中国药物警戒 ›› 2025, Vol. 22 ›› Issue (2): 147-154.
DOI: 10.19803/j.1672-8629.20230800

• 雷公藤肝损伤相关研究专栏 • 上一篇    下一篇

iNKT细胞体外刺激方法筛选及雷公藤甲素在体外活化iNKT细胞的条件

梁晓婧1, 陈欣1, 农程1, 李亮2, 丁健2, 张陆勇1,3, 江振洲1,4#, 王欣之1,2,*   

  1. 1中国药科大学多靶标天然药物全国重点实验室新药筛选与药效评价中心,江苏 南京 210009;
    2南京中医药大学镇江附属医院/镇江市中医院,镇江市中医脾胃病临床医学研究中心,江苏 镇江 212003;
    3广东药科大学新药研发中心,广东 广州 510006;
    4江苏省药效研究与评价服务中心,江苏 南京 210009
  • 收稿日期:2023-12-21 出版日期:2025-02-17 发布日期:2025-02-17
  • 通讯作者: *王欣之,女,副研究员·博导,分子药理学与毒理学。E-mail:wangxz@cpu.edu.cn;#为共同通信作者。
  • 作者简介:梁晓婧,女,硕士,分子药理学与毒理学。
  • 基金资助:
    国家自然科学基金资助项目（81703626、82073948、82274200）; 南京中医药大学镇江附属医院/镇江市中医院,镇江市中医脾胃病临床医学研究中心资助项目（SSPW2023-KF08,镇江市科技局SS2021005）

Screening of in vitro Stimulation Methods for iNKT Cells and Conditions for in vitro Activation of iNKT Cells by Triptolide

LIANG Xiaojing1, CHEN Xin1, NONG Cheng1, LI Liang2, DING Jian2, ZHANG Luyong1,3, JIANG Zhenzhou1,4#, WANG Xinzhi1,2,*   

  1. 1New Drug Screening and Pharmacodynamics Evaluation Center, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing Jiangsu 210009, China;
    2Zhenjiang Hospital Affiliated to Nanjing University of Chinese Medicine, Zhenjiang Hospital of Traditional Chinese Medicine, Zhenjiang Traditional Chinese Medicine Spleen and Stomach Disease Clinical Medicine Research Center, Zhenjiang Jiangsu 212003, China;
    3Center for Drug Research and Development, Guangdong Pharmaceutical University, Guangzhou Guangdong 510006, China;
    4Jiangsu Center for Pharmacodynamics Research and Evaluation, Nanjing Jiangsu 210009, China
  • Received:2023-12-21 Online:2025-02-17 Published:2025-02-17

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摘要： 目的 雷公藤甲素（Triotolide, TP）活化恒定自然杀伤T（iNKT）细胞导致小鼠肝损伤。因此,考察iNKT细胞的体外刺激方法和TP在体外活化iNKT细胞的条件。方法 使用佛波酯（PMA）与离子霉素（ION）组合、抗CD3e与CD28抗体组合的2种方式,流式细胞术检测iNKT细胞系DN32.D3细胞表面活化标志物CD69和分泌的IL-2;使用不同浓度PMA刺激小鼠肝脏非实质细胞,流式细胞术检测iNKT细胞表面CD69和分泌IFN-γ、IL-4、IL-17、IL-10水平,分析体外活化iNKT细胞的最佳方法条件,进而分析TP体外活化iNKT细胞的最佳方法条件。结果 PMA和ION联合刺激,PMA为50 ng·mL-1时,iNKT细胞活化效果最佳。当加入抗原递呈细胞（APC）RBL-CD1d共培养时,TP可活化DN32.D3细胞。结论 PMA和ION组合,PMA为50 ng·mL-1时,iNKT细胞活化效果最好;且TP活化iNKT细胞需要APC的存在。

关键词: 雷公藤甲素, 佛波酯, 恒定自然杀伤T细胞, 肝脏, DN32.D3细胞, 体外活化, 流式细胞术, 小鼠

Abstract: Objective To study the methods of activating iNKT cells and the conditions for TP's activation of iNKT cells in vitro. Methods Flow cytometry was performed to detect the activation marker CD69 and cytokine IL-2 of iNKT cell line DN32.D3 cells using either a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) or a combination of CD3e and CD28 antibodies. The liver nonparenchymal cells of mice were stimulated under different concentrations of PMA. The levels of CD69, IL-4, IL-17 and IL-10 in iNKT cells were detected by flow cytometry to screen the optimal methodological condition for the in vitro activation before the conditions for in vitro activation of DN32.D3 cells by TP were investigated. Results The best activation of iNKT cells was achieved when stimulated by the combination of PMA and ION with the concentration of PMA at 50 ng·mL-1. TP activated DN32.D3 cells when antigen-presenting cell (APC) RBL-CD1d cells were added for co-culture. Conclusion INKT cells are most activated when the PMA concentration is 50 ng·mL-1 with the combination of PMA and ION. TP activation of iNKT cells requires the presence of APCs.

Key words: Triptolide, Phorbol 12-Myristate 13-Acetate, Invariant Natural Killer T Cells (iNkT Cells), Liver, DN32.D3 Cells, in vitro Activation, Flow Cytometry, Mice

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