REGULATION OF STROMAL WOUND HEALING BY GROWTH FACTORS

在准分子激光治疗后经常发生的主要临床问题是 上皮下混浊或基质瘢痕的发展。 组织学检查显示 瘢痕似乎主要由I型和III型胶原组成， 蛋白聚糖，推测是由基质成纤维细胞合成的 和上皮细胞。 没有任何治疗方法被证明是临床上 有效减少上皮下混浊 皮质类固醇治疗经常使用， 青光眼和继发性白内障。 因此，需要治疗 这将选择性地和安全地减少疤痕形成后， 根据PI的初步实验结果， 大鼠角膜，他假设角膜炎后的基质瘢痕 主要由TGF-β系统刺激。 这表明， TGF-β系统是减少角膜炎的治疗的主要靶点。 疤痕 为了验证这个假设，目标1将测量 TGF-β亚型和TGF-β IIR的mRNA和蛋白质沿着 以下关键时间大鼠角膜中ECM的重要成分 使用竞争性RT-PCR和ELISA进行检测。 这些数据将 确定大鼠中TGF-β系统最活跃的时间范围 因此，这将有助于设计重组 腺相关病毒（rAAV）-反义载体，将开发 目的3是选择性地减少TGF-β II受体的合成。 目的 2号将使用原位杂交和免疫组织化学， 鉴定角膜细胞合成ECM瘢痕蛋白， 通过TGF-β系统激活。 这些数据将表明哪些角膜 细胞是rAAV-反义载体转导的靶， 如目标4中的体内实验所述。 目标3将 合成表达反义RNA定向的不同rAAV载体 针对TGF-β IIR mRNA，并将评估 载体在降低TGF-B IIR mRNA和蛋白质水平的培养物中的作用 转染的角膜成纤维细胞。 具体目标4号将在 准分子激光消融大鼠角膜的体内实验。 rAAV反义 含有最佳启动子和反义序列的载体， 特定目标编号3将用于培养大鼠角膜细胞 继。 rAAV反义转染对人肝癌细胞系的影响 将评估TGF-β II型受体mRNA和蛋白的表达 以及转染对TGF-β II型表达的影响 受体mRNA和蛋白质将与对 大鼠角膜混浊的形成。 核酶有潜力 比反义RNA更有效。 随着发展的 rAAV-反义质粒，我们将合成rAAV-核酶载体， 靶向切割TGF-B II型受体mRNA的序列， 评估它们在减少TGF-B系统表达方面的有效性， 调节间质瘢痕形成，并将开发新的方法， 预防角膜切削术后的角膜混浊。

A major clinical problem that occurs frequently after excimer PRK is the development of subepithelial haze or stromal scar. Histologically, the scar appears to be composed primarily of collagen types I and III and proteoglycans that presumably are synthesized by stromal fibroblasts\s and epithelial cells. No treatment has been proven to be clinically effective for reducing subepithelial haze although long term topical corticosteroid treatment is frequently used in spite of the risks of glaucoma and secondary cataracts. Thus, there is a need for treatments that will selectively and safely reduce scar formation following PRK. Based on the PI initial experimental results with excimer PRK ablated rat corneas, he hypothesize that stromal scarring following PRK is stimulated predominately by the TGF-B system. This suggest that the TGF-B system is a prime target for treatments to reduce corneal scarring. To test this hypothesis, Aim number 1 will measure the levels of mRNAs and proteins for the TGF-B isoforms and TGF-B IIR along with important components of the ECM in rat corneas at key times following PRK using competition-based RT-PCR and ELISAs. These data will determine the time frame in which the TGF-B system is most active in rat corneas after PRK and thus will help in the design of recombinant adeno-associated virus (rAAV)-antisense vectors that will be developed in Aim number 3 to selective reduce TGF-B II receptor synthesis. Aim number 2 will use in situ hybridization and immunohistochemistry to identify corneal cells synthesizing ECM scar proteins in response to activation by the TGF-B system. These data will indicated which corneal cells are targets for transduction by the rAAV-antisense vectors as described in the in vivo experiments in Aim number 4. Aim number 3 will synthesized different rAAV-vectors expressing antisense RNA directed against TGF-B IIR mRNA and will evaluate the effectiveness of the vectors in reducing levels of TGF-B IIR mRNA and protein in cultures of transfected corneal fibroblasts. Specific Aim number 4 will perform in vivo eperiments on excimer PRK ablated rat corneas. rAAV-antisense vectors containing optimal promoter and antisense sequences developed in specific Aim number 3 will be used to transduce rat corneal cells following PRK. The effects of the rAAV-antisense transfection on expression of TGF-B type II receptor mRNA and protein will be assessed together with the effects on transfection on expression of TGF-B type II receptor mRNA and protein will be assessed together with effects on formation of corneal haze in the rats. Ribozymes have the potential to be more effective than antisense RNA. Following the development of the rAAV-antisense plasmid, we will synthesize rAAV-ribozyme vectors that are targeted to cut sequences for TGF-B type II receptor mRNA and evaluate their effectiveness in reducing expression of TGF-B system in regulating stromal scarring and will develop new approaches for prevention of corneal haze following PRK ablations.