一种基于A549细胞与内皮细胞共培养的新型气液培养模型的建立

Establishment of a novel air-liquid model based on the co-culture of A549 cells and endothelial cells

目的通过气-液界面(ALI)培养技术将人Ⅱ型肺泡上皮细胞A549与内皮细胞共培养,探究内皮细胞存在条件下A549细胞表型和功能的变化。方法基于transwell培养体系建立气液共培养模型,A549细胞接种于transwell膜上表面,内皮细胞接种于transwell膜下表面,实时定量PCR(RT-qPCR)检测A549细胞经过传统ALI培养和ALI与内皮细胞共培养后的肺表面活性蛋白、细胞间连接以及与新冠病毒入侵细胞相关分子的表达,流式细胞术检测新冠病毒S蛋白截短体重组慢病毒的细胞感染率。结果通过比较分析,发现EA.hy926内皮细胞系较原代内皮细胞HUVEC更加适合建立新型ALI共培养模型。A549细胞经过传统ALI培养后肺表面活性蛋白SP-B、SP-D的表达上调,促进上皮钙黏蛋白E、紧密连接蛋白表达,新冠病毒受体神经紧张素1、去唾液酸糖蛋白受体1、含kringle跨膜蛋白1显著上调;而在ALI与内皮细胞共培养体系中,上述基因的上调更加显著(P<0.05),模型病毒粒子感染率较传统ALI培养的A549细胞显著上升(P<0.05)。结论成功构建A549细胞和EA.hy926内皮细胞共培养模型,为相关肺部疾病病理研究和药物筛选提供了新的研究模型。

Objective To investigate the effects of air-liquid interface(ALI)co-culture of human typeⅡalveolar epithelial cell A549 and endothelial cells on the phenotype and function of A549 cells in the presence of endothelial cells.Methods An air-liquid co-culture model was established using the transwell culture system,in which A549 cells were cultured on the upper surface of transwell membrane and endothelial cells on the bottom surface of transwell membrane.Real-time quantitative PCR(RT-qPCR)was performed to detect the expression levels of surfactant associated proteins,intercellular junctions and proteins associated with SARS-CoV-2 invasion in A549 cells after ALI co-culture with or without endothelial cells,while flow cytometry was performed to detect the infection rate of recombinant lentivirus particles encoding the truncated S protein in the cells.Results Comparative analysis suggested that the EA.hy926 endothelial cell line was preferable for establishing a new ALI co-culture model than primary endothelial cells HUVEC.The traditional ALI culture up-regulated the expressions of surfactant protein SP-B,SP-D,E-cadherin and tight junction protein 1(ZO-1),and SARS-CoV-2 receptorsneuropilin 1(NRP1),asialoglycoprotein receptor 1(ASGR1)and kringle containing transmembrane protein 1(Kremen1),while ALI co-culture with endothelial cells significantly up-regulated these genes(P<0.05).The cell infection rate of model virus particles increased significantly compared with traditional ALI culture in A549 cells(P<0.05).Conclusion An air-liquid co-culture model of A549 cells and EA.hy926 cells has been established,which provides a new pathological model for the investigation of lung diseases and drug screening.