Shinjo K, Takeshita A, Higuchi M, Ohnishi K, Ohno R
Department of Medicine III, Hamamatsu University School of Medicine, Japan.
Br J Haematol. 1997 Mar;96(3):551-8. doi: 10.1046/j.1365-2141.1997.d01-2071.x.
In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine-RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by 125I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34+CD38 cells (approximately 1600 sites/cell), and decreased in the following order: CD34+CD38- cells > CD34+CD38+ cells > CD34-CD38+ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34+ fraction expressed more EpoR than their CD34- fraction. However, the expression levels of EpoR of these fractions were lower than CD34+CD38- cells. These results indicated that EpoR was highly expressed on CD34+ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.
为了开发一种用于检测人细胞上促红细胞生成素（Epo）受体（EpoR）的非同位素定量分析方法，我们设计了一种流式细胞术分析方法，使用生物素标记的细胞和链霉亲和素-RED670偶联物进行染色。为了进行定量，我们应用了柯尔莫哥洛夫-斯米尔诺夫检验并计算了D值。根据生物素标记的Epo与EpoR特异性结合导致的荧光强度增加，从两个曲线的偏移程度评估D值。通过¹²⁵I-Epo结合试验计算的EpoR数量与D值之间观察到良好的相关性。然后，通过三色流式细胞术研究了正常个体骨髓细胞上的EpoR表达。在正常骨髓中，细胞上EpoR的数量在CD34⁺CD38⁻细胞中最高（约1600个位点/细胞），并按以下顺序降低：CD34⁺CD38⁺细胞＞CD34⁻CD38⁺细胞。血型糖蛋白A（GpA）阳性的红系细胞也表达EpoR，其CD34⁺部分比CD34⁻部分表达更多的EpoR。然而，这些部分的EpoR表达水平低于CD34⁺CD38⁻细胞。这些结果表明，EpoR在分化非常早期的CD34⁺造血祖细胞上高表达，且不表达CD38抗原，并且在人类骨髓细胞中，随着红系分化以及各种谱系的定向分化，EpoR的表达水平降低。
