Study on Liposomes for Transfection of Hepatoma Cells in vitro: Charged and Asialofetuin Labeled Liposomes.

• Main Authors: Liu, Nai-Jing, 劉乃菁
• Other Authors: Yu Hsiu-Ying
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/36512822302859329971

碩士 === 國立臺灣大學 === 藥學系 === 85 === The prospect of alleviating genetic disorders by the corrective introduction of exogenous genes has made gene therapy the ambition of immense medical and biotechnological research. However, several current DNA transfection techniques suffer from cellular toxicity and/or poor efficiency, and are most often inapplicable for use in vivo. Among the conventional reagents such as calcium phosphate, DEAE-dextran and other particulate reagents, liposomes have become increasingly acceptable as a convenient and reproducible reagent for DNA-mediated transfection. There are generally two classes of liposomal transfection reagents: those which are cationic and those which are anionic.The purposes of the present study are to investigate the better liposome system which could deliver the reporter gene into hepatoma cell lines. Three hepatoma cell lines HepG2, Hepa1-6, Gp7TB and a fibroblast cell line NIH 3T3 were used for our experimental system. The cationic lipid, DC-Chol (3b [N-(N'', N''-dimethylaminoethane)-carbamoyl]) cholesterol, was synthesized by the reaction of cholesteryl chloroformate and N,N- dimethylethylenediamine. The liposome system included DC-Chol- liposome (DOPE(Dioleoyl phosphatidyl ethanolamine)/DC-Chol = 1/1), AF-DC-liposome (AF (asialofetuin) labeled liposome, prepared from DC-liposome by using the detergent dialysis method), Lipofectin (purchased from GIBCO) and AF-Lipofectin. The method of DNA complexed with, or encapsulated in liposomes was applied to transfer the fluorescent gene (pGreen Lantern-1) into hepatoma cell lines. The transfection efficiency was observed by fluorescence microscopy and quantitated by analyzing the detected fluorescent intensity.Our result showed that the cationic liposomes delivered gene into cells successfully, but not the anionic liposomes. AF-DC-liposome and AF-Lipofectin had much higher transfection efficiency than DC-liposome and Lipofectin, respectively. Moreover, those plasmid DNA that formed a complex with cationic liposomes had better transfection efficieay than those that entrapped in liposomes. However the serum-induced reduction in transfection efficiency are more significant in those complexed plasmid DNA than those non- complexed ones. Gramicidin S (GrS), a peptide, enhanced the transfection efficiency of encapsulation method to a level almost equivalent to the complex method possibly by destabilizing the membrane.In summary, our result demonstrated that the modified cationic AF-DC-liposome was a better transfection reagent for gene delivery than the cationic DC- liposome. And the transfection by anionic liposome was not detectable. The complex method showed higher transfection efficiency in vitro, but the GrS-DNA complex entrapped in liposome might be the better way for transfection in vivo.