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

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(P<0.05)。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。

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 ofhTERT 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 （P〈0.05）. Conclusion 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.