Constrution And Expression Of Bfgf Molecules Mediated By Lentivirus Vector In Rat Bone Marrow Stem Cells

Objective To construct the recombinant lentirvirus vector carrying human basic fibroblast growth factor(bFGF) and to observe the expression of adenovirus transfected basic fibroblast growth factor in rat mesenchymal stem cells (MSCs).Methods huamn bFGF and FGF4 were obtained by usmg RT-PCR amplification,and then cloned into the pGC-FU vector,which contained green fluorescent protein (GFP). The resulting lentiviral vector containing FGF4 and bFGF was named pGC FU- FGF4-bFGF.After identificationb by PCR and sequencing,it was transfected into 293T cells by lipofectamine 2000 mediation, together with lentivirus-packaging plasmid pHelper 1.0 and pHelper 2.0. The newly constructed recombinant lentivirus and titration value were confirmed by Real time-PCR.Then to observe the expression of lentirvirus transfected basic fibroblast growth factor in rat mesenchymal stem cells. MTT assay detected the growth situation of Mesenchymal stem cells which were genetically modified and which were unmodified stem cellsResults1. Rat BMSCs at the passage 3 were positive for CD44 and CD90 (97.8%±0.9%,96.9%±1.5%,respectively), but negative for CD11b/c and CD34 (14.2%±0.7% and 1.3%±0.5%, respectively).2. The newly constructed recombinant lentivirus and titration value were confirmed by Real time-PCR and its titration value was 2.00E+9 TU/ml .3. After it was transfected into Rat BMSCs ,the gene was about 500bp that confirmed by RT-PCR .4. The expression of GFP was observed by using fluorescence microscope; Western blot showed FGF4-BFGF-GFP expression in the transfected cells and the protein was between 36～55KDr that almost coincided with the protein of FGF4-BFGF-GFP(21+28KDr=49KDr)(ps:the gene of constructed FGF4-BFGF-GFP was about 549bp).ELISA detection showed that the concentration of bFGF in the supernatant was obviously higher than those in the control group .5.The growth situation of Mesenchymal stem cells which were genetically modified,are much better than unmodified stem cells by the way of MTT assay.Conclusions The recombinant lentivirus carrying human FGF4 and bFGF was successfully constructed.The newly constructed lentivirus can produce a sufficiently high titration value in 293T cells and it was transfected into Rat BMSCs,providing a reliable tool for genetic modification of bone marrow stem cells in treatment of hind limb ischemia.