气管上皮细胞的分离培养及其在呼吸道冠状病毒感染中作用的研究进展

doi:10.16431/j.cnki.1671-7236.2025.04.035

摘要/Abstract

摘要： 近年来呼吸道冠状病毒成为研究热点，其主要感染宿主气管的纤毛细胞和杯状细胞，使细胞间丧失紧密连接，损害呼吸系统屏障的完整性，进而引发下呼吸道感染。气管上皮细胞(trachea epithelium cells,TECs)作为机体内阻挡病原入侵的重要屏障，同时也是许多病原体的靶细胞，是研究呼吸道冠状病毒的重要模型。在宿主与病毒的相互作用中，呼吸道冠状病毒优先攻击纤毛细胞，导致纤毛清除功能障碍，破坏气管上皮的防御屏障，从而加剧继发性感染。笔者概述气管上皮细胞的形态结构和生理功能，重点总结了气管上皮细胞体外分离培养的方法及其优缺点，并进一步探讨了呼吸道病原体与宿主之间的作用机制，以期为气管上皮细胞的分离、培养提供参考，同时为研究呼吸道冠状病毒的感染机制和防治策略提供支撑。

关键词: 气管上皮细胞; 分离; 培养; 呼吸道冠状病毒

Abstract: In recent years,respiratory Coronaviruses have become a focus of research.They mainly infect ciliated cells and goblet cells of the host trachea,resulting in loss of tight connections between cells,damage the integrity of the respiratory system barrier,and then cause low respiratory tract infection.The trachea epithelium cells (TECs),as an important barrier against pathogen invasion in the body,are also the target cells of many pathogens,and are an important model for the study of respiratory Coronaviruses.During the host-virus interactions,respiratory Coronaviruses preferentially attack ciliary cells,resulting in ciliary clearance dysfunction and destruction of the defensive barrier of the tracheal epithelium,thus exacerbating secondary infection.The authors summarized the morphological structure and physiological function of tracheal epithelial cells,and focused on the methods of isolation and culture of tracheal epithelial cells in vitro,as well as their advantages and disadvantages.The mechanism of interaction between respiratory pathogens and their hosts was further discussed，in order to provide a reference for the isolation and culture of tracheal epithelial cells,and provide support for the study of the infection mechanism and prevention strategy of respiratory Coronavirus.

Key words: tracheal epithelial cells; isolation; culture; respiratory Coronaviruses

中图分类号:

S852.65⁺9.6

郭倩倩, 汤雨晴, 齐振昌, 李庆豪, 金鑫, 马俊兴, 孙娟. 气管上皮细胞的分离培养及其在呼吸道冠状病毒感染中作用的研究进展[J]. 中国畜牧兽医, 2025, 52(4): 1836-1843.

GUO Qianqian, TANG Yuqing, QI Zhenchang, LI Qinghao, JIN Xin, MA Junxing, SUN Juan. Research Progress on Isolation and Culture of Tracheal Epithelial Cells and Their Role in Respiratory Coronaviruses Infection[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1836-1843.

