Establishment and characterization of constitutively expressed BDNF in genetically modified 3T3 cell line

• Main Authors: Tsung-Yi Lin, 林宗逸
• Other Authors: Chung-Liang Chien
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/01968570373489628636

碩士 === 國立臺灣大學 === 解剖學研究所 === 94 === Brain-derived neurotrophic factor (BDNF) influences almost all aspects in central nervous system (CNS). Recently, BDNF was discovered as an inducer for adult stem cells and considered as a potential therapeutic agent for the brain injury. However, there are several drawbacks about using injections or minipumps for long-term BDNF treatment. Alternatively, cell mediated BDNF delivery could be one of solutions for it. In this study, we transfected cDNAs of mouse BDNF and enhanced green fluorescent protein (EGFP) into Swiss albino mouse 3T3 cell line. After selection of neomycin analogue G418, two stable 3T3-BDNF-EGFP cell clones constitutively expressing BDNF and EGFP independently were established. Stable 3T3-BDNF-EGFP cell clones were analyzed by immunocytochemistry, Western blot, and Enzyme-Linked Immunosorbent Assay (ELISA). Besides, the functional test of secreted BDNF activity was also assayed via the viability of chicken DRG neurons. Immunostaining patterns and Western blots of 3T3-BDNF-EGFP cells that we obtained showed more BDNF products than controls. In the ELISA study, the amount of BDNF secreted from 3T3-BDNF-EGFP cells (339 ± 50 pg/ml) was also showed significantly larger than others. Furthermore, we found the higher survival rate in the functional assay of embryonic chicken DRG neurons with BDNF secreted from 3T3-BDNF-EGFP cells. From the present study, we conclude that the stable 3T3-BDNF-EGFP cells could constitutively express functional BDNF in vitro. These BDNF-enriched cells with strong green fluorescence will be useful for further studies, such as the intracerebral cell grafting for brain injuries.