Research Of The Signal Conduct Mechanism And Effects Of Cerebral Crtex Cell’s Conditioned Mediums On NSCs’ Proliferation And Differentiation

Objective: The aim of this study was to analysis the effects of different hypoxicconditioned mediums-cultured cerebral cortex cells on the proliferation anddifferentiation of neural stem cells (NSCs) and to clarify the PI3-K and JNKsignaling pathways transduction mechanism on the two processes.Methods: This research was mainly divided into five parts.⑴Prepared thehypoxic conditioned mediums: Separated and primarily cultured SD rat cerebralcortex cells, then made the cells inoculated in the24-well plates with the densityof(6.5‐7.0)×105cells/ml which were coated with0.01%poly-L-lysine in advance,and changed the full medium after5days. Then the cells were cultured inenvironments of4%O2、1%O2and normal oxygen concentration, respectively, for6hours. The culture mediums were collected and centrifuged as the hypoxicconditioned mediums (HCMs) and normoxic conditioned medium (NCM).⑵Observed and analyzed the influences of different HCMs on the NSCs，proliferation.The cerebral cortex cells of newborn24h rats were separated and respectivelysuspension cultured with HCMs and NCM in the normoxic environment with thedensity of (6.0‐6.5)×105cells/ml in the48-well plates. The experiment groups werecultured with HCMs, and the control group was cultured NCM. Counting the amountof neurospheres to analyze the proliferation efficiency and measuring theneurospheres，diameters at24h、48h and72h respectively to analyze theproliferation speed. Immunohistochemical staining was finished after suspensionculture72h for identification of NSCs in the neurospheres.⑶Observed andanalyzed the influences of different HCMs on the NSCs，differentiation. First,neurospheres were cultured with serum-free Neurobasal medium which was addedinto2%B27and20ng/ml bFGF, then they were transferred to the48-well platescoated with0.01%poly-L-lysine after48h and changed the full medium. Theexperiment groups were cultured with HCMs, and the control group was culturedwith NCM. Double immunofluorecence staining was used after adherence culturing48h to identify neurons and astrocytes in the progeny of NSCs.⑷Analyzed theinhibitors of LY294002and SP600125in the4%HCM to the influence of NSCs，proliferation. The separation method was the same to⑵. The experiment wasdivided into four groups:①HCM group;②DMSO group (DMSO was addedinto the HCM and keep the ultimate density was0.5%);③LY294002group；④SP600125group. The two inhibitors before using were diluted with DMSO, thenwere added into the HCM with the ultimate density10μmol/L, DMSO ultimate density was0.5%. The methods of culturing cells, counting the amount andmeasuring the diameters of neurospheres was the same to⑵.⑸Analyzed theinhibitors of LY294002and SP600125in the4%HCM to the influence of NSCs，differentiation. The methods of separating NSCs, culturing cells and counting werethe same to⑶. The method of experimental group was the same to⑷. Each groupof⑵、⑶、⑷was set3duplication wells. All of the cells were placed in the cellculture chamber of37℃、5%CO2、95%air. The experiment was repeated3timesindependently.Results:1. The influences of different HCMs on the NSCs，proliferation:①Theproliferation efficiency of4%HCM and NCM was higher than1%HCM (P<0.01),but there was no difference in the first two groups;②The proliferation speed oftwo HCMs groups was faster than the NCM group (P<0.01), but the speed of4%HCM was the fastest.2. The influences of different concentrations HCM on the NSCs，differentiation:NSCs in the cerebral cortex could differentiate into β-TublinIII immunoreactiveneurons and GFAP immunoreactive astrocytes in three conditioned mediums, andthe neurons proportion in progeny of NSCs was higher than astrocytes in all threegroups. The proportion of neurons in4%HCM was higher than in NCM (P<0.01).But the proportion of neurons in1%HCM was less than that in NCM (P<0.01).3. The influences of the two inhibitors of LY294002and SP600125in the4%HCMon NSCs，proliferation: The two inhibitors both inhibited NSCs proliferation whichwas induced by4%NCM (P<0.01), but the inhibitory effect of LY294002wasstronger than SP600125(P<0.01).4. The influences of the two inhibitors of LY294002and SP600125in the4%HCMon NSCs，differentiation: The both LY294002and SP600125inhibited NSCs todifferentiate into high proportion neurons induced by4%NCM (P<0.01), but theinhibitory effect of LY294002was stronger than SP600125(P <0.01).Conclusion: Cerebral cortex cells4%HCM can promote NSCs proliferate speedand induce NSCs to differentiate into more neurons, PI3-K signaling pathwaymainly mediate the two processes.