Regulation of Stromal Wound Healing by Growth Factors

描述(申请人提供)：感染后的角膜疤痕， 创伤或手术会导致视力受损，但人们对此知之甚少 关于哪些基因导致角膜瘢痕形成或哪些分子调节 这些纤维化基因表达的变化。我们的总体目标是更多 全面明确了角膜创伤愈合的分子调控，并发展 能够控制惊吓的特工。在我们之前的拨款中，我们假设 转化生长因子β系统在调节角膜瘢痕形成中起主导作用，并且 我们测量了标准化的TGFbeta系统在愈合过程中的变化 大鼠角膜准分子激光切割损伤模型有力地支持这一点 假设。我们还开发了试剂(反义寡核苷酸和核酶)。 在体外选择性地抑制了转化生长因子β系统，我们建立了 人角膜基质细胞体内转染法的基本参数 寡核苷酸和质粒。在我们对TGFbeta系统的研究中， 我们鉴定了一种新的下游生长因子结缔组织生长因子 (CTGF)介导转化生长因子β对角膜胶原合成的影响 成纤维细胞。在这项拨款中，我们将先前的假设扩大到包括 探讨CTGF在角膜瘢痕形成中的作用。具体来说，我们 假设结缔组织生长因子增加角膜瘢痕形成。我们进一步假设 即：TGFbeta将上调CTGF表达；CTGF是下游 转化生长因子β对角膜成纤维细胞的许多作用的中介体； 结缔组织生长因子和结缔组织生长因子受体的mRNA和蛋白在角膜瘢痕形成过程中增加； II型IGF受体是CTGF受体；CTGF将调节多个基因 参与瘢痕形成；反义选择性降低CTGF和CTGF-R 寡核苷酸或核酶将减少角膜瘢痕形成；基因表达 在角膜愈合过程中，伤口会以簇状图案变化， 由基因的功能和分子调节器决定。我们 我将在5个具体目标上检验这些假设。具体目标#1将衡量 9次大鼠角膜愈合过程中CTGF及其受体水平的变化 准分子激光消融术后90天。具体目标#2将决定 CTGF诱导培养的人角膜组织中基因表达的变化 应用Affymetrix微阵列培养成纤维细胞。具体目标#3将确定 CTGF受体与反义寡核苷酸和核酶的研制和检测 体外靶向CTGF和CTGF-R基因。特定目标#4将测试 靶向TGFβ、CTGF和CTGF-R的反义寡核苷酸和核酶 准分子激光角膜切削术中瘢痕形成的在体减少。具体目标#5将 确定愈合过程中12次基因表达的变化模式 使用Affymetrix微阵列对大鼠角膜伤口进行检测，然后使用聚集的数据 为了验证先前关于角膜相关基因作用的假说 疤痕形成，产生了关于角膜疤痕调节的新假说 形成，并确定新的靶基因，以减少疤痕的形成。这些 综合的、基于假设的实验将提高我们的总体 了解角膜疤痕是如何在分子水平上调节的，将 验证CTGF调节角膜重要方面的新假说 并将评估新的选择性抗疤痕药物。

DESCRIPTION (provided by applicant): Corneal scarring following infection, trauma, or surgery can lead to impaired vision, yet relatively little is known about which genes contribute to corneal scarring or what molecules regulate changes in expression of these fibrotic genes. Our-overall objective is to more fully define the molecular regulation of corneal wound healing, and to develop agents that will control scaring. In our previous grant, we hypothesized that the TGFbeta system played a dominant role in regulating corneal scarring, and we measured changes in the TGFbeta system during healing of a standardized excimer ablation wound model in rat corneas that strongly support this hypothesis. We also developed agents (antisense oligonucleotides and ribozymes) that selectively inhibited the TGFbeta system in vitro, and we established basic parameters for in vivo transfection of corneal cells with oligonucleotides and plasmids. During our investigations of the TGFbeta system, we identified a new downstream growth factor connective tissue growth factor (CTGF) that mediated the effects of TGFbeta on synthesis of collagen in corneal fibroblasts. In this grant, we enlarge our previous hypothesis to include investigating the actions of CTGF on corneal scarring. Specifically, we hypothesize that CTGF increases corneal scar formation. We further hypothesize that: TGFbeta will upregulate CTGF expression; that CTGF is the down stream mediator of many of the actions of TGFbeta on corneal fibroblasts; levels of CTGF and CTGF-R mRNA and protein will increase during corneal scarring; the type II IGF receptor is the CTGF receptor; CTGF will regulate multiple genes involved in scarring; selective reduction of CTGF and CTGF-R by antisense oligonucleotides or ribozymes will reduce corneal scarring; expression of genes during healing of corneal wounds will change in clustered patterns that are determined by the function and by the molecular regulators of the genes. We will test these hypotheses in 5 specific aims. Specific Aim #1 will measure the changes in CTGF and CTGF-R levels in rat corneas during healing at 9 time points up to 90 days after excimer ablation. Specific Aim #2 will determine CTGF-induced changes in gene expression in cultures of human corneal fibroblasts using Affymetrix microarrays. Specific Aim #3 will identify the CTGF receptor and develop and test antisense oligonucleotides and ribozymes targeting CTGF and CTGF-R genes in-vitro. Specific Aim #4 will test the antisense oligonucleotides and ribozymes targeting TGFbeta, CTGF and CTGF-R for in vivo reduction of scarring in excimer ablated corneas. Specific Aim #5 will determine the patterns of changes in gene expression at 12 times during healing of rat corneal wounds using Affymetrix microarrays then use the clustered data to test previous hypotheses about the roles of genes implicated in corneal scarring, generate new hypotheses about the regulation of corneal scar formation, and identify new target genes to reduce scar formation. These integrated, hypothesis-based experiments will increase our general understanding of how corneal scarring is regulated at the molecular level, will test the new hypotheses that CTGF regulates important aspects of corneal scarring and will evaluate new selective anti-scarring agents.