| Summary: | 碩士 === 國立成功大學 === 臨床藥學研究所 === 93 === Gene therapy refers to the transfection of DNA encoding a therapeutic gene into the targeted cells or organs with expression of the transgene. Recently, gene therapy has received attention due to its potential application in the replacement of dysfunctional gene and treatment of acquired diseases. The electronegativity of the naked DNA tends to inhibit itself from entering most negatively-charged cell membrane. Moreover, the unprotected DNA is rapidly degraded by nuclease. The most important issue in gene therapy is to develop a safe and effective gene transfection system. Several methods have been developed to enhance the transfer of DNA into cells for gene therapy. Virus vectors are very effective, but they have fatal drawbacks such as immune response and oncogenic effects when used in vivo. Non-viral vectors, generally consisting of cationic liposomes and linear cationic polymers, have several advantages over viral vectors. However, non-viral gene delivery has its limitations such as toxicity and low transfection efficiency. Chitosan is a cheap, biocompatible, biodegradable, and non-toxic polymer that forms polyelectrolyte complex with DNA.
In this study, three different chitosans, chitosan 100, chitosan 500, and low molecular weight chitosan, were characterized and evaluated as a gene delivery vector in four different human cell lines. The objectives were to investigate the formation of plasmid/chitosan complex, and to evaluate the transfection efficiency of the complexes which chitosans formed with plasmid, pCMV-β, in human cell lines, including basal cell carcinoma (BCC), human immortalized keratinocytes (HaCaT), human hepatoma cells (Huh7), and human gastric carcinoma cells (AGS). The cytotoxicity of three different chitosans was determined by MTT assay, and localization of reporter gene, pEGFP-C1, expression in the transfected cells was examined using microscopes.
The reaults showed that all chitosans formed complex with plasmids in appropriate weight ratios except water soluble chitosan. At a plasmid/chitosan weight ratio between 1:1~1:10, the most effective transfection efficiency was obtained at weight ratio of 1:5 in BCC cells, and the transfection efficiency of chitosans in Huh7 was lower than other cells. The transfection efficiency of chitosans in skin cell lines (BCC and HaCaT) is higher than other cell lines (Huh7 and AGS). The influence of molecular weight of chitosans in transfection efficiency appeared to be cell-dependent. In addition, DNA/chitosans complex showed negligible cytotoxicity for four different cell lines. Microscopic examination revealed that the gene expression products were distributed in the entire cell including cytosol and nuclei.
In conclusion, this study demonstrated that plasmid DNA formed complexes with chitosan 100, chitosan 500, and low molecular weight chitosan, and was effectively transfected into four different human cells lines. Therefore, the non-cytotoxic chitosans have the potential for safe and efficient non-viral gene delivery.
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