CONTROL OF CORNEAL EPITHELIAL CELL PROLIFERATION

角膜上皮细胞增殖的控制

• 批准号: 6125179
• 负责人: Peter S Reinach
• 金额: $ 23.71万
• 依托单位: STATE COLLEGE OF OPTOMETRY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6125179
• 关键词: animal tissue; calcium indicator; cell cell interaction; cell differentiation; cell morphology; cell proliferation; corneal epithelium; endothelin; epidermal growth factor; ion transport; mathematical model; membrane transport proteins; organ culture; single cell analysis; video microscopy

The corneal epithelium provides an essential barrier function which protects the cornea against pathogenic invasion and loss of its optical transparency. Following corneal epithelial wounding, the cytokine endothelial accelerates healing. This hastening of wound closure may include increases in migration, proliferation and differentiation. Even though there is endothelin receptor subtype heterogeneity in the corneal epithelium, little is known about the cell signaling mechanisms which link receptor stimulation to the mediation of these responses. The focus of this proposal is to determine if there is any difference between the cell signaling mechanisms of the endothelin receptors in the proliferating basal and differentiating suprabasal layers of the corneal epithelium. To evaluate this question, we will determine if endothelin has different effects on cell volume regulatory responses in primary subconfluent proliferating cultures of the bovine corneal epithelium and in differentiating suprabasal cells of the intact bovine corneal epithelium. Their effects on cell volume regulation are evaluated because this response is a reflection of individual ion fluxes which account for osmotically driven water flow. The measurements of volume changes in the cultured cells are made simultaneous to those of changes in intracellular [Ca2+] and pH. To monitor these changes, the technique uses microfluorometric measurements of changes in the concentration of intracellularly trapped dyes to report relative volume. By using pH or Ca2+ sensitive dyes and measuring the fluorescence resulting from excitation at the ion sensitive and isosbestic wavelengths with an imaging system, cell volume changes and intracellular ion activities are concomitantly measured. Preliminary results show that the technique is appropriate for characterizing volume regulatory responses which occur subsequent to dilution of the bathing solution by 55%. These regulatory volume decreases (RVD) reflect increases in net ion transport which restore the match between intracellular and extracellular osmolality. Simultaneous to this response there are large transient changes in intracellular (Ca2+] whereas in other nonregulating cells these transients are not seen. To characterize cell volume regulation in the intact suprabasal epithelial layers of the cornea, cell height is used as an index of volume and it is measured with a video microscopy technique. In another preliminary study, it is apparent that these cells perform RVD and can also restore themselves to their control volume during exposure to a hypertonic stress (i.e. regulatory volume increase, RVI). To ascertain if membrane ion transport changes are part of the linkage involved in the control of proliferation by endothelin receptors, ionic transport will be perturbed. If such an alteration affects this control, membrane ion transport changes contribute to endothelin mediated cell signaling and stimulation of proliferation in cultured cells. Taken together, these results could impact on the development of drugs which can selectively stimulate cell proliferation.

角膜上皮提供了必不可少的屏障功能 保护角膜免受致病性入侵和光学的丧失 透明度。角膜上皮损伤后，细胞因子 内皮加速愈合。这种加速伤口闭合可能 包括迁移，增殖和分化的增加。甚至 尽管角膜中有内皮素受体亚型异质性 上皮，对连接的细胞信号传导机制知之甚少 受体刺激这些反应的介导。重点 该建议是确定单元格之间是否有任何差异 增殖中内皮素受体的信号传导机制 角膜上皮的基底和区分上层。到 评估这个问题，我们将确定内皮素是否有不同 对主要亚汇合中细胞体积调节反应的影响 牛角膜上皮的增殖培养物和 分化完整牛角膜上皮的上prapabasal细胞。 评估它们对细胞体积调节的影响，因为这 响应反映了单个离子通量的反映 渗透驱动的水流。 体积变化的测量 培养的细胞与细胞内变化同时。 [Ca2+]和pH。为了监视这些更改，该技术使用 浓度变化的微荧光测量 细胞内捕获的染料以报告相对体积。通过使用pH或 Ca2+敏感染料并测量由 带有成像的离子敏感和同质波长的激发 系统，细胞体积变化和细胞内离子活性是 同时测量。初步结果表明该技术是 适合表征发生的音量调节响应 在沐浴溶液稀释之后降低了55％。这些监管 体积减少（RVD）反映了净离子运输的增加 恢复细胞内和细胞外渗透压之间的匹配。 同时与此响应有很大的瞬态变化 细胞内（Ca2+]，而在其他非调节细胞中这些瞬态 没有看到。 表征完整的细胞体积调节 角膜上皮上皮层，用作细胞高度 音量索引，并通过视频显微镜技术进行测量。在 另一项初步研究，显然这些细胞执行RVD，并且 在暴露于一个 高渗应力（即调节体积增加，RVI）。确定是否 膜离子运输变化是涉及的连锁的一部分 通过内皮素受体控制增殖，离子运输将是 忐忑。如果这种改变会影响这种控制，则膜离子 运输变化有助于内皮素介导的细胞信号传导和 刺激培养细胞中增殖。总的来说，这些 结果可能会影响可以选择性地的药物开发 刺激细胞增殖。