Zhang Lei, Zhao Junfang, Liang Chenmeizi, Liu Mingyao, Xu Feng, Wang Xin
a East China Normal University and Shanghai Fengxian District Central Hospital Joint Research Center for Translational Medicine , Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University , Shanghai , China.
b Department of Pharmacy , Shanghai Fengxian District Central Hospital , Shanghai , China.
Drug Deliv. 2017 Nov;24(1):1453-1459. doi: 10.1080/10717544.2017.1381199.
Breast cancer resistance protein (BCRP), a key drug efflux transporter, significantly affects the therapeutic efficacy of many drugs. Thus, screening specific BCRP inhibitors and distinguishing between substrates and non-substrates of BCRP are valuable in drug discovery and development. This study presents a novel BCRP biosensor based on intestinal 3D organoids for rapid and sensitive detection of BCRP function. First, the crypts were isolated from mouse small intestine, and cultured in advanced DMEM/F12 medium to develop intestinal 3D organoids. Second, immunohistochemical studies demonstrated that BCRP protein in the organoids presented a similar expression and physiologic position to the small intestinal epithelium. Finally, the cultured organoids were treated in BCRP fluorogenic probe substrate Hoechst 33342 with or without BCRP inhibitor Ko143 and YHO-13177. The fluorescence intensity of Hoechst 33342 released from inner of the organoids was detected by microplate reader and the concentrations were calculated. Ko143 and YHO-13177 significantly inhibited the BCRP-mediated Hoechst 33342 transport in the 3D organoids. Consequently, a rapid and efficient biosensor has been successfully established to study BCRP, especially screening BCRP inhibitors in a high-throughput way.
乳腺癌耐药蛋白（BCRP）是一种关键的药物外排转运蛋白，对许多药物的治疗效果有显著影响。因此，筛选特异性BCRP抑制剂并区分BCRP的底物和非底物在药物研发中具有重要价值。本研究提出了一种基于肠道3D类器官的新型BCRP生物传感器，用于快速、灵敏地检测BCRP功能。首先，从小鼠小肠分离隐窝，并在先进的DMEM/F12培养基中培养以发育肠道3D类器官。其次，免疫组织化学研究表明，类器官中的BCRP蛋白在表达和生理位置上与小肠上皮相似。最后，将培养的类器官用BCRP荧光探针底物Hoechst 33342处理，同时加入或不加入BCRP抑制剂Ko143和YHO-13177。通过酶标仪检测从类器官内部释放的Hoechst 33342的荧光强度，并计算其浓度。Ko143和YHO-13177显著抑制了3D类器官中BCRP介导的Hoechst 33342转运。因此，已成功建立了一种快速有效的生物传感器来研究BCRP，特别是以高通量方式筛选BCRP抑制剂。
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