年龄相关性黄斑变性(AMD)是老龄社会最重要致盲疾病，发病机制复杂，治疗措施有限。近年临床研究发现AMD患眼中较多合并玻璃体黄斑粘连（VMA），引发科学界对VMA与AMD发病关系的重视，但至今缺少相关实验证据支持。我们推测VMA可能通过产生牵拉力对视网膜细胞细胞骨架重塑产生影响，引起细胞骨架信号调控通路激活，进而导致细胞因子和炎性因子异常激活，参与AMD病理发展。本课题采用体外培养视网膜色素上皮细胞、Müller细胞和血管内皮细胞，建立两种机械力作用模型，观察外力对细胞骨架重塑过程的作用，和对细胞生理代谢及存活的影响。本研究旨在通过探讨外力作用下视网膜相关细胞是否发生与AMD相关的细胞因子和细胞状态变化，揭示VMA参与AMD发病机制的可能性，为AMD预防和治疗提供新思路。

Age-related macular degeneration (AMD) is one of the most important blinding disease of the aging society, the pathogenesis is complex and treatment methods are limited. In recent years, clinical studies found a higher prevalence of vitreomacular adhesion (VMA) in eyes with AMD, which caused an attention in the scientific community to the relation between VMA with AMD incidence, but we still lack supports of relevant experimental evidence. Here we hypothesizes that VMA may produce mechanical force to impact on cytoskeleton remodeling of retinal cells, to cause abnormal activation of cytoskeleton signal transduction pathways, to result in abnormal activation of cytokines and inflammatory factor, which participating in pathological development of AMD. Therefore, in this project, using in vitro retinal pigment epithelial cells, Muller cells and vascular endothelial cells, two kinds of mechanical force model will be set up, to observe the role of different external force on cytoskeleton remodeling process, and its influence on cell physiological metabolism and survival. This study aims to, through the investigation whether there are changes of AMD-related cytokines and cell state in the associated retinal cells under the action of external force, reveal the possibility of VMA participating in the pathogenesis of AMD, and ultimately provide new thoughts for the prevention and treatment of AMD.

年龄相关性黄斑变性(AMD)是老龄社会最重要致盲疾病，发病机制复杂，治疗措施有限。近年临床研究发现AMD患眼中较多合并玻璃体黄斑粘连（VMA），引发科学界对VMA与AMD发病关系的重视，但至今缺少相关实验证据支持。我们推测VMA可能通过产生牵拉力对视网膜细胞细胞骨架重塑产生影响，引起细胞骨架信号调控通路激活，进而导致炎性细胞因子异常激活，参与AMD病理发展。结果显示，应用力学加载平台给予体外培养的人视网膜色素上皮施加牵张力，细胞骨架发生重塑障碍，细胞逐渐由伸展状态变为收缩状态，在拉伸20h时，收缩状态的细胞比例增加到70%左右。通过Western蛋白印迹方法，检测了参与细胞骨架调节的integrin-FAK-rac/cdc42信号通路，integrinβ3表达基本维持稳定，但其下游重要的调节蛋白pFAK397表达在牵拉8h时升高，并维持在较高水平一直到14h时；Rac/cdc42的磷酸化水平与pFAK397表达趋势一致。应用Western蛋白印迹方法证实，视网膜色素上皮细胞在牵张14h后高表达炎性细胞因子IL-8和血管内皮生长因子，并在20h达到峰值。应用流式细胞术AnnexinV-PI双染法检测了细胞的凋亡水平，牵拉14h时细胞出现明显的凋亡现象，撤除牵拉力后6h时可凋亡率可恢复至正常，这一过程与Western免疫蛋白电泳证实的Bcl-2、Bax、p53等凋亡调控蛋白表达趋势改变一致。研究结果提示，力学刺激确能影响视网膜色素上皮细胞细胞骨架重塑及细胞存活状态改变，揭示VMA参与AMD发病机制的可能性，为AMD预防和治疗提供新思路。