Transfection of basic fibroblast growth factor gene into rabbit bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Basic fibroblast growth factor (bFGF) has significant promotion effects on repair in trauma, but local application cannot play a role for a long time.OBJECTIVE: To observe expression of bFGF gene in rabbit bone marrow mesenchymai stem cells (BMSCs) following transfection.DESIGN, TIME AND SETTING: The cytogene in vitro observation was performed at the College of Life Science, Yunnan University from March to August 2009.MATERIALS: One Japan flap-eared rabbit was purchased from the Department of Animal, Kunming Medical College. pCDNA3.1plasmid (Invitrogen, USA) was used in this study.METHODS: Bone marrow was extracted from rabbit anterior superior iliac spine. BMSCs were harvested by the adherent method.Cells were digested and subcultured when 80% confluent. According to GeneBank bFGF cDNA sequence, gene was designed and synthesized. Following electropherosis, the gel was retrieved using xhol I, BamH I enzyme digestion. Restriction enzyme was used to perform enzyme digestion, electropherosis and gene recovery in PcDNA Vector plasmid. bFGF DNA was connected with PcDNA Vector plasmid. PcDNA-bFGF eukaryotic expression vector was constructed. Recombinant was transfected into rabbit BMSCs using liposome infection protocol, and stable transfected line was screened.MAIN OUTCOME MEASURES: BMSC surface antigen expression was measured. Western blot was utilized to determine the expression of target protein.RESULTS: Results of flow cytometry showed that cultured cells were positive for CD90 and CD44, but negative for CD45.Results of immunohistochemistry demonstrated that vessel-derived BMSCs were negative for CD34, but positive for CD44. In cell disruption liquid of bFGF-transfected BMSCs, a significant positive zone of hybridization was visible at M, 23 000. However, no positive band was found in protein from pCDNA3.1(-) blank vector-transfected BMSCs.CONCLUSION: The bFGF gene was successfully transfected into BMSCs, and this target gene can stably express.