Wada H, Suda T, Miura Y, Kajii E, Ikemoto S, Yawata Y
Department of Medicine, Kawasaki Medical School, Kurashiki, Japan.
Blood. 1990 Jan 15;75(2):505-11.
In order to examine the sequential expression of major blood group antigens on human erythroblasts, a selective two phase liquid culture system for erythroid progenitors was established. After mononuclear cells obtained from peripheral blood were cultured in the presence of phytohemagglutinin stimulated-leukocyte conditioned medium (PHA-LCM) for 7 days (the first phase), nonphagocytic cells were recultured under hypoxic culture conditions containing 30% fetal calf serum, 1% bovine serum albumin, 300 micrograms/mL transferrin and 2 U/mL recombinant erythropoietin (the second phase). Mature (orthochromatic) erythroblasts were observed on day 4 of the second phase, and reached 57.1 +/- 3.1% of total cells on day 8, followed by the appearance of denucleated red cells, equivalent to mature red cells in peripheral blood. Hemoglobin contents reached the level of 16.8 +/- 0.7 micrograms/10(6) cells on day 8. Flow cytometric analyses revealed that, on day 3 of the second phase, cells became blood type M-positive, corresponding to the maturation of erythroid cells. Regarding the expression of ABH blood group antigens, a small number of blood type H-positive cells were initially detected on day 0 of the second phase, while blood type A-positive cells, which essentially were not observed on day 0, increased gradually corresponding to the extent of erythroid maturation. In the present system, Lewis and P1 blood group antigens were expressed at day 5 of the second phase, although autologous plasma was required to determine the expression of Lewis blood group antigens. This culture system is beneficial for studies on normal and abnormal human red cell membranes, because the erythroid progenitors in human peripheral blood were used, and a reasonable number of erythroid cells (0.5 to 1.5 x 10(7] was obtained with good maturation.
为了检测人类成红细胞上主要血型抗原的顺序表达，建立了一种用于红系祖细胞的选择性两阶段液体培养系统。从外周血获得的单核细胞在植物血凝素刺激的白细胞条件培养基（PHA-LCM）存在下培养7天（第一阶段）后，非吞噬细胞在含有30%胎牛血清、1%牛血清白蛋白、300微克/毫升转铁蛋白和2单位/毫升重组促红细胞生成素的低氧培养条件下重新培养（第二阶段）。在第二阶段第4天观察到成熟（正染）成红细胞，在第8天达到总细胞数的57.1±3.1%，随后出现无核红细胞，相当于外周血中的成熟红细胞。血红蛋白含量在第8天达到16.8±0.7微克/10⁶细胞的水平。流式细胞术分析显示，在第二阶段第3天，细胞变为M血型阳性，这与红系细胞的成熟相对应。关于ABH血型抗原的表达，在第二阶段第0天最初检测到少量H血型阳性细胞，而在第0天基本未观察到的A血型阳性细胞随着红系成熟程度逐渐增加。在本系统中，Lewis和P1血型抗原在第二阶段第5天表达，尽管需要自体血浆来确定Lewis血型抗原的表达。该培养系统有利于对正常和异常人类红细胞膜的研究，因为使用了人类外周血中的红系祖细胞，并且获得了数量合理（0.5至1.5×10⁷）且成熟良好的红系细胞。
