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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 ,并对HBV基因表达和复制产生特异性抑制作用
Objective To investigate the specific inhibitory effect of Gal PLL targeting antisense oligonucleotides on hepatitis B virus (HBV) gene expression. Methods According to the direct sequencing results of polymerase chain reaction (PCR) products, a 16-mer polyphosphoric acid antisense oligonucleotide was synthesized in the HBV U5-like sequence region and ligated with the target ligand Gal PLL. 15 cells compared to observe their impact on HBV gene expression. Results HBVDNA was found to belong to the ayw1 subtype of HBV surface antigen in 2 215 cells by sequencing and was used for the sequence design of antisense oligonucleotide. Fluorescence histochemical analysis showed that Gal PLL was selective for rat liver tissue The optimal molar ratio of Gal PLL to DNA was 2:1. Under the same experimental conditions, the inhibition rate of HBsAg and HBeAg by phosphorothioate antisense oligonucleotides was 70% and 58% , While the inhibitory rates of Gal PLL phosphorothioate antisense oligonucleotides to HBsAg and HBeAg were 96% and 82%, respectively. Meanwhile, the content of HBVDNA in culture supernatant and cells was significantly decreased, and the unrelated sequence oligonucleotides had no significant effect, Oligonucleotides are not toxic to cells. Conclusion The complexes of liver targeting ligands and antisense oligodeoxynucleotides can target into 2 2 15 cells via asialoglycoprotein receptor and have specific inhibitory effect on HBV gene expression and replication