【摘要】：目的:構(gòu)建攜帶HBVx基因的慢病毒表達(dá)載體,感染14-19肝前體細(xì)胞使其穩(wěn)定表達(dá)HBVx基因。通過(guò)門靜脈注射肝前體細(xì)胞入動(dòng)物體內(nèi),將HBVx基因?qū)雱?dòng)物體內(nèi)后建立動(dòng)物模型,分別檢測(cè)小鼠各個(gè)組織中HBVx目的基因的表達(dá)情況,重點(diǎn)探討在HBVx基因?qū)Ω闻K的作用以及發(fā)生的一系列生物學(xué)變化。方法:(1)通過(guò)PCR擴(kuò)增HBVx目的基因,使之與慢病毒表達(dá)載體(p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro)連接,后通過(guò)酶切、菌落PCR、測(cè)序驗(yàn)證質(zhì)粒是否成功構(gòu)建。(2)病毒構(gòu)建成功后,感染14-19肝前體細(xì)胞,用嘌呤霉素(puromycin)連續(xù)篩選4周攜帶HBVx 14-19肝前體細(xì)胞,使用PCR、western blot進(jìn)行細(xì)胞HBVx目的基因、蛋白質(zhì)檢測(cè)。(3)同時(shí)進(jìn)行門靜脈動(dòng)物模型構(gòu)建,分別在第3、7、14、30、60天用定量PCR方法進(jìn)行檢測(cè)小鼠各個(gè)組織器官中的HBVx的表達(dá)情況,同時(shí)使用酶聯(lián)免疫吸附法測(cè)定小鼠血清內(nèi)AFP和ALT的表達(dá)量。結(jié)果:(1)成功構(gòu)建攜帶HBVx的慢病毒表達(dá)質(zhì)粒,并用慢病毒(p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro)成功感染14-19肝前體細(xì)胞,通過(guò)使用嘌呤霉素(puromycin)連續(xù)四周的篩選,成功構(gòu)建了穩(wěn)定攜帶HBVx的14-19肝前體細(xì)胞。(2)成功構(gòu)建門靜脈動(dòng)物模型。并分別于第3、7、14、30、60天用定量PCR方法進(jìn)行檢測(cè)小鼠各個(gè)組織器官中的HBVx的表達(dá)情況,發(fā)現(xiàn)HBVx只在在肝臟中表達(dá),不在其他組織內(nèi)表達(dá);使用Elisa方法分別測(cè)定第3、7、14、30、60天的小鼠血清中AFP、ALT的表達(dá)含量,同對(duì)照組相比的結(jié)果顯示,實(shí)驗(yàn)組小鼠血清內(nèi)的AFP、ALT含量均升高。結(jié)論:攜帶HBVx的肝前體細(xì)胞可在體內(nèi)穩(wěn)定表達(dá)。通過(guò)檢測(cè)各組織HBVx的表達(dá)量,說(shuō)明HBVx具有嗜肝性,不累及其他臟器。構(gòu)建了一個(gè)長(zhǎng)期表達(dá)HBVx的動(dòng)物模型,為本課題組后續(xù)研究HBVx導(dǎo)致肝癌發(fā)生發(fā)展的機(jī)制研究奠定了一個(gè)良好基礎(chǔ)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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