摘要
在IL-5靶点药物的发现阶段,为评价候选药物的阻断活性,需建立依赖IL-5增殖且信噪比高、重现性好的检测用细胞株。TF-1是人白血病细胞,其生长完全依赖IL-3或GM-CSF,而IL-5与IL-3和GM-CSF共用β(βc)受体,因此,研究以GM-CSF依赖的TF-1细胞株为来源,用IL-5替换TF-1细胞生长必需的生长因子GM-CSF,降低血清含量进行细胞驯化培养传代。通过35 d的细胞驯化及3~5次降血清传代培养,采用有限稀释法分离单克隆,一共得到14个单克隆细胞株。对14株细胞进行FACS检测,其中,有9个细胞株IL-5Rα表达量升高。对IL-5Rα过表达的细胞株进行IL-5增殖检测,结果表明,相比8%FBS得到的单克隆,6%FBS得到的单克隆对IL-5刺激更敏感。通过单因素优化实验,细胞增殖检测最优的细胞接种量为2×10^(4)个/孔,FBS含量为3%,血清品牌为Gibco。驯化后的细胞TF-1-9E3对IL-5的剂量-效应曲线的信噪比为3.19,Emax区间为2.15,可用于IL-5的生物学活性检测及IL-5与IL-5Rα的抗体阻断活性检测。
In the discovery phase of IL-5-targeted drugs,in order to assess the blocking activity of the drug candidates,it is necessary to establish a cell line with high signal-to-noise ratio and good reproducibility for the assay depending on IL-5 proliferation.TF-1 growth is completely dependent on IL-3 or GM-CSF,while IL-5 sharesβc receptors with IL-3 and GM-CSF.GM-CSF-dependent TF-1 cell line was used in this study.GM-CSF,a growth factor essential for TF-1 cell growth,was replaced with IL-5,and the serum content was reduced for cell domestication and culture passaging.By 35 days of cell domestication and 3-5 times of reduced FBS passages in culture,14 monoclonal cell lines were obtained by the limited dilution method.FACS assays were performed on 14 cell lines,9 of which had elevated IL-5Rαexpression.IL-5 proliferation assay was performed on the cell lines with IL-5Rαoverexpression,and the results showed that monoclonal clones obtained with 6%FBS were more sensitive to IL-5 stimulation compared to monoclonal clones obtained with 8%FBS.The optimal cell number for the cell proliferation assay was 2×10^(4) cells/well,the brand of FBS was Gibco at a concentration of 3%.The signal-to-noise ratio and Emax of dose-response curve of IL-5-dependent TF-1 cells against IL-5 were 3.19 and 2.15 respectively.The TF-1-9E3 could be used for the biological activity assay of IL-5 and blocking assay of IL-5 and IL-5Rα.
作者
李诗洁
代维燕
王雪莲
柯文锋
韩飞
陈永奇
刘畅
白仲虎
LI Shijie;DAI Weiyan;WANG Xuelian;KE Wenfeng;HAN Fei;CHEN Yongqi;LIU Chang;BAI Zhonghu(Zhuhai Resproly Biopharmaceutical Co.,Ltd.,Zhuhai 519040,China;National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;The Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China;The Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《生物学杂志》
CAS
CSCD
北大核心
2023年第6期109-114,共6页
Journal of Biology
基金
珠海市产学研合作及基础与应用基础研究项目(ZH2201700220000ZPWC)。
