ABSTRACT
BACKGROUND: It is one of hot topics for the application of neurotrophic factors including neurenergen-3 to promote peripheral neural regeneration nowadays; however, clinical application is restricted to safety and effective administration route. Gene transfection brings a novel thinking and pathway for neurotrophic factors used in clinic.OBJECTIVE: To observe the expression of neural stem cells modified by neurenergen-3 gene after transfection.DESIGN: Completely randomized study.SETTING: Department of Traumatic Orthopaedics and Hand Surgery, the First Affiliated Hospital, Guangxi Medical University;Department of Hand Surgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Three healthy SD rats of four months old and either gender were selected from Animal Center, Tongji Medical College, Huazhong University of Science and Technology. Recombinant adenoviral expressing vector for transfection of neural stem cells was constructed in Laboratory of Orthopaedics, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, and the concentration was 0.15 g/L.METHODS: The experiment was carried out in the Orthopaedic Laboratory and Central Laboratory, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology from December 2002 to March 2004. Recombinant plasmid pAD-neurenergen-3 containing with green fluorescent protein (GFP) gene was transfectd into primarily cultured neural stem cells by using cationic tiposome interventional method. At 72 hours after transfection, fluorescent inverted microscope was used to observe GFP expression in neural stem cells, and transfection efficiency was measured simultaneously. Expression and transcription of neurenergen-3 gene in neural stem cells were detected at 72 hours, 1 and 5 weeks after transfection by using immunocytochemical stain and reverse transcription polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: Expression of neurenergen-3 gene in transfected neural stem cells.RESULTS: ① Neural stem cells transfected by recombinant plasmid pAD-neurenergen-3: GFP expressed on partial neural stem cells at 72 hours after transfection, and the transfection efficiency was 40%. Five weeks later, GFP expression was still observed. ②Transcription and expression of neurenergen-3 gene in neural stem cells: Transcription of neurenergen-3 mRNA was observed in neural stem cells at 72 hours, 1 and 5 weeks after transfection, and expression of neurenergen-3 mRNA was still observed in 5 weeks after trans fection.CONCLUSION: As the carrier of cationic liposome, neurenergen-3 gene can effectively transfect, culture and long-term express neural stem cells via the introduction of adenoviruses.