参考文献

[1] PARK S E.Epidemiology,virology,and clinical features of Severe acute respiratory syndrome-coronavirus-2(SARS-CoV-2;Coronavirus disease-19)[J].Clinical and Experimental Pediatrics, 2020,63(4):119-124.
[2] HU B,GUO H,ZHOU P,et al.Characteristics of SARS-CoV-2 and COVID-19[J].Nature Reviews Microbiology,2021,19(3):141-154.
[3] KITANO M,OHNISHI H,MAKINO A,et al.An infection model for SARS-CoV-2 using rat transplanted with hiPSC-airway epithelial cells[J].Tissue Engineering Part A,2024.Doi:10.1089/ten.TEA.2024.0016.Online ahead of print.
[4] CHIU M C,LI C,LIU X,et al.Human nasal organoids model SARS-CoV-2 upper respiratory infection and recapitulate the differential infectivity of emerging variants[J]. mBio,2022,13(4):e0194422.
[5] KEEP S,CARR B V,LEAN F Z X,et al.Porcine respiratory coronavirus as a model for acute respiratory Coronavirus disease[J]. Frontiers in Immunology,2022,13:867707.
[6] PENG J Y,PUNYADARSANIYA D,SHIN D L,et al.The cell tropism of Porcine respiratory coronavirus for airway epithelial cells is determined by the expression of porcine aminopeptidase N[J].Viruses-Basel,2020,12(11):1211.
[7] RUIZ GARCÍA S,DEPREZ M,LEBRIGAND K,et al.Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures[J].Development,2019,146(20):dev177428.
[8] RYU J H,KIM C H,YOON J H.Innate immune responses of the airway epithelium[J]. Molecular Cells,2010,30(3):17-83.
[9] KRASTEVA G,KUMMER W. "Tasting" the airway lining fluid[J].Histochemistry and Cell Biology, 2012,138(3):365-383.
[10] PARKER D,PRINCE A.Innate immunity in the respiratory epithelium[J]. American Journal of Respiratory Cell and Molecular Biology,2011,45(2):189-201.
[11] 黄海碧,郑佳琪,王晓晖,等.绵羊气管上皮细胞的分离培养及绵羊肺炎支原体对其的吸附[J].中国兽医学报,2015,35(8):1239-1243.HUANG H B,ZHENG J Q,WANG X H,et al.Isolation and culture of sheep tracheal epithelium and establishment of adherence of Mycoplasma ovipneumoniae to host cells[J].Chinese Journal of Veterinary Medicine,2015,35(8):1239-1243.(in Chinese)
[12] 孙裴,张彦明,张浩,等.鸡气管上皮细胞分离培养及传支病毒在其培养特性研究[J].中国农业科学,2008,3:861-867.SUN P,ZHANG Y M,ZHANG H,et al.Isolation and culture of chicken trachea epithelium and culture characteristics of IBV in chicken trachea epithelium[J].Scientia Agricultura Sinica,2008,3:861-867.(in Chinese)
[13] ENGLER A E,MOSTOSL AVSKY G,MILLER L,et al.Maintenance and differentiation of primary tracheal basal cells from adult rhesus macaque[J].Methods and Protocols, 2019,2(4):79.
[14] UALIYEVA S,YOSHIMOTO E,BARRETT N A,et al.Isolation and quantitative evaluation of brush cells from mouse tracheas[J].Journal of Visualized Experiments, 2019,148:10.3791/59496.
[15] UALIYEVA S,BANKOVA L G.Isolation,ex vivo culture,and stimulation of tracheal and nasal chemosensory cells[J].Methods in Molecular Biology, 2022,2506:151-165.
[16] 孙裴,张彦明,杨小云,等.猪支气管黏膜上皮细胞的分离培养和传代研究[J].畜牧兽医学报,2008,39(6):7.SUN P,ZHANG Y M,YANG X Y,et al.Research of dissociation culture and subculture of epithelium cell from swine bronchia[J].Acta Veterinaria et Zootechnica Sinica,2008,39(6):7.(in Chinese)
[17] 张瑜,韩彪,陈昶,等.大鼠气管上皮细胞培养:3种方法的比较[J].中国组织工程研究与临床康复,2009,13(5):825-828.ZHANG Y,HAN B,CHEN C,et al.Culture of tracheal epithelial cells in rats:Comparison of three methods[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2009,13(5):825-828.(in Chinese)
[18] 邓三明,汪健,连耀国,等.四种气管上皮细胞分离培养方法的比较研究[J].医学研究生学报,2005,10:870-871.DENG S M,WANG J,LIAN Y G,et al.Comparison of tracheal epithelial cells culture methods in vitro[J].Journal of Graduate Medical Students,2005,10:870-871.(in Chinese)
[19] 杜永成,许建英,延峰.胰酶冷消化加刮刷法分离培养气管上皮细胞的实验研究[J].中国病理生理杂志,2003,9:135-137.DU Y C,XU J Y,YAN F.Study of enzyme digestion in low temperature with crushing for isolating and culturing tracheal epithelia cell[J].Chinese Journal of Pathophysiology,2003,9:135-137.(in Chinese)
[20] SHI H C,LU D,LI H J,et al.In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique[J].In Vitro Cellular Developmental Biology Animal, 2013,49(4):245-249.
[21] 韩石.兔气管黏膜上皮细胞和骨髓间充质干细胞的体外培养与鉴定[D].扬州:扬州大学,2013.HAN S.In vitro culture and identification of rabbit tracheal epithelial cells and bone marrow mesenchymal stem cells[D].Yangzhou:Yangzhou University,2013.(in Chinese)
[22] 李海甲.兔气管黏膜上皮细胞原代培养的研究[D].扬州:扬州大学,2013.LI H J.A primary culture model of rabbit tracheal epithelial cells with tissue culture technique[D].Yangzhou:Yangzhou University,2013.(in Chinese)
[23] 唐隽,马玲国,何发尧,等.气管黏膜上皮细胞无血清培养的初步实验研究[J].山东大学耳鼻喉眼学报,2005,6:403-406.TANG J,MA L G,HE F Y,et al.Primary study of tracheal membrane epithelium cells cultured by serum freemedium[J]. Journal of Otolaryngology and Ophthalmology of Shandong University,2005,6:403-406.(in Chinese)
[24] FARSANI S M,DEIJS M,DIJKMAN R,et al.Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses[J].Influenza Other Respiraytory Viruses, 2015,9(1):51-57.
[25] UPADHYAY S,PALMBERG L.Air-liquid interface:Relevant in vitro models for investigating air pollutant-induced pulmonary toxicity[J].Toxicological Sciences, 2018,164(1):21-30.
[26] 冀晓丽,胡玥,盛云华,等.气液界面培养小鼠气管上皮细胞模型构建[J].中国药理学通报,2021,37(2):282-288.JI X L,HU Y,SHENG Y H,et al.Culture and differentiation of mouse tracheal epithelial cells at air-liquid interface[J].Chinese Pharmacological Bulletin,2021,37(2):282-288.(in Chinese)
[27] 黄宁,唐彬,吴琦,等.人气管上皮细胞气液界面无血清培养[J].基础医学与临床,2000,2:89-92.HUANG N,TANG B,WU Q,et al.Serum-free culture of human tracheal epithelial cells with gas-liguid interface biphasic chamber system[J].Basic Medicine&Clinical,2000,2:89-92.(in Chinese)
[28] 孙裴,张彦明,魏建忠,等.2种气管黏膜上皮细胞体外培养技术的研究[J].中国微生态学杂志,2010,22(2):164-168.SUN P,ZHANG Y M,WEI J Z,et al.Research on two culture for bronchia epithelia[J].Chinese Journal of Microecology,2010,22(2):164-168.(in Chinese)
[29] YOU Y,BRODY S L.Culture and differentiation of mouse tracheal epithelial cells[J]. Methods in Molecular Biology,2013,945:123-143.
[30] 肖开颜,周广东,刘伟,等.猪气管上皮细胞的分离、培养和鉴定[J].中华胸心血管外科杂志,2006,5:326-328.XIAO K Y,ZHOU G D,LIU W,et al.Growth and proliferous environment for porcine tracheal epithelial cell in vitro[J].Chinese Journal of Thoracic and Cardiovascular Surgery,2006,5:326-328.(in Chinese)
[31] 陈思,肖华,张伟,等.小鼠气管-支气管上皮细胞的气-液界面培养[J].中国呼吸与危重监护杂志,2019,18(4):362-368.CHEN S,XIAO H,ZHANG W,et al.Air-liquid interface culture of mouse tracheal-bronchial epithelial cells[J].Chinese Journal of Respiratory and Critical Care,2019,18(4):362-368.(in Chinese)
[32] ROWE R K,BRODY S L,PEKOSZ A.Differentiated cultures of primary hamster tracheal airway epithelial cells[J].In Vitro Cellular Developmental Biology Animal, 2004,40(10):303-311.
[33] O'BOYLE N,SUTHERLAND E,BERRY C C,et al.Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface[J].PLoS One, 2017,12(7):e0181583.
[34] O'BOYLE N,SUTHERLAND E,BERRY C C,et al.Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface[J].PLoS One, 2018,13(3):e0193998.
[35] RUNFT S,FÄRBER I,KRVGER J,et al.In vitro characteristics of canine primary tracheal epithelial cells maintained at an air-liquid interface compared to in vivo morphology[J].International Journal of Molecular Sciences, 2023,24(5):4987.
[36] 孙裴,张彦明,杨小云,等.鸡胚成纤维细胞与鸡气管黏膜上皮细胞共培养体系的建立[J].西北农林科技大学学报(自然科学版),2010,38(3):40-46.SUN P,ZHANG Y M,YANG X Y,et al.Establishment of co-culture system of chicken embryo fibroblasts and chicken trachea epithelium cells[J].Journal of Northwest A&F University (Natural Science Edition),2010,38(3):40-46.(in Chinese)
[37] 李茂中,庞立丽,王宏,等.人气管上皮细胞的原代分离及气液界面培养[J].中国生物制品学杂志,2016,29(1):65-69.LI M Z,PANG L L,WANG H,et al.Isolation of primary human bronchial epithelial cells and culture at an air-liquid interface[J].Chinese Journal of Bioproducts,2016,29(1):65-69.(in Chinese)
[38] KESHEH M M,HOSSEINI P,SOLTANI S,et al.An overview on the seven pathogenic human Coronaviruses[J].Reviews in Medical Virology, 2022,32(2):e2282.
[39] TOYOSHIMA Y,NEMOTO K,MATSUMOTO S,et al.SARS-CoV-2 genomic variations associated with mortality rate of COVID-19[J].Journal of Human Genetics, 2020,65(12):1075-1082.
[40] DIJKMAN R,JEBBINK M F,KOEKKOEK S M,et al.Isolation and characterization of current human Coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism[J].Journal of Virology, 2013,87(11):6081-6090.
[41] IBRICEVIC A,PEKOSZ A,WALTER M J,et al.Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells[J].Journal of Virology, 2006,80(15):7469-7480.
[42] SIMS A C,BURKETT S E,YOUNT B,et al.SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium[J].Virus Research, 2008,133(1):33-44.
[43] THALER M,WANG Y,VAN DER DOES A M,et al.Impact of changes in human airway epithelial cellular composition and differentiation on SARS-CoV-2 infection biology[J].Journal of Innate Immunity, 2023,15(1):562-580.
[44] GONZALEZ-RUBIO J,LE-TRILLING V T K,BAUMANN L,et al.SARS-CoV-2 particles promote airway epithelial differentiation and ciliation[J].Frontiers in Bioengineering and Biotechnology, 2023,11:1268782.
[45] ZHU N,WANG W,LIU Z,et al.Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells[J].Nature Communications, 2020,11(1):3910.
[46] HAO S,NING K,KUZ C A,et al.Long-term modeling of SARS-CoV-2 infection of in vitro cultured polarized human airway epithelium[J].mBio, 2020,11(6):e02852-20.
[47] RAVINDRA N G,ALFAJARO M M,GASQUE V,et al.Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells,alterations in gene expression,and cell state changes[J].PLoS Biology, 2021,19(3):e3001143.
[48] SCHREINER T,ALLNOCH L,BEYTHIEN G,et al.SARS-CoV-2 infection dysregulates cilia and basal cell homeostasis in the respiratory epithelium of hamsters[J].International Journal of Molecular Sciences, 2022,23(9):5124.
[49] ZIEGLER C G K,MIAO V N,OWINGS A H,et al.Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19[J].Cell,2021,184(18):4713-4733.e22.
[50] XU J B,GUAN W J,ZHANG Y L,et al.SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl^- concentration[J].Signal Transduction and Targeted Therapy,2024,9(1):74.
[51] LI S,LU Y,YANG S,et al.Porcine lung tissue slices:A culture model for PRCV infection and innate immune response investigations[J].AMB Express, 2024,14(1):57.
[52] ROBERTS A,DEMING D,PADDOCK C D,et al.A mouse-adapted SARS-Coronavirus causes disease and mortality in BALB/c mice[J].PLoS Pathogens, 2007,3(1):e5.
[53] MITCHELL J,BROOKS H,SZLADOVITS B,et al.Tropism and pathological findings associated with Canine respiratory coronavirus (CRCoV)[J].Veterinary Microbiology, 2013,162(2-4):582-594.