乙型肝炎病毒S／C基因位点反义锁核酸在小鼠体内抗病毒疗效研究

Inhibition of hepatitis B virus （HBV） replication using antisense LNA targeting to both S and C genes in HBV

目的探讨针对HBV S／C双基因位点反义锁核酸对乙型肝炎转基因小鼠HBV复制和表达的影响。方法将30只HBV转基因小鼠随机分为5组，每组6只。分别为5％葡萄糖液对照组、空脂质体对照组、单靶区S组、单靶区C组、双靶区SC组。反义锁核酸片段经尾静脉注入小鼠体内，采用时间分辨免疫荧光技术定量检测血清HBsAg；实时荧光聚合酶链反应定量检测血清HBV DNA含量；逆转录聚合酶链反应检测肝组织HBV C-mRNA的表达；免疫组织化学法检测肝细胞HBsAg、HBcAg的表达；自动生物化学分析仪检测血清白蛋白、ALT、尿素氮、肌酐；小鼠肝、肾脏做常规病理切片，观察反义锁核酸对小鼠脏器的影响。应用SPSS12．0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Kruskat Wallis H检验。结果注射锁核酸后，对HBsAg的表达均显示有较强的抑制作用，单靶区S组、单靶区C组和双靶区SC组的平均抑制率分别为36．6％、31．5％和54．9％；对HBV DNA的复制也有抑制作用，平均抑制率分别为24．0％、21．1％和35．8％。注射后l、3、5d，HBsAg的平均抑制率分别为14．4％、25．6％和31．3％；HBV DNA的平均抑制率分别为11．0％、19．2％和24．1％；血清中自蛋白、ALT、尿素氮、肌酐等指标，各组结果与对照组比较，差异均无统计学意义；小鼠肝细胞的HBsAg，HBcAg阳性细胞数均较对照组明显减少。小鼠肝、肾脏组织学表现未见异常。结论HBV S／C基因位点反义锁核酸对乙型肝炎病毒转基因鼠HBV复制和表达有显著抑制作用，且双基因靶位优于单基因靶位。

Objective To investigate the inhibitory effect on HBV replication of antisense locked nucleic acid （LNA） targeting to both S and C genes in HBV transgenic mice. Methods Thirty HBV transgenic mice were randomly divided into five groups （n = 6）： glucose control group were treated with 5% glucose solution, liposome control group were treated with liposome alone, S group were treated with LNA targeting to S gene, C group were treated with LNA targeting to C gene, and dual-target group were treated with LNA targeting to both S and C genes, Antisense LNA was injected into mice via the tail vein. Serum HBsAg was quantified by TRFIA. Serum HBV DNA was quantified by real-time PCR. The expression of HBV C-mRNA in the liver was detected by RT-PCR. The expression of HBsAg and HBcAg in the liver was detected by immunohistochemistry. Serum ALB, ALT, BUN and CR were measured using an antomatic biochemical analyzer. The effects of antisense LNA on mouse organs were investigated by HE staining. Results 5 days after LNA injection, serum HBsAg levels in the dual-target group were reduced by 72.8%, and serum HBV DNA levels were decreased by 52.9%. These values were significantly higher than those in the control groups （all P 〈 0.05）. No significant differences were noted in serum ALB, ALT, BUN and CR between the experiment groups and the control groups （all P 〉 0.05）. The expression levels of HBsAg and HBcAg in the liver of dual-target group were significantly lower than those in the control groups. No significant histopathological abnormality was found in liver and kidney tissues in all groups. Conclusion Antisense LNA targeting to both S and C genes can significantly inhibit HBV replication in transgenic mice.