Eukaryotic Expression Of BDNF Gene Of Rats And Committed Differentiation Effects Of BMSCs On Its Production

Brain-derived neurotrophic factor (BDNF) belongs to nerve growth factors.The relation between BDNF and development of the nerve system is very osculating. BDNF act on neuron's growth, multiplication, deposit and repairment. A large number of members of nerve growth factor family are cloned and used to cure nerve system disease recently.The objective is that BDNF eukaryotic expression plasmid pEGFP (N1) -BDNF is constructed by RT-PCR and gene cloned. The recombined pEGFP (N1) -BDNF plasmid is transfected into bone marrow stromal cells (BMSCs) with lipofectamine. We check up constructed gene engineering cell by fluo-microscope and immunocytochemitry. And we observe if BMSCs can transform nerve cell or neuroglial cells by its expressed BDNF protein stably.The construction of pEGFP (N1) -BDNF plamid: extracting total RNA from the brain of 2-3 days mouse as templates, the BDNF gene was cloned using RT-PCR and T-Vector cloning methods, the positive clone was identified by the restriction enzymes. After recombinant plasmid BDNF-T and plasmid pEGFP (N1) were digested by two restriction enzymes, the BDNF fragments were linked to the linear pEGFP (N1) by T4 DNA ligase, the eukaryotic expression vector (pEGFP (N1) -BDNF) was constructed. The positive clone was identified by the restriction enzymes and sequenced. Compared the cloned BDNF gene with the GenBank sequence, we demonstrate that both of sequence were from the initiation codon ATG to the termination TAG identically. The cloned mouse BDNF gene is 750 bp.The expression of pEGFP (N1) -BDNF in BMSCs: At first, the isolation and attachment culture of rat bone marrow stromal stem cells, and the expansion and purification of rat bone marrow stromal stem cells to provide higher purification seed cells for experiment, the recombined pEGFP (N1)-BDNF plasmid was transfected into BMSCs with lipofectamine. To observe transfected BMSCs and identify BDNF by immunofluorescence. Eighteen hour after transfected plasmid into BMSCs, we could see green fluorescence in pEGFP (N1) - BDNF groups and pEGFP (N1) empty carrier. In reverse fluomicroscope, GFP positive BMSCs is about 60 percent, transfected BMSCs is green fluorescence by immunofluorescence. Positive BMSCs is about 30 percent, BDNF can express protein in transfected cell. The immunocytochemitry of the results, transfected cell can express NSE.Conclusion is that the eukaryotic expression vector pEGFP (N1) -BDNF are constructed and confirmed with the restriction enzymes and sequencing. The recombined pEGFP (N1) BDNF plasmids are transfected into BMSCs using lipofectamine. We prove that transfected BMSCs can express BDNF protein by immunofluorescence. At the same time, The BMSCs express NSE. It indicate BMSCs can differentiate towards nerve cell by its self secretary BDNF protein.