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Journal of Integrative Agriculture  2022, Vol. 21 Issue (2): 504-520    DOI: 10.1016/S2095-3119(21)63644-4
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection
XIE Xing1, HAO Fei1, WANG Hai-yan1, PANG Mao-da2, GAN Yuan1, LIU Bei-bei1, ZHANG Lei1, WEI Yan-na1, CHEN Rong1, ZHANG Zhen-zhen1, BAO Wen-bin3, BAI Yun1, SHAO Guo-qing1, XIONG Qi-yan1, FENG Zhi-xin1
1 Key Laboratory for Veterinary Bio-Product Engineering of Ministry of Agriculture and Rural Affairs, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China
2 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China
3 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China
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摘要  

猪的呼吸道是多种病原微生物的定殖场所,包括常见的四种猪源支原体,分别是猪肺炎支原体(Mhp),猪鼻支原体(Mhr),猪絮状支原体(MF)和猪滑液支原体(MHS)。猪源支原体主要寄居于猪气管黏膜表面,最具代表性的Mhp是引起猪气喘病的主要病原,并易与其他猪呼吸道病原混合或继发感染,引发猪呼吸道疾病综合征,使得感染猪产生慢性持续性呼吸系统疾病,给养猪业带来巨大的经济损失。沿着猪呼吸道上皮细胞的纤毛的黏附是猪源支原体成功感染的前提。猪原代气管上皮细胞(PTEC)是研究包括猪源支原体的各种猪呼吸道病原发病机制的合适模型,但原代PTEC细胞短暂的寿命极大地限制了其应用前景。因此,构建永生化的猪气管上皮细胞,对包括Mhp在内的所有猪源支原体等猪呼吸道病原,具有较高的应用价值。

我们首先提供了详细的分离和培养原代PTEC的方法步骤。随后通过用含有人端粒酶逆转录酶(hTERT)的重组构建的质粒pEGFP-hTERT转染原代PTECs,通过两轮G418的抗性筛选,建立永生化的猪气管上皮细胞系(hTERT-PTECs),并可传代至60代以上。通过比较原代与永生化细胞的上皮细胞表面标志物角蛋白18的表达,细胞周期,细胞生长曲线,端粒酶活性,染色体核型分析,端粒酶基因的蛋白质印记检测,软琼脂和裸鼠成瘤试验,增殖能力等相关基因的定量PCR检测,不仅证实了永生化细胞具备了原代细胞的形态及功能特性,与原代PTECs相比,hTERT-PTEC也具有更长的寿命,更高的端粒酶活性和增殖活性。裸鼠体内未表现出恶性表型,表明该细胞系不具有致瘤性。将不同的猪源支原体菌株感染hTERT-PTECs,通过颜色变化单位CCU50黏附率的定量计算,hTERT-PTECs对所有猪源支原体易感,且原代和永生化细胞之间的黏附能力无显著差异。而对于代表性的MhpDNA拷贝定量实时PCR测定,间接免疫荧光测定和蛋白质印迹分析表明hTERT-PTECs能够粘附不同毒力的Mhp菌株。总之,与原代PTECs类似,hTERT-PTECs可以广泛用作猪源支原体的粘附细胞模型,并可用于多种猪呼吸道病原体的感染研究。



Abstract  Primary porcine tracheal epithelial cells (PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respiratory tract of pigs and mainly found on the mucosal surface surrounding swine trachea.  However, the short proliferation ability of primary PTECs greatly limits their lifespan.  In this study, primary PTECs were carefully isolated and cultured, and immortal PTECs were constructed by transfecting primary PTECs with the recombinant constructed plasmid pEGFP-hTERT containing human telomerase reverse transcriptase (hTERT).  Immortal PTECs (hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cell-cycle analysis, proliferation assay, Western blotting, telomerase activity assay, karyotype analysis and quantitative RT-PCR.  Compared to primary PTECs, hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity.  In addition, this cell line resulted in a lack of transformed and grown tumors in nude mice, suggesting that it could be safely applied in further studies.  Moreover, hTERT-PTECs were vulnerable to all swine-origin mycoplasmas through quantitative analysis as indicated by 50% color changing unit (CCU50) calculation, and no significant differences of adhesion ability between primary and immortal PTECs were observed.  For the representative swine mycoplasma Mycoplasma hyopneumoniae (Mhp), except for DNA copies quantitative real-time PCR assay, indirect immunofluorescence assay and Western blotting analysis also depicted that hTERT-PTECs was able to adhere to different Mhp strains of different virulence.  In summary, like primary PTECs, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in infection studies of various porcine respiratory pathogens.  
Keywords:  porcine tracheal epithelial cells (PTECs)       hTERT-PTECs       swine-origin mycoplasmas       adhesion       cell model 

  
Received: 02 September 2020   Accepted: 01 February 2021
Fund: This work was supported by the National Natural Science Foundation of China (31800161, 31700157, 31800160, 31900159, and 31770193), the Natural Science Foundation of Jiangsu Province, China (BK20180297 and BK20170600), and the Independent Research Project Program of Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology, China (2019sy004).
About author:  XIE Xing, Tel: +86-25-84390881, E-mail: yzxx1989@163.com; Correspondence FENG Zhi-xin, Tel: +86-25-84391320, E-mail: fzxjaas@163.com; XIONG Qi-yan, E-mail: qiyanxiongnj@163.com

Cite this article: 

XIE Xing,  HAO Fei, WANG Hai-yan, PANG Mao-da, GAN Yuan, LIU Bei-bei, ZHANG Lei, WEI Yan-na, CHEN Rong, ZHANG Zhen-zhen, BAO Wen-bin, BAI Yun, SHAO Guo-qing, XIONG Qi-yan, FENG Zhi-xin. 2022. Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection. Journal of Integrative Agriculture, 21(2): 504-520.

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