生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (6): 109-.doi: 10.3969/j.issn.2095-1736.2023.06.109

• 技术方法 • 上一篇    下一篇

依赖IL-5增殖的细胞株TF-1-9E3的驯化及验证

李诗洁1,2,3,4， 代维燕1， 王雪莲1， 柯文锋2,3,4， 韩　飞2,3,4， 陈永奇1， 刘　畅1， 白仲虎2,3,4   

  1. 1. 珠海瑞思普利医药科技有限公司， 珠海 519040； 2. 江南大学 粮食发酵工艺与技术国家工程
    实验室， 无锡 214122； 3. 江南大学 生物工程学院 工业生物技术教育部重点实验室， 无锡 214122；
    4. 江南大学 生物工程学院 糖化学与生物技术教育部重点实验室， 无锡 214122
  • 出版日期:2023-12-18 发布日期:2023-12-18
  • 通讯作者: 刘畅，副高级研究员，主要从事细胞生物学研究，E-mail:changliu@resproly.com;白仲虎，教授，博士生导师，主要从事微生物学、分子生物学、发酵工程研究，E-mail:baizhonghu@jiangnan.edu.cn；刘畅和白仲虎为共同通信作者
  • 作者简介:李诗洁，博士，主要从事分子生物学研究，E-mail:shijieli2018@163.com
  • 基金资助:
    珠海市产学研合作及基础与应用基础研究项目（ZH2201700220000ZPWC）

Adaptive evolution and validation of TF-1-9E3 dependent on IL-5 for proliferation

LI Shijie1,2,3,4, DAI Weiyan1, WANG Xuelian1, KE Wenfeng2,3,4, HAN Fei2,3,4,CHEN Yongqi1, LIU Chang1, BAI Zhonghu2,3,4   

  1. 1. Zhuhai Resproly Biopharmaceutical Co., Ltd.，Zhuhai 519040, China; 2. National Engineering Laboratory for
    Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China; 3. The Key Laboratory of
    Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;
    4. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology,
    Jiangnan University, Wuxi 214122, China
  • Online:2023-12-18 Published:2023-12-18

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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2×104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。

关键词: IL-5, TF-1 cells, proliferation, blocking activity, cell domestication

Abstract: 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 shares βc 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-5Rα expression. IL-5 proliferation assay was performed on the cell lines with IL-5Rα overexpression, 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 2×104cells/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.

Key words: IL-5, TF-1细胞, 增殖, 阻断活性, 细胞驯化

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