Mechanisms Of Snail Regulation On The Spinal Cord Regeneration Of Gekko Japonicus Following Tail Amputation

Objective Cloning the full length cDNA of gecko snail family members,and characterizing their coding amino acid sequences;to examine the expression changes of snail family members during the spinal cord regeneration following the gecko tail amputation,and then,the colocalization of snails with the different nerve cells;we also attempt to understand the roles of snails on the colocalized nerve cells by tansfection method,focusing on the proliferation and migration of astrocytes,as well as the anti-apoptosis to the injured neurons;by using various techniques,we will elucidate the mechanisms of snail regulation on the cell events.The aim of this study is to unveil the mechanisms of snail regulation on the promoting the spinal cord regeneration of gekko japonicus..Method To obtain the full-length of snail cDNA in gekko japonicus by using Rapid Amplification of cDNA Ends(RACE)technique;The expression changes of snails in the spinal cord and their tissue distribution were examined by Real-time PCR,Western blot and immunohistochemistry;by construction of the lentivirus vectors of snail overexpression,the gecko astrocyte cell line GSN1,and neuron cell line SH-SY5 Y were thus transfected;the effects of snails on the proliferation and migration of astrocytes were examined by EDU,transwell or the wound healing experiments;the activation of small G-protein was detected by pull-down experiments according to the instruction of manufacturer;the roles of snail anti-apoptosis on the SHSY5 Y were examined by Western blot method.Result(1)We have cloned the full-length cDNAs of snail family members.The full-length cDNA of gecko snail1 is 1292 bp,encoding a protein of 259 amino acids;The full-length cDNA of gecko snail2 is 1959 bp,encoding a protein of 268 amino acids;and the full-length cDNA of gecko snail3 is 1590 bp,encoding a protein of 300 amino acids.(2)All the snail family members were ubiquitously expressed in the tissues examined,with high abundance in the ovary and moderate expression in the spinal cord;The expression of snail1,snail2 and snail3 were upregulated in the spinal cord following gecko tail amputation.(3)The results of immunohistochemistry demonstrated that all snail family members colocalized with astrocytes and neurons.(4)Overexpression of snail family members results in the promoting migration of astrocytes,but showing no effects on the proliferation of astrocytes.(5)Overexpression of snail family members can activate small GTPase proteins,cdc-42 and Rac1,which are involved in the regulation of cell migration.(6)Overexpression of snail family members in the astrocytes promoted the expression of epithelial-mesenchymal transitions(EMT)markers.(7)TGF?1 promoted the expression of snail genes in the astrocytes.(8)Overexpression of snail family members was able to promote the anti-apoptosis of injured neurons,Western blots have revealed that snail can increase the expression of the P-Erk,P-Akt and BCL-XL,and decrease the levels of Cleaved Caspase 3,which are involved in the regulation of apoptotic signaling.Conclusion1.We obtained the full length cDNA of gecko snail family members.2.Gecko snail family members play roles on promoting spinal cord regeneration.3.Gecko snail family members promote the migration of astrocytes by the activation of cdc-42 and Rac1.4.The overexpression of snail genes in astrocytes were involved in the EMT process.5.TGF-? activated the expression of snail genes in the spinal cord of gecko.6.Gecko snail family members can decrease the apoptosis of injured neurons following gecko tail amputation,by activating the expression of P-Erk,P-Akt and BCL-XL.