GROWTH AND DIFFERNTIATION OF OCULAR SURFACE EPITHELIA

描述（改编自申请人的摘要）：本申请的目的 研究建议是继续探索致病机制， 各种眼表疾病。 几项体内研究发现， 破坏包含茎的角膜缘上皮部分 角膜上皮细胞（SC）群体启动角膜上皮细胞的类型。 以结膜上皮向内生长为特征的疾病，延迟 愈合和新血管形成。 因为SC是细胞的起源 谱系和细胞增殖和分化的最终来源， 研究SC如何维持上皮表型是非常重要的。 SC 和瞬时扩增细胞（TAC），另一种短寿命的增殖性细胞， 细胞，可以通过其独特的不同解剖部位，角膜缘， vs.角膜 能够调节SC激活或更新的因子，以及 TAC的产生或扩增，将在无血清 允许单细胞克隆生长的培养系统。 关于3 T3 饲养层培养系统，一种负责 SC更新的旁分泌控制将使用肝素亲和纯化 和HPLC柱。 由于SC耐药，而TAC对肿瘤敏感， 启动子（TPA）处理，SC和TAC将在以下方面有所区别： 蛋白激酶C介导的促有丝分裂途径。这两 亚群将分别免疫单克隆抗体（MNAb） 发展 几项体内研究也证实，维生素A， 或维甲酸，是保持正常的终末分化， 转化为异常鳞状化生，这是一种常见的特征， 眼表疾病的数量。 由于早期鳞状上皮化生 显示杯状细胞丢失，因此重要的是要确定是否 结膜SC，如果存在，常见于杯状细胞和非杯状细胞 分化 这将使用无血清单细胞 结膜上皮与AM3的克隆生长培养系统 MNAb与眼粘蛋白。 对最高法院和最高上诉法院职能的监管 将结膜上皮与角膜/角膜缘上皮进行比较 上皮细胞 作为正常终末分化的标志物， MNAb AK 1将被表征，AK 1阳性128 K 还将研究粘蛋白结合中的蛋白质复合物。 的假设 维甲酸剂量依赖性地影响EGF受体的相对水平， vs. TGFB m-RNA，从而调节增殖和分化， 结合MNAb标志物检测正常和异常终末 分化 最后，为了证明SC的寿命与有限 TAC的寿命，并以明确的方式解决结膜SC是否 可转分化为角膜上皮，体外单细胞 移植将成立。 通过探索监管 在细胞和分子水平上的机制，申请人然后可以 更好地了解各种眼表疾病的发病机制， 因此申请人治疗这些疾病的治疗设备 困难条件可以改善。 这位研究者已经从对宇宙的现象学的描述 从利姆布斯的伤口更新的细胞生物学，表征 细胞通过其行为和单克隆抗体标记呈现， 组织培养模型的发展，现在将使他们能够解剖 细胞控制和受体机制控制愈合 角膜上皮细胞。 申请人现在提出将其描述性结果逻辑地扩展到 所涉及的细胞机制。 在初步结果中，他报告说， 他在无血清培养基中建立了一种新的组织培养系统 以及3 T3饲养层培养支持系统， 生长因子的条件下，允许单细胞克隆生长。 与 这两个系统，他应该能够研究 干细胞和瞬时扩增细胞（TAC）的特性。 使用SC而不是TAC对肿瘤促进剂（TPA）的抗性 处理后，两种细胞系将在细胞分化方面发生分化。 蛋白激酶C介导的促有丝分裂途径。

DESCRIPTION (adapted from applicant's abstract): The objective of this research proposal is to continue to explore the pathogenic mechanisms of various ocular surface disorders. Several in vivo studies have found that destruction of the portion of limbal epithelium which contains the stem cell (SC) population of corneal epithelium initiates the types of corneal disorders characterized by conjunctival epithelial ingrowth, delayed healing, and neovascularization. Because SC is the origin of the cell lineage and the ultimate source of cell proliferation and differentiation, it is fundamental to study how SC maintain the epithelial phenotype. SC and the transient amplifying cell (TAC), another short-living proliferative cell, can be separated by their unique different anatomical sites, limbal vs. corneal. Factors able to modulate the activation or renewal of SC, and the generation or amplification of TAC, would be surveyed in a serum-free culture system that allows single-cell clonal growth. From the 3T3 feeder-layer culture system, a FGF-like growth factor responsible for the paracrine control of SC renewal would be purified using heparin-affinity and HPLC columns. Because SC is resistant, but TAC is sensitive to tumor promoter (TPA) treatment, SC and TAC would be differentiated with respect to the mitogenic pathway mediated by protein kinase C. These two subpopulations would be immunized separately for monoclonal antibody (MNAb) development. Several in vivo studies have also confirmed that vitamin A, or retinoids, is crucial for keeping normal terminal differentiation from transforming into aberrant squamous metaplasia, a common feature of a number of ocular surface disorders. Since early squamous metaplasia manifests goblet cell loss, it is thus important to determine whether conjunctival SC, if present, is common for goblet cell and non-goblet cell differentiation. This would be examined using a serum-free single-cell clonal growth culture system for conjunctival epithelium together with AM3 MNAb to ocular mucin. The regulation of SC and TAC functions of conjunctival epithelium would be compared to those of corneal/limbal epithelia. As a marker for normal terminal differentiation, the epitope of MNAb AK1 would be characterized and the role of the AK1-positive 128K protein complex in mucin binding would also be studied. The hypothesis that retinoids dose-dependently affect the relative level of EGF receptor vs. TGFB m-RNA, thereby modulating proliferation and differentiation would be tested in conjunction with MNAb markers for normal and abnormal terminal differentiation. Finally, to prove the longevity of SC vs. the limited lifespan of TAC, and to resolve in a definitive way whether conjunctival SC can be transdifferentiated into corneal epithelium, in vitro single-cell transplantation would be established. By exploring the regulatory mechanism at the cellular and molecular level, the applicant can then understand better the pathogenesis of various ocular surface disorders and thereby the applicant's therapeutic armamentarium in treating these difficult conditions can be enhanced. This investigator has moved from a description of the phenomenology of the cell biology of wound renewal from the limbus, characterizing the types of cells present by their behavior and by monoclonal antibody labeling to the development of tissue culture models which will now enable them to dissect out the cell control and receptor mechanisms underlying control of healing of the corneal epithelium. The applicant now proposes to extend logically his descriptive results to the involved cellular mechanisms. In the preliminary results, he reports that he has established a novel tissue culture system in serum-free medium as well as a 3T3 feeder-layer culture support system providing a FGF-like growth factor under conditions which allow single-cell clonal growth. With these two systems, the he should be able to study the growth characteristics of the stem cell and the transient amplifying cell (TAC). Using the resistance of the SC but not the TAC to tumor-promoter (TPA) treatment, the two cells lines will be differentiated with respect to the mitogenic pathway mediated by protein kinase C.