Gastric Mesenchymal Myofibroblasts Maintain Stem Cell Activity and Proliferation of Murine Gastric Epithelium in Vitro
Gastric Mesenchymal Myofibroblasts Maintain Stem Cell Activity and Proliferation of Murine Gastric Epithelium in Vitro
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DOI：
10.1016/j.ajpath.2014.11.007
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发表时间：
2015-03-01
影响因子：
6
通讯作者：
Joh, Takashi
中科院分区：
文献类型：
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作者：
Katano, Takahito;Ootani, Akifumi;Joh, Takashi
干细胞受到包括间充质细胞在内的微环境生态位的影响。我们建立了一种新的长期方法，用间充质肌成纤维细胞进行原代小鼠腺胃培养，以研究胃上皮-间质相互作用。从小鼠腺胃中建立胃间充质肌成纤维细胞（GMF）细胞系。来自新生小鼠的腺胃细胞和 GMF 细胞在胶原蛋白中共培养。培养的胃细胞产生膨胀的球状结构。在 GMF 共培养系统中，与对照培养物相比，胃球的数量和大小有所增加（分别为 P = 0.009 和 0.008）。免疫组织化学显示细胞对人胃粘蛋白、HIK1083和嗜铬粒蛋白A呈阳性，表明细胞分别分化为表面粘液细胞、粘液颈细胞和肠内分泌细胞。 Lgr5的RNA原位杂交显示培养的胃球中有Lgr5(+)干细胞。在GMF共培养体系中培养2个月后，胃球上皮中持续观察到Lgr5(+)细胞。 GM Fs 使培养的胃上皮保持与体内相似的活跃增殖。实时定量RT-PCR显示，GMF中Gas1表达较高（P=0.0445），肠间充质肌成纤维细胞中Hoxc8、Notch1和Sox10表达较高（分别为P=0.0003、0.0143和0.0488）。我们展示了 GMF 在维持 Lgr5(+) 干细胞活性和影响正常胃上皮分化和增殖方面的潜在作用。
Stem cells are influenced by a microenvironmental niche that includes mesenchymal cells. We established a novel Long-term method for primary mouse glandular stomach culture with mesenchymal myofibroblasts to investigate gastric epithelial-mesenchymal interactions. A gastric mesenchymat myofibroblast (GMF) cell Line was established from mouse glandular stomach. Glandular stomach cells from neonatal mice and GMF cells were co-cultured in a collagen get. Cultured stomach cells yielded expanding sphere-Like structures. In the GMF co-culture system, the number and size of gastrospheres were increased compared with control cultures (P = 0.009 and 0.008, respectively). Immunohistochemistry showed cells positive for human gastric mucin, HIK1083, and chromogranin A, indicating differentiation into surface mucous cells, mucous neck cells, and enteroendocrine cells, respectively. RNA in situ hybridization for Lgr5 showed Lgr5(+) stem cells in the cultured gastrospheres. Lgr5(+) cells were observed persistently in the epithelium of gastrospheres in the GMF co-culture system for 2 months. GM Fs allowed the cultured gastric epithelium to maintain active proliferation similar to that seen in vivo. Real-time quantitative RT-PCR showed that Gas1 expression was higher in GMFs (P = 0.0445), and Hoxc8, Notch1, and Sox10 expressions were higher in intestinal mesenchymal myofibroblasts (P = 0.0003, 0.0143, and 0.0488, respectively). We show the potential role of GMFs in sustaining Lgr5(+) stem cell activity and affecting normal gastric epithelial differentiation and proliferation.