Pharmaceutical Research on Antisense Origonucleotide

探索了阳离子脂质体作为非病毒载体，合成了6种含不同叔胺的胆固醇衍生物作为脂质组分。检测由二油基磷脂酰乙醇胺（dioleoylphosphatidylethanolamine， DOPE）及其胆固醇衍生物组成的阳离子脂质体pSV2 cat转染频率及相应反义基因（5′-GATCCCCCCCTCCAT-3′）对质粒DNA的抑制作用。澄清如下：(1)荧光研究表明，质粒DNA与脂质体以适当的比例（0.15 DNA/脂质，w/w）在这些组分上形成较大的复合物，过量的质粒DNA降低了转染效率。(2)反义寡核苷酸也与脂质体形成大复合体，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与反义DNA与脂质体之间形成的复合物的结构不同。(4)共聚焦显微镜观察显示，整个复合物被带入细胞。为了建立一种新的基因传递检测系统，我们选择了一株已知il - 1能刺激il -6释放的人黑色素瘤细胞系（a - 376 -6），并研究了反义蛋白（5'-TGTGGAGAAGGAGTT3'）对il -6的抑制作用。(1)阳离子脂质体促进il - 6的释放。(2)反义抑制脂质体的增强作用，但不能抑制a - 376 -6受il - 1刺激后的净释放。

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.