Nakahara Masanori, Isozaki Koji, Vanderwinden Jean-Marie, Shinomura Yasuhisa, Kitamura Yukihiko, Hirota Seiichi, Matsuzawa Yuji
Department of Internal Medicine and Molecular Science, Osaka University Graduate School of Medicine, Suita, Japan.
Life Sci. 2002 Apr 5;70(20):2367-76. doi: 10.1016/s0024-3205(02)01517-5.
Interstitial cells of Cajal (ICCs) play a role as pacemakers for gastrointestinal movement. Although some in vivo experiments showed that the c-kit receptor tyrosine kinase (KIT) and its ligand, stem cell factor (SCF), might be required for the development of murine ICCs near birth, in vitro experiments would be useful to clarify the role of SCF-KIT system for the development of ICCs. We attempted to establish a culture system in order to investigate the proliferation of ICCs. Murine gastrointestinal cells from embryos or neonates were cultured with SCF and stained with anti-KIT antibody and/or alcian-blue. The numbers of KIT+ cells a n d alcian-blue+ cells we re counted, and the number of KIT+.alcian-blue- cells, which represent ICCs was calculated. Clusters containing KIT+ cells were formed in culture. The number of KIT+.alcian-blue- cells from day-18 post coitum embryos increased in response to SCF up to a concentration of 50 ng/ml or for 8 days. The number of cells from day-2 post-partum neonates increased for 4 days, and then remained constant in the presence of SCF. In contrast, the number of cells from day-6 post-partum neonates did not increase and remained constant, even in the presence of SCF. ICCs showed a dose-dependent and time-limited proliferation in response to SCF in the in vitro culture system used here in.
Cajal间质细胞（ICCs）作为胃肠运动的起搏细胞发挥作用。尽管一些体内实验表明，c-kit受体酪氨酸激酶（KIT）及其配体干细胞因子（SCF）可能是出生前后小鼠ICCs发育所必需的，但体外实验有助于阐明SCF-KIT系统在ICCs发育中的作用。我们试图建立一种培养系统，以研究ICCs的增殖情况。将胚胎或新生小鼠的胃肠细胞用SCF进行培养，并用抗KIT抗体和/或阿尔新蓝染色。对KIT+细胞和阿尔新蓝+细胞的数量进行计数，并计算代表ICCs的KIT+、阿尔新蓝-细胞的数量。培养物中形成了含有KIT+细胞的集落。来自受孕后18天胚胎的KIT+、阿尔新蓝-细胞数量在SCF浓度高达50 ng/ml时或培养8天时会随着SCF的作用而增加。来自产后第2天新生小鼠的细胞数量增加4天，然后在有SCF存在的情况下保持稳定。相比之下，来自产后第6天新生小鼠的细胞数量即使在有SCF存在的情况下也不会增加，而是保持稳定。在此处使用的体外培养系统中，ICCs对SCF表现出剂量依赖性和时间限制性增殖。
