论文部分内容阅读
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 a natural host for many respiratory virus infections and share similarities with humans in lung physiology, respiratory morphology and respiratory cell types as well as receptor distribution. In order to understand the mechanisms of respiratory virus infection and to screen for respiratory diseases, a porcine airway epithelial cell (PAEC) differentiation model was established by using porcine lung tissue as raw material and serum-free gas-liquid interface culture method. Microscopy, electrophysiological and immunohistochemical methods such as identification of the model. Recombinant adeno-associated virus rAAV6-GFP (rAAV6-GFP) was constructed by co-transfection of three plasmids and was used to infect the PAEC model by apical surface to explore the application of AAV6 in gene therapy in PAEC. The results showed that the differentiated mature PAEC model is a multilayer epithelial structure with ciliated cells, mucus-secreting cells and basal cells on the apical surface. rAAV6-GFP infects PAECs via the apical surface and effectively mediates the long-term expression of foreign genes. The in vitro differentiation model of porcine respiratory epithelial cells established in this paper laid a good foundation for the research of respiratory tract infection and respiratory diseases drug screening and gene therapy.