| Summary: | 碩士 === 長庚大學 === 生化與生醫工程研究所 === 99 === Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for gene therapy and transcient gene expression. Cationic carriers including polyethyleneimine, liposomes, and chitosan have been used to deliver plasmid DNA in vitro by condensing anionic DNA. Such non-viral vectors offer considerable advantages of safety and easy production over viral vectors. In this study, several polycations including poly-l-lysine, protamine sulfate, polyethylenimine (PEI), and oligochitosan (OC) were screened by using a statistical approach. Transfection efficacy, toxicity, and expression of green fluorescence protein in four cell lines were evaluated. Plasmid (pEGFP) containing green fluorescent protein gene was used as a reporter gene. The transfection efficacy and cell viability of the transfection vehicles were analyzed by using IN Cell Analyzer 1000 (GE Healthcare). We showed that polyplexes formed by PEI, OC, and plasmid could be efficiently delivered into the tested cells and elevate the fluorescent protein expression. By using the response surface methodology and steepest ascent technology, the optimal concentration of OC and PEI were found to be 28 mg/ml and 23 μg/ml, respectively. High doseage of PEI in transfection could significantly enhance cell toxicity. The introduction of OC in transfection could increase the transfection efficacy and protein expression and could reduce the toxicity of PEI. Additionally, the synergistic effects of PEI and OC were confirmed in CHO, Caco2, SK, and 3T3 cell lines. The detailed mechanism of PEI and OC on transfection is investigated herein. PEI/OC reagent could not further increase the uptake of plasmid complex into the cells as compared with the PEI reagent. PEI/OC could reduce the plasmid complex size and could protect the plasmid complex from DNase in the intracellular trafficking. Additionally, the toxicity of PEI could be reduced in four cell lines by supplement of OC. In conclusion, OC is a novel and potent reagent in transgene and transient expression.
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