INTESTINAL CELL DIFFERENTIATION AND GROWTH REGULATION

拟议研究的总体目标是：1)研究 表面膜在分化过程中的变化，胚胎和 肠上皮细胞的出生后发育；2)鉴定 触发和调节分化基因表达的因素 培养的肠道细胞的功能；3)生长的研究 培养的肠隐窝细胞的特性；4)比较肿瘤 具有正常细胞表面的肠道细胞系 膜抗原和生长特性。在下一年 支持，我们的主要努力将指向净化一个 明显特异的肠道生长抑制物，以前被证明是 存在于绒毛细胞匀浆和胎儿废液中 肠道类器官培养及其制备和表征 抗肠道表面膜和细胞骨架的单抗 组件。绒毛细胞抑制因子的纯化 直到最近，匀浆一直进展缓慢，因为它显然是 在各种实验条件下的异质性行为，但 最近发现一种相似或相同的抑制物是由胎儿分泌的 将肠道器官培养物放入培养基向我们表明 这可能代表了一个更好的抑制剂来源，从那里它的 可以在没有流明的情况下尝试和进行纯化 蛋白酶和粘液成分。许多计划进行的研究 这项提案的一部分集中在使用单克隆抗体作为 用于体内和体外研究的特定试剂。大量的 我们实验室已经有了特性良好的抗体。 在下一年的支持期间，我们计划准备新的抗体 使用不同的抗原进行免疫：表面膜组分 从肠道隐窝细胞提纯，刷状缘膜从胎儿和 新生(5-9日龄)大鼠，从成年大鼠中提纯细胞骨架成分 绒毛细胞刷缘，和培养的人肿瘤细胞(Caco-2细胞)。 将用这些不同的抗原免疫小鼠，并进行测试：这两种 将从每组中选择血清效价最高的小鼠，并 他们的脾细胞将与NS1小鼠骨髓瘤细胞融合。杂交瘤 用HAT培养基挑选的培养物将用于生产 用酶联免疫吸附试验和放射免疫结合试验检测特异性抗体。中国的文化 利息将被克隆并用于生产大量的 单克隆抗体。抗体将被提纯并用于 识别的抗原的鉴定和表征。

The overall objectives of the proposed research are: 1) the study of the surface membrane changes occurring during differentiation, embyonic and postnatal development in the intestinal epithelium; 2) the identification of the factors which trigger and modulate the expression of differentiated functions in cultured intestinal cells; 3) the study of the growth characteristics of cultured intestinal crypt cells; and 4) to compare tumor intestinal cell lines with normal cells with respect to their surface membrane antigens and growth characteristics. During the next year of support, our major efforts will be directed towards the purification of an apparently specific intestinal growth inhibitor, previously shown to be present in villus cell homogenates and in the spent culture medium of fetal intestinal organoid cultures, and the preparation and characterization of monoclonal antibodies to intestinal surface membrane and cytoskeletal components. Purification of the inhibitory factor from villus cell homogenates has progressed slowly until recently, due to its apparently heterogeneous behaviour under a variety of experimental conditions, but the recent finding that a similar or identical inhibitor is secreted by fetal intestinal organoid cultures into the culture medium suggests to us that this may represent a much better source of inhibitor, from which its purification may be attempted and carried out in the absence of luminal proteases and mucus components. Many of the studies which are planned as part of this proposal are centered on the use of monoclonal antibodies as specific reagents for both in vivo and in vitro studies. A large number of well characterized antibodies are already available in our laboratory. During the next year of support we are planning to prepare new antibodies using different antigens for immunization: surface membrane fractions purified from intestinal crypt cells, brush border membranes from fetal and newborn (5-9 days old) rats, cytoskeletal components purified from adult villus cell brush borders, and cultured human tumor cells (CaCo-2 cells). Mice will be immunized with these different antigens, and tested: the two mice from each group having the highest serum titer will be selected, and their spleen cells will be fused with NS1 mouse myeloma cells. Hybridoma cultures, selected with HAT medium, will be tested for the production of specific antibodies by ELISA and radioimmunobinding assays. Cultures of interest will be cloned and used for the production of large amounts of monoclonal antibodies. Antibodies will be purified and used for the identification and characterization of the antigens recognized.