METHODS FOR THE ISOLATION OF INTACT EPITHELIUM FROM THE MOUSE INTESTINE
METHODS FOR THE ISOLATION OF INTACT EPITHELIUM FROM THE MOUSE INTESTINE
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DOI：
10.1002/ar.1091990412
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发表时间：
1981-01-01
影响因子：
--
通讯作者：
CHENG, H
中科院分区：
文献类型：
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作者：
BJERKNES, M;CHENG, H
描述了分离几种新形式的完整肠上皮的方法。所描述的第一种方法适用于必须在不杀死动物的情况下研究的肠段，例如切除的肠段。在这种方法中，将分离的节段外翻到玻璃棒上，并在37℃下孵化。C在新鲜的30 mM EDTA中，在无钙镁绞车‘’S平衡盐液中放置15或20分钟。在孵化结束时，通过振动将上皮分离到一管冷的CMF中。如果孵育20分钟，整个上皮以单个隐窝绒毛单位的形式被分离。如果孵育15分钟，上皮细胞以小片完整的形式被分离，但不是所有的上皮细胞都被移除。第二种方法要求动物死亡，但效果更好。麻醉后的动物通过左心室以37度的新鲜温度灌流。EDTA(0.01-30 mM)CMF溶液。然后取出肠段，外翻到玻璃棒上，通过振动将上皮分离到冰冷的CMF中。用这个程序分离整个上皮，在EDTA浓度较高的情况下，它以大片(1-2 cm2)的形式被分离。无论采用哪种方法，都可以获得结构完整的单元。没有潜在的非上皮性成分的污染。用30 mM乙二胺四乙酸乙二酯灌注法测定细胞的形态和活力，结果最佳。在这种浓度下，整个过程，从动物的牺牲到上皮的分离，只需不到4分钟。还描述了从小肠分离纯完整隐窝或纯完整绒毛的程序。所描述的每一种方法都可以同样容易地从肠道的任何区域分离上皮。文中还讨论了这些方法的一些应用。
Methods are described for the isolation of intact intestinal epithelium in several novel forms. The 1st method described is for application to segments of bowel that must be studied without killing the animals, e.g., a resected length of bowel. In this method, the isolated segment is everted onto a glass rod and incubated at 37.degree. C in fresh 30 mM EDTA in Ca-Mg-free Hank''s balanced salt solution (CMF) for either 15 or 20 min. At the end of the incubation, the epithelium is isolated by vibration into a tube of cold CMF. If it is incubated for 20 min, the entire epithelium is isolated in the form of single crypt-villus units. If it is incubated for 15 min, the epithelium is isolated in the form of small intact sheets, but not all of the epithelium is removed. The 2nd method requires the death of the animals but yields better results. The anesthetized animal is perfused through the left ventricle with a fresh 37.degree. solution of EDTA (0.01-30 mM) in CMF. Then the segment of gut is removed, everted onto a glass rod, and the epithelium isolated by vibration into cold CMF. The whole of the epithelium is isolated with this procedure and, and with higher concentrations of EDTA, it is isolated in the form of large sheets (1-2 cm2). Structurally intact units could be obtained with either procedure. There was no contamination with underlying nonepithelial elements. The best results, as determined by morphology and viability, were obtained with the perfusion method using 30 mM EDTA. With this concentration, the whole procedure, from sacrifice of the animal to isolation of the epithelium, requires less than 4 min. Procedures are also described for the isolation of either pure intact crypts or of pure intact villi from small intestine. Each of the methods described could be used to isolate the epithelium from any region of the intestinal tract with equal ease. Some applications of the methods are also discussed.