针对HBV S基因mRNA反义锁核酸设计及其在2.2.15细胞内的抗病毒作用

Antiviral effects of locked nucleic acid antisense oligonucleotides targeting HBV S gene mRNA in HepG2 2.2.15 cells

目的:探讨针对乙型肝炎病毒(HBV)S基因翻译起始区反义锁核酸(LNA)片段在HepG22.2.15细胞内抗病毒效果及有效LNA序列筛选.方法:设计合成互补于HBVS基因mRNA翻译起始区同一位点的4条不同序列长度LNA片段及无关对照序列,以阳离子脂质体介导,作用于HepG22.2.15细胞,采用ELISA法和FQ-PCR法分别监测24、48和72h细胞培养上清液中HBsAg和HBVDNA的含量;MTT法检测LNA对细胞代谢的影响.结果:4条不同序列长度(10、15、20及25个碱基)的反义LNA对HBsAg的表达和HBVDNA的复制均有显著性抑制作用,72h后的抑制率分别为46.58%、54.38%、72.43%、69.92%及27.09%、28.77%、34.71%、32.68%,且抑制率随时间呈增高趋势.LNA对细胞代谢无明显影响.结论:针对HBVS基因mRNA翻译起始区的反义LNA短序列体外能显著抑制HBV基因的表达,且抑制作用最强的序列长度应在15-25个碱基之间.

AIM： To investigate the inhibitory effects of locked nucleic acid （LNA） antisense oligonucleotides targeting hepatitis B virus （HBV） S gene in HepG2 2.2.15 cells, and screen effective LNA antisense oligonucleotides. METHODS： Four LNA antisense oligonucleotides of different lengths that are complementary to the translation initiation region of HBV S 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 supernatant were tested by enzyme-linked immunosorbent assay （ELISA） and fluorescent quantitative-polymerase chain reaction （FQPCR） 24, 48 and 72 hours after transfection. The cell toxicity of LNA antisense oligonucleotides was detected by methyl thiazolyl tetrazolium （MTT） assay. RESULTS： All four LNA antisense oligonucleotides （10, 15, 20 and 25 base, respectively） could inhibit the expression of HBsAg and the replication of HBV DNA. Seventy-two hours after transfection, the reduced rates of HBsAg and HBV DNA levels were 46.58%, 54.38%, 72.43% and 69.92% as well as 27.09%, 28.77%, 34.71% and 32.68%, respectively. No obvious cell toxicity of LNA antisense oligonucleotides was noted. CONCLUSION： LNA antisense oligonucleotides targeting HBV S gene show strong inhibitory effects on HBV replication in vitro. The optimal length of LNA antisense oligonucleotides ranges from 15 to 25 base. LNA antisense oligonucleotides targeting HBV S gene have a therapeutic potential in patients infected with HBV.