Regulation of Stromal Wound Healing by Growth Factors

描述（由申请人提供）：感染后角膜瘢痕， 创伤或手术可能导致视力受损，但相对而言知之甚少 哪些基因导致了角膜瘢痕形成，哪些分子调节了 这些纤维化基因表达的变化。我们的总体目标是 完全确定角膜伤口愈合的分子调控，并开发 控制惊吓的代理人在之前的研究中，我们假设 TGF β系统在调节角膜瘢痕形成中起主导作用， 我们测量了标准化的创伤愈合过程中TGF β系统的变化， 大鼠角膜中的准分子消融伤口模型强烈支持这一点 假说.我们还开发了试剂（反义寡核苷酸和核酶） 在体外选择性抑制TGF β系统， 角膜细胞的体内转染的基本参数 寡核苷酸和质粒。在我们对TGF β系统的研究中， 我们发现了一种新的下游生长因子结缔组织生长因子 CTGF介导TGF β对角膜胶原合成的影响 成纤维细胞在本研究中，我们扩大了我们之前的假设， 研究CTGF对角膜瘢痕形成的作用。我们特别 假设CTGF增加角膜瘢痕形成。我们进一步假设 TGF β将上调CTGF表达; CTGF是下游 TGF β对角膜成纤维细胞的许多作用的介质; CTGF和CTGF-R的mRNA和蛋白在角膜瘢痕形成过程中会增加， II型IGF受体是CTGF受体; CTGF将调节多个基因 参与瘢痕形成;通过反义核酸选择性减少CTGF和CTGF-R 寡核苷酸或核酶将减少角膜瘢痕形成;基因表达 在角膜伤口愈合期间， 由基因的功能和分子调节器决定。我们 将在5个具体目标中测试这些假设。具体目标#1将衡量 大鼠角膜愈合过程中CTGF和CTGF-R的变化 在准分子消融术后90天内。具体目标#2将决定 CTGF诱导人角膜上皮细胞基因表达的变化 成纤维细胞使用Affyellow微阵列。具体目标#3将确定 CTGF受体和开发和测试反义寡核苷酸和核酶 体外靶向CTGF和CTGF-R基因。具体目标#4将测试 靶向TGF β、CTGF和CTGF-R的反义寡核苷酸和核酶， 在体内减少准分子消融的角膜中的瘢痕形成。具体目标#5 确定愈合过程中12次基因表达变化的模式 然后使用聚类数据， 为了测试之前关于角膜相关基因作用的假设 产生了关于角膜瘢痕调节的新假设 形成，并确定新的靶基因，以减少疤痕形成。这些 综合的，基于假设的实验将增加我们的一般 了解角膜瘢痕形成是如何在分子水平上调节的， 测试新的假设，CTGF调节角膜的重要方面， 并将评估新的选择性抗瘢痕形成剂。

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.