The cryofixation of isolated rat gastric mucosa provides new insights into the functional transformation of gastric parietal cells:: an in vitro experimental model study
The cryofixation of isolated rat gastric mucosa provides new insights into the functional transformation of gastric parietal cells:: an in vitro experimental model study
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DOI：
10.1679/aohc.68.151
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发表时间：
2005-09-01
影响因子：
--
通讯作者：
Suganuma, T
中科院分区：
文献类型：
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作者：
Sawaguchi, A;Aoyama, F;Suganuma, T
目前冷冻固定被认为是最好的初始固定步骤，不仅可以保留生物样品的精细结构，还可以保留其抗原性。为了阐明胃壁细胞的功能转化，我们新开发了一种体外实验模型，命名为离体胃粘膜。在这项研究中，用组胺刺激壁细胞的酸分泌或用西咪替丁抑制壁细胞的酸分泌，并且通过用于光学显微镜的骤冷或用于电子显微镜的高压冷冻来冷冻样品。结果，分离后至少2小时内，腺细胞的组织保存完好，与新鲜切除的大鼠胃粘膜非常相似。 H+/K+-ATP酶的免疫组织化学证明H+/K+-ATP酶从细胞质易位至与组胺刺激相关的顶膜。在西咪替丁处理的粘膜中，大多数壁细胞在形态上处于静息状态，在其细胞质中显示出大量的管状小泡。相反，组胺刺激的壁细胞表现出发育良好的细胞内小管，内衬长微绒毛。据我们所知，本研究首先展示了一张电子显微照片，该照片强烈表明管网和顶膜之间存在膜融合。此外，清楚地显示了与刺激相关的埃兹蛋白从细胞质到顶端区域的易位，对应于离体胃粘膜模型中顶端微绒毛的发育。我们在这里描述了离体大鼠胃粘膜模型的制备，该模型为应用冷冻技术对壁细胞的功能转化提供了新的见解。
Cryofixation is currently accepted as the best initial fixation step to preserve not only the fine structure but also the antigenicity of biological samples. To elucidate the functional transformation of gastric parietal cells, we have newly developed an in vitro experimental model, named the isolated gastric mucosa. In this study, acid secretion of the parietal cell was stimulated with histamine or inhibited with cimetidine, and the samples were cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, the organization of glandular cells was well-preserved and quite similar to freshly excised rat gastric mucosa for at least 2 h after isolation. Immunohistochemistry of H+/K+-ATPase demonstrated a translocation of H+/K+-ATPase from the cytoplasm to the apical membrane associated with histamine-stimulation. In cimetidine-treated mucosa, most of the parietal cells were morphologically in the resting state, showing numerous tubulovesicles in their cytoplasm. In contrast, histamine-stimulated parietal cells exhibited well-developed intracellular canaliculi lined with long microvilli. To the best of our knowledge, the present study is first to demonstrate an electron micrograph that strongly suggests a membrane fusion between the tubulovescile and the apical membrane. Moreover, a stimulation-associated translocation of ezrin was clearly shown from the cytoplasm to the apical region, corresponding to apical microvilli development in the isolated gastric mucosa model. We here describe the preparation of the isolated rat gastric mucosa model, which provides new insights into the functional transformation of parietal cells by the application of cryotechniques.