潜在腺病毒感染的肺泡上皮细胞模型的构建

Establishment of Cell Model with Latent Adenovirus Infection

为了探讨腺病毒潜伏感染对正常气道上皮细胞生理功能的影响以及与呼吸道疾病发生、发展的关系,构建了含腺病毒最主要效应蛋白E1A的高效真核表达载体,转染正常大鼠肺泡上皮细胞(CCL149),经过长期抗性筛选获得多个抗性细胞克隆,应用PCR、RT-PCR、Western印记和免疫组化的方法对抗性克隆进行鉴定,最终获得不同E1A蛋白表达强度的阳性细胞克隆。通过细胞增殖曲线和周期分析,观察E1A基因对细胞基本功能的影响。结果显示,E1A基因明显抑制细胞增殖周期,引起细胞S期比例下降,G1期延长,同时E1A能明显增强TNFα诱导下的细胞凋亡。

The influences of latent adenovirus infection on the rat alveolar epithelial cells, and the relationship between latent adenovirus infection and airway disorder have been well documented. For illustrating the roles of adenovirus infection in the process of airway disorder, we constructed a eukaryotic expression vector for expressing adenovirus E1A protein and transfected into CCL149 cell. Cells stably expressing E1A protein were selected by G418 resistance. 109 G418-resistant clones were obtained, those clones were identified by PCR, RT-PCR, Western blot and immunocytochemistry. Among them four E1A-positive clones and nine control clones were verified as stable transfected clones. Primary study showed that E1A gene could inhibit the proliferation of CCL149 and reduce the cell of S phase and prolong the cells in the G1 phase at the same time E1A sensitizes cell to TNFα induced apoptosis of CCL149 cell.