摘要
BACKGROUND： Brain-derived neurotrophic factor （BDNF） provides nourishment to injured neurons. Neural stem cells can differentiate into neurons to repair neuronal injury in vivo. It has been hypothesized that continuous secretion of BDNF from neural stem cells could benefit brain injury repair. OBJECTIVE： To transfect BDNF and enhanced green fluorescent protein （EGFP） into neural stem cells with adenovirus vector and to observe expression of BDNF and EGFP in transfected neural stem cells. DESIGN, TIME AND SETTING： Observational, cellular, molecular study was performed at the Biochemistry Laboratory, Tongji University School of Medicine, China from July 2004 to September 2006. MATERIALS： Neural stem cells were provided by the Anatomy and Histoembryology Laboratory of Fudan University Medical School, China. METHODS： BDNF cDNA was extracted by reverse transcription polymerase chain reaction from the rat hippocampus. Following gene cloning and packaging by HEK293.BDNF, the EGFP gene was transfected into cultured neural stem cells with the Ad-EGFP-BDNF vector. BDNF-expressing neural stem cell clones were selected by G418 selection. MAIN OUTCOME MEASURES： EGFP expression and cell morphology were observed by fluorescent microscopy; neural stem cell expressing BDNF mRNA was examined by reverse transcription polymerase chain reaction; BDNF expression was detected by enzyme-linked immunosorbent assay from supematant of infected neural stem cells. RESULTS： High transfection efficiency was obtained using 5×10^8 virus titers to transfect neural stem cells. G418-resistant neural stem cell clones integrated BDNF mRNA fragments. Enzyme-linked immunosorbent assay results showed that BDNF expression in the supernatant increased with increasing culture time and peaked at 72 hours. CONCLUSION： Adenovirus-mediated BDNF and EGFP genes were successfully transfected into neural stem cells and were expressed in neural stem cells for a long period of time.
BACKGROUND： Brain-derived neurotrophic factor （BDNF） provides nourishment to injured neurons. Neural stem cells can differentiate into neurons to repair neuronal injury in vivo. It has been hypothesized that continuous secretion of BDNF from neural stem cells could benefit brain injury repair. OBJECTIVE： To transfect BDNF and enhanced green fluorescent protein （EGFP） into neural stem cells with adenovirus vector and to observe expression of BDNF and EGFP in transfected neural stem cells. DESIGN, TIME AND SETTING： Observational, cellular, molecular study was performed at the Biochemistry Laboratory, Tongji University School of Medicine, China from July 2004 to September 2006. MATERIALS： Neural stem cells were provided by the Anatomy and Histoembryology Laboratory of Fudan University Medical School, China. METHODS： BDNF cDNA was extracted by reverse transcription polymerase chain reaction from the rat hippocampus. Following gene cloning and packaging by HEK293.BDNF, the EGFP gene was transfected into cultured neural stem cells with the Ad-EGFP-BDNF vector. BDNF-expressing neural stem cell clones were selected by G418 selection. MAIN OUTCOME MEASURES： EGFP expression and cell morphology were observed by fluorescent microscopy; neural stem cell expressing BDNF mRNA was examined by reverse transcription polymerase chain reaction; BDNF expression was detected by enzyme-linked immunosorbent assay from supematant of infected neural stem cells. RESULTS： High transfection efficiency was obtained using 5×10^8 virus titers to transfect neural stem cells. G418-resistant neural stem cell clones integrated BDNF mRNA fragments. Enzyme-linked immunosorbent assay results showed that BDNF expression in the supernatant increased with increasing culture time and peaked at 72 hours. CONCLUSION： Adenovirus-mediated BDNF and EGFP genes were successfully transfected into neural stem cells and were expressed in neural stem cells for a long period of time.
基金
the Natural Science Foundation of Shanghai,No.04ZR14107
the Science and Technology Developmental Fund of Shanghai Railway Station,No.2003Y04
