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作 者:丁惠如 赵敏[1] 程宁宁 付远辉[1] 彭向雷[1] 虞结梅 郑妍鹏[1] 何金生[1] DING Huiru;ZHAO Min;CHENG Ningning;FU Yuanhui;PENG Xianglei;YU Jiemei;ZHENG Yanpeng;HE Jinsheng(College of Life Sciences and Bioengineering,Beijing Jiaotong University,Beijing 100044,China)
机构地区:[1]北京交通大学生命科学与生物工程研究院,北京100044
出 处:《生物学杂志》2024年第1期20-25,共6页Journal of Biology
基 金:国家自然科学基金项目(81771777)。
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,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.
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
分 类 号:R373[医药卫生—病原生物学]
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