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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。
In order to investigate whether HBVx affects other tissues and organs, a lentiviral vector carrying HBVx gene was constructed to infect 14-19 hepatic progenitor cells to stably express HBVx gene. A portal vein animal model was constructed in which hepatic precursor The cells survived and further differentiated in mice, and the expression of HBVx gene in each tissue of mice was detected at different time points. The HBVx gene was amplified by PCR and ligated with the retrovirus expression vector (p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro). The plasmid was verified by restriction enzyme digestion, colony PCR and sequencing . After successful construction, 14-19 hepatic progenitor cells were infected with HBVx retrovirus, and puromycin was continuously screened for 4 weeks to detect the target gene of HBVx. At the same time, the construction of portal vein animal model was established. The expression of HBVx in various tissues and organs of mice was detected on day 3, day 7, day 14, day 30 and day 60 respectively. Successfully constructed lentiviral expression plasmid carrying HBVx, stably carrying 14-19 hepatic precursor cells carrying HBVx, a portal vein animal model. At the same time, we detected the expression of HBVx in various tissues and organs. HBVx could grow and differentiate in the liver and could be expressed for a long time in the liver, while the other tissues did not express. Hepatic precursor cells carrying HBVx can grow and differentiate in the liver, and HBV has hepatotropic and does not affect other organs. Constructed a long-term expression of HBVx in animal models for the follow-up study of our group HBVx lead to the occurrence and development of liver cancer has laid a good foundation.