Experimental Study On The Transfection And Expression Of SOD1 Gene Mediated By Non-viral Vector In Vitro Cells

Object:To construction of eukaryotic expression vector of the human SOD1 (Cu/Zn superoxide dismutase, SOD1) and expression in HeLa cells.Methods:The open reading frame (ORF) of SOD1 was amplified from human peripheral blood by RT-PCR. TA cloning strategy was used to insert the target fragments into pUCm-T vector. The recombinant plasmid was identified and noted as pUCm-T-SOD1. Then, SOD1 was subcloned into pTracer-CMV/Bsd, a eukaryotic expression vector. The plasmid of pTracer-CMV/Bsd-SOD1 was sequenced and was introduced into Hela cells by LipofectamineTM 2000. The expression of green fluorescence protein (GFP) was observed by the fluorescence microscope. The expression of SOD1 was detected by western blot after screening by blasticidin for 4 weeks.Result:The eukaryotic expression plasmid pTracer-CMV/Bsd-SOD1 was successfully constructed. The GFP was observed in transfected cells by the fluorescence microscope. The expression of SOD1 was detected in transfected cells by western blot.Conclusion:The recombinant eukaryotic expression vector of pTracer-CMV/Bsd-SOD1 has been constructed successfully which could express GFP and SOD1, respectively, providing a tool for further gene therapy study. Chapter Two Surface modification and DNA-binding assessment of nano-hydroxyapatiteObject:To evaluate the impact of surface modification on the DNA-binding ability of nano-hydroxyapatite(nHA).Methods:Chemical co-precipitation-hydrothermal synthesis was utilized to prepare the nHA particles, and polyethylenimine (PEI) was used for surface modification of the nHA. Transmission electron microscopic (TEM) observation and zeta potential detection of the nHA were carried out before and after surface modification. The abilities of the nanoparticles, at different pH values and different concentrations, for DNA-binding and DNA protection against nuclease digestion were assessed before and after surface modification by electrophoresis.Result:TEM observation showed a short rod-like morphology of PEI-modified nHA with uniform particle size and good dispersion; the nHA without the modification tended to aggregate with poor dispersion. With a positive zeta potential, the PEI-modified nHA showed an obviously enhanced ability of DNA binding at different pH values and concentrations, with strong capacity to protect the DNA against Dnase I digestion. At the concentration of 250?g/mL and a pH value of 7.0, the nHA-PEI showed an optimal efficiency of DNA-binding and DNA protection.Conclusion:nHA with surface modification by PEI can serve as an effective vector for DNA binding and transfer. Chapter Three Transfection of SOD1 gene in HeLa cells mediated by nano-hydroxyapatite with PEI surface modificationObject:To investigate the efficiency of nano-hydroxyapatite with surface modification by Polyethylenimine (PEI) mediating SOD1 gene transfection in vitro cells.Method:Polyethylenimine (PEI) was used for surface modification of nHA(abbreviation:nHA-PEI);The cytotoxicity of nHA-PEI and nHA of without surface modification to Hela cells were assessed by the colony formation experiment. Transfection of SOD1 gene into Hela was mediated by nHA-PEI, nHA and LPTM 2000,and its efficiency of transfection was evaluated; The expression of SOD1 were further detected by RT-PCR and western-blot after transfecting Hela cells by mediating of nHA-PEI, nHA and LPTM2000.Result:The colony formation experiment showed the inhibition of nHA-PEI to Hela cells no obvious at concentration of 31.25?500?g/mL, more than 500?g/mL it would displayed obviously cytotoxicity, while nHA of without surface modification showed well biocompatibility and no cytotoxicity at the whole concentration ranges;The expression of green fluorescence protein (GFP) was observed by the fluorescence microscope, the efficiency of transfection of no modificated nHA showed much lower about 5%?8%,while along with the increasing of the concentration of modification,the efficiency of transfection of nHA-PEI also would increase and reach a maximum about 30%,but it was inferiored to the liposome about 80%; The expression of SOD1 in the nHA-PEI group was obviously higher than the nHA group by detectiong of RT-PCR and western blot,but its expression was lower than the liposome group.Conclusion:The appropriate surface modification of nHA by PEI can become a kind of effective vector for gene transfer, but currently the efficiency of transfectionis low, it needs to be further research.