生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (1): 20-.doi: 10.3969/j.issn.2095-1736.2024.01.020

• 研究报告 • 上一篇    下一篇

人原代呼吸道上皮细胞培养及抗呼吸道合胞病毒活性

丁惠如， 赵　敏， 程宁宁， 付远辉， 彭向雷， 虞结梅， 郑妍鹏， 何金生
  

  1. 北京交通大学 生命科学与生物工程研究院， 北京 100044
  • 出版日期:2024-02-18 发布日期:2024-02-18
  • 通讯作者: 郑妍鹏，博士，副教授，研究方向为生物药物基础与应用，E-mail:ypzheng@bjtu.edu.cn；何金生，博士，教授，研究方向为新型疫苗研究，E-mail:jshhe@bjtu.edu.cn；郑妍鹏和何金生为共同通信作者
  • 作者简介:丁惠如，硕士研究生，研究方向为生物药物基础与应用，E-mail:20121606@bjtu.edu.cn；赵敏，硕士研究生，研究方向为生物药物基础与应用，E-mail:18435178508@163.com；丁惠如和赵敏为共同第一作者
  • 基金资助:
    国家自然科学基金项目（81771777）

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

DING Huiru, ZHAO Min, CHENG Ningning, FU Yuanhui, PENG Xianglei, YU Jiemei, ZHENG Yanpeng, HE Jinsheng   

  1. College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing 100044, China
  • Online:2024-02-18 Published:2024-02-18

PDF (PC)

59

摘要： 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，IC50）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, CC50）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。

关键词: 人原代呼吸道上皮细胞, 呼吸道合胞病毒, 抗病毒化合物, 抗病毒活性, 抗病毒机制

Abstract: 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 (IC50) 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 (CC50) 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.

Key words: human airway epithelial cell, human respiratory syncytial virus, antiviral compound, antiviral activity, antiviral mechanism

中图分类号: