HBV preS1反基因锁核酸的设计及体外对病毒复制的抑制

Antiviral effects of locked nucleic acid antisense oligonucleotides targeting the HBV preS1 gene in HepG2 2.2.15 cells

目的:探讨针对乙型肝炎病毒(hepatitis Bvirus,HBV)preS1 dsDNA同聚嘌呤区设计反基因锁核酸分子,并观察其在HepG2 2.2.15细胞内抑制病毒复制的效果.方法:针对HBVpreS1 dsDNA的2941-2962 nt、3 015-3 036 nt和3 089-3 110 nt三个同聚嘌呤区,利用RNA structure软件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,以阳离子脂质体介导转染HepG22.2.15细胞,采用荧光定量聚合酶链反应技术(FQ-PCR)和时间分辨免疫荧光技术(TRFIA)分别监测1、3、5和7 d细胞培养上清液中HBV DNA和HBsAg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:反基因锁核酸对细胞内的HBV DNA复制与HBsAg表达有明显的抑制作用,且抑制率随时间呈增高趋势,7 d后抑制率分别为64.32%和67.51%.各实验组与对照组比较差异均具有统计学意义(均P<0.05),而封闭2 941-2 962 nt同聚嘌呤靶区的LNA抑制作用最强,且最适序列长度为20-30 bp.LNA对细胞代谢无明显影响.结论:针对preS1 dsDNA同聚嘌呤区的反基因锁核酸分子,体外能有效抑制HBV的复制,以封闭2 941-2 962 nt靶位效果最强,且合适序列长度为20-30 bp.

AIM： To investigate the inhibitory effects of locked nucleic acid（LNA） antisense oligonucleotides targeting the purine region of the hepatitis B virus（HBV） preS1 gene in HepG2 2.2.15 cells,and to screen effective LNA anti-gene oligonucleotides.3 015-3 036 nt and 3 089-3 110 nt） of the HBV preS1 gene were designed,synthesized,and introduced into HepG2 2.2.15 cells by cationic liposome-mediated transfection.Hepatitis B surface antigen（HBsAg） and HBV DNA levels in cell supernatants were tested by time-resolved fluorescence immune assay（TRFIA） and fluorescent quantitative polymerase chain reaction（FQ-PCR） 1,3,5 and 7 d after transfection.The cell toxicity of LNA anti-gene oligonucleotides was detected by methyl thiazolyl tetrazolium（MTT） assay.RESULTS： LNA anti-gene oligonucleotides targeting the HBV preS1 gene showed strong inhibitory effects on HBV DNA replication and HBsAg expression in vitro,and the effects were time-dependent.Seven days after transfection,the reduced rates of HBV DNA and HBsAg levels were 64.32% and 67.51%,respectively.The inhibitory effects were significantly different between each experimental group and control group（all P 〈 0.05）.The inhibitory effect of the LNA anti-gene oligonucleotide targeting the region 2 941-2 962 nt was most strong.The optimal length of LNA anti-gene oligonucleotides ranges from 20 to 30 bases.No obvious cell toxicity was observed with LNA anti-gene oligonucleotides.CONCLUSION： LNA anti-gene oligonucleotides targeting the HBV preS1 gene showed strong inhibitory effects on HBV replication in vitro.The inhibitory effect of the LNA anti-gene oligonucleotide targeting the region 2 941-2 962 nt was most strong,and the optimal length of LNA anti-gene oligonucleotides ranges from 20 to 30 bases.