HBVx基因在体内对肝组织的影响及作用研究

发布时间：2018-12-30 16:56

【摘要】：目的:构建携带HBVx基因的慢病毒表达载体,感染14-19肝前体细胞使其稳定表达HBVx基因。通过门静脉注射肝前体细胞入动物体内,将HBVx基因导入动物体内后建立动物模型,分别检测小鼠各个组织中HBVx目的基因的表达情况,重点探讨在HBVx基因对肝脏的作用以及发生的一系列生物学变化。方法:(1)通过PCR扩增HBVx目的基因,使之与慢病毒表达载体(p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro)连接,后通过酶切、菌落PCR、测序验证质粒是否成功构建。(2)病毒构建成功后,感染14-19肝前体细胞,用嘌呤霉素(puromycin)连续筛选4周携带HBVx 14-19肝前体细胞,使用PCR、western blot进行细胞HBVx目的基因、蛋白质检测。(3)同时进行门静脉动物模型构建,分别在第3、7、14、30、60天用定量PCR方法进行检测小鼠各个组织器官中的HBVx的表达情况,同时使用酶联免疫吸附法测定小鼠血清内AFP和ALT的表达量。结果:(1)成功构建携带HBVx的慢病毒表达质粒,并用慢病毒(p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro)成功感染14-19肝前体细胞,通过使用嘌呤霉素(puromycin)连续四周的筛选,成功构建了稳定携带HBVx的14-19肝前体细胞。(2)成功构建门静脉动物模型。并分别于第3、7、14、30、60天用定量PCR方法进行检测小鼠各个组织器官中的HBVx的表达情况,发现HBVx只在在肝脏中表达,不在其他组织内表达;使用Elisa方法分别测定第3、7、14、30、60天的小鼠血清中AFP、ALT的表达含量,同对照组相比的结果显示,实验组小鼠血清内的AFP、ALT含量均升高。结论:携带HBVx的肝前体细胞可在体内稳定表达。通过检测各组织HBVx的表达量,说明HBVx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导致肝癌发生发展的机制研究奠定了一个良好基础。
[Abstract]:Aim: to construct a lentivirus expression vector carrying HBVx gene and infect 14-19 liver precursor cells to stably express HBVx gene. The HBVx gene was introduced into the animal model by portal vein injection of liver precursor cells, and the expression of HBVx target gene in various tissues of mice was detected. Focus on the role of HBVx gene in the liver and a series of biological changes. Methods: (1) the target gene of HBVx was amplified by PCR and ligated with lentivirus expression vector (p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro), then digested by enzyme, colony PCR, was obtained. (2) after the successful construction of the virus, 14-19 liver precursor cells were infected. Purine mycin (puromycin) was used to screen the HBVx 14-19 liver precursor cells for 4 weeks, and PCR,western blot was used to carry the HBVx target gene. (3) the animal model of portal vein was constructed at the same time. The expression of HBVx in various tissues and organs of mice was detected by quantitative PCR method on the 3rd day of 71430D. Enzyme linked immunosorbent assay (Elisa) was used to detect the expression of AFP and ALT in serum of mice. Results: (1) Lentivirus expression plasmid carrying HBVx was successfully constructed, and 14-19 liver precursor cells were successfully infected with lentivirus (p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro). Purine mycin (puromycin) was used to screen for four weeks. Stable 14-19 liver precursor cells carrying HBVx were successfully constructed. (2) Portal vein animal model was successfully constructed. The quantitative PCR method was used to detect the expression of HBVx in various tissues and organs of mice on the 60th day of the 3rd day. It was found that HBVx was expressed only in the liver, not in other tissues. Elisa method was used to determine the expression of AFP,ALT in the serum of mice on the 30th day of the 3rd day. Compared with the control group, the results showed that the AFP,ALT content in the serum of the experimental group was higher than that of the control group. Conclusion: liver precursor cells carrying HBVx can express stably in vivo. By detecting the expression of HBVx in various tissues, it was proved that HBVx was hepatophilic and did not involve other organs. An animal model expressing HBVx for a long time was constructed, which laid a good foundation for our team to study the mechanism of occurrence and development of liver cancer caused by HBVx.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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