Liesveld J L, Broudy V C, Harbol A W, Abboud C N
Department of Medicine, University of Washington, Seattle.
Exp Hematol. 1995 Mar;23(3):202-9.
Hematopoiesis is influenced by the presence of the hematopoietic microenvironment, and Dexter-type liquid culture systems represent an in vitro representation of some aspects of the microenvironment that are optimal for the propagation of myeloid progenitors. Marrow stromal layers, which constitute part of these culture systems, produce growth factors, including stem cell factor (SCF), a ligand for the c-kit proto-oncogene that has been found to increase detection of myeloid, erythroid, and megakaryocytic progenitors in short-term marrow colony assays. In this work, the role of SCF in Dexter-type culture systems was examined to better define its contribution to steady-state myelopoiesis. When cultured in the continued presence of 100 ng/mL SCF, both primary and recharged cultures demonstrated significantly greater CFU-GM output, with quantitative differences noted throughout culture duration (up to 6 weeks). This increase in CFU-GM could be inhibited specifically with the addition of 1:1500 SR-1, a neutralizing anti-c-kit monoclonal antibody (MAb) that neutralizes the biological effects of SCF, and the increase was noted both with recharged light-density marrow cells and purified CD34+ progenitor cells. On the other hand, when primary or recharged marrow cultures were established in the absence of exogenous SCF, but in the continuous presence of SR-1, no inhibition of CFU-GM output was observed. When light-density marrow cells were purged of pre-existing CFU-GM by 4-hydroperoxycyclophosphamide (4-HC) and were seeded over irradiated stromal layers, exogenous SCF resulted in detection of CFU-GM from 4-HC-treated cells as early as 1 week of culture, as compared to the lack of significant emergence of CFU-GMs at 4 weeks in the control cultures. This SCF effect was also inhibited by SR-1. Purified CD34+ progenitor cells did not adhere to SCF immobilized to tissue culture plates, and the adhesion of such progenitors to murine Steel lines transfected with membrane-bound SCF was not greater than to the parent nontransfected Steel line, suggesting that the effect of SCF was not on CD34+ cell adhesion. These studies confirm the action of SCF at a pre-CFU level, and they demonstrate the ability of SCF to stimulate increased production of myeloid progenitors in long-term liquid culture systems.
造血作用受造血微环境的影响，德克斯特型液体培养系统代表了微环境某些方面的体外模型，这些方面对于髓系祖细胞的增殖最为适宜。构成这些培养系统一部分的骨髓基质层可产生生长因子，包括干细胞因子（SCF），它是c-kit原癌基因的配体，已发现在短期骨髓集落试验中可增加髓系、红系和巨核系祖细胞的检出率。在本研究中，对SCF在德克斯特型培养系统中的作用进行了研究，以更好地确定其对稳态髓系造血的贡献。当在持续存在100 ng/mL SCF的条件下培养时，原代培养和再充质培养的CFU-GM产量均显著增加，在整个培养期间（长达6周）均存在定量差异。CFU-GM的这种增加可通过添加1:1500的SR-1特异性抑制，SR-1是一种中和抗c-kit单克隆抗体（MAb），可中和SCF的生物学效应，再充质低密度骨髓细胞和纯化的CD34+祖细胞均出现这种增加。另一方面，当在无外源性SCF但持续存在SR-1的情况下建立原代或再充质骨髓培养时，未观察到CFU-GM产量受到抑制。当用4-氢过氧环磷酰胺（4-HC）清除低密度骨髓细胞中预先存在的CFU-GM，并将其接种到经辐照的基质层上时，外源性SCF导致早在培养1周时就能从4-HC处理的细胞中检测到CFU-GM，而对照培养在4周时CFU-GM未显著出现。这种SCF效应也被SR-1抑制。纯化的CD34+祖细胞不粘附于固定在组织培养板上的SCF，并且此类祖细胞对转染了膜结合SCF的小鼠Steel系的粘附力并不大于对亲本未转染的Steel系的粘附力，这表明SCF的作用不是对CD34+细胞的粘附。这些研究证实了SCF在CFU前水平的作用，并证明了SCF在长期液体培养系统中刺激髓系祖细胞产量增加的能力。
