Rottem M, Okada T, Goff J P, Metcalfe D D
Allergic Diseases Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Blood. 1994 Oct 15;84(8):2489-96.
Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL-3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4-week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.
在重组人干细胞因子（rhSCF）存在的情况下，肥大细胞可从人外周血中培养获得。外周血中产生肥大细胞的细胞特性尚不清楚。由于人类骨髓中的肥大细胞前体是CD34+，因此对肥大细胞增多症患者和正常对照的人外周血单个核细胞进行基于CD34表达的分选，并将阳性和阴性细胞群体在rhSCF、重组人白细胞介素-3（rhIL-3）或两者中培养6周。每2周检查一次细胞培养物，使用瑞氏吉姆萨染色和酸性甲苯胺蓝染色以及针对肥大细胞类胰蛋白酶和糜蛋白酶、细胞相关组胺的抗体，通过流式细胞术分析总细胞数、肥大细胞数和细胞分化情况，以及CD34、c-kit、FcεRI和FcγRII的表达。通过电子显微镜检查肥大细胞超微结构解剖。在rhSCF中培养的外周血CD34+细胞，无论有无rhIL-3，到6周时都能产生由超过80%肥大细胞组成的细胞培养物。单独在rhIL-3中培养的CD34+细胞不会产生肥大细胞，而与单独在rhSCF中培养的细胞相比，rhIL-3加rhSCF可使最终肥大细胞数量增加8倍。肥大细胞数量增加的同时，未分化的大单核细胞数量减少。CD34-细胞不会产生肥大细胞。6周时每个细胞的组胺含量约为5皮克。对4周龄培养物的电子显微镜检查显示，未成熟肥大细胞主要含有类胰蛋白酶阳性颗粒，这些颗粒要么均匀一致，要么含有晶格结构、部分卷轴模式或中央致密核心以及囊泡、细颗粒物质和颗粒的混合物。通过荧光激活细胞分选分析，第0天的CD34+群体表达Kit（65%）和FcγRII（95%），但不表达FcεRI。6周时，源自CD34+的肥大细胞除了表达Kit和FcγRII外，还表达FcεRI，并且对CD34抗原呈阴性。与正常对照相比，肥大细胞增多症患者培养得到的每个CD34+细胞中的肥大细胞数量更多。因此，人外周血中的肥大细胞前体是CD34+/FcεRI-，在有或无rhIL-3的rhSCF存在下可产生肥大细胞，并且与正常受试者相比，从肥大细胞增多症患者获得的CD34+细胞在培养中每个CD34+细胞产生的肥大细胞数量更多。
