逆转录病毒介导双靶区反义RNA在转基因小鼠肝细胞中的表达及对HBV DNA复制的影响

Retrovirus-mediated expression of dual-target antisense RNA in liver cells of transgenic mice and its influence on replication of hepatitis B virus

目的:探讨逆转录病毒介导的乙型肝炎病毒(HBV)X,P单、双靶区的反义RNA在乙型肝炎转基因小鼠肝细胞中HBV复制的影响．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,注入小鼠体内,第8周时取小鼠肝脏,提取肝组织总RNA,进行逆转录,将逆转录后的cDNA用PCR扩增,琼脂凝胶电泳和DNA测序定性,荧光聚合酶链反应定量:提取肝组织基因组DNA,利用荧光聚合酶链反应定量测定HBV DNA含量.结果:经过PCR扩增,琼脂凝胶电泳和DNA测序定性,荧光聚合酶链反应定量等方法检测到小鼠肝脏内反义RNA得到高效表达;8 wk后,对HBV DNA(×105 copies／g)的作用:与对照组相比(7．24±1．62),空质粒组(9．49±3．34)及正义RNA组(6．52±1．53)之间无明显差异,单(2．60±1．13,2．83±1．67)、双靶区反义RNA组(3．24±0．45)与对照组之间有明显差异(P<0．05),但单、双靶区反义RNA组间无明显差异(P>0．05)．结论:将脂质体包裹逆转录病毒载体(质粒)介导的单、双靶区乙肝病毒反义RNA经iv导入转基因小鼠细胞的方法可行;反义RNA对乙型肝炎病毒转基因小鼠有显著抗HBV复制作用;对转基因小鼠肝脏中HBV DNA的复制有明显的抑制作用,但单、双靶区反义RNA组间无明显差异．

AIM： To detect the expression of X and P region dual-target antisense RNA of hepatitis B virus （HBV） mediated by ratroviral vector in the liver cells of transgenic mice and its effect on the replication of HBV DNA. METHODS： The recombinant retroviral vector plasmids expressing dual-target antisense RNA complementary to HBV X and P region were constructed and then injected into the transgenic mice. The experimental mice were divided into blank, pLXSN, pLXSN-asX, pLXSN-asP, pLXSN-asXP, and pLXSN-seXP group. The total RNA, extracted from the mice at the 8th wk, was reversely transcribed, and then the cDNA was amplified with polymerase chain reaction （PCR）. Thereafter, agarose gel electrophoresis, sequencing analysis and fluorescent PCR were performed. The replication of HBV DNA was tested by fluorescence quantitative PCR. RESULTS： The HBV dual-target antisense RNA was expressed in the liver cells of mice and with high efficiency. After 8 wk, the replications of HBV DNA （×10^5 copies/g） were 7.24 ± 1.62, 9.49 ± 3.34, 2.60 ± 1.13, 2.83 ± 1.67, 3.24 ± 0.45, 6.52 ± 1.53 in blank, pLXSN, pLXSN-asX, pLXSN-asP, pLXSN-asXP and pLXSN-seXP group. The levels of HBV DNA were significantly higher in single-or dual-target antisense RNA groups than those in other groups （P 〈 0.05）. However, there were no marked differences between control groups as well as between single-target and dual-target group （P 〉 0.05）. CONCLUSION： The hepatitis B virus dual-target antisense RNA can be expressed in the liver cells of transgenic mice with high efficiency, and it can inhibit the replication of HBV DNA in mice liver. However, there is no significant difference between single-target and dual-target RNA.