Simon-Assmann Patricia, Bolcato-Bellemin Anne-Laure, Klein Annick, Kedinger Michèle
Inserm U682, Development and Physiopathology of the Intestine and Pancreas, 67200, Strasbourg, France.
Methods Mol Biol. 2009;522:309-18. doi: 10.1007/978-1-59745-413-1_20.
Several techniques have been used to study the expression of basement membranes molecules but none of them allow distinguishing the cellular origin of the deposition of a single molecule at the subepithelial basement membrane. For this purpose, we designed an experimental model using recombinants between chick and mouse embryonic intestines. Following constructions of interspecies endodermal/mesenchymal associations in culture, developmental growth was achieved by in vivo transplantation in the chick embryo. Immunocytochemistry, using species-specific antibodies recognizing either chick or mouse basement membrane molecules, was then performed on cryosections made through the developed hybrid intestines.The use of this experimental design permits determination of the precise expression/secretion in the intestinal basement membrane region of the individual constituents: interestingly some of them are strictly of epithelial or of mesenchymal origin, while others are of dual origin. Furthermore, we could show that each of these molecules is expressed in a peculiar development-dependent pattern. Such interspecies as well as heterotopic recombinants (from different levels of the gastrointestinal tract) can also be used successfully to approach the regulation of the expression of functional markers, i.e., digestive enzymes.
已经使用了几种技术来研究基底膜分子的表达，但没有一种技术能够区分单个分子在亚上皮基底膜沉积的细胞起源。为此，我们设计了一个使用鸡和小鼠胚胎肠道重组体的实验模型。在培养物中构建种间内胚层/间充质联合体后，通过在鸡胚中进行体内移植实现发育生长。然后，使用识别鸡或小鼠基底膜分子的种特异性抗体对通过发育的杂种肠道制作的冰冻切片进行免疫细胞化学分析。这种实验设计的使用允许确定单个成分在肠道基底膜区域的精确表达/分泌：有趣的是，其中一些严格来源于上皮或间充质，而其他一些则具有双重起源。此外，我们可以表明这些分子中的每一种都以一种特殊的发育依赖性模式表达。这种种间以及异位重组体（来自胃肠道的不同水平）也可以成功地用于研究功能标记物即消化酶表达的调节。
