and significantly increased the cell viability,cell cycle test showed that 0 ng/ml group,125 ng/ml group,250 ng/ml group,500 ng/ml group,S phase cells accounted for(%):7.03 +0.59,8.76 +0.86,11.15 +0.56,13.10 +0.88,the proliferation index PI respectively(%):17.62+2.12,19.98+2.04,21.71+1.76,25.48+1.01.Western Blot results also confirmed that with the increase of the concentration of IGFBP-2,PCNA,Ki-67 expression increased;and found that C17.2 induced by IGFBP-2 after 5 min,no significant changes in the expression of t-ERK1/2,p-ERK1/2 expression increased rapidly.3.NSCs induced differentiation of IGFBP-2:The D/F12 culture medium group:Western Blot,flow cytometry analysis showed that the concentration of group GFAP,the expression of TUJ-1 was no significant difference;the OCM culture medium group:immunofluorescence counting revealed that the 0 ng/ml group,125 ng/ml group,250 ng/ml group,500 ng/ml group,the number of GFAP positive cells respectively(%):6.87+1.34,5.24+6.04,28.14+6.38,2.97+10.03;the number of TUJ-1 positive cells(%)were:44.00 +6.48,25.24 +5.85,20.25+7.59,11.50+4.13.Western Blot also confirmed that,with the increase of IGFBP-2 concentration,GFAP expression increased,TUJ-1 expression decreased gradually,each concentration group and the control group and the concentration between group differences were statistically significant.Conclusion:1.IGFBP-2 can promote the proliferation of C17.2,which is positively correlated with the concentration of IGFBP-2,and its mechanism may be related to the activation of ERK 1/2 pathway;2.IGFBP-2 had no effect on C17.2 differentiation;3.Under the condition of OCM induction,IGFBP-2 could promote the differentiation of C17.2 into glial cells,which was positively correlated with the concentration of IGFBP-2.