[The effects of brain-derived neurotrophic factor gene-modified neural stem cells on primary cultured dorsal root ganglion cells in vitro].
OBJECTIVE
To gain stable genetic modification of neural stem cells (NSC) that constitutively secrete brain-derived neurotropic factor (BDNF) and to explore the biological role on the survival and neurite outgrowth of cultured dorsal root ganglion (DRG) neurons.


METHODS
BDNF gene fragment from human brain cDNA libraries was obtained by using PCR. With molecular cloning technique, the recombinant stem cell viral vector with report gene was constructed, which is that MSCV-BDNF-IRES(2)-EGFP vector could encode Polycistronic mRNA. Viral particle was packaged by PT67 cell line and infected neural stem cells (mouse clone: C17.2). After selection with cloning cylinder, the expression of BDNF was assessed by immunohistochemistry enzyme linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The effects of stable gene-modified neural stem cells on embryonic mouse DRG neurons were evaluated in a dual-chambered cocultivation system at 3th, 10th day.


RESULTS
RT-PCR analysis demonstrated expression of mRNA for human-BDNF. ELISA confirmed the presence of secreted BDNF 24 h after transfection and showed that the level of BDNF production by NSC-BDNF transfected C17.2 was at a rate of (14.6 +/- 0.8) ng x d(-1) x 10(-6) cells even after 3 months. With immunohistochemical analysis, compared with the control, the longer neurite outgrowth of cultured DRG cells and the more survival neurons were observed in NSC-BDNF transfected cells group.


CONCLUSIONS
BDNF gene could be stably expressed in C17.2 cell line by MSCV, and the BDNF gene-modified NSC markedly enhances the survival and neurite outgrowth of cultured DRG neurons.
Jiang
Hui
H
Department of Spine Surgery, Southern Medical University, Nanfang Hospital, Guangzhou 510515, China.
Jin
Da-di
DD
Qu
Dong-bing
DB
Wang
Chun-ting
CT
Ju
Gong
G
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis / Q000235:genetics*; D018929:Cell Culture Techniques; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D005727:Ganglia, Spinal / Q000166:cytology*; D006801:Humans; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
D019208:Brain-Derived Neurotrophic Factor