慢病毒介导过表达端粒酶逆转录酶的 人口腔黏膜上皮稳定细胞系的构建
Construction of human mucosa oral epithelial cell lines overexpressing telomerase reverse transcriptase gene mediated by lentivirus
作者：曾飒 秦晓东 何祥一 车春晓 张潇 解斯羽 孙贵军 王立鹤
Author：Zeng Sa, Qin Xiaodong, He Xiangyi, Che Chunxiao, Zhang Xiao, Xie Siyu, Sun Guijun Wang Lihe.
收稿日期：2016-04-15 年卷（期）页码：2016,34(5):443-443-447
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：口腔黏膜上皮细胞,人端粒酶逆转录酶,慢病毒载体,稳定表达,永生化,
Key words：oral mucosa epithelial cells,human telomerase reverse transcriptase,lentivirus,stable expression,immortalization,
基金项目：
甘肃省自然科学基金（1308RJZA248）
中文摘要
目的 通过慢病毒法建立稳定表达外源性人端粒酶逆转录酶（hTERT）基因的口腔黏膜上皮细胞（OMECs），探索构建高效、稳定的永生化OMECs细胞系的方法。方法 提取293T细胞总RNA，应用聚合酶链反应（PCR）法扩 增hTERT基因全长，构建重组慢病毒载体pLVX-puro-hTERT。包装慢病毒颗粒后感染人正常OMECs，经嘌呤霉素抗 性筛选获得阳性克隆，采用实时荧光定量PCR法和Western blot法检测hTERT基因mRNA和蛋白的表达水平。结果 成功构建了pLVX-puro-hTERT过表达慢病毒载体并感染到OMECs中；感染细胞与正常OMECs形态相似，呈铺路石 样生长；实时荧光定量PCR和Western blot结果均显示，hTERT在感染细胞中高表达，与正常细胞相比差异有统计学 意义（P结论 通过慢病毒法成功建立了过表达hTERT的OMECs稳定细胞系，为构建高效、稳定增殖的人 永生化OMECs细胞系奠定了实验基础。
英文摘要
Objective To construct a cell line of oral mucosa epithelial cells that stably express human telomerase reverse transcriptase (hTERT) by lentiviral vectors, approaches for the establishment of stable and efficient immortalized oral mucosa epithelial cell lines were explored.Methods Whole RNA was extracted from 293T cells. The hTERT gene was amplified by polymerase chain reaction (PCR) and cloned into the lentiviral vector as pLVX-puro-hTERT. The lentivirus particles were successfully packaged and used to infect primary oral epithelial cells. The positive cell clones were selected by puromycin. Finally, the expression of hTERT was examined by real-time fluorescent quantitative PCR (qRT-PCR) and Western blot analysis.Results The sequencing results confirmed the construction of the recombinant lentivirus pLVX-puro-hTERT. The morphology of infected cells was similar to that of normal oral mucosal epithelial cells, with a cobble stone-like appearance. The qRT-PCR and Western blot results showed that hTERT was overexpressed in infected cells compared with the normal group (PConclusion The oral epithelial cell line with stable expression of hTERT was successfully established by the lentivirus, which provides an experimental basis for the establishment of a highly efficient and stable oral epithelial immortalized cell line.
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