REGULATION OF STROMAL WOUND HEALING BY GROWTH FACTORS

准分子激光原位角膜磨镶术后经常出现的一个主要临床问题是 上皮下混浊或基质瘢痕的形成。从组织学上讲， 疤痕似乎主要由I型和III型胶原蛋白组成， 推测由基质成纤维细胞合成的蛋白多糖\S 和上皮细胞。目前还没有临床证明的治疗方法。 有效减少上皮下雾霾，尽管是长期局部应用 皮质类固醇治疗经常被使用，尽管存在以下风险 青光眼和继发性白内障。因此，有必要进行治疗。 这将选择性和安全地减少PRK后疤痕的形成。 基于准分子激光消融的PI初步实验结果 大鼠角膜，他假设PRK后的基质疤痕是 主要由转化生长因子-β系统刺激。这表明， 转化生长因子-β系统是减少角膜病变治疗的主要靶点 伤痕累累。为了检验这一假设，目标1号将测量水平 转化生长因子-β亚型和转化生长因子-βIIR的mRNAs和蛋白的表达 大鼠角膜中细胞外基质的重要成分 PRK使用基于竞争的RT-PCR和ELISA。这些数据将 确定转化生长因子-β系统在大鼠体内最活跃的时间框架 角膜PRK术后将有助于重组的设计 腺相关病毒(RAAV)-即将开发的反义载体 目的3选择性减少转化生长因子-βII受体的合成。目标 2号将使用原位杂交和免疫组织化学来 鉴定合成细胞外基质疤痕蛋白的角膜细胞 由转化生长因子-β系统激活。这些数据将显示哪一种角膜 细胞是rAAV反义载体转导的靶点，如 在活体实验中描述的目标4。目标3将 合成表达反义RNA的不同重组腺相关病毒载体 抗转化生长因子-βIIR mRNA，并将评估 表达载体降低人肾小管上皮细胞转化生长因子-βIIR基因和蛋白水平 转染型角膜成纤维细胞。具体目标4号将在 准分子激光切割大鼠角膜的活体实验。RAAV-反义 含有最佳启动子和反义序列的载体 特异靶3号将用于转导大鼠角膜细胞 继PRK之后。重组腺相关病毒反义基因转染人卵巢癌细胞的实验研究 将评估转化生长因子-βII型受体mRNA和蛋白的表达 转染法对转化生长因子-βII表达的影响 受体的信使核糖核酸和蛋白质将与影响一起评估。 在大鼠体内形成角膜混浊。核酶有可能 比反义RNA更有效。随着世界经济的发展， RAAV-反义质粒，我们将合成rAAV-核酶载体 靶向于切割转化生长因子-βII型受体mRNA和 评价其降低肾小管上皮细胞转化生长因子-β系统表达的效果 调节基质瘢痕形成，并将开发新的治疗方法 PRK术后角膜混浊的预防。

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.