Kanakura Y, Sonoda S, Nakano T, Fujita J, Kuriu A, Asai H, Kitamura Y
Institute for Cancer Research, Osaka University Medical School, Japan.
Am J Pathol. 1987 Oct;129(1):168-76.
Connective tissue-type mast cells, other than those located in the serosal cavity, are fixed in the tissues. For study of the differentiation processes of mast cells in connective tissue, an in vitro method for producing mast-cell colonies is required. The authors enzymatically dispersed the cells from the skin of either neonatal or adult mice and plated them in methylcellulose containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). More than 97% of the colonies that developed consisted of mast cells alone. The clonal nature of the mast-cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when a mixture of skin cells from C57BL/6-bgJ/bgJ and C57BL/6-+/+ mice was plated, most of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. In spite of depletion of T-cell-derived factors, concentrations of mast cells and mast cell colony-forming units (CFU-Mast) in the skin of nude athymic mice are normal. However, PWM-SCM was necessary for in vitro development of mast-cell colonies from the skin of nude mice. The concentration of CFU-Mast in the skin of genetically mast-cell-deficient WBB6F1-W/Wv mice was negligible when compared with the value observed in the skin of control WBB6F1-+/+ mice. Individual mast-cell colonies derived from the skin of neonatal WBB6F1-+/+ mice were lifted from the methylcellulose, and cells from each colony were injected into the wall of the glandular stomach and the skin of WBB6F1-W/Wv mice. Most of the mast cell that appeared at the skin injection sites of the WBB6F1-W/Wv mice stained with berberine sulfate, indicating that they contained heparin. In contrast, the mast cells that appeared in the stomach mucosa of the recipient WBB6F1-W/Wv mice did not stain. This suggests that CFU-Mast located in the skin have not been committed to the connective tissue type. The present method may be useful for investigation of the mechanisms of mast-cell differentiation in connective tissue other than the serosal cavity.
除位于浆膜腔的肥大细胞外，结缔组织型肥大细胞固定于组织中。为研究结缔组织中肥大细胞的分化过程，需要一种体外产生肥大细胞集落的方法。作者用酶法从新生或成年小鼠的皮肤中分散细胞，并将其接种于含有商陆有丝分裂原刺激的脾细胞条件培养基（PWM-SCM）的甲基纤维素中。形成的集落中超过97%仅由肥大细胞组成。肥大细胞集落的克隆性质通过使用C57BL/6-bgJ/bgJ小鼠的巨大颗粒作为标志物来确定：即使将C57BL/6-bgJ/bgJ和C57BL/6-+/+小鼠的皮肤细胞混合接种，大多数产生的集落也仅由bgJ/bgJ型肥大细胞或+/+型肥大细胞组成。尽管T细胞衍生因子耗竭，但裸无胸腺小鼠皮肤中肥大细胞和肥大细胞集落形成单位（CFU-Mast）的浓度正常。然而，PWM-SCM对于从裸鼠皮肤体外培养肥大细胞集落是必需的。与对照WBB6F1-+/+小鼠皮肤中观察到的值相比，遗传性肥大细胞缺陷的WBB6F1-W/Wv小鼠皮肤中CFU-Mast的浓度可忽略不计。从新生WBB6F1-+/+小鼠皮肤衍生的单个肥大细胞集落从甲基纤维素中取出，每个集落的细胞注射到WBB6F1-W/Wv小鼠的腺胃壁和皮肤中。在WBB6F1-W/Wv小鼠皮肤注射部位出现的大多数肥大细胞用硫酸小檗碱染色，表明它们含有肝素。相反，在受体WBB6F1-W/Wv小鼠胃黏膜中出现的肥大细胞未染色。这表明位于皮肤中的CFU-Mast尚未定向分化为结缔组织型。本方法可能有助于研究浆膜腔以外的结缔组织中肥大细胞分化的机制。
