CORNEAL WOUND HEALING--REGULATION BY GROWTH FACTOR

需要有效的治疗方法来增强角膜溃疡的愈合， 手术、创伤或疾病造成的伤口。 我们的长期目标是 确定肽生长因子及其受体在 自发角膜伤口愈合和基于此开发的治疗 知识，以促进这一进程。 我们的假设是， 因子及其受体调节角膜伤口愈合的关键方面 通过自分泌和旁分泌机制。 我们会调查， 通过关注角膜创伤的三个方面， 愈合：（1）确定生长因子及其受体的概况 在角膜伤口的自发愈合过程中;（2）评价一种新的合成 基质金属蛋白酶（SIMMP）抑制剂用于治疗碱和 细菌诱导的角膜溃疡;（3）合成嵌合融合蛋白 由EGF和纤连蛋白的胶原结合结构域组成，和 测量生物化学特性和对角膜伤口愈合的影响。 在第一个项目中，我们将重点关注可能发生的变化， TGF-α、TGF-β和FGF系统中的角膜伤口愈合。 我们 最近报道角膜上皮和内皮细胞合成 大鼠眼房水中TGF-α mRNA和TGF-α蛋白水平 猫内皮损伤后增加12倍。 我们将确定是否 上皮细胞TGF-α mRNA水平发生变化， 内皮细胞在器官培养的人伤口愈合过程中的作用， 用32 P标记的牛角膜进行定量原位杂交 TGF-α正义和反义RNA探针。 TGF-α蛋白的变化 将通过免疫组织化学评估上皮和内皮细胞水平 使用我们产生的特异性TGF-α抗体， TGF-α受体水平的变化将通过定量测定来确定。 放射自显影125 I-TGF-α与受伤角膜切片的结合。 我们 还将确定加入TGF-α中和抗体是否 抑制体外角膜伤口愈合。 的第二部分 项目，我们将扩大我们最近的发现，局部治疗与 重度碱烧伤后即刻SIMMP完全阻断角膜 溃疡 我们将确定是否与SIMMP治疗可以停止溃疡 一旦溃疡开始，我们将评估碱伤眼睛的情况， SIMMP治疗假单胞菌感染性角膜溃疡 色葡萄 我们将确定SIMMP对趋化性的影响， 炎症细胞的吞噬反应和测量药代动力学 14 C标记的SIMMP参数。 在第三个项目中，我们将解决一个 角膜创伤生长因子治疗的主要局限性：快速丧失 为了克服这个问题，我们将 合成由人EGF基因偶联 与人胶原蛋白结合结构域（EGF-FnCol）。 我们将测量 包括EGF的EGF-FnCol融合蛋白的生物化学性质 受体结合、促有丝分裂活性、趋化活性和可逆性 与胶原蛋白结合。 嵌合体融合蛋白的生物学效应 将在上皮和内皮伤口愈合模型中进行评价。

There is a need for effective treatments which enhance healing of corneal wounds caused by surgery, trauma or disease. Our long-term goals are to determine the roles peptide growth factors and their receptors play in spontaneous corneal wound healing and develop treatments based on this knowledge to enhance this process. Our hypothesis is that peptide growth factors and their receptors regulate key aspects of corneal wound healing by the autocrine and paracrine mechanisms. We will investigate and further enlarge this concept by focusing on three aspects of corneal wound healing: (1) determine the profiles of growth factors and their receptors during spontaneous healing of corneal wounds; (2) evaluate a new synthetic inhibitor of matrix metalloproteinases (SIMMP) for treatment of alkali and bacterial induced corneal ulcers; (3) synthesize a chimeric fusion protein consisting of EGF and the collagen binding domain of fibronectin, and measure the biochemical properties and effect on corneal wound healing. For the first project, we will focus on the changes which may occur during corneal wound healing in the TGF-alpha, TGF-beta, and FGF systems. We recently reported that corneal epithelial and endothelial cells synthesize TGF-alpha mRNA and that levels of TGF-alpha protein in aqueous fluid of cats increased 12-fold after endothelial injury. We will determine if changes occur in the levels of TGF-alpha mRNA of epithelial and endothelial cells during healing of wounds in organ cultured human and bovine corneas by quantitative in situ hybridization with 32P-labeled TGF-alpha sense and antisense RNA probes. Changes in TGF-alpha protein levels epithelial and endothelial will be assessed by immunohistochemistry of specimens using a specific TGF-alpha antibody we have generated, and changes in TGF-alpha receptor levels will be determined by quantitative autoradiography 125I-TGF-alpha binding to sections of wounded corneas. We will also determine if addition of a TGF-alpha neutralizing antibody will inhibit corneal wound healing in vitro. In the second part of this project, we will extend our recent finding that topical treatment with SIMMP immediately after severe alkali injury completely blocked corneal ulceration. We will determine if treatment with SIMMP can halt ulceration of alkali injured eyes once ulceration has begun, and we will evaluate SIMMP in the treatment of corneal ulcers induced by Pseudomonas or Staphylococcus. We will determine the effect of SIMMP on chemotactic and phagocytic responses of inflammatory cells and measure pharmacokinetic parameters of 14C labeled SIMMP. In the third project, we will address a major limitation of growth factor treatment of cornea wounds: rapid loss of growth factors from a wound site To overcome this problem, we will synthesize a chimeric protein consisting of the human EGF gene coupled with the human collagen binding domain (EGF-FnCol). We will measure biochemical properties of the EGF-FnCol fusion protein including EGF receptor binding, mitogenic activity, chemotactic activity, and reversible binding to collagen. Biological effects of the chimera fusion protein will be evaluated in epithelial and endothelial wound healing models.