Rubin D C, Roth K A, Birkenmeier E H, Gordon J I
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Cell Biol. 1991 Jun;113(5):1183-92. doi: 10.1083/jcb.113.5.1183.
Transgenes consisting of segments of the rat liver fatty acid-binding protein (L-FABP) gene's 5' non-transcribed domain linked to the human growth hormone (hGH) gene (minus its regulatory elements) have provided useful tools for analyzing the mechanisms that regulate cellular and spatial differentiation of the continuously renewing gut epithelium. We have removed the jejunum from normal and transgenic fetal mice before or coincident with, cytodifferentiation of its epithelium. These segments were implanted into the subcutaneous tissues of young adult CBY/B6 nude mouse hosts to determine whether the bipolar, migration-dependent differentiation pathways of gut epithelial cells can be established and maintained in the absence of its normal luminal environment. Immunocytochemical analysis of isografts harvested 4-6 wk after implantation revealed that activation of the intact endogenous mouse L-FABP gene (fabpl) in differentiating enterocytes is perfectly recapitulated as these cells are translocated along the crypt-to-villus axis. Similarly, Paneth and goblet cells appear to appropriately differentiate as they migrate to the crypt base and villus tip, respectively. The enteroendocrine cell subpopulations present in intact 4-6-wk-old jejunum are represented in these isografts. Their precise spatial distribution along the crypt-to-villus axis mimics that seen in the intact gut. A number of complex interrelationships between enteroendocrine subpopulations are also recapitulated. In both "intact" and isografted jejunum, nucleotides -596 to +21 of the rat L-FABP gene were sufficient to direct efficient expression of the hGH reporter to enterocytes although precocious expression of the transgene occurred in cells located in the upper crypt, before their translocation to the villus base. Inappropriate expression of hGH occurred in a high percentage (greater than 80%) of secretin, gastrin, cholecystokinin, and gastric inhibitory peptide producing enteroendocrine cells present in the intact jejunum of 4-6-wk-old L-FABP-596 to +21/hGH transgenics. Addition of nucleotides -597 to -4,000 reduced the percentage of cells co-expressing this reporter four- to eightfold in several of the subpopulations. Jejunal isografts from each transgenic pedigree studied contained a lower percentage of hGH positive enteroendocrine cells than in the comparably aged intact jejunum.(ABSTRACT TRUNCATED AT 400 WORDS)
由大鼠肝脏脂肪酸结合蛋白（L-FABP）基因5'非转录区片段与人类生长激素（hGH）基因（不含其调控元件）组成的转基因，为分析调节不断更新的肠道上皮细胞和空间分化机制提供了有用工具。我们在正常和转基因胎鼠的空肠上皮细胞分化之前或同时，将其空肠取出。将这些片段植入年轻成年CBY/B6裸鼠宿主的皮下组织，以确定在没有正常管腔环境的情况下，肠道上皮细胞的双极、迁移依赖性分化途径是否能够建立和维持。对植入后4-6周收获的同基因移植组织进行免疫细胞化学分析发现，随着分化的肠细胞沿隐窝至绒毛轴移位，完整的内源性小鼠L-FABP基因（fabpl）在这些细胞中的激活得到了完美重现。同样，潘氏细胞和杯状细胞在分别迁移到隐窝底部和绒毛顶端时似乎也能正常分化。完整的4-6周龄空肠中存在的肠内分泌细胞亚群在这些同基因移植组织中也有体现。它们沿隐窝至绒毛轴的精确空间分布与完整肠道中的情况相似。肠内分泌亚群之间的一些复杂相互关系也得到了重现。在“完整”和同基因移植的空肠中，大鼠L-FABP基因的核苷酸-596至+21足以将hGH报告基因高效表达导向肠细胞，尽管转基因在位于隐窝上部的细胞中过早表达，然后才迁移到绒毛底部。在4-6周龄L-FABP-596至+21/hGH转基因小鼠完整空肠中，产生促胰液素、胃泌素、胆囊收缩素和胃抑制肽的肠内分泌细胞中，有很高比例（超过80%）出现hGH的不适当表达。添加核苷酸-597至-4000可使几个亚群中同时表达该报告基因的细胞百分比降低4至8倍。所研究的每个转基因谱系的空肠同基因移植组织中，hGH阳性肠内分泌细胞的百分比低于同龄完整空肠。（摘要截短于400字）
