反基因锁核酸体内抑制HBV S基因的表达

Inhibition of hepatitis B virus S gene expression by anti-gene locked nucleic acid in vivo

目的目前针对乙型肝炎的治疗仍缺乏有效治疗手段,探讨反基因锁核酸在转基因小鼠体内抑制HBV S基因的表达和抗病毒效果。方法采用随机数字表法将HBV转基因小鼠分为5组,每组6只,分别为空白组、无关序列组、拉米夫定组、反义锁核酸组、反基因锁核酸组。空白组、无关序列组、反义锁核酸组、反基因锁核酸组于第1、3、5 d经尾静脉分别注射5%GLU-脂质体、5%GLU-脂质体-无关序列、5%GLU-脂质体-反义锁核酸、5%GLU-脂质体-反基因锁核酸各500μL。拉米夫定组以2 mg/kg剂量每天灌胃2次,连续7 d。采用RT-PCR检测血清HBV DNA;酶联免疫法检测血清HBs Ag;逆转录PCR检测肝HBV S mRNA水平;免疫组化检测肝细胞HBs Ag水平;血清肝肾功能检查和组织细胞HE染色监测反基因锁核酸的毒副作用。结果给药后第3、5、7天,反基因锁核酸组对HBV DNA复制的的平均抑制率分别为37.18%、50.27%和61.46%,第7天的抑制作用较空白组、无关序列组、拉米夫定组、反义锁核酸组明显增强,差异有统计学意义(P<0.05)。给药后第3、5、7天,反基因锁核酸对HBs Ag表达的平均抑制率分别为30.17%、44.00%和57.76%,第7天的抑制作用较空白组、无关序列组、拉米夫定组、反义锁核酸组明显增强,差异有统计学意义(P<0.05)。与空白组、无关序列组、拉米夫定组和反义锁核酸组比较,反基因锁核酸更能抑制HBV S基因mRNA的表达(P<0.05)。空白组、无关序列组、拉米夫定组、反义锁核酸组、反基因锁核酸组阳性细胞率分别为93%、92%、78%、47%和31%,反基因锁核酸组肝组织切片中HBV HBs Ag阳性细胞率明显小于拉米夫定组及反义锁核酸组(P<0.05)。各组肝细胞切片中,肝组织结构和肝细胞形态正常,肾细胞切片中,各组肾小球结构完整,未见肾小球肥大,肾小球基膜和系膜基质未见明显病理改变和炎细胞浸润。结论反基因锁核酸在转基因小鼠体内能有效抑制HBV S基因表达,具有明显的抗病毒效果,为乙肝病毒的基因治疗提供理论依据和实验基础。

Objective There is still a lack of effective treatment for hepatitis B.The article aimed to investigate the inhibitory and antiviral effects of hepatitis B virus（HBV） S gene expression by anti-gene locked nucleic acid（LNA） in vivo in transgenic mice.Methods The HBV transgenic mice were divided into 5 groups by random number,6 in each group.They were blank group,irrelevant sequence group,lamivudine group,antisense locked nucleic acid group,and anti-gene locked nucleic acid group respectively.The lamivudine group was treated by oral gastric lavage,with 2 mg/kg dose2 times per day for continuous 7 d,and the rest groups were injected with 500 m L by tail vein injection at 1,3,5 d after administration.HBV DNA was tested by real-time quantitative polymerase chain reaction（q PCR）; HBs Ag was tested by ELISA; mRNA of HBV S gene was detected by reverse transcription PCR（RT-PCR）; the positive rate of HBs Ag in liver cells was detected by immunohistochemistry.Results After the treatment of anti-gene locked nucleic acid on 3 rd,5 th,7 thday,the inhibition rate of HBV DNA were 37.18%,50.27%,61.46%,and HBs Ag were 30.17%,44%,57.76%.The inhibitory effect on 7 thday was more obvious than those of the blank group,the unrelated sequence group,the lamivudine group and the antisense lock nucleic acid group,and the difference was statistically significant（P0.05）.The expression of mRNA S gene HBV（0.33） and liver cells HBs Ag positive rate（31%） was significantly reduced compared with control group（P0.05）.No abnormal change was found in the function of liver and kidney tests and tissue HE staining.Conclusion Anti-gene lock nucleic acid targeting to S gene has strong inhibitory effects on HBV replication and expression in transgenic mice,which provides theoretical and experimental knowledge for HBV gene therapy.