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目的探讨针对乙型肝炎病毒(HBV)X、P双靶区反义RNA对乙型肝炎转基因小鼠HBV复制和表达的影响。方法构建表达HBV X、P双靶区正、反义RNA的重组载体质粒,与脂质体混合,经尾静脉注入小鼠体内,用酶联免疫法检测血清HBsAg;用荧光聚合酶链反应定量法检测血清HBV DNA含量;用免疫组化法检测肝细胞HBsAg、HBcAg的表达;小鼠心脏、肝脏、脾脏、肺脏、肾脏常规病理切片,观察反义RNA对小鼠脏器的影响。结果小鼠血清HBsAg表达,pLXSN-asX组和pLXSN-asXP组均于第四周明显低于给药前,最高抑制率分别达24%、27%;其余组未出现明显变化。与给药前相比,小鼠血清HBV DNA的复制,在pLXSN-asX组和pLXSN-asP组分别于第2周、第8周达到抑制高峰,抑制率均为58%;在pLXSN-asXP组于第1周、第8周出现两次抑制高峰,抑制率分别为66%、77%;其余组未出现明显变化。8周后,pLXSN-asX组、pLXSN-asP组和pLXSN-asXP组小鼠肝细胞HBsAg、HBcAg的表达显著低于其他组。小鼠脏器组织学观察未见异常。结论HBV X、P双靶区反义RNA对乙型肝炎病毒转基因鼠有显著抗HBV作用,且在作用效率和作用时间上优于单靶区反义RNA。
Objective To investigate the effect of anti-sense RNA targeting HBV X and P double targets on HBV replication and expression in hepatitis B transgenic mice. Methods Recombinant plasmids expressing both positive and negative RNA of HBV X and P target regions were constructed, mixed with liposomes and injected into mice via the tail vein to detect the serum HBsAg by enzyme-linked immunosorbent assay. The contents of serum HBV DNA were detected by ELISA. The expression of HBsAg and HBcAg in hepatocytes was detected by immunohistochemistry. The histopathology of heart, liver, spleen, lung and kidney of mice was observed. Results The expression of HBsAg in mice serum was significantly lower than that in the pLXSN-asX group and the pLXSN-asXP group by 24% and 27%, respectively, before the administration in the fourth week. The other groups showed no significant changes. Compared with pre-dose, the replication of serum HBV DNA in mice reached the peak inhibition at week 2 and week 8, respectively, and the inhibition rates were both 58% in pLXSN-asX group and pLXSN-asP group. In pLXSN-asXP group The inhibition peak appeared at the first week and the eighth week respectively, with the inhibition rates of 66% and 77% respectively. No significant changes were observed in the remaining groups. After 8 weeks, the expression of HBsAg and HBcAg in hepatocytes of pLXSN-asX group, pLXSN-asP group and pLXSN-asXP group was significantly lower than that of other groups. Histological observation of mouse organs no abnormalities. Conclusion The antisense RNA targeting HBV X and P double targets has a significant anti-HBV effect on hepatitis B virus transgenic mice and is superior to single-target antisense RNA in efficiency and duration of action.