乳糖化多聚赖氨酸导向反义寡核苷酸抗乙型肝炎病毒的作用

Inhibition of HBV gene expression by antisense oligonucleotides using galactosylated poly(L-lysine) as a hepatotropic carrier

目的 研究乳糖化多聚赖氨酸 (Gal PLL)导向反义寡核苷酸对乙型肝炎病毒 (HBV)基因表达的特异性抑制作用。方法 根据聚合酶链反应 (PCR)产物直接测序结果 ,在HBVU5样序列区合成了一段 16聚硫代磷酸反义寡核苷酸 ,并将其与肝靶向配体Gal PLL连接 ,在 2 2 15细胞比较 ,观察了它们对HBV基因表达的影响。结果 通过测序确定了 2 2 15细胞中HBVDNA属于HBV表面抗原ayw1亚型 ,并将此用于反义寡核苷酸的序列设计 ;经荧光组化分析表明 ,Gal PLL对大鼠肝组织有选择性亲和力 ;Gal PLL与DNA形成复合物的最佳摩尔比为 2∶1;在相同实验条件下 ,硫代磷酸反义寡核苷酸对HBsAg和HBeAg的抑制率分别为 70 %和 5 8% ,而Gal PLL硫代磷酸反义寡核苷酸对HBsAg和HBeAg的抑制率分别为 96 %和 82 % ,同时培养上清液和细胞中HBVDNA的含量明显下降 ,无关序列寡核酸无明显效果 ,寡核苷酸对细胞未产生毒性。结论 肝靶向配体和反义寡核酸的复合物可以通过无唾液酸糖蛋白受体靶入 2 2 15细胞内 。

Objective To study the specific inhibition of HBV gene expression by antisense oligonucleotide (ASON) targeted by galactosylated poly (L\|lysine)(Gal PLL).Methods According to the result of direct sequencing of PCR amplified products,a 16 mer phosphorothioate analogue of the antisense oligonucleotide (PS\|ASON) directed against the HBV U5\|like region was synthesized and then linked with one liver\|targeting ligand,the Gal PLL. Using the 2.2.15 cells compared the effect of them on the expression of HBV gene.Results We identified that HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw2 by sequencing. The fluorescent histochemistry test indicted that Gal PLL had a selective affinity to the rat liver tissues. A 2∶1 molar ratio of the Gal PLL to DNA optimized the complex formation. In the same experimental conditions, the inhibitory effects of HBsAg and HBeAg by PS\|ASON were 70% and 58%,respectively at a concentration of 10 μmol/L,while by ligand\|PS\|ASON were 96% and 82%,respectively,and the amount of HBV DNA in culture supernatant and cells was depressed significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotide had no cytotoxicity. Conclusion Antisense oligonucleotides complex with the liver\|targeting ligand can be targeted to cells via asialoglycoprotein receptors resulting in specific inhibition of HBV gene expression and replication.