Study for gene transfection and intracellular distribution of transfected DNA.

DNA介导的转基因技术已成为现代生物学的重要工具。我们研究了阳离子脂质体在基因转染中的应用。结果表明，在8种阳离子胆固醇衍生物中，含有阳离子胆固醇衍生物-3β-羧胺乙基二甲胺的阳离子脂质体效果最好。同时，用激光多普勒法测定了脂质体的Zeta电位。脂质体的Zeta电位值与pSV2CAT对细胞的转染率具有良好的一致性。结果表明，阳离子脂质体的Zeta电位是控制基因转移的重要因素之一。其次，我们研究了一种新型的含羟乙氨基的阳离子胆固醇衍生物。新型胆固醇衍生物的转染率远高于上述含有二烷氨基阳离子衍生物的阳离子脂质体的转染率。此外，在有血清存在的情况下，脂质体的效率不会降低太多。这表明一种新型的阳离子胆固醇衍生物在脂质体介导质粒和反义DNA进入靶细胞的基因转移中具有很好的应用前景。此外，利用原子力显微镜(AFM)研究了含有阳离子胆固醇衍生物的阳离子脂质体介导的基因转移。AFM图像显示，脂质体/DNA复合体形成的囊泡直径不同，取决于每个具有不同间隔臂的阳离子胆固醇衍生物。结果表明，复合体的大小与质粒DNA对培养细胞的转染活性有很好的相关性。结果发现，中等大小(0.4~1.4微米)的囊泡对靶细胞的基因转导效果最好。

DNA-mediated transfection has become an important tool in modern biology. We have studied the use of the cationic liposomes for gene transfection. The results showed that the cationic liposomes containing a cationic cholesterol derivative, cholesteryl-3 beta -carboxyamido ethylenedimethylamine, were most effective among eight derivatives of cationic cholesterol. At the same time we measured zeta potential of the liposomes by laser Doppler spectroscopy. Values of zeta potential of the liposomes were well consistent with those of transfection efficiency of pSV2CAT into the cells. The results showed that zeta potential of cationic liposomes was one of the important factors which control gene transfection.Next, we investigated that a novel cationic cholesterol derivative with a hydroxyethylamino head group. The transfection efficiency by the new cholesterol derivative was much higher than those by the cationic liposomes containing cationic derivatives with a dialkylamino head group described above. Further, the efficiency by the liposomes was not so much decreased in the presence of serum. This suggested that a novel cationic cholesterol derivative should be very promising in liposome-mediated gene transfection of plasmid and antisense DNA into target cells.Further, atomic force microscopy (AFM) was used for studying gene transfection mediated by cationic liposomes which contain a cationic cholesterol derivative. AFM images showed that vesicles made of the liposome/DNA complex had various diameters depending on each cationic cholesterol derivative with a different spacer arm. The results showed that the diameter of the complex was well related to the transfection activity of plasmid DNA to cultured cell lines. From the results it was found that the vesicles with moderate diameters (from 0.4 to 1.4 mum) were most effective for gene transfection of plasmid DNA into the target cells.