Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-city, Yamanashi, 409-3898, Japan.
Histochem Cell Biol. 2013 Oct;140(4):491-505. doi: 10.1007/s00418-013-1113-x. Epub 2013 Jun 23.
Intestinal ischemia and ischemia-reperfusion rapidly progress to tissue destruction and reconstruction of functional organs. To date, precise immunolocalizations and the timing of appearance of cell signaling components under such conditions have not been well visualized. Mitogen-activated protein kinase (MAPK) signal transduction pathways have been reported to be activated under various types of cell damage, and cyclic AMP response element-binding protein (CREB) was directly phosphorylated with various cellular stimuli. In this study, both the expression and the immunolocalization of ERK1/2, a member of the MAPK family, were examined in mouse intestinal tissues by in vivo cryotechnique, which is useful to retain soluble molecules including cell signaling molecules. Under normal conditions, although ERK was widely immunolocalized in the cytoplasm of epithelial cells, phosphorylated (p) ERK1/2 was slightly detected in a small amount of epithelial cells in crypt and top parts of the villi. In 5 min ischemia, more pERK1/2 immunolocalization was detected in epithelial cells of the crypt part. Up to 60 min, the pERK1/2 immunoreactivity was remarkably increased in wide areas of epithelial cells. In the 20 and 60 min ischemia groups, phosphorylated CREB was also immunostained in the nuclei of the same epithelial cell areas of pERK1/2. In 20 min ischemia with 60 min reperfusion experiments, pERK1/2 immunointensity was reduced in the crypt areas. In 60 min ischemia with 60 min reperfusion, however, it was still strongly immunolocalized in epithelial cells of the crypts. Thus, rapidly changing ERK1/2 phosphorylation was visualized in the intestinal epithelial stem cells of mouse small intestine.
肠缺血和缺血再灌注迅速导致组织破坏和功能器官重建。迄今为止，这种情况下细胞信号成分的精确免疫定位和出现时间尚未得到很好的观察。有报道称，丝裂原活化蛋白激酶（MAPK）信号转导途径在各种类型的细胞损伤下被激活，而环腺苷酸反应元件结合蛋白（CREB）则直接被各种细胞刺激物磷酸化。在这项研究中，通过体内冷冻技术检查了 MAPK 家族成员 ERK1/2 在小鼠肠组织中的表达和免疫定位，这对于保留包括细胞信号分子在内的可溶性分子非常有用。在正常情况下，尽管 ERK 广泛存在于上皮细胞的细胞质中，但在隐窝和绒毛顶部的少量上皮细胞中仅检测到少量磷酸化（p）ERK1/2。在 5 分钟缺血时，隐窝部分的上皮细胞中检测到更多的 pERK1/2 免疫定位。直到 60 分钟，上皮细胞中 pERK1/2 的免疫反应性显著增加。在 20 分钟和 60 分钟缺血组中，磷酸化 CREB 也在 pERK1/2 的同一上皮细胞区域的核中被免疫染色。在 20 分钟缺血 60 分钟再灌注实验中，pERK1/2 免疫强度在隐窝区减少。然而，在 60 分钟缺血 60 分钟再灌注中，它仍然在上皮细胞的隐窝中强烈免疫定位。因此，在小鼠小肠的肠上皮干细胞中观察到 ERK1/2 磷酸化的快速变化。
