Establishment and expression of recombinant human glial cell line-derived neurotrophic factor and TNF α receptor in human neural stem cells
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Published on: March 17, 2023
Ke-Xiong Zhuang1, Wei Huang, Bin Yan
1Department of Orthopedic Surgery, Shenzhen Guanlan People's Hospital Affiliated to Guangdong Medical College, Shenzhen 518000, China. szns120@126.com
Objective:
To investigate the interference and expression of human glial cell line-derived neurotrophic factor (hGDNF) and soluble TNF alpha (sTNFR I) 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 (558 bp) and sTNFR I cDNA (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 cells. The infection and expression of gene were tested under immunofluorescence, ELISA and Western-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 RFP were observed in neural stem cells, which indicated the expression of GDNF and sTNFR I. After transfection of GDNF and sTNFR I genes, 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 sTNFR I genes expressing in neural stem cells, which is useful for differentiation of neural stem cell.
