Raaijmakers H G P, Van Den Bosch G, Boezeman J, De Witte T, Raymakers R A P
Department of Hematology, University Medical Center Nijmegen, Nijmegen, The Netherlands.
Cytometry. 2002 Dec 1;49(4):135-42. doi: 10.1002/cyto.10157.
Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux.
We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively).
The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively).
The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.
正常和恶性造血干细胞的特征在于其主动排出荧光染料的能力。由于干细胞数量少限制了使用标准方法评估功能性外排，不同的ATP结合盒（ABC）转运蛋白对这一现象的贡献在很大程度上尚不清楚。
我们使用落射荧光显微镜（EFM）结合单细胞图像分析，研究在粘附生物层上分选的高度纯化、有活力的CD34+CD38-细胞中ABC转运蛋白介导的外排。使用荧光底物（罗丹明-123和钙黄绿素乙酰甲酯[钙黄绿素-AM]）和特异性抑制剂（分别为维拉帕米和丙磺舒）对P-糖蛋白和多药耐药蛋白（MRP）介导的外排进行定量。
在细胞系中显示了使用单细胞EFM进行罗丹明-123外排定量的可行性、敏感性和可重复性，并与标准流式细胞术评估进行了比较。P-糖蛋白介导的转运在CD34+CD38-细胞中高于分化程度更高的祖细胞（平均外排指数分别为2.24±0.35和1.14±0.11；P = 0.01）。P-糖蛋白介导的转运是这些细胞罗丹明“暗淡”表型的主要决定因素。此外，在CD34+CD38-和CD38+细胞中证实了显著的MRP介导的外排（平均外排指数分别为1.42±0.19和1.28±0.18）。
所描述的方法是评估高度纯化单细胞中ABC转运蛋白介导的外排的有价值工具。P-糖蛋白和MRP介导的外排均存在于人类CD34+CD38-造血干细胞中。
