本文關(guān)鍵詞： HBVx 肝前體細胞 慢病毒 動物模型　出處：《基因組學與應用生物學》2017年04期 　論文類型：期刊論文

【摘要】：為了探究HBVx是否累及其他組織器官,本研究構(gòu)建了攜帶HBVx基因的慢病毒表達載體,感染14-19肝前體細胞,使其能穩(wěn)定表達HBVx基因,并構(gòu)建了門靜脈動物模型,使肝前體細胞在小鼠體內(nèi)存活并能夠進一步分化,并在不同時間段分別檢測小鼠各個組織中HBVx目的基因的表達情況。通過PCR擴增HBVx目的基因,使之與逆轉(zhuǎn)錄病毒表達載體(p Lenti-CMV-HA-3FLAG-PGK-EGFP-F2A-Puro)連接,后通過酶切、菌落PCR、測序驗證質(zhì)粒是否構(gòu)建成功。構(gòu)建成功后用攜帶HBVx逆轉(zhuǎn)錄病毒感染14-19肝前體細胞,嘌呤霉素(puromycin)連續(xù)篩選4周后進行細胞HBVx目的基因檢測。同時進行門靜脈動物模型構(gòu)建,分別在第3天、7天、14天、30天、60天進行檢測小鼠各個組織器官中的HBVx的表達情況。成功構(gòu)建攜帶HBVx的慢病毒表達質(zhì)粒、穩(wěn)定攜帶HBVx的14-19肝前體細胞、門靜脈動物模型。同時檢測了各個組織器官HBVx的表達情況,HBVx可在肝臟內(nèi)生長分化且可在肝臟內(nèi)長時間高表達,其余組織不表達。攜帶HBVx的肝前體細胞可在肝內(nèi)生長分化,且HBV具有嗜肝性,不累及其他臟器。構(gòu)建了一個長期表達HBVx的動物模型,為本課題組后續(xù)研究HBVx導致肝癌發(fā)生發(fā)展奠定了良好基礎(chǔ)。
[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.

【作者單位】： 重慶醫(yī)科大學;分子醫(yī)學與腫瘤研究中心;
【基金】：國家自然科學基金面上項目(81201679,81071770)資助
【分類號】：R512.62;R735.7

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本文編號：1543392