Research Of Glutamic Acid/NMDA1, Shh Signaling Pathway In Adult NSC Proliferation And Differentiation

Background In recent years, the repair of damaged brain tissue has been the focus and difficulty of research at home and abroad. The study found that Glutamic can promote neural stem cells (NSC:Neural Stem Cells) on the proliferation and differentiation, providing a new theoretical basis and ideas for the repair of damaged brain tissue. Cerebral infarction and brain tissue can lead to cell apoptosis after cerebral ischemia, then the amount of glutamic acid (glutamic acid) outflowed, causing high local Glutamic. Although high concentration of Glutamic increased the toxic effects on the organization, but it can promote the proliferation and differentiation of NSC. With the deepening of the research, we found that Glutamic mediated the proliferation and differentiation of NSC by its receptor and Shh (Sonic hedgehog) signaling pathway. Compared to previous cytology point of view, in recent years, the continuous progress of the molecular biology technology provided new methods and ideas for us. In this article, we further studied the proliferation and differentiation of NSC mediated by Glutamic and its receptor and Shh (Sonic hedgehog) signal pathway at the molecular level..Objective Age of SD rat hippocampus in vitro 3 generation of neural stem cells, use neural stem cells extended more than three generations to screen the optimal concentration of purpose gene. Join in three generations of neural stem cell gene. After observing Shh gene silencing, under the Glutamic acid stimulation neural stem cells in the expression of nesting, Shh signaling pathways proliferation capacity of neural stem cells, at the same time after blocking upstream Glutamic NMDA receptors, under the Glutamic acid stimulation, in neural stem cells Shh, Nestin mRNA and protein expression changes and their effects on neural stem cells related to proliferation.Methods1.5 um Glutamic was added in rat neural stem cells, then, mRNA and protein expression of Shh were detected by fluorescence quantitative PCR and Western blot;2 The Shh-shRNA plasmid was transfected into rat neural stem cells, then 5 um glutamic acid was add after screening positive NSCs, at last mRNA and protein expression of Shh and Nestin were detected by fluorescence quantitative PCR and Western blot;3 After Glutamic receptor inhibitor Conantokin NMDAR1-R blocking in rat neural stem cells,5 um Glutamic acid was added, then mRNA and protein expression of Shh and Nestin were detected by fluorescence quantitative PCR and Western blot;Results1. Due to Shh-shRNA plasmid with green fluorescent protein (eGFP) gene, transfection after 24 hours, cells were observed under fluorescence microscope. A small amount of positive cells showed green fluorescence were observed. Most of the Shh-shRNA plasmid transfected cells were screened by puromycin after 48 hours and a large number of positive cells were viable.2. Real-time fluorescent quantitative PCR detection results showed that amplification and melting curves of Shh and Nestin are specific, without no specific amplification. Compared with normal culture groups of neural stem cells, expression of shh and nestin were significantly increased(P< 0.05) in 5 nmol Glutamic acid groups. There were no significantly increased(P> 0.05) in Conantokin-R groups. In Shh shRNA groups, shh was significantly suppressed (P< 0.05), but nestin was not significant (P>0.05). In NC-shRNA groups, shh and nestin were significantly increased (P<0.05).3. Compared with the normal culture of neural stem cells, Western blot results showed that protein expression of Shh and Nestin were significantly increased (P< 0.05) in 5 uM Glutamic acid groups. No significances (P> 0.05) were observed in Conantokin-R groups. Protein Shh was obviously suppressed (P< 0.05) in Shh shRNA groups, while the protein Nestin was not obviously influenced (P> 0.05). NC-protein of Shh and Nestin were significantly increased (P< 0.05) in NC-shRNA groups.ConclusionsShh signaling pathways and NMDA receptors played an important role in neural stem cells and Glu can activate Shh signaling pathway by NMDAR1,resulting in stimulation the proliferation of NSC.