Experimental Study On The Recombinant PcDNA₃-β-NGF Transfecting BMSCs Of GFP Transgenic Mice

Objective: To construct the eukaryotic expression vector of human nerve growth factor beta by applying β-NGF gene and to explore the new method of cultivating and purifying BMSCs of GFP transgenic mice for further observing the expression pcDNA₃-β-NGF in BMSCs of GFP transgenic mouse .Methods: Human β-NGF was amplified from mRNA of human brain tumor adjacent tissue by RT-PCR. The RT-PCR products and pcDNA₃ plasmid DNA were simultaneously digested by Hindâ…¢ and ApaI, then purified, ligased, and transformated to construct the recombinant vector pcDNA₃-β-NGF. The constructed recombinant vector which contains the inserted sequence was identified by the restricted enzymes and the sequence analysis.Bone marrow cells isolated GFP transgenic mice are directly planted in culture flask and exchanged the total volume medium in different time, then differently counted according to the cell types and examined by immunohistochemistry using the antibodies of CD₄₄, CD₄₅ and CD₅₄ in three days. Selecting cells exchanged the total volume medium in four hours, eight hours and twenty four hours are successively subcultured and in the fifth passage calculated the amplified folds and examined byimmunohistochemistry using the antibodies of CD44 ^ CD45 and CD54.The constructed recombinant vector pcDNA3- |3 -NGF was transfected into BMSCs in the fifth passage of GFP transgenic mouse . The BMSCs expressing NGF were established by G418 selection ,and the expression of NGF was confirmed by western blot and immunohistochemistry .Results: The result of RT-PCR product in the agarose gel elctrophrosis show that there had positive strap, which matched the expected size. Through digested and sequenced, the inserted DNA sequence was the gene of premature gene of $ -NGF. In time of primary culture , with the firstly exchanged medium time extending, the density of adhering to culture plastic cells increases accordingly, but the BMSCsproportion decreases.The purification of BMSCs is high in 2 4hours firstly exchanged medium time but the cell quantity low, and while the BMSCs cell quantity is high, the purification is low. Nevertheless, in 8 ^lOhours exchanged medium time, both of cell purification and cell quantity are high, and after subcultured, their amplification is better than others. After transfected the recombinant vector, BMSCs can expresss the NGF protein, as well as compared with the vector control and BMSCs no transfected that showed no expression or expressed relatively low protein.Conclusion: The recombinant vector pcDNA3- P -NGF has successfully constructed. The method of adhering to culture plastic in different time for cultivating and purifying BMSCs of GFP transgenic mice is effective. It is suitable to exchanged firstly total volume medium in 8—10 hours. The subcultured cell has amplying capacity and can probably be a seed cell for the research of tissue engineering and gene therapy. Theresults suggest that the recombinant expression vector could be expressed in BMSCs successfully. This study establishes the foundation for the research of combining cellular transplantation by BMSCs and gene therapy byP-NGF.