Dynamics of gastric mucous gel layer in vivo and in vitro using high pressure freezing method

冷冻固定是目前公认的最好的初始固定步骤，不仅可以保留生物样品的精细结构，而且可以保留其抗原性。为了阐明胃壁细胞的功能转化，我们建立了一种新的体外实验模型，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，并通过用于光镜检查的急冷冷冻或用于电子显微镜检查的高压冷冻将样品冷冻固定。结果，腺细胞的组织保存完好，分离后至少2小时与新鲜切除的大鼠胃粘膜非常相似。H+/K+-ATP酶的免疫组化显示了与组胺刺激相关的酶从细胞质到顶端膜的易位。在西咪替丁处理的粘膜，大多数壁细胞的形态处于静息状态，在其细胞质中显示出许多tubulovesicle。而组胺刺激的壁细胞则显示出细胞内小管发达，排列着长的微绒毛。

Cryofixation is currently accepted as the best initial fixation step to preserve not only the fine structure but also the antigenicity of biological specimens. 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 the enzyme 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.Further study should be required to clarify the functional morphological dynamics of gastric gland.