CHARACTERIZATION OF FUNDIC MUCOSAL CELLS IN VITRO

犬眼底粘膜细胞体外增殖培养体系的建立 对与生理相关的生长因子(GF)的反应 发展起来的。我们假设粘膜生长调节是一个过程 对于维持粘膜完整性和愈合来说，需要一个 内分泌、神经分泌和神经递质分泌的生长因子之间的复杂相互作用 自分泌/旁分泌路线。我们提出了一种简化论的方法来 阐明生长调节的细胞机制。条件 允许在无血清、无生长因子的培养液(Ro)中培养分裂细胞 都被开发出来了。复制细胞的上皮性将是 用[~3H]-胸腺嘧啶核苷(THM)确认和监测细胞增殖 成立为法团。分化细胞标记物(胃蛋白酶原、H+/K+-ATPase和 粘蛋白)将通过免疫组织化学和原位杂交检测 和[~3H]-THM放射自显影和掺入鉴定的分裂细胞 溴脱氧尿嘧啶核苷免疫组织化学。我们将延长初步的 研究表明，大多数分裂细胞是未分化的，而 少数含有胃蛋白酶原。在RO中，胃泌素和蛙皮黄素可以刺激生长。 这些多肽的受体将被表征和定位到 使用放射性配基技术的特定细胞类型。类似物和 拮抗剂将被用来建立受体特异性。我们会 生长因子对未分化分裂细胞与主细胞的分化作用 细胞前体以定位其促有丝分裂作用的部位。增长 RO依赖于电镀细胞密度，提示自分泌和/或 控制旁分泌生长我们将寻求初步数据表明 转化生长因子α(TGFα)和胰岛素样生长因子-I(IGF-I)参与其中 在自分泌/旁分泌调节中评估如下：(1)生产 在复制细胞自身中的GF含量(通过放射免疫分析的GF含量， (2)，(2) 胰岛素样生长因子受体的定位与鉴定(3)胰岛素样生长因子释放 (4)特异性免疫吸附抑制Ro的生长。 抗体；(5)潜在生长调节生长因子基因的表达。 调制器。将调控事件本地化到特定细胞类型将 需要细胞分离技术和/或以下各项的组合： (1)溴脱氧尿苷免疫组织化学[~3H]-THM放射自显影 鉴定分裂细胞，(2)免疫组织化学或原位杂交 (3)免疫组织化学或原位染色。 用于分化细胞标记的杂交。我们还将确认 转化生长因子β和生长抑素抑制粘膜细胞增殖的研究 并使用免疫中和法确定转化生长因子β是否发挥自分泌作用 增长控制。从这些研究中，我们将开发一个新的模型系统来 在细胞水平上评估粘膜生长的调节。这些 粘膜细胞复制迅速，对生理反应非常灵敏 GF的浓度，这表明这种简化论的方法可能允许 阐明与生长控制相关的基本机制。

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.