Establishment and characterization of a telomerase-immortalized porcine bronchial epithelial cell line
Establishment and characterization of a telomerase-immortalized porcine bronchial epithelial cell line
复制标题
端粒酶永生化猪支气管上皮细胞系的建立和表征。
DOI：
10.1002/jcp.26942
复制
发表时间：
2018-12-01
影响因子：
5.6
通讯作者：
Feng, Zhixin
中科院分区：
文献类型：
--
作者：
Xie, Xing;Gan, Yuan;Feng, Zhixin
原代猪支气管上皮细胞是研究猪呼吸道病原菌分子生物学和致病机制的理想模型。然而，初级PBEC的短寿命极大地限制了其应用。本研究分离培养了人外周血内皮细胞，并将含有人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）的重组质粒pEGFP-hTERT转染到人外周血内皮细胞上，建立了永生化的人外周血内皮细胞。细胞角蛋白18表达、端粒酶活性测定、增殖测定、核型分析和定量逆转录聚合酶链反应显示，永生化PBECs（hTERT-PBECs）保留了原代PBECs的形态和功能特征。hTERT-PBECs较原代PBECs具有更高的端粒酶活性，延长了复制寿命，并显示出增强的增殖活性。此外，该细胞系在体外不转化，并且在体内不表现出恶性表型，这表明它可以安全地用于进一步的研究。此外，hTERT-PBECs对H3 N2亚型猪流感病毒和猪圆环病毒2型易感。结论：永生化hTERT-PBECs是一种有价值的体外模型，可广泛应用于猪呼吸道病原性感染的研究。
Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP-hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT-PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse-transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT-PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT-PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT-PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.