REGULATION OF CORNEAL MYOFIBROBLAST TRANSFORMATION

描述：继续指导PI研究的长期目标 了解基质伤口愈合的基本机制， 影响角膜形状，从而导致角膜损伤后的屈光 或屈光手术。 PI发表的数据表明，角膜伤口 愈合成纤维细胞发展成肌成纤维细胞表型，其特征在于 α-平滑肌肌动蛋白（a-SM）的表达被组织成一个假定的 由微丝束（应力纤维）组成的收缩装置， 整合膜受体α 5 β 1整合素和细胞外纤连蛋白 （FN）。 正在进行的研究表明，TGFB诱导a-SM的表达， 在无血清培养的角膜基质细胞中通过由外向内的信号传导 细胞-基质相互作用介导的级联反应。 PI的调查结果导致 我认为肌成纤维细胞转化涉及 TGFB对角膜基质细胞的作用导致α 5 β 1整联蛋白的表达， 局灶性接触形成和应力纤维组装（假设#1），随后 通过a5 B1介导的独特信号肽的产生， 酪氨酸磷酸化导致a-SM的表达（假设#2），和 在细胞外产生增加的回缩、“牵引”力 矩阵（假设#3）。 为了进一步研究这个模型， 实验上，他（1）开发了一种无血清培养系统， 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 转化;（2）建立SV 40转化的肌成纤维细胞和 角膜细胞染色剂，其可以被分子调节以确定 特异性细胞骨架、受体和信号蛋白对 a-SM表达和基质组织;和（3）开发了一种新的生物物理 体外伤口收缩模型，以评估力的产生 单个细胞，以进一步表征 基质组织和整体伤口收缩。 具体目标是： (1)表征TGFB对肌成纤维细胞转化与 （2）探讨a5 B1与角膜基质细胞活化的关系。 整合素诱导酪氨酸磷酸化和应力纤维组装 肌成纤维细胞转化;和（3）表征潜在机制 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.