GROWTH AND DIFFERNTIATION OF OCULAR SURFACE EPITHELIA

描述(改编自申请人的摘要)：本报告的目的 研究建议是继续探索其发病机制。 各种眼表疾病。几项体内研究发现， 破坏包含干部的部分角膜缘上皮 角膜上皮细胞(SC)群启动角膜类型 以结膜上皮向内生长为特征的疾病，延迟 愈合和新生血管。因为SC是细胞的原点 血统和细胞增殖和分化的最终来源， 研究SC如何维持上皮细胞表型是非常重要的。SC 以及另一种短暂的增殖物--瞬时放大细胞(TAC) 细胞，可以由它们独特的不同解剖位置分开，角膜缘 对阵角膜。能够调节SC激活或更新的因素，以及 TAC的产生或扩增将在无血清中进行检测 允许单细胞克隆生长的培养系统。从3T3开始 饲养层培养系统，一种成纤维细胞生长因子样生长因子，负责 利用肝素亲和力纯化SC更新的旁分泌控制 和高效液相色谱柱。因为SC是耐药的，但TAC对肿瘤敏感 促进剂(TPA)治疗、SC和TAC在方面将有所区别 蛋白激酶C介导的有丝分裂途径，这两个 将对亚群单独接种单抗(MNAb)。 发展。几项体内研究也证实，维生素A、 或维甲酸，对于保持正常的末端分化是至关重要的 转化为异常的鳞状化生，这是 眼表疾病的数量。自早期鳞状化生以来 表现为杯状细胞丢失，因此确定 结膜SC在杯状细胞和非杯状细胞中很常见 差异化。这将使用无血清单细胞进行检测。 结膜上皮联合AM3的克隆性生长培养体系 MNAb对眼粘液的影响。对SC和TAC功能的调节 结膜上皮可与角膜/角膜缘进行比较。 上皮细胞。作为正常末端分化的标志，其表位 MNAb AK1的特征和AK1阳性的128K的作用 还将研究粘蛋白结合中的蛋白质复合体。假说 维甲酸对EGF受体相对水平的影响呈剂量依赖性 与TGFb m-RNA相比，从而调节增殖和分化将 与MNAb标志物联合检测正常和异常末端 差异化。最后，为了证明供应链与有限的长寿 TAC的寿命，并最终解决结膜SC 可转分化为角膜上皮细胞，体外单细胞 移植就会建立起来。通过探索监管机构 在细胞和分子水平上的机制，然后申请人可以 加深对各种眼表疾病的发病机制的了解 因此，申请人的治疗性医疗设备在治疗这些疾病时 困难的条件可以改善。 这位调查者已经从描述人类的现象学 角膜缘创面再生的细胞生物学特征 细胞通过它们的行为存在，并通过对 开发组织培养模型，现在将使他们能够解剖 阐明了控制愈合的细胞控制和受体机制 角膜上皮细胞。 申请人现在建议将他的描述性结果逻辑地扩展到 涉及到的细胞机制。在初步结果中，他报告说 他已经建立了一种新的无血清培养体系 以及3T3饲养层培养支持系统，提供类似于成纤维细胞生长因子的 生长因子在允许单细胞克隆生长的条件下。使用 这两个系统，他应该能够研究成长 干细胞和瞬时扩增细胞(TAC)的特征。 利用SC而不是TAC对肿瘤促进剂(TPA)的抗性 处理后，这两种细胞系将发生分化 蛋白激酶C介导的有丝分裂途径

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.