CORNEAL WOUND HEALING--REGULATION BY GROWTH FACTOR

需要有效的治疗方法来促进角膜的愈合。 因手术、创伤或疾病造成的伤口。我们的长期目标是 确定多肽生长因子及其受体在 角膜伤口的自发愈合和在此基础上开发的治疗方法 加强这一进程的知识。我们的假设是多肽生长 因子及其受体调控角膜伤口愈合的关键因素 通过自分泌和旁分泌机制。我们将调查并 通过关注角膜创伤的三个方面，进一步扩展这一概念 愈合：(1)测定生长因子及其受体的分布 在角膜伤口自然愈合过程中；(2)评价一种新的合成材料 基质金属蛋白酶抑制物(SIMMP)治疗碱烧伤 细菌诱导的角膜溃疡；(3)合成嵌合融合蛋白 由表皮生长因子和纤维连接蛋白的胶原结合域组成，以及 测定其生化特性及对角膜创面愈合的影响。 对于第一个项目，我们将重点放在可能发生的变化上 在转化生长因子-α、转化生长因子-β和成纤维细胞生长因子系统中的角膜伤口愈合。我们 最近报道，角膜上皮细胞和内皮细胞合成 大鼠房水中转化生长因子-αmRNA及蛋白水平变化 在内皮损伤后，CAT增加了12倍。我们将确定是否 上皮细胞和上皮细胞的转化生长因子-αmRNA水平发生变化。 血管内皮细胞在器官培养和创面愈合中的作用 ~(32)P标记牛角膜的定量原位杂交 转化生长因子α正义和反义RNA探针。转化生长因子-α蛋白的变化 上皮细胞和内皮细胞的水平将通过免疫组织化学进行评估 使用我们产生的特定转化生长因子-α抗体的样本，以及 转化生长因子-α受体水平的变化将通过定量测定 放射自显影125I-转化生长因子-α与损伤角膜切片结合。我们 还将确定添加转化生长因子-α中和抗体是否会 体外抑制角膜伤口愈合。在本部分的第二部分 项目，我们将扩展我们最近的发现，局部治疗 严重碱损伤后即刻SIMMP完全阻断角膜 溃疡。我们将确定使用SIMMP治疗是否可以阻止溃疡 碱损伤的眼睛一旦开始溃烂，我们将进行评估 SIMMP治疗假单胞菌引起的角膜溃疡 葡萄球菌。我们将确定SIMMP对趋化和 炎性细胞的吞噬反应及药代动力学测定 ~(14)C标记SIMMP的参数。在第三个项目中，我们将解决一个 生长因子治疗角膜创伤的主要局限性：迅速丧失 从伤口部位提取生长因子来克服这个问题，我们将 人表皮生长因子基因偶联嵌合蛋白的合成 与人胶原结合结构域(EGF-FnCol)结合。我们将衡量 含EGF的EGF-FnCol融合蛋白的生化性质 受体结合、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估。

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.