人原代呼吸道上皮细胞培养及抗呼吸道合胞病毒活性

Anti-respiratory syncytial virus activity evaluation via primary human airway epithelial cell culture

为建立人原代呼吸道上皮细胞(Human airway epithelial cell,hAEC)的培养方法,并在hAEC体系上探讨3-硫代吲哚类化合物RSV-A-4和免疫抑制剂代谢产物6-MMPr的抗呼吸道合胞病毒(Respiratory syncytial virus,RSV)活性及其机制,从而构建可用于RSV药物筛选及药效评价的细胞模型。采集人呼吸道上皮细胞样本,分离细胞后建立hAEC的培养方法,并进行细胞形态和活力鉴定;在hAEC体系上检测小分子化合物RSV-A-4和6-MMPr的抗RSV活性及其细胞毒性;采用实时荧光定量PCR(RT-qPCR)与Time-of-addition assay技术,探讨RSV-A-4和6-MMPr的抑制RSV复制的作用机制。培养的hAEC经鉴定:其体外培养存活率可达93.51%;RSV-A-4和6-MMPr的半数抑制浓度(Half maximal inhibitory concentration,IC_(50))分别为(207.30±4.77)μmol/L和(3191.00±6.11)μmol/L,6-MMPr的半数细胞毒性浓度(Half maximal cytotoxic concentration,CC_(50))为(95526.00±10.97)μmol/L,而RSV-A-4对hAEC未见明显细胞毒性;RSV-A-4和6-MMPr均在RSV病毒基因组复制阶段抑制RSV复制。成功建立可用于抗RSV药物筛选及体外评价的hAEC培养体系,RSV-A-4和6-MMPr在细胞水平均能有效抑制RSV复制。

This study aims to establish the culture method of human primary airway epithelial cell(hAEC)and to investigate the anti-respiratory syncytial virus(RSV)activity and mechanism of 3-thioindole compound RSVA-4 and immunosuppressive metabolite 6-MMPR using hAEC system,which intends to construct a cell model for RSV drug screening and efficacy evaluation.The respiratory tract epithelial cells from volunteers were collected and cultured,then the morphology,activity and purity were identified.The anti-RSV activity and cytotoxicity of RSVA-4 and 6-MMPR were further verified in hAEC system.The mechanism of RSVA-4 and 6-MMPR accounting for the suppression of RSV replication on hAEC was explored by using fluorescence real-time quantitative PCR(RT-qPCR)and time-of-addition assay.The survival rate of cultured hAEC was 93.51%as determined by trypan blue staining.The half maximal inhibitory concentrations(IC_(50))of RSV-A-4 and 6-MMPR were(207.30±4.77)μmol/L and(3191.00±6.11)μmol/L,respectively.The half maximal cytotoxic concentration(CC_(50))of 6-MMPR was(95526.00±10.97)μmol/L,while no toxicity of RSVA-4 was observed on hAEC.Mechanistically,RSV-A-4 and 6-MMPr inhibited RSV replication in the genome replication/transcription phase.The hAEC culture method was successfully established,which could be used to screen and evaluate the anti-RSV drugs in vitro.RSVA-4 and 6-MMPR could effectively inhibit RSV replication at the cellular level.Altogether,the result could provide an experimental basis for the research and development of RSV drug and pathogenesis.