The Effect And Mechanism Of Involvement Of PTEN Silencing In The Regulation Of Neurite Outgrowth And Apoptosis Of Cortical Neurons

?Objective?This study screened for specific inhibitor targeted against the PTEN,mTOR and PI3 K respectively.We investigated the regenerative effects of these protein inhibited on axon regeneration and anti-apotosis after spinal cord injury(SCI).And we also investigated the function of PI3K-AKT-mTOR signal pathway during this proceduce.?Methods?1.The experiment separate,culture and purify the neuron from cerebral cortex of Wistar rats,and then we observe morphology of neuron in vitro,then we use the Immuno-cytochemical staining of anti-?-tubulin III(Nestin)to identify and analyse neuron.2.In this experiment,primary rat cerebral cortical neurons were separated.Then the neurons were plated on culture dishes which had been coated with poly-L-lysine(PLL).The neurons were cultured in plating medium for 4 hours,and serum-free medium for 7 days.Western blotting was used to analyse the expression of PI3K-AKT-m TOR signal pathway associated protein.3.Primary rat cerebral cortical neurons were separated and then plated on six well plates which both were coated with PLL,CSPGs,and laminin.The neurons were cultured in plating medium for 4 hours,and serum-free medium for 10 days.Immunofluorescent staining(IF)was performed to identify the neurites and CSPGs.The maximum axon length,the number of neurites per neuron,and the percentage of neurites that cross the CSPGs border was measured by Image-Pro Plus 6.0.4.Primary rat cerebral cortical neurons were separated.Then the neurons were plated on culture ninety six well plates and culture dishes which had been coated with PLL.The neuron were cultured in plating medium for 4 hours,then cultured in serum-free medium for 7 days.WB and Tunel were performed to identify the expression of cell apoptosis associated protein and the percentage of apoptosis.?Results?1.We successfully isolate and culture the neuron from cerebral cortex of Wistar rats,and we observe the morphology of the cells and use immunocytochemical staining to prove this.2.The function of specific inhibitor was proved by the WB.And the m TOR is a key factor.3.Neurons with PTEN inhibition gain significant longer neurites than other groups(p < 0.001).Meanwhile,it showed a stronger ability to cross the CSPGs border in bpv(pic)group(p < 0.001).4.Neurons with PTEN inhibition had a significantly high rate of cell viability.?Conclusion?This experiment proved that the PTEN inhibition can promote the neuron from cerebral cortex of Wistar Rat to regenerated longer neurites,and ability to cross the CSPGs border.It regulated through the PTEN-PI3K-AKT signaling pathway.The mTOR was an important factor in PTEN-PI3K-AKT signaling pathway.This study provided a certain amount of experimental evidence and theoretical foundation for further studing treatment of spinal cord injury.