CHARACTERIZATION OF FUNDIC MUCOSAL CELLS IN VITRO

犬胃底粘膜细胞的培养体系， 对生理相关的生长因子（GF）的反应一直是 开发 我们假设粘膜生长调节， 对于维持粘膜完整性和愈合是不可或缺的， 由内分泌、神经分泌和 自分泌/旁分泌途径。 我们提出了一种简化的方法， 阐明生长调节的细胞机制。 条件 允许在无血清、无GF培养基（Ro）中培养分裂细胞 被开发出来。 复制细胞的上皮性质将是 确认并通过[3 H]-胸苷（THM）监测细胞增殖 合并。 分化细胞标志物（胃蛋白酶原、H+/K+-ATP酶和 粘蛋白）将通过免疫组织化学和原位杂交检测 通过[3 H]-THM放射自显影和掺入鉴定和分裂细胞 溴脱氧尿苷免疫组化。 我们将延长初步 研究表明，大多数分裂细胞是未分化的， 少数含有胃蛋白酶原。 在Ro中，胃泌素和蛙皮素刺激生长。 这些肽的受体将被表征和定位于 使用放射性配体技术的特定细胞类型。 类似物和 拮抗剂将用于建立受体特异性。 我们将 GF对未分化分裂细胞的分化作用与主要 细胞前体定位其促有丝分裂作用的位点。 增长 Ro依赖于平板细胞密度，表明自分泌和/或 旁分泌生长控制我们将追踪初步数据， 转化生长因子α（TGF α）和胰岛素样生长因子-I（IGF-I）参与 在自分泌/旁分泌调节评估以下：（1）生产 复制细胞本身中的GF（通过放射免疫测定的GF含量， GF mRNA表达，GF mRNA原位杂交定位），（2） GF受体的定位和表征（3）GF释放到 （4）通过特异性免疫吸附抑制Ro的生长 抗体，和（5）通过潜在的生长调节GF mRNA的表达 调制器。 将调节事件定位于特定细胞类型将 需要细胞分离技术和/或以下的组合： (1)溴脱氧尿苷免疫组化[3 H]-THM放射自显影 鉴定分裂细胞，（2）免疫组织化学或原位杂交 用于GF含量和表达，和（3）免疫组织化学或原位 用于分化细胞标记物的杂交。 我们还将确认 发现TGF β和生长抑素抑制粘膜细胞增殖 并使用免疫中和法来确定TGF β是否自分泌 生长控制 从这些研究中，我们将开发一个新的模型系统， 评估细胞水平上对粘膜生长的调节。 这些 粘膜细胞复制迅速，并且对生理反应非常敏感。 浓度的GF，这表明这种简化的方法可能允许 阐明与生长控制有关的基本机制。

A culture system of canine fundic mucosal cells that proliferate in response to growth factors (GF) of physiologic relevance has been developed. We hypothesize that mucosal growth regulation, a process integral to the maintenance of mucosal integrity and to healing, entails a complex interplay between GF delivered by endocrine, neurocrine and autocrine/paracrine routes. We propose a reductionist approach to elucidate cellular mechanisms underlying growth regulation. Conditions permitting culture of dividing cells in serum-free, GF-free medium (Ro) were developed. The epithelial nature of the replicating cells will be confirmed and cell proliferation monitored by [3H]-thymidine (THM) incorporation. Differentiated cell markers (pepsinogen, H+/K+-ATPase, and mucin) will be detected by immunohistochemistry and in situ hybridization and dividing cells identified by [3H]-THM autoradiography and incorporation of bromodeoxyuridine immunohistochemistry. We will extend preliminary studies which indicate that most dividing cells are undifferentiated, while a few contain pepsinogen. In Ro, gastrin and bombesin stimulate growth. Receptors for these peptides will be characterized and localized to specific cell types using radioligand techniques. Analogues and antagonists will be used to establish receptor specificity. We will differentiate effects of GF on undifferentiated dividing cells vs. chief cell precursors to localize the site of their mitogenic action. Growth in Ro is dependent upon plating cell density, suggesting autocrine and/or paracrine growth control We will pursue preliminary data indicating that transforming GFalpha (TGFalpha) and insulin-like GF-I(IGF-I) are involved in autocrine/paracrine regulation assessing the following: (1) production of GF in the replicating cells themselves (GF content by radioimmunoassay, GF mRNA expression, GF mRNA localization by in situ hybridization), (2) localization and characterization of GF receptors (3) release of GF into medium, (4) inhibition of growth in Ro by immunoabsorption with specific antibodies, and (5) regulation of expression of GF mRNA by potential growth modulators. Localization of regulatory events to a specific cell type will entail cell separation techniques and/or a combination of the following: (1) bromodeoxyuridine immunohistochemistry of [3H]-THM autoradiography to identify dividing cells, (2) immunohistochemistry or in situ hybridization for GF content and expression, and (3) immunohistochemistry or in situ hybridization for differentiative cell markers. We will also confirm findings that TGFBeta and somatostatin inhibit mucosal cell proliferation and use immunoneutralization to determine if TGFBeta exerts autocrine growth control. From these studies we will develop a new model system to evaluate the regulation of mucosal growth at a cellular level. These mucosal cells replicate rapidly and are very responsive to physiologic concentrations of GF, suggesting that this reductionist approach may allow elucidation of basic mechanisms relevant to growth control.