Construction Of Eukaryotic Expression Vector PcDNA3.1-LIF PcDNA3.1-bFGF And Stable Expression Of The Vector In The Bone Marrow Derived Mesenchymal Stem Cells

It is an important task to promote peripheral nerve regeneration after injury in neuroscience. Recent technical progress of molecular biology,cell biology and microsurgery results in better outcome, but not all patients have satisfactory results. It is a nodus how to improve the recovery after nerve injury in peripheral nerve surgery.The objective task is to solve that bioprotein factor simply applied cure to peripheral nerve injury and surgery couldn't satisfy the patient's recovery of the never injury. To reconstitute the part of function and improve the past of never injury, the long time recovery make people suffering. The ideal of this experiment is to rebuilt the peripheral never injury and promote the recovery of never injury.Our experimental study were divied into three partsPart I: Construction of eukaryotic expression vector pcDNA3.1-LIF,pcDNA3.1-bFGF LIF/bFGF gene was cloned from pregnant uterine deciduas/placenta tissues by using RT-PCR method and was inserted into pcDNA3.1 to construct eukaryotic expression vector consisting of LIF/bFGF gene. Identified by restriction enzyme analysis and PCR amplification.Part II: MSCs(mesenchymal stem cells) derived from rat bone marrow were isolated and culture expanded efficiently in vitro and its biological features Ficoll separating medium was added to rat bone marrow mixed liquor that should be centrifuge at 2000rmp×15min soon after it was taken out, then absorb the white layer. Wash the cells twice with PBS and seed cells at a density of 1×106cells per well (6-well format) in 1ml of 15% DMEM of FBS medium. Incubate cells at 37℃in a CO2 incubator. Passage cell was seeded at a density 2～4×104/ml.Bone marrow mesenchymal stem cells (BMSCs) is one kind of adult stem cells that have self-renewal and multilineage differentiation potential. It has recently been reported that BMSCs can differentiate into neural cells, opening new frontiers in therapy for nerve injury. The morphology of BMSCs was observed with invert-microscope constantly. BMSCs' curve of growth was depicted and cell cycle was analyzed by flowing cytometry. BMSCs were induced to differentiate into osteoblasts and adipocytes in induction medium containing desamethasone,β-Glycerophosphate and horse serum. Then differentiated cells were identificated by AKP and oil O stain. Results showed BMSCs could be isolated and proliferated by density gradient centrifugation adherent method in vitro. The appearance of BMSCs were fibroblast-like cells and its growth curves was like shape of S. Cell cycle analysis showed that most of BMSCs were in G1/G0 phase. BMSCs can be differentiated into osteoblasts and adipocytes in vitro.Part III: LIF/bFGF was successfully expressed in BMSCsTransfected by Lipofectmine into BMSCs. The eukaryotic expression vector pcDNA3.1-LIF/pcDNA3.1-bFGF, which can be expressed stablely in BMSCs, has been successfully constructed. LIF/bFGF expression was analyzed using Western blot and RT-PCR. LIF/bFGF was successfully expressed in BMSCs.Conclude: LIF/bFGF gene was cloned from pregnant uterine deciduas/ placenta tissues by using RT-PCR method and was inserted into pcDNA3.1 to construct eukaryotic expression vector consisting of LIF/bFGF gene. The recombinant vector pcDNA3.1-LIF/ pcDNA3.1- bFGF was identified by restriction enzyme analysis and PCR amplification. Then transfected by Lipofectmine into BMSCs. LIF/bFGF expression was analyzed using Western blot and RT-PCR. LIF/bFGF was successfully expressed in BMSCs. The eukaryotic expression vector pcDNA3.1-LIF/bFGF, which can be expressed stablely in BMSCs, has been successfully constructed.