| Objective To investigate the feasibility, safety, transfe ction efficiency, and delivery method of a novel Nanosphere com plexes-Based gene therapy, compared with the Cationic Lipid, an d plasmid DNA. Methods After Reconstructing plasmid pcDNA3.1/m yc-hisA-VEGF165, Non-virual carriers was used to transfect the VE GF165 gene into the mice Vascular Endothelium Cell line(NIH3T3)b y nanosphere and cation lipofectin.The expression of VEGF165 gen e was examined by immunohistochemistry and fluorescence-microsc opy and structurally morphological features of the transfected cells were also observed. Localization the transfected VEGF165 g ene and its expression product in the NIH3T3 cell line was exam ined through immunohistochemistry and Western blot assay. The 1 evel of time-course of transgene expression was measured by ELI SA. Cell proliferation assays (tetrazolium salt conversion to f ormazan colorimetric assay) were performed beginning 1-3 days a fter plating the VEGF gene was transfected into the mice fibrob lasts Cell line(NIH3T3) by nanosphere and cation lipofectin. R esults The selected positive cell clone by G418 system was con firmed to be VEGF165 highly expressing Vascular Endothelium Cell and the gene expression products were localized in the cytopla sm, by using immunohistochemical staining and fluorescence-micr oscopy techniques. It was found that nanosphere -complexes hada significantly inhibitory effect on nanosphere - NIH3T3 cell 1 ines in by using MTT method. The level of gene expression by nan osphere -complexes was detectable using ELISA assays, but no mor e than 4 weeks, duration of transgene expression was transient, and decreased over time , but 2-3 fold higher than Cationic Lip id in day 3-day 5. Conclusion Nanosphere complexes-Based gene d elivery system as non-virual vector may provide a novel gene t herapentic strategy for Vascular Reconstruction, can possibly ap ply for clinic usage.
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