LNAzyme特异性阻断丙肝病毒5'-NCR/C基因表达  

LNAzyme specific targeting against to both 5'-NCR and C gene expression of hepatitis C virus

作　　者：邓益斌[1] 农乐根[1] 梁祚仁[1] 覃羽华[1] 

机构地区：[1]右江民族医学院附属医院医学检验科,广西百色533000

出　　处：《基础医学与临床》2015年第7期938-942,共5页Basic and Clinical Medicine

基　　金：广西自然科学基金(2011GXNSFA018285)

摘　　要：目的探讨针对HCV 5'-NCR/C双靶基因位点的锁核酸核酶对病毒基因复制与表达的特异性抑制作用。方法实验分对照组和实验组。对照组分别为空白对照组和脂质体对照组。实验组分别为单靶区NCR组、单靶区C组和双靶区NCR/C组。半乳糖配体介导转染Hep G2.9706细胞,用荧光定量PCR检测细胞培养液中HCV mRNA表达;化学发光技术检测细胞培养液中荧光素酶基因表达;荧光显微镜系统检测细胞内荧光蛋白表达;四甲基偶氮唑蓝法检测细胞代谢。应用SPSS 19.0统计学软件分析。各组间比较采用重复测量方差分析的SNK检验和Kruskal Wallis H检验。结果加入锁核酸核酶后,对HCV RNA的复制均显示有较强的抑制作用(F=77.50,P<0.05),单靶区NCR组、单靶区C组和双靶区NCR/C组的平均抑制率分别为62.12%、61.39%和75.37%;对荧光素酶的表达有抑制作用(F=48.65,P<0.05),平均抑制率分别为66.49%、65.06%和73.30%。给药后24、48和96 h,HCV mRNA的平均抑制率分别为52.36%、66.81%和75.05%;荧光素酶的平均抑制率分别为53.02%、62.98%和79.45%;细胞内的荧光蛋白表达阳性细胞数均较对照组明显减少(P<0.05)。结论针对5'-NCR/C基因位点的LNAzyme能特异性抑制丙型肝炎病毒的复制与表达,且双基因靶位优于单基因靶位。Objective To investigate the inhibitory effects on HCV replication of LNAzyme targeting at both 5＇-NCR and C genes in HepG2.9706 cells.Methods The experimental groups were divided into five groups：blank control group was treated with DMEM （dulbecco＇s modified eagle medium） solution,galactose ligand control group was treated with galactose ligand alone,NCR group was treated with LNAzyme targeting to NCR gene,C group was treated with LNAzyme targeting to C gene,and dual-target group was treated with LNAzyme targeting at both NCR and C genes.LNAzyme was transfected into HepG2.9706 cells by galactose ligand.The level of HCV RNA was quantified by fluorescence quantitative PCR.The expression of luciferase gene was detected by chemiluminescence technique.LNAzyme＇s cytotoxicity on cell was evaluated by MTT method.Results After LNAzyme transfection,the levels of HCV RNA in the NCR group,C group and dual-target group were reduced by 62.12％,61.39％ and 75.37％,respectively,and the luciferase gene expression also decreased by 66.49％,65.06％ and 73.30％,respectively.These values were significantly higher than those in the control groups （all P ＜ 0.05）.The expression level of fluorescent protein in all experimental groups was significantly lower than that in the control groups.Conclusions LNAzyme targeting to both 5＇-NCR and C gene can significant inhibit effect on HCV replication and expression in vitrol,and dual-target is stronger than single target.

关 键 词：锁核酸核酶 丙型肝炎病毒 非编码区 基因治疗 

分 类 号：R373[医药卫生—病原生物学]