反基因锁核酸体外阻断乙肝病毒前S2基因表达  

Antiviral Effects of Locked Nucleic Acid Anti-gene Oligonucleotides Targeting HBV preS2 Gene in vitro

作　　者：邓益斌[1] 罗艳红[1] 邹佳峻[1] 王春芳[1] 温旺荣[2] 

机构地区：[1]右江民族医学院临床学院医学检验中心,广西百色533000 [2]暨南大学第一临床学院临床检验中心,广东广州510070

出　　处：《现代生物医学进展》2013年第19期3641-3644,共4页Progress in Modern Biomedicine

基　　金：广西壮族自治区教育厅基金项目(200911MS187)

摘　　要：目的:探讨针对乙肝病毒前S2基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的作用。方法:针对乙肝病毒前S2基因同聚嘌呤区,分别设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以半乳糖配体介导转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术(FQ-PCR)、时间分辨免疫荧光技术(TRFIA)和酶联免疫法(ELISA)分别监测1、3、5和7 d细胞培养上清液中HBV DNA、HBsAg和前S2抗原的含量;四甲基偶氮唑蓝(MTT)法检测锁核酸对细胞代谢的影响。结果:加入锁核酸后,对HBV DNA复制、HBsAg和前S2抗原表达均显示有较强的抑制作用,且抑制率随时间呈增高趋势,7 d后抑制率分别达61.56%、68.18%和72.82%。各实验组与对照组比较差异均具有统计学意义(均P<0.05)。LNA对细胞代谢无明显影响。结论:针对乙肝病毒前S2基因同聚嘌呤区的反基因锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗提供理论和实验依据。Objective： To investigate the inhibitory effects of locked nucleic acid （LNA） anti-gene oligonucleotides targeting hep- atitis B virus（HBV） preS2 gene purine region in HepG2 2.2.15 cells, and screen effective LNA anti-gene oligonucleotides. Methods： The LNA anti-gene oligonucleotides of different lengths that were complementary to the purine rich region of HBV preS2 gene of double stranded DNA, were designed, synthesized, and introduced into HepG2 2.2.15 cells by galactose ligand-mediated transfection. HBV DNA, HBsAg and preS2-Ag levels in cell supematant were tested by Fluorescent Quantitative Polymerase Chain Reaction（FQ-PCR）, Time-resolved Fluorescence Immune Assay （TRFIA） and enzyme linked immunosorbent assays （ELISA） at 1,3,5 and 7 d after transfec- tion. The cell toxicity of LNA anti-gene oligonucleotides was detected by methyl thiazolyl tetrazolium （MTT） assay. Results： The anti- gene LNA that targeting on the purine rich region ofHBV preS2 gene showed strong inhibitory effects on replication ofHBV DNA and the expression of HBsAg and preS2-Ag with the inhibition rates of 61.56%, 68.18% and 72.82% respectively after 7 days. The difference of each experimental group compared with the controls was significant （P〈0.05）. There＇s no obvious toxicity on cell. Conclusions： LNA anti-gene oligonucleotides targeting HBV preS2 gene purine rich region of dsDNA shows strong inhibitory effects on HBV replication in vitro.

关 键 词：半乳糖配体 乙型肝炎病毒 锁核酸 反基因治疗 

分 类 号：R512.62[医药卫生—内科学] Q78[医药卫生—临床医学]