Construction Of VEGF165Lentiviral Vector And Its Expression In Mesenchymal Stem Cells

Objective：In order to find new gene therapy of prevention of peridural fibrosis,We extract people of vascular endothelial growth factor165(VEGF165) genesequences,to construct the Lentiviral vector and infect mesenchymal stem cells.Use Western Blot technology to observe the expression of VEGF165in mesenchymalstem cells.Methods:(1).Extract VEGF165sequence: VEGF165sequence comes from NCBI(GeneBank:AF486837.1), then design primer attB1-k-VEGF165-F and VEGF165-attB2-R.(2).Construction of vector: Take the advantage of PCR method, mixed withprimer, DNA template, substrate and so on for reaction. Then we get the DNA ofVEGF165, and use Gateway technology clone VEGF165gene into entry vector.Finally clone it into destination vector. After positive selection, we get the expressionvector.(3).Lentiviral’s envelop: Collect293FT cells in good condition and logarithmicgrowth phase, counting cells and cultivate it overnight. Change the medium into newone and waiting for infection in the second day. Add DNA-Lipofectamine2000mixture, which contains pLV/helper-SL3, pLV/helper-SL4, pLV/helper-SL5, destinyvector, medium and so on into medium which contains293FTcell.Collect liquid andconcentrate, then add it into10ml fresh medium for further cultivate, recollect it after72hours of infection, divide and conserve in-80℃.Inspect titer.(4).Extract and culture of mesenchymal stem cell: Prepare a SD rat (male,4-5weeks old), collect the rat femurs and tibiae, change the medium every24h in the firstthree days in early culture. After that, we change the medium about5to7days a timesince the cell propagating slow in earlier stage. The prophase culture needs40to60 days and change medium5to7days a time. Waiting for mesenchymal stem cellpurified and subculture one generation, and use it to transfect.(5).Western Blot check: The samples which used to check are divided into threegroups: SD MSC, SD MSC/eGFP, SD MSC/VEGF165.After contract protein, we usenucleic acid protein detector to check the total concentration of protein andSDS-PAGE, then we going to do protein electrotransfer and memlbrane close.Antigen antibody reaction and color development are last procedures. We observe andtake pictures in the Fiuorchem HD2gel image analysis system.Results: Take the advantage of site-specific recombination technology, weconstruct the Lentiviral vector contained VEGF165successfully. After virus envelop,TCID of VEGF165group is4.7×10~7, while eGFP group is6.0×10~7.Transduction rateof VEGF165is40-50%while eGFP is more than90%after48h of transfection.Compare transfected and untransfect mesenchymal stem cell, transfected cell canexpress VEGF165.Conclusion: constructed VEGF165Lentiviral vector transfect mesenchymalstem cell successful and can express VEGF165, which provide the basic theory ofgene therapy to prevent peridural fibrosis.