LNAzyme设计及其体外特异性抑制丙型肝炎病毒C基因的表达

Hepatitis C virus gene-specific locked nucleic acid enzyme significantly inhibits C gene expression in vitro

目的:探讨针对丙型肝炎病毒(hepatitis C virus,HCV)C基因的锁核酸核酶对病毒RNA复制与表达的特异性抑制作用.方法:设计合成能切割HCV C基因位点的DNAzyme、硫代DNAzyme和LNAzyme.实验设对照组和实验组.对照组包括空白对照组、脂质体对照组和脂质体-无关LNAzyme对照组.实验组包括脂质体-DNAzyme、脂质体-硫代DNAzyme组和脂质体-LNAzyme组.以阳离子脂质体介导转染hepG2.9706细胞.采用荧光定量PCR和化学发光技术分别监测24、48、96 h细胞培养上清液中HCV RNA含量及荧光素酶基因表达;四甲基偶氮唑蓝(MTT)法监测细胞代谢.结果:加入药物后,脱氧核酶、硫代脱氧核酶及锁核酸核酶组对HCV RNA复制和荧光素酶基因表达的抑制作用均较对照组强(P<0.01),其中,锁核酸核酶的抑制作用均较脱氧核酶及硫代脱氧核酶明显(P<0.05),平均抑制率分别达47.55%和52.44%,且随用药时间延长,抑制率呈增高趋势,96 h后,HCV RNA复制和荧光蛋白表达的下降率均较用药前明显(P<0.01),其中,锁核酸核酶的抑制作用均较脱氧核酶及硫代脱氧核酶明显(P<0.05),平均下降率分别达79.40%和80.05%.而LNAzyme对细胞活性基本无影响.结论:LNAzyme能特异性抑制HCV C基因的复制与表达,且优于硫代修饰的DNAzyme.

AIM： To investigate the inhibitory effects of locked nucleic acid enzyme targeting the hepatitis C virus（HCV） C gene on HCV RNA replication and expression in HepG2.9706 cells.METHODS： The sequences encoding DNAzyme, thiolmodificated DNAzyme and LNAzyme targeting the HCV C gene were designed andsynthesized. The following experimental groups were set up： lipo-DNAzyme, lipo-S-DNAzyme, lipo-LNAzyme, blank control, empty liposomes, and lipo-random-LNAzyme. Transfection was performed using cationic liposomes. The level of HCV RNA and luciferase gene expression in supernatants were tested by real-time fluorescent quantitative PCR and chemiluminescence technique 24, 48 and 96 h after treatment, respectively. Cytotoxicity of LNAzyme was evaluated by MTT assay.RESULTS： Significant down-regulation of HCV RNA replication and luciferase gene expression was noted in the lipo-LNAzyme group, lipoDNAzyme group and lipo-S-DNAzyme group compared with the control group（P 0.05 for all）. Relative to the lipo-DNAzyme group and lipo-S-DNAzyme group, the average inhibition rates in the lipo-LNAzyme group were 47.55% and 52.44%, respectively. With the prolongation of the treatment time, the inhibition rate increased. At 96 h, HCR RNA replication and fluorescent protein expression were significantly lower than those before treatment in the lipoLNAzyme group（P 0.01 for both）, and the average inhibition rates were 79.40% and 84.05%, respectively. No obvious toxicity was observed.CONCLUSION： LNAzyme has a significant inhibitory effect on HCV C gene replication and expression in vitro, which is stronger than that of the thiolmodificated DNAzyme.