INTESTINAL CELL DIFFERENTIATION AND GROWTH REGULATION

本研究的总体目标是：1）研究 在分化、胚泡和 出生后肠上皮的发育; 2）鉴定 触发和调节分化的表达的因素， 功能的研究; 3）生长的研究 培养的肠隐窝细胞的特征;和4）比较肿瘤 肠细胞系与正常细胞的表面 膜抗原和生长特性。 在接下来的一年里， 支持，我们的主要努力将是针对净化一个 明显特异的肠道生长抑制剂，以前被证明是 存在于绒毛细胞匀浆和胎儿的用过的培养基中 肠类器官培养物，以及 肠表面膜和细胞骨架单克隆抗体 件. 绒毛细胞抑制因子的纯化 直到最近，匀浆进展缓慢，由于其明显的 在各种实验条件下的非均相行为，但 最近发现胎儿分泌类似或相同的抑制剂 肠类器官培养物进入培养基中向我们表明， 这可能是一种更好的抑制剂来源， 可以尝试纯化，并在不存在鲁米诺的情况下进行纯化。 蛋白酶和粘液成分。 许多研究计划， 这一建议的一部分集中在使用单克隆抗体， 用于体内和体外研究的特定试剂。 大量的 我们的实验室已经有了经过充分鉴定的抗体。 在下一年的支持期间，我们计划制备新的抗体 使用不同的抗原进行免疫：表面膜组分 从肠腺细胞、胎儿和胎儿的刷状缘膜中纯化， 新生（5-9天）大鼠，从成年大鼠中纯化的细胞骨架成分 绒毛细胞刷状缘和培养的人肿瘤细胞（CaCo-2细胞）。 小鼠将用这些不同的抗原免疫，并进行测试： 从每组中选择具有最高血清滴度的小鼠， 它们的脾细胞将与NS 1小鼠骨髓瘤细胞融合。 杂交瘤 将检测用HAT培养基选择的培养物是否产生 通过ELISA和放射免疫结合试验检测特异性抗体。 文化 将被克隆并用于生产大量的 克隆抗体 抗体将被纯化并用于 识别和表征所识别的抗原。

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.