微环DNA载体介导的shRNA在HBV转基因小鼠中的抗病毒作用

发布时间：2018-03-31 12:51

本文选题：微环载体　切入点：shRNA　出处：《解放军医学杂志》2017年11期

【摘要】：目的探讨微环DNA载体介导的shRNA对HBV转基因小鼠体内乙肝病毒复制及表达的抑制效应。方法采用分子克隆技术制备靶向HBV基因的shRNA通用质粒p U6-shRNA HBV和微环shRNA质粒p MC-U6-shRNA HBV。将HBV转基因小鼠随机分为微环shRNA组、通用质粒shRNA组和无关质粒对照组,利用水动力转染技术分别导入p MCU6-shRNA HBV、p U6-shRNA HBV及对照载体p U6-control。于转染后第1、7、14、21、28、35天,分别采用Real-time PCR及化学发光微粒子免疫分析法(CMIA)检测转基因小鼠血清HBV DNA及HBs Ag水平,免疫组化染色观察转基因小鼠肝脏HBc Ag的表达。结果质粒注射后第7~21天,p U6-shRNA HBV及p MC-U6-shRNA HBV对转基因小鼠血清HBs Ag的表达及HBV DNA的复制均有明显抑制作用,与p U6-control相比差异有统计学意义(P0.05);21d后p U6-shRNA HBV组血清HBs Ag及HBV DNA均有所回升,至第35天基本回复至对照水平;至注射后35d,p MC-U6-shRNA HBV依然保持较强的体内抑制作用,与其他两组比较差异有统计学意义(P0.05)。免疫组化结果显示,与p U6-shRNA HBV组相比,p MC-U6-shRNA HBV组小鼠肝组织内HBc Ag阳性细胞数明显减少。结论与p U6-shRNA HBV质粒相比,微环shRNA质粒p MC-U6-shRNA HBV对HBV转基因小鼠体内乙肝病毒的抑制作用持续时间更长久。采用微环DNA作为shRNA的体内递送载体与普通的质粒载体相比具有一定优势。
[Abstract]:Objective to investigate the inhibitory effect of microloop DNA vector mediated shRNA on HBV replication and expression in HBV transgenic mice.Methods the shRNA general plasmid p U6-shRNA HBV targeting HBV gene and the microloop shRNA plasmid p MC-U6-shRNA HBV were prepared by molecular cloning technique.HBV transgenic mice were randomly divided into three groups: microloop shRNA group, general plasmid shRNA group and unrelated plasmid control group. Hydrodynamic transfection technique was used to introduce p MCU6-shRNA HBV HBV and control vector pU6-control. respectively.Real-time PCR and chemiluminescence microparticle immunoassay were used to detect the levels of HBV DNA and HBs Ag in serum of transgenic mice. The expression of HBc Ag in liver of transgenic mice was observed by immunohistochemical staining.Results the expression of HBs Ag in serum and the replication of HBV DNA in transgenic mice were significantly inhibited by p#en10# HBV and p MC-U6-shRNA HBV on the 7th day after injection. The serum HBs Ag and HBV DNA in p U6-shRNA HBV group increased significantly after 21 days compared with p U6-control.At the 35th day, the control level was basically restored to the control level, and the inhibitory effect of MC-U6-shRNA HBV was still strong at 35d after injection, which was significantly different from that of the other two groups (P 0.05).The results of immunohistochemistry showed that compared with p U6-shRNA HBV group, the number of HBc Ag positive cells in liver tissue of mice in p U6-shRNA HBV group was significantly lower than that in p U6-shRNA HBV group.Conclusion compared with p U6-shRNA HBV plasmid, the inhibitory effect of microloop shRNA plasmid p MC-U6-shRNA HBV on hepatitis B virus in HBV transgenic mice is longer than that of p U6-shRNA HBV plasmid.Using microloop DNA as the delivery vector of shRNA in vivo has some advantages compared with the ordinary plasmid vector.
【作者单位】：解放军第458医院全军肝病中心、全军转基因动物重点实验室;
【基金】：广东省科技计划项目(2014A020212639) 广州市科技计划项目(201510010004,201604046020)~~
【分类号】：R512.62

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