Uoshima N, Ozawa M, Kimura S, Tanaka K, Wada K, Kobayashi Y, Kondo M
First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Japan.
Br J Haematol. 1995 Sep;91(1):30-6. doi: 10.1111/j.1365-2141.1995.tb05240.x.
We analysed c-Kit expression during erythroid differentiation using immunocytochemical staining and flow cytometric analysis. Burst-forming units-erythroid (BFU-E)-derived cell aggregates were identified in methylcellulose cultures containing human umbilical cord blood CD34+ cells and were stained by the indirect immunoalkaline phosphatase method. To investigate the changes in levels of cell-surface c-Kit expression, we subjected progenitor cells in liquid culture to flow cytometric analysis. In addition, the effects of stem cell factor (SCF) on cell-surface c-Kit expression were analysed in these two culture systems and the effects of SCF on erythroid colony formation were studied in a methylcellulose culture. c-Kit was expressed on the cell surface from BFU-E to erythroid precursors recognized morphologically as basophilic erythroblasts. Flow cytometric analysis showed that c-Kit expression increased until 6 d in liquid culture, and that decreased expression of c-Kit was associated with the increased expression of glycophorin A. Moreover, SCF increased the size of erythroid colonies when added at days 0, 4 and 8 in methylcellulose cultures. These results indicate that the c-Kit/SCF system still plays in proliferation of erythroid progenitor cells at the colony-forming units-erythroid stage. Finally, expression of c-Kit in erythroid progenitor cells cultured without SCF showed a diffuse pattern on the cell surface, whereas we observed positive c-Kit immunoreactivity in the region of the Golgi apparatus of these cells cultured with SCF. Flow cytometric analysis also showed that the levels of cell-surface c-Kit expression decreased in the presence of SCF. These results suggest that SCF induced down-modulation of cell-surface c-Kit expression, despite continuous synthesis of c-Kit protein.
我们使用免疫细胞化学染色和流式细胞术分析，研究了红系分化过程中的c-Kit表达。在含有人类脐带血CD34+细胞的甲基纤维素培养物中，鉴定出爆式红系集落形成单位（BFU-E）衍生的细胞聚集体，并采用间接免疫碱性磷酸酶法进行染色。为了研究细胞表面c-Kit表达水平的变化，我们对液体培养中的祖细胞进行了流式细胞术分析。此外，在这两种培养系统中分析了干细胞因子（SCF）对细胞表面c-Kit表达的影响，并在甲基纤维素培养中研究了SCF对红系集落形成的影响。从BFU-E到形态上被识别为嗜碱性成红细胞的红系前体细胞，c-Kit均在细胞表面表达。流式细胞术分析显示，在液体培养中，c-Kit表达在6天前增加，而c-Kit表达的降低与血型糖蛋白A表达的增加相关。此外，在甲基纤维素培养中于第0、4和8天添加SCF时，SCF增加了红系集落的大小。这些结果表明，c-Kit/SCF系统在红系祖细胞于红系集落形成单位阶段的增殖中仍发挥作用。最后，在无SCF条件下培养的红系祖细胞中，c-Kit在细胞表面呈弥漫性表达，而在用SCF培养的这些细胞的高尔基体区域，我们观察到了阳性的c-Kit免疫反应性。流式细胞术分析还显示，在有SCF存在的情况下，细胞表面c-Kit表达水平降低。这些结果表明，尽管c-Kit蛋白持续合成，但SCF诱导了细胞表面c-Kit表达的下调。
