感染HBVx的肝前体细胞的小鼠体内稳定表达模型构建

发布时间：2018-02-27 16:15

  本文关键词： HBVx 肝前体细胞 慢病毒 动物模型　出处：《基因组学与应用生物学》2017年04期 　论文类型：期刊论文

【摘要】：为了探究HBVx是否累及其他组织器官,本研究构建了携带HBVx基因的慢病毒表达载体,感染14-19肝前体细胞,使其能稳定表达HBVx基因,并构建了门静脉动物模型,使肝前体细胞在小鼠体内存活并能够进一步分化,并在不同时间段分别检测小鼠各个组织中HBVx目的基因的表达情况。通过PCR扩增HBVx目的基因,使之与逆转录病毒表达载体(p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro)连接,后通过酶切、菌落PCR、测序验证质粒是否构建成功。构建成功后用携带HBVx逆转录病毒感染14-19肝前体细胞,嘌呤霉素(puromycin)连续筛选4周后进行细胞HBVx目的基因检测。同时进行门静脉动物模型构建,分别在第3天、7天、14天、30天、60天进行检测小鼠各个组织器官中的HBVx的表达情况。成功构建携带HBVx的慢病毒表达质粒、稳定携带HBVx的14-19肝前体细胞、门静脉动物模型。同时检测了各个组织器官HBVx的表达情况,HBVx可在肝脏内生长分化且可在肝脏内长时间高表达,其余组织不表达。携带HBVx的肝前体细胞可在肝内生长分化,且HBV具有嗜肝性,不累及其他脏器。构建了一个长期表达HBVx的动物模型,为本课题组后续研究HBVx导致肝癌发生发展奠定了良好基础。
[Abstract]:In order to explore whether HBVx involvement of other organs, this study constructed HBVx gene lentiviral vector infection, 14-19 hepatic progenitor cells, the stable expression of HBVx gene, and constructed the animal model of portal vein, the hepatic progenitor cells in vivo, and can be further divided, and in different time some were detected the expression of HBVx gene in various tissues of mice. The HBVx gene was amplified by PCR, the carrier and retroviral expression (P Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro) after connected by enzyme digestion, colony PCR, sequencing plasmid is constructed successfully. The successful construction of the use of portable HBVx retrovirus infection in 14-19 liver precursor cells. Puromycin (puromycin) after 4 weeks of continuous screening test cell HBVx gene. At the same time to build the animal model of portal vein, respectively in third days, 7 days, 14 days, 30 days, 6 The expression of 0 days were detected in HBVx tissues and organs of mice. Lentivirus carrying HBVx expression plasmid is successfully constructed, stable HBVx carrying 14-19 hepatic progenitor cells, animal model of portal vein. At the same time to examine the expression of various tissues and organs of HBVx, HBVx and growth differentiation in the liver in the liver of long time high expression, no expression. The remaining tissue carrying HBVx hepatic progenitor cells growth and differentiation in the liver, and the HBV is hepatophilic, not involving other organs. Construct an animal model of long-term expression of HBVx, the research group for further study of HBVx leads to the occurrence and development of HCC has laid a good foundation.

【作者单位】： 重庆医科大学;分子医学与肿瘤研究中心;
【基金】：国家自然科学基金面上项目(81201679,81071770)资助
【分类号】：R512.62;R735.7

【相似文献】

相关硕士学位论文 前1条

1 钟沁;HBx转染肝前体细胞后的体内作用研究[D];重庆医科大学;2015年

，

本文编号：1543392