Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of thes...Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of these proteins in the genetic treatment of spinal cord injury.Methods:Full-length of GDNF cDNA(538 bp) and sTMFRⅠcDNA(504 bp) were inserted into the early 1 region of adenovirus genomic DNA respectively and were immediated by the human cytomegalovirus(gene promoter/enhancer). These adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo,then they were used to infect human neural stem ceils.The infection and expression of gene were tested under immunofluorescence.ELISA and Westem-blot after 48 hours.Results:Almost all the cultured cells showed the nestin immunofluorescence positive staining,which was the characteristics of neural stem cell.A great quantity of EGFP and KFP were observed in neural stem cells,which indicated the expression of GDNF and sTMFRⅠ.After transfection of GDNF and sTMFRⅠgenes,many neural stem cells show GFAP and tubulin immunofluorescence positive staining,which meant that most neural stem cells differentiated into neuron at that condition.Conclusions:The infective efficiency of adenovirus is greatly acceptable to neural stem cell,thus adenovirus provide a useful vector for exogenous GDNF and sTMFRⅠgenes expressing in neural stem cells,which is useful for differentiation of neural stem cell.展开更多
基金Shenzhen Science and Technology Project(No.201103061)
文摘Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of these proteins in the genetic treatment of spinal cord injury.Methods:Full-length of GDNF cDNA(538 bp) and sTMFRⅠcDNA(504 bp) were inserted into the early 1 region of adenovirus genomic DNA respectively and were immediated by the human cytomegalovirus(gene promoter/enhancer). These adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo,then they were used to infect human neural stem ceils.The infection and expression of gene were tested under immunofluorescence.ELISA and Westem-blot after 48 hours.Results:Almost all the cultured cells showed the nestin immunofluorescence positive staining,which was the characteristics of neural stem cell.A great quantity of EGFP and KFP were observed in neural stem cells,which indicated the expression of GDNF and sTMFRⅠ.After transfection of GDNF and sTMFRⅠgenes,many neural stem cells show GFAP and tubulin immunofluorescence positive staining,which meant that most neural stem cells differentiated into neuron at that condition.Conclusions:The infective efficiency of adenovirus is greatly acceptable to neural stem cell,thus adenovirus provide a useful vector for exogenous GDNF and sTMFRⅠgenes expressing in neural stem cells,which is useful for differentiation of neural stem cell.
