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

发布时间：2018-01-02 03:02

本文关键词：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　出处：《华西口腔医学杂志》2016年05期 　论文类型：期刊论文

【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(h TERT)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h TERT基因全长,构建重组慢病毒载体p LVX-puro-h TERT。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h TERT基因m RNA和蛋白的表达水平。结果成功构建了p LVX-puro-h TERT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,h TERT在感染细胞中高表达,与正常细胞相比差异有统计学意义(P0.05)。结论通过慢病毒法成功建立了过表达h TERT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。
[Abstract]:Objective to establish oral mucosal epithelial cells (OMECs) stably expressing exogenous human telomerase reverse transcriptase (hTERT) gene by lentivirus method and to explore the construction efficiency. Methods Total RNAs of 293T cells were extracted and h TERT gene was amplified by polymerase chain reaction (PCR). To construct recombinant lentivirus vector p LVX-puro-h TERT. after packaging lentivirus particles and infecting human normal OMECs, positive clones were obtained by purine mycin resistant screening. The expression of h TERT gene m RNA and protein was detected by real-time fluorescence quantitative PCR and Western blot. TERT overexpression of lentivirus vector and infection into OMECs; The morphology of infected cells was similar to that of normal OMECs, and the infected cells grew like paving stones. The results of real-time fluorescence quantitative PCR and Western blot showed the high expression of TERT in infected cells. Conclusion the stable cell line of OMECs expressing h TERT was successfully established by lentivirus method. The stable proliferation of human immortalized OMECs cell line laid the experimental foundation.
【作者单位】：兰州大学口腔医学院;中国农业科学院兰州兽医研究所;
【基金】：甘肃省自然科学基金(1308RJZA248)~~
【分类号】：R781
【正文快照】： epithelial cells,OMECs)多因素、多步骤致病机制,建立人永生化OMECs细胞系是非常必要的。正常情况下,OMECs在体外仅能传代培养5~6代,无法满足研究需求。有学者[1]对人永生化OMECs细胞系进行了探索,如采用16型人乳头状瘤病毒E6和E7诱导培养永生化口腔黏膜上皮细胞系,但培养的

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