Construction Of BFGF Gene Fluorescent Recombinatant Eukaryotic Expression Vector And Its Expression In Mesenchymal Stem Cells

Basic fibroblast growth factor(bFGF) is a kind of neurotrophic factor with many biologic activities. It can active the multiplication of cells coming from the ectomesoblast, the mesoblast and the endoblast, and it has close relation to the upgrowth of body. bFGF can be found in various tissues of body. It has important role in keeping the normal founctions of the central nervous system. In ischemic or traumatic conditions, bFGF can rehabilitate the damaged tissues, but the increase of bFGF in brain after the damage is not enough. So it is necessary to give the exotic bFGF to treat the diseases of central nervous system. However, the exotic bFGF is expensive, it is difficult to go through the brain-blood-barrier, and it will be degraded in the body quickly, which will limit the direct use of exotic bFGF in nervous system. So we will construct bFGF gene fluorescent recombinatant eukaryotic expression vector for gene therapy of central nervous system diseases.Now in the stem cell's study, mesenchymal stem cells (MSCs) is a hotspot. Thesecells can transdifferentiate into multiple cell lineages. The character of MSCs make them ideal engineering cells in cell and gene therapies such as the multipotention, easy isolation, cultivation and proliferation, low immunogenicity, easy be transfected and expression exotic gene and so on. In this study, MSCs of rat is used as engineering cells to express the bFGF.Objective:To construct bFGF gene fluorescent recombinatant eukaryotic expression plasmid and transfect it to MSCs for gene therapy of central nervous system diseases.Methods:(1) We established the focal cerebral ischemic model of mature Sprague-Dawley rat, took the ischemic tissues of brain quickly and amplified the coding sequence of bFGF by reverse transcription-polymerase chain reaction (RT-PCR).(2) The bFGF gene was cloned into pGEM -T Easy vector and was selected and identified by PCR amplication, restriction enzyme analysis.(3) The bFGF gene was subcloned into pEGFP -N3 plasmid by means of gene clone. And the recombinant vector was selected and identified by PCR amplication, restriction enzyme analysis and nucleotide sequence determination.(4) MSCs coming from rat were tuansfected with bFGF eukaryotic cell expression vector by lipofectamine mediated gene transfer. 18h after the transfection the expression was detected by green fluorescent protein, and 36h later the expression was detected by immunocytochemisty.Results:(1) Correct construction of pEGFP -N3-bFGF was identified by methods of PCR amplication, restriction enzyme analysis and nucleotide sequence determination.(2) 18h after the transfection, with the fluorescent microscope we can see green fluorescence. And 36h later immunocytochemisty showed that transfected cells expressed bFGF (brown staining ) protein.Conclusions:(1) The bFGF gene fluorescent eukaryotic expression plasmid was constructed successfully.(2) The constructed bFGF gene fluorescent eukaryotic expression plasmid can expresse bFGF in rat MSCs.(3) The present study provides the expremental evidences for the gene therapy of central nervous system diseases.