Differentiation of immature enterocytes into enteroendocrine cells by Pdx-1 overexpression.

人们对肠道内多种内分泌细胞的发育情况知之甚少。我们测试了未成熟的肠道干细胞是否通过体外转移同源盒基因而转变为多个肠内分泌激素产生细胞。我们将胰腺-十二指肠同源盒1基因(Pdx1)导入来源于正常大鼠的胚胎肠上皮细胞系IEC-6，并筛选出Pdx1高表达150倍的细胞。免疫细胞化学和电子显微镜观察细胞分化为肠内分泌细胞的情况。在微孔滤膜上培养的转基因细胞形成堆积在单层细胞上的小梁网络。这些小梁细胞显示Pdx1蛋白的核定位，胞质内含有发达的粗面内质网和许多多形性的分泌颗粒。抗嗜铬粒素A、5-羟色胺、缩胆囊素、胃泌素和生长抑素的抗体染色这些胞浆内的分泌颗粒。此外，免疫荧光双染色分析表明，细胞内产生了不同的激素。这些结果为未成熟的肠上皮细胞可以在Pdx1过表达的情况下分化为多种激素分泌的内分泌细胞提供了证据。

The development of a variety of enteroendocrine cells of the gut is poorly understood. We tested whether immature intestinal stem cells were switched to multiple enteroendocrine hormone-producing cells by in vitro transfer of a homeobox gene. We transfected the pancreatic-duodenal homeobox 1 gene (Pdx1) into IEC-6 cells, an embryonic intestinal epithelial cell line derived from a normal rat, and selected the cells that overexpressed Pdx1 by 150-fold compared with control. The cells were examined for differentiation into enteroendocrine cells by immunocytochemical and electron microscopic analyses. Transfected cells cultured on micropore filters formed a trabecular network piled up on monolayer cells. These trabecular cells showed nuclear localization of Pdx1 protein and contained well-developed rough endoplasmic reticulum as well as many secretory granules of pleomorphic shape in the cytoplasm. Antibodies against chromogranin A, serotonin, cholecystokinin, gastrin, and somatostatin stained these secretory granules in the cytoplasm. Furthermore, immunofluorescence double staining analysis showed that different hormones were produced within a cell. These results provide the evidence that immature intestinal epithelial cells can differentiate into multiple hormone-producing enteroendocrine cells in response to overexpression of Pdx1.