Effects Of Hypoxia On Proliferation And Differentiation Of Rat’s Neural Stem Cells And Analysis Of Signaling Pathway

ObjectiveTo observe the effects of hypoxia on proliferation and differentiation of Neural StemCells which cultured under1%O2,4%O2and normal oxygen. Then role of PI3K,JNK and Notch on proliferation and differentiation of rat’s Neural Stem Cells innormoxia and hypoxia is further compared.Methods1. NSCSof newborn SD rat’s cerebral cortex in24h were isolated, then cultured48hours under1%O2,4%O2and normal oxygen. The number of neurospheres iscounted in24for analysis of effects of hypoxia on proliferation efficiency.2. NSCSof newborn SD rat’s cerebral cortex in24h were isolated. NSCScultured24h in vitro is transferred to adherent culture with polylysine in4%O2and normoxia.Then β-TubulinIII and GFAP was stained with immune fluorescence. Then number ofneurons and astrocytes is counted and its proportion is calculated respectively.3. NSCSof newborn SD rat’s cerebral cortex in24h were isolated. NSCSwerecultured48hours in Neurabasal which is added with inhibitor of PI3K, JNK andNotch in4%O2and normoxia. Then diameters of neurospheres were measured at24h/36h/48h for analysis of effects on hypoxia on proliferation rate.4. NSCScultured24h in vitro is transferred to adherent culture in4%O2andnormoxia. After differentiation medium is replaced, LY294002/SP600125/DAPT isadded into the medium andβ-TubulinIII and GFAP is stained with immunefluorescence. Then number of neurons and astrocytes is counted and its proportion iscalculated respectively.Results1. Effects of hypoxia on proliferation of NSCS.（1） Effects of hypoxia onproliferation efficiency. The number of neurospheres of1%group is significantlylower than the control group, and there is significant difference between the twogroups（P＜0.05）. The number of neurospheres of4%group is significantly higherthan control group, but there is not difference between the two groups.（2）Effects ofhypoxia on proliferation rate. The diameter of1%group is shorter than control groupin24h,36h and48h. There are significant difference between the two groups in24hand36h（P＜0.05）,and extremely significant difference between group in48h（P＜0.01）.The diameter of4%group is longer than control group in24h,36h and48h.There are significant difference between the two groups in36h and extremelysignificant difference between group in48h (P＜0.01).2. Effects of hypoxia on differentiation of NSCS.(1) Proportion of neurons of groupof1%O2was higher than its control group（P＜0.01）, while proportion of astrocytesof group of1%O2was lower than its control group（P＜0.01）.(2) Proportion ofneurons of group of4%O2was higher than its control group（P＜0.01）,while proportion of astrocytes of group of4%O2was lower than its control group（P＜0.01）.3. Effects of inhibitor of PI3K, JNK and Notch on proliferation of NSCS. underhypoxia and normoxia. Diameters of neurospheres treated with LY294002(inhibitor ofPI3K/Akt), SP600125(inhibitor of JNK), DAPT(inhibitor of Notch) under normaloxygen were smaller than those of DMSO group in normal oxygen in36h and48h. In36h and48h, diameters of neurospheres treated with LY294002/SP600125have nosignificance compared to DMSO group, but Diameters of neurospheres treated withDAPT were much smaller than DMSO group.4. Effects of inhibitor of PI3K, JNK and Notch on differentiation of NSCS. underhypoxia and normoxia. In normal oxygen concentration，proportion of neurons N+LYwas lower than N+DMSO（P＜0.01）,while proportion of astrocytes was higher thanN+DMSO（P＜0.01）.The proportion of neurons and astrocytes of N+SP600125andN+DAPT was no significant difference compared to N+DMSO; In hypoxia, theproportion of neurons and astrocytes of H+LY294002and H+SP600125was nosignificant different compared to H+DMSO. The proportion neurons of H+DAPT wasextremely higher than H+DMSO（P＜0.01）, while there was no significance onastrocytes between the two groups.Conclusion1.4%O2promotes proliferation of NSCS, while1%O2inhibits proliferation ofNSCS.2. Both4%O2and1%O2promote the neuronal differentiation while inhibit NSCStoward astrocytes direction.3. PI3K/AKT, JNK and Notch signaling pathway all play important roles in theproliferation of NSCS, but Notch pathway mainly regulates the proliferation of NSCSinduced by hypoxia.4. It is suggested that PI3K/Akt mediates neuronal differentiation in normoxia,while hypoxia may promote the neuronal differentiation through Notch pathway.