微环DNA载体介导的shRNA在HBV转基因小鼠中的抗病毒作用  

Antiviral activity of minicircle DNA-mediated shRNA in HBV transgenic mice

作　　者：李秀梅[1] 刘光泽[1] 谢勇 孔祥平[1] 

机构地区：[1]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,广州510600

出　　处：《解放军医学杂志》2017年第11期979-984,共6页Medical Journal of Chinese People's Liberation Army

基　　金：广东省科技计划项目(2014A020212639);广州市科技计划项目(201510010004,201604046020)~~

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(P<0.05);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(P<0.05)。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。Objective To investigate whether minicircle DNA-mediated shRNA can inhibit the replication and expression of HBV in HBV transgenic mice. Methods First, a universal plasmid shRNA, pU6-shRNA HBV and a minicircle shRNA vector pMCU6-shRNA HBV targeting HBV gene was prepared by molecular cloning technique. Then, the HBV transgenic mice were divided into three groups and the pMC-U6-shRNA HBV, pU6-shRNA HBV and control vector pU6-control were transfected into them respectively by hydrodynamic injection（HDI）. Sera were collected at different time points after injection. The changes of HBV DNA and HBsAg in serum of transgenic mice were detected by real-time PCR and chemiluminescence microparticle immunoassay（CMIA）.Immunohistochemistry was used to analyze the expression of HBcAg in the liver of transgenic mice. Results Compared with pU6-control group, HBsAg and HBV DNA in serum of transgenic mice were significantly inhibited by pU6-shRNA HBV and pMC-U6-shRNA from day 7 to day 21 after HDI（P〈0.05）. After that, the serum HBsAg and HBV DNA recovered and returned to the control level on the 35 th day in pU6-shRNA HBV group. However, pMC-U6-shRNA HBV still maintained a strong inhibitory effect in vivo until 35 days post-injection compared with the other two groups（P〈0.05）. Immunohistochemical results also suggest that pMCU6-shRNA HBV could more significantly inhibit HBcAg expression than pU6-shRNA HBV in mouse liver tissue. Conclusion The minicircle DNA-based shRNA vector pMC-U6-shRNA HBV is of longer inhibitory effect on HBV replication and gene expression in HBV transgenic mice than pU6-shRNA HBV. Minicircle DNA is superior to pRNAT-U6.1/Neo for shRNA delivery in vivo.

关 键 词：微环载体 SHRNA 转基因小鼠 乙型肝炎 

分 类 号：R512.62[医药卫生—内科学]