| Summary: | 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.
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