Experimental Study On Human Umbilical Cord Mesenchymal Stem Cells Transfected By Lentiviral Expression Vector Carrying Recombinant Human Insulin Gene

Experimental study on human umbilical cord mesenchymal stem cells transfected by lentiviral expression vector carrying recombinant human insulin geneCandidate master:XUE MeisiTutor:LIU YiCenter of burns and plastic surgery of CPLA,Lanzhou general hospital, lanzhou command, lanzhou 730050, ChinaObjective:1, To explore isolation, and purification culture conditions of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, and its biological characteristics; 2,To experimentally construct containing human recombinant insulin (insulin) and enhanced green fluorescent protein (EGFP) gene lentiviral vector pLenti6.3-insulin-IRES-EGFP, and carry out packaging, purification, concentration, and titer determination of slow virus-like particles; 3 To regard GFP as a reporter gene, screening Lentiviral transfection optimal MOI value so as to further carrying exogenous gene transfection to explore conditions; 4 To study expression of genes and protein levels, carrying recombinant human insulin Lentiviral transfection transfected human umbilical cord mesenchymal stem cells,in order to further body planting research.Method:1, removing arteries and veins of fresh umbilical cord tissue, cuting small pieces of culture (1cm x 1cm x 1cm), obtained after 1-2 weeks culture adherent cells were observed under inverted phase contrast microscope, cell morphology, cell count drawn growth curve; applicating of flow cytometry identified cell surface markers; staining in vitro-induced adipogenic and osteogenic differentiation capacity; 2, applicatiing PCR amplified insulin gene sequences in the target gene plus the upstream BamHI, downstream AscI two restriction sites; PCR products were cloned T vector and transformed into Competent DH5a cells, by means of screening recombinant plasmid pMD18T-insulin, correct sequencing of the selected clone sequences extracted plasmid; restricting endonuclease digested pMD18T-insulin, respectively, and pLenti6.3-IRES-EGFP plasmid, the insulin gene fragment and pLenti6.3-IRES-EGFP vector ligation, transformation into the feeling state of DH5a cells, by means of screening lentiviral expression vector pLenti6.3 insulin-IRES-EGFP, and sequenced. In the amount of extraction lentiviral vector, lentiviral vector will be transfected 293T cells, packaging the virus, purification and concentration, and determination of virus titers.3 To regard GFP as a reporter gene, MOI values 0,1,3,5,7,10,15,20 transfected cells were observed for 48 hours,72 hours, 96 hours, fluorescence expression, screening lentivirus transfection human umbilical cord mesenchymal stem cells, optimal transfection time and MOI values; 4, transfecting human umbilical cord mesenchymal stem cells, applicating qPCR and RT-PCR method detection recombinant human insulin gene expression, applicating westen bloting detect protein expression of insulin gene.Results:culturing 1 week in vitro, cells climbing from the tissue; cells of cultured 10 generations have no significant change in shape and value-added; cells positive expression of CD44, CD106, while CD34, HLA-DR expression was negative. In vitro experiments confirmed that hUCMSCs with adipogenic and osteogenic differentiation capacity.2, polymerase chain reaction to obtain the length of 347bp with BamHI and AscI restriction sites of two target gene sequences, connecting the cloning vector pMD18-T vector and lentiviral vector pLenti6.3-IRES-EGFP, building lentivirus expression vector pLenti6.3-insulin-IRES-EGFP. Successful packaging, purification and concentration and determination of virus titers.3, when the transfection complex (MOI) value is 10 recombinant lentiviral vector transfection efficiency was high, the transfection efficiency can reach 90%. Applicating qPCR and RT-PCR method to expression of recombinant human insulin gene, confirming the level of gene expression, transfection group was positive, non-transfected group was negative. Applicating westen bloting to detect the protein expression of insulin, confirming transfected group cells and supernatants positive expression; expression of non-transfected group were negative. Conclusion:1, hUCMSCs can cultivate and expanse in vitro, the biological properties similar to bone marrow-derived mesenchymal stem cells; 2, successfully constructing lentiviral vector pLenti6.3-insulin-IRES-EGFP, packing lentiviral particle purification and determing virus titer; 3, Screening to determine the optimal MOI= 10, carrying recombinant human insulin gene transfection hUCMSCs slow virus particles, its gene expression and protein levels of insulin.