| Background:With the development of modern biotechnology and the improvement of the human gene pool, gene therapy offers a new approach for treatment of many human diseases after the exploration in the theory and practice. Gene therapy is an approach for treatment of diseases by inserting functional gene or other gene into patients in order to produce normal, none or low expression of exogenous gene. The basic criteria for gene therapy is the acquisition of the targeted gene, the selection of targeted cells and the delivery system of the targeted gene into the targeted cells. Gene delivery systems are classified into viral and non-viral systems. The non-viral system is an very important system for its safety and low immunogenicity. But how to improve the transfection efficiency is a very difficult problem. In this study we choose the gene gun as an delivery system in order to offer a convenient and efficient way.Objective:To synthesize dextran-spermine polycation (DSP)as a gene vector, amplifly and extract plasmid DNA pmcherry-cl and pEGFP-c3, investigate the methods of preparation of gene gun bullets based on DSP as gluing agent, compare with the gene gun and ordinary delivery systems of the transfection efficiency in vitro, compare with the DSP and spermidine as gluin agent effecting on the transfection efficiency in vitro, and evaluate the gene gun whether or not enhance the transfection efficiency.Methods:40 KDa Dextran was oxidized under room temperature for 6h using potassium periodate to obtain oxidized dextran, which was then reacted with spermine(1 to 1.25 mole ratio)to form imine-based polycation. The Schiff base polycation was reduced to the stable amine-based dextran-spermine polycation by sodium borohydride. The total nitrogen and primary amine content were determined by elemental analysis and TNBS, respectively. The chemical structure of DSP was elucidated by 1H-NMR and FT-IR.Plasmid pmcherry-cl with red fluorescent protein and plasmid pEGFP-c3 with green fluorescent protein was ampliflied and extracted by alkaline lysis. Firstly, the plasmid were inserted into the E.coli for amplifling, and then were extracted by alkaline lysis from the E.coli and purified using plasmid extraction kit in order to get rid of the cell debris and endotoxin, eventually measured by UV and identified by agarose gel electrophoresis (AGE).The gene gun bullets which used pmcherry-cl as reported gene were based on DSP as gluing agent instead of spermidine and breast cancer cells (MCF-7) as the targeted cells, and the transfection efficiency were examined by fluorescence microscopy after transfection, investigated the influence on the cell transfection efficiency and cell survival rate of the gene gun-mediated gene transfection, simultaneously the central composite design-response surface methodology was used to optimize the conditions of delivery. The cytotoxicity of polyplexes was evaluated by MTT assay, with the spermidine for the control. Further more, the gene gun bullets constituted of DSP were cotransfected with plasmid pmcherry-cl and PEGFP-c3 compared with that of constituted of spermidine.Results:The aldehyde content of oxidized dextran was 7.27 mmol/g and the content of grafting of DSP was 0.0214mg/ml.The purity (ratio between 1.8 to 2.0) and the content of plasmid DNA pmcherry-cl and pEGFP-c3 (A260/A280) was 1.94,1.92, 492.8ug/ml,498.1ug/ml,respectively.The transfection efficiency and cell survival rate was 14.8% and 83.2% which based on DSP as gluing agent and transferred by chemical methods. The preparation of gene gun bullets used the plasmid pmcherry-cl as reported gene and the synthetized DSP as gluing agent instead of spermidine. The transferred conditions of gene gun- mediated gene transfection were optimized by the central composite design-response surface methodology, the results were:the content of tungsten in each bullet was 1.0mg; the content of plasmid DNA was 1.2ug; the pressure of the gene gun was 1.5kpa; the gene gun bombardment distance was 2cm. Based on these conditions, the transfection efficiency and the cell survival rate was 22.1% and 52.2% which with the absolute deviation of 8.3% and 6.1% compared with predicted model value. In the same conditions, the transfection efficiency and the cell survival rate based on spermidine as the gluing agent was 15.8% and 54.5%,respectively.the transfection efficiency of plasmid pmcherry-cl and pEGFP-c3 was 21.0% and 19.9%,and the cotransfection efficiency of the plasmid pmcherry-cl and pEGFP-c3 that prepared by DSP was 23.0%.However,the transfection efficiency of plasmid pmcherry-cl and pEGFP-c3 that prepared with spermidine was only 15.5% and 15.2%,and the cotransfection efficiency was 17.9%.Conclusion:This research indicates that the gene gun bullets prepared with DSP showed a better transfection efficiency in vitro compared with that prepared by spermidine. and chemical method Further more, the cotransfection with plasmid pmcherry-cl and pEGFP-c3 that prepared with DSP was also better than that prepared with spermidine. The central composite design-response surface methodology was well applied to the optimization of the conditions of delivery, it was feasible for description of the relationship between the factors and effects.The synthesized dextran-spermine polycation (DSP) was easily prepared as the gluing agent of gene gun bullets and shows low toxicity. |
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