Pharmaceutical Research on Antisense Origonucleotide

探索了阳离子脂质体作为非病毒载体，其中新合成了六种具有各种叔胺的胆固醇衍生物作为脂质组分。用脂质体转染pSV 2 cat质粒，观察脂质体对pSV 2 cat的转染率和相应反义核酸的抑制作用。（5 ′-GATCCCCCCCTCCAT-3 ′）对质粒DNA的作用。（1）荧光研究表明，质粒DNA与脂质体以适当的比例形成相对大的复合物（0.15DNA/脂质，w/w）的转染，过量的质粒DNA降低了转染效率。(2)反义寡核苷酸也能与脂质体形成大的复合物，抑制质粒DNA的基因表达。(3)质粒DNA与脂质体之间形成的复合物的结构可能不同于反义核酸与脂质体之间形成的复合物的结构。(4)共聚焦显微镜研究表明，整个复合物被带入细胞。为了建立一种新的基因传递系统，我们选择了一种人黑色素瘤细胞系（A-375-6），该细胞系中IL-1可以刺激IL-6的释放，并研究了IL-6的反义（5 ′-TGTGGAGAAGGAGTT 3 ′）的作用。(1)阳离子脂质体可促进IL 6的释放。(2)反义核酸可抑制脂质体对A-375-6细胞IL-6的增强作用，但不能抑制IL-1刺激的A-375-6细胞IL-6的净释放。

Cationic liposomes were explored as non-viral vectors for which six cholesterol derivatives with various tertiary amines were newly synthesized as a lipid component. Cationic liposomes consisting of dioleoylphosphatidylethanolamine (DOPE) and the cholesterol derivatives were tested in terms of the frequency of transfection of plasmid pSV2 cat and the suppressing effect of the corresponding antisense (5'-GATCCCCCCCTCCAT-3') against the plasmid DNA.The following were clarified : (1) Fluorescence studies showed that the plasmid DNA forms a relatively large complex with the liposomes at an appropriate ratio (0.15 DNA/lipid, w/w) on these components and excess plasmid DNA lowered the transfection efficiency. (2) Antisense oligonucleotide also formed a large complex with the liposomes, which suppressed the gene expression of the plasmid DNA.(3) The structure of ths complexes formed between plasmid DNA and the liposomes may be different from that formed between the antisense and the liposomes. (4) A confocal microscope study showed that the whole complexes were taken into cells. To establish a new assay system for gene delivery, a human melanoma cell line (A-375-6) to which IL1 is known to stimulate the release of IL6 was selected and the effect of antisense (5'-TGTGGAGAAGGAGTT3') against IL6 was investigated. (1) Cationic liposome enhanced the release of IL6. (2) Antisense suppressed the enhancement by liposome, however, could not depress net release of IL6 stimulated with IL1 from A-375-6.