HCV LNAzyme对病毒5'非编码区基因调控的特异性抑制作用  

Hepatitis C virus( HCV) 5' non-coding region( NCR) genespecific LNAzyme significantly inhibits HCV replication and expression in vitro

作　　者：邓益斌[1] 农乐根[1] 梁祚仁[1] 张梁[1] 覃羽华[1] 何平[1] 

机构地区：[1]右江民族医学院附属医院医学检验中心,广西百色533000

出　　处：《基础医学与临床》2014年第10期1363-1366,共4页Basic and Clinical Medicine

基　　金：广西自然科学基金(2011GXNSFA018285)

摘　　要：目的探讨针对丙肝病毒5'非编码区内源性核糖体进入位点的锁核酸核酶(LNAzyme)对病毒基因复制与表达的特异性抑制作用。方法设计合成能切割HCV-5'-NCR-IRES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、脂质体对照组和无关LNAzyme对照组。实验组包括DNAzyme、硫代DNAzyme组和LNAzyme组。以阳离子脂质体介导转染hep G2.9706细胞,用荧光定量PCR和化学发光技术分别监测24、48和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)法监测细胞活性。结果加入核酶后,LNAzyme对HCV RNA复制和荧光素酶基因表达的抑制作用最强(P<0.05),平均抑制率分别为48.02%和53.05%,且随用药时间延长,抑制率呈增高趋势,96 h后,平均抑制率分别为81.21%和84.25%。LNAzyme对细胞活性无影响。结论 LNAzyme能特异性抑制丙型肝炎病毒5'非编码区的基因调控,且优于硫代修饰的DNAzyme。Objective To investigate the inhibitory effects of LNAzyme targeting the hepatitis C virus （HCV） 5＇non-coding region internal ribosome entry site（IRES） on HCV RNA replication and expression in cells.Methods The sequence encoding DNAzyme,thiolmodificated DNAzyme and LNAzyme targeting against the HCV IRES（located in the 5＇ non-coding region） were designed and synthesized.The following experimental groups were set up：DNAzyme group,S-DNAzyme group,LNAzyme group,blank control group,empty liposomes group,and randomLNAzyme group.The level of HCV RNA and luciferase gene expression of supernatant was tested by real-time fluorescent quantitative PCR and chemiluminescence technique at 24,48 and 96 hours after treatment.LNAzyme＇s cyto-toxicity on cell was evaluated by MTT method.Results Significant down-regulation of HCV RNA replication and luciferase gene expression were noted in the LNAzyme group when compared with other groups （P ＜ 0.05）.The average inhibition rates were 48.02％ and 53.05％,respectively,and with the treatment time prolonged,the inhibition rate was increasing,96 hours later,the average inhibition rates were 81.21％ and 84.25 ％ respectively.There＇s no toxicity on cell.Conclusions LNAzyme has a significant inhibitory effect on HCV RNA replication and expression in vitrol as compare to the thiolmodificated DNAzyme.

关 键 词：丙型肝炎 丙型肝炎病毒 锁核酸核酶 非编码区 基因调控 

分 类 号：R373.21[医药卫生—病原生物学]