REGULATION OF CORNEAL MYOFIBROBLAST TRANSFORMATION

描述：国际和平研究所的长期目标继续被引导 有助于理解基质伤口愈合的基本机制 影响角膜形状，从而影响屈光，如角膜损伤后发生的 或者做屈光手术。PI公布的数据表明，角膜创伤 愈合性成纤维细胞形成一种肌成纤维细胞表型，其特征是 α-平滑肌肌动蛋白(a-SM)的表达 由微丝束(应力纤维)组成的收缩装置， 整合膜受体a5B1整合素和细胞外纤维连接蛋白 (FN)。正在进行的研究表明，TGFb诱导a-SM的表达 无血清培养角膜基质细胞中由外向内的信号转导 细胞-基质相互作用介导的级联反应。PI调查结果导致了 认为肌成纤维细胞的转化涉及到细胞的初始激活 TGFb诱导角膜基质细胞表达A5B1整合素， 焦点接触形成和应力纤维组装(假设1)，随后 通过5B1介导的独特的信号肽的产生 酪氨酸磷酸化导致α-SM的表达(假设2)和 对细胞外力产生越来越大的回缩力、拉力 矩阵(假设3)。为了进一步研究这一提出的模型 在实验方面，他(1)开发了一种无血清培养系统，它模仿 正常的静止期角膜细胞生长，使我们能够系统地评估 角膜基质细胞活化和肌成纤维细胞的分子机制 (2)建立了SV-40转化的肌成纤维细胞； 角质细胞染色可以通过分子调控来确定 特定的细胞骨架、受体和信号转导蛋白在 A-SM的表达和基质组织；以及(3)开发了一种新的生物物理 体外创面收缩模型评估力的产生 以进一步描述细胞之间的关键相互作用 矩阵组织和整体伤口收缩。具体目标是： (1)研究TGFb对成肌细胞转化的影响。 角质形成细胞活化，(2)表征A5B1之间的关系 整合素诱导的酪氨酸磷酸化和应力纤维组装 肌成纤维细胞转化；以及(3)表征 肌成纤维细胞介导的基质重组。

DESCRIPTION: The long-range goal of the PI studies continues to be directed toward understanding the basic mechanisms of stromal wound healing that effect corneal shape, hence refraction, as occurs following corneal injury or refractive surgery. The PI published data indicates that corneal wound healing fibroblasts develop a myofibroblast phenotype characterized by the expression of a-smooth muscle actin (a-SM) organized into a putative contractile apparatus comprised of microfilament bundles (stress fibers), the integral membrane receptor a5B1 integrin, and extracellular fibronectin (FN). On-going studies suggest that TGFB induces the expression of the a-SM in serum-free cultured corneal keratocytes by an outside-in' signalling cascade mediated by cell-matrix interactions. The PI findings have lead to propose that myofibroblast TRANSFORMATION involves the initial ACTIVATION of corneal keratocytes by TGFB leading to the expression of a5B1 integrin, focal contact formation and stress fibers assembly (Hypothesis #1), followed by the generation of unique signaling peptides through a5B1 mediated tyrosine phosphorylation resulting in expression of a-SM (Hypothesis #2) and generation of increasing retractive, 'pulling', forces on the extracellular matrix (Hypothesis #3). In order to further investigate this proposed model experimentally he has (1) developed a serum-free culture system which mimics normal quiescent keratocyte growth and allows us to evaluate systematically the molecular mechanisms underlying keratocyte activation and myofibroblast transformation; (2) established SV 40 transformed myofibroblast and keratocyte cell stains which can be molecularly-modulated to determine the importance of specific cytoskeletal, receptor, and signaling proteins on a-SM expression and matrix organization; and (3) developed a new biophysical in vitro wound contraction model to assess the generation of force by individual cells to further characterize the critical interactions between matrix organization and overall wound contraction. The Specific Aims are: (1) Characterize the effect of TGFB on myofibroblast TRANSFORMATION vs keratocyte ACTIVATION, (2) Characterize the relationship between a5B1 integrin induced tyrosine phosphorylation and stress fiber assembly on myofibroblast TRANSFORMATION; and (3) Characterize the mechanism underlying myofibroblast mediated matrix reorganization.