Hypoxic conditioned medium from rat cerebral cortical cells enhances the proliferation and differentiation of neural stem cells mainly through PI3-K/Akt pathways.
OBJECTIVE
To investigate the effects of hypoxic conditioned media from rat cerebral cortical cells on the proliferation and differentiation of neural stem cells (NSCs) in vitro, and to study the roles of PI3-K/Akt and JNK signal transduction pathways in these processes.


METHODS
Cerebral cortical cells from neonatal Sprague-Dawley rat were cultured under hypoxic and normoxic conditions; the supernatant was collected and named 'hypoxic conditioned medium' (HCM) and 'normoxic conditioned medium' (NCM), respectively. We detected the protein levels (by ELISA) of VEGF and BDNF in the conditioned media and mRNA levels (by RT-PCR) in cerebral cortical cells. The proliferation (number and size of neurospheres) and differentiation (proportion of neurons and astrocytes over total cells) of NSCs was assessed. LY294002 and SP600125, inhibitors of PI3-K/Akt and JNK, respectively, were applied, and the phosphorylation levels of PI3-K, Akt and JNK were measured by western blot.


RESULTS
The protein levels and mRNA expressions of VEGF and BDNF in 4% HCM and 1% HCM were both higher than that of those in NCM. The efficiency and speed of NSCs proliferation was enhanced in 4% HCM compared with 1% HCM. The highest percentage of neurons and lowest percentage of astrocytes was found in 4% HCM. However, the enhancement of NSCs proliferation and differentiation into neurons accelerated by 4% HCM was inhibited by LY294002 and SP600125, with LY294002 having a stronger inhibitory effect. The increased phosphorylation levels of PI3-K, Akt and JNK in 4% HCM were blocked by LY294002 and SP600125.


CONCLUSIONS
4%HCM could promote NSCs proliferation and differentiation into high percentage of neurons, these processes may be mainly through PI3-K/Akt pathways.
Shanghai Key Laboratory of Human Sport Competence Development and Maintenance, Shanghai University of sport, Shanghai, China.
Ming
M
Cai
Shanghai Key Laboratory of Human Sport Competence Development and Maintenance, Shanghai University of sport, Shanghai, China.
Yuehui
Y
Zhou
Department of Physiology, Brody school of medicine, East Carolina University, Greenville, North Carolina, United States of America.
Bin
B
Zhou
Shanghai Key Laboratory of Human Sport Competence Development and Maintenance, Shanghai University of sport, Shanghai, China.
Shujie
S
Lou
D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D000860:Hypoxia / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D000873:Anthracenes; D019208:Brain-Derived Neurotrophic Factor; D002867:Chromones; D017077:Culture Media, Conditioned; D009025:Morpholines; D042461:Vascular Endothelial Growth Factor A; C432165:pyrazolanthrone; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt