猪呼吸道上皮细胞体外分化模型的建立及其应用研究

Establishment and Application of a Model of Well-differentiated Porcine Airway Epithelial Cells in Vitro

猪是许多呼吸道病毒感染的天然宿主,其与人类在肺生理学、呼吸道形态学和呼吸道细胞类型以及受体分布都有相似之处。为了解呼吸道病毒感染机制和筛选呼吸系统疾病药物,选择以猪肺组织为原料采用无血清气液界面培养法构建一种猪呼吸道上皮细胞(Porcine airway epithelial cell,PAEC)体外分化模型,然后通过扫描电子显微镜、电生理和免疫组织化学等方法鉴定该模型。采用三质粒共转染法构建重组腺相关病毒rAAV6-GFP(rAAV6-green fluorescent protein,rAAV6-GFP),通过顶端表面感染PAEC模型,探讨AAV6在PAEC模型中基因治疗领域的应用。结果显示分化成熟的PAEC模型为多层上皮结构,顶端表面含有纤毛细胞、黏液分泌细胞和基底细胞。rAAV6-GFP能通过顶端表面感染PAEC,有效地介导外源基因的长期表达。本文建立的猪呼吸道上皮细胞体外分化模型为呼吸道病原体感染和呼吸系统疾病药物筛选和基因疗法等研究奠定了良好的基础。

Pigs are increasingly recognized as＂natural＂hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology,airway morphology,cell types,and distribution of cell receptors in the respiratory tract.We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases.Hence,we developed a model of well-differentiated porcine airway epithelial cells（PAECs）derived from pig-lung tissue and cultured them with serum-free medium under an air–liquid interface condition in vitro.We identified the PAEC model using scanning electron microscopy,electrophysiology,and immunohistology.To evaluate application of gene therapy of adeno-associated virus（AAV）6on the PAEC model,we generated recombinant adeno-associated virus 6-green fluorescent protein（rAAV6-GFP）using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP.Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells,with basal cells located directly beneath the multilayer.rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene.Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens,as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.