药学学报, 2020, 55(2): 265-271
引用本文:
王梦梦, 张旭霞, 尹俊, 任湘祎, 李欣悦, 陈红红. 基于竞争ELISA法的铀促排螯合剂体外筛选模型的建立[J]. 药学学报, 2020, 55(2): 265-271.
WANG Meng-meng, ZHANG Xu-xia, YIN Jun, REN Xiang-yi, LI Xin-yue, CHEN Hong-hong. Establishment of an in vitro screening model for uranium decorporation chelators based on the competitive ELISA method[J]. Acta Pharmaceutica Sinica, 2020, 55(2): 265-271.

基于竞争ELISA法的铀促排螯合剂体外筛选模型的建立
王梦梦, 张旭霞, 尹俊, 任湘祎, 李欣悦, 陈红红
复旦大学上海医学院放射医学研究所, 上海200032
摘要:
血液中的铀[U(Ⅵ)]与去铁转铁蛋白(apotransferrin,apo-Tf)形成稳定的络合物在U(Ⅵ)进入细胞致细胞毒性中发挥重要作用。本研究通过探讨螯合剂与apo-Tf竞争结合U(Ⅵ)的作用,建立了一种基于酶联免疫吸附实验(enzyme linkedimmunosorbent assay,ELISA)的U(Ⅵ)促排螯合剂体外筛选新模型,通过方法学研究确定了该检测方法的最佳包被抗原apo-Tf、Tf抗体、二抗稀释比例和U(Ⅵ)处理的浓度,验证了该方法的稳定性和重现性,并采用该模型考察了4种螯合剂邻苯二酚-3,6二甲撑亚氨基四乙酸(CBMIDA)、Tiron、DTPA-CaNa3、DTPA-ZnNa3与apo-Tf竞争结合U(Ⅵ)的能力,其强弱顺序为:CBMIDA ≈ Tiron > apo-Tf > DTPA-CaNa3 ≈ DTPA-ZnNa3;采用动物实验观察以上螯合剂的排U(Ⅵ)效果发现,CBMIDA和Tiron立即给药能显著提高U(Ⅵ)内污染小鼠24 h尿U(Ⅵ)排出、明显降低肾脏和股骨U(Ⅵ)蓄积;而DTPA-CaNa3和DTPA-ZnNa3则无明显促排效果,与竞争ELISA法的检测结果相一致。动物实验符合复旦大学药学院动物伦理委员会规程。以上结果表明,该方法快速、简便、重现性好,能够用于U(Ⅵ)促排螯合剂的快速筛选。
关键词:          螯合剂      去铁转铁蛋白      竞争酶联免疫吸附实验      筛选方法     
Establishment of an in vitro screening model for uranium decorporation chelators based on the competitive ELISA method
WANG Meng-meng, ZHANG Xu-xia, YIN Jun, REN Xiang-yi, LI Xin-yue, CHEN Hong-hong
Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China
Abstract:
Uranium[U(Ⅵ)] in the blood is known to form stable complexes with apotransferrin (apo-Tf), which plays an important role in mediating the cytotoxicity induced by U(Ⅵ) transported to cells. The present study aimed to establish an new in vitro screening model of U(Ⅵ) decorporation agents through exploring the capability of chelating agents competing with U(Ⅵ) binding to apo-transferrin based on enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of apo-Tf coated antigen, Tf antibody, secondary antibody and U(Ⅵ) treatment were achieved and the stability and reproducibility of this method were validated by methodology study. Using this model, the ability of four chelating agents to mobilize the U(Ⅵ) binding to apo-Tf was evaluated, and the rank of competitiveness was catechol-3,6-bis(methyleiminodiacetic acid) (CBMIDA) ≈ Tiron > apo-Tf > DTPA-CaNa3 ≈ DTPA-ZnNa3. The efficacy of these chelating agents in removal of U(Ⅵ) was tested by animal experiments. The results showed that immediate administration of CBMIDA or Tiron after injection of U(Ⅵ) in mice significantly promoted urinary U(Ⅵ) excretion and reduced U(Ⅵ) accumulation in kidneys and femurs, while DTPA-CaNa3 and DTPA-ZnNa3 have no obvious effects as compared to U(Ⅵ)-exposed mice alone, which was consistent with the results of competitive ELISA method. The animal experiments conform to the rules of the Animal Research Ethics Committee of School of Pharmacy of Fudan University. These results show that the new proposed method is rapid, simple and convenient with good reproducibility and has the potential to be used for in vitro screening of U(Ⅵ) decorporation agents.
Key words:    uranium    chelating agent    apotransferrin    competitive enzyme linked immunosorbent assay    screening method   
收稿日期: 2019-08-27
DOI: 10.16438/j.0513-4870.2019-0687
基金项目: 国家自然科学基金资助项目(81972971).
通讯作者: 陈红红,Tel/Fax:86-21-64437130,E-mail:hhchen@shmu.edu.cn
Email: hhchen@shmu.edu.cn
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