MYOFIBROBLASTS AND CORNEAL WOUND CONTRACTION

肌成纤维细胞和角膜伤口收缩

• 批准号: 2161459
• 负责人: James V Jester
• 金额: $ 22.24万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2161459
• 关键词: cell transformation; collagen; connective tissue cells; cornea; cytochalasins; fibroblasts; gene expression; laboratory rabbit; mechanical stress; morphology; muscle cells; muscle contraction; muscle proteins; papaverine; serotonin; smooth muscle; tissue /cell culture; wound healing

In studies of radial keratotomy, we have demonstrated that changes in corneal shape are regulated by the cellular events of corneal wound healing and wound contraction. These events include: (1) the transformation of adjacent keratocytes to a myofibroblast-like cell containing a putative contractile apparatus comprised of alpha-smooth muscle (alpha-SM) actin, non-muscle myosin and alpha-actinin (ie stress fibers); and (2), the progressive alignment of stress fibers parallel to the long axis of the wound. The major objective of this proposal is to clearly identify the cellular mechanism underlying changes in wound gape. We propose as an overall mechanisms that corneal myofibroblasts actively participate in wound contraction by exerting a contractile (pulling) forces, which are critically influenced by the mechanical state or STRESS distribution of the surrounding tissue. In order to test this hypothesis we have: (1) developed an in vivo model of corneal wound contraction using a rabbit full-thickness corneal injury in which both the direction and amount of stress is altered by the placement of intracorneal compression sutures; (2) established an in vitro rabbit keratocyte model of wound contraction which shares essential pathobiologic features to in vivo wounds using a collagen lattice culture system under non-stressed (free floating lattice) or stressed (attached lattice) conditions; and (3) established morphologic and biochemical techniques to assess the effects of STRESS on the development and organization of myofibroblasts. Our specific aims are: (1) To determine the effect of STRESS on myofibroblast organization by comparing morphologically the orientation of stress fibers under (a) normal STRESS and (b) altered STRESS (compression sutures); (2) To identify the contractile characteristics of myofibroblasts by determining their morphologic response to released STRESS (suture removal) and by correlating changes in wound gape to the changes in orientation of stress fiber bundles (a) under normal conditions, and (b) after cytochalasin D treatment; (3) To correlate the organizational changes in type I collagen with changes in stress fiber orientation during (a) contraction of normal, altered and released STRESS wounds following injection of DTAF-labeled soluble collagen, and (b) in vitro contraction of attached and free floating lattice cultures; and (4) To determine the effect of STRESS on myofibroblast transformation by: a) comparing and contrasting the expression of smooth muscle specific proteins (ie alpha-SM actin) in normal keratocytes, myofibroblasts and attached and free floating lattice cultures; and, b) determining the effects of muscle antagonists (papaverine) and agonists (serotonin) on wound and lattice contraction. These studies should provide important insights into fundamental questions concerning the mechanism of wound contraction and the role of the myofibroblast in corneal wound repair.

在径向角化术的研究中，我们已经证明了 角膜形状受角膜伤口的细胞事件调节 愈合和伤口收缩。这些事件包括：（1） 将相邻角膜细胞转化为肌纤维细胞样细胞 包含由α-光滑的推定收缩设备 肌肉（α-SM）肌动蛋白，非肌肉肌球蛋白和α-肌动蛋白（即胁迫 纤维）; （2），应力纤维的进行性比对平行于 伤口的长轴。该提议的主要目的是 清楚地识别伤口炎症的细胞机制。 我们提出了一种积极的角膜肌纤维细胞的总体机制 通过施加收缩（拉）来参加伤口收缩 受到机械状态或压力严重影响的力 周围组织的分布。为了检验这一假设 我们有：（1）使用使用的体内角膜伤口收缩模型 兔子全厚性角膜损伤，方向和 压力的放置改变了压力的量 缝线； （2）建立了一种体外兔子的伤口模型 在体内具有重要病原体特征的收缩 使用胶原蛋白晶格培养系统的伤口在无压力下（免费 浮动晶格）或压力（附着的晶格）条件； （3） 建立的形态学和生化技术来评估影响 对肌纤维细胞的发展和组织压力。我们的 具体目的是：（1）确定压力对肌纤维细胞的影响 通过在形态学上比较压力纤维的方向来组织 在（a）正常应力和（b）改变应力（压缩缝合线）之下； （2） 确定肌纤维细胞的收缩特征 确定其对释放应力的形态反应（缝合缝线） 并通过将伤口毛发的变化与方向的变化相关联 应力纤维束（a）在正常条件下，（b） 细胞切拉斯蛋白D治疗； （3）将组织变化相关联 I型胶原蛋白随着（a）期间应力纤维方向的变化 遵循正常，改变和释放的压力伤的收缩 注射DTAF标记的可溶性胶原蛋白和（b）体外收缩 附着和自由浮动的晶格文化； （4）确定 压力对肌纤维细胞转化的影响：a）比较和 对比平滑肌特异性蛋白的表达（即α-SM 肌动蛋白）在正常的角膜细胞，肌纤维细胞中，并附着并免费 浮动晶格文化； b）确定肌肉的影响 拮抗剂（爸爸）和激动剂（5-羟色胺）在伤口和晶格上 收缩。这些研究应该为 有关伤口收缩机制的基本问题和 肌纤维细胞在角膜伤口修复中的作用。