HSP47在近视眼巩膜重塑中的作用与机制

Scleral remodeling which relates to the reducing of collagen synthesis and increasing of the collagen degradation plays a key role in the onset and progression of myopia. HSP47 is a collagen-specific molecular chaperone that assists in the correct folding and stabilization of triple-helical procollagen molecules. Ablation of HSP47 results in reducing of collagen synthesis and increasing susceptibility to protease digestion of collagen. It is probable that HSP47 contributes to the sclera remodeling in myopia, but so far the direct evidence still unavailable. Our previous study showed that the expression of HSP47 mRNA of sclera was decreased in form-deprive myopia in guinea pig. Therefore the present study is designed to firstly characterize the expression of HSP47 in retina, choroid and sclera of form-deprive myopia. The changes of cell biomechanics of sclera fibroblasts in myopia are investigated using atomic force microscopy; and then explore the influence of HSP47 on modulation the biomechanical properties and the synthesis and secretion of collagen of sclera fibroblasts transfected with HSP47 recombinant plasmids. On the basis of the results, it is investigated that the effect of HSP47 which is modulated through injecting HSP47 recombinant plasmids in Sub-Tenon’s capsule on the refraction and synthesis of scleral collagen in guinea pig under the normal and form-deprived environment respectively. The purpose is to clarify the effect and mechanism of HSP47 on the scleral remodeling during myopic eye growth, and to provide theoretical basis for the following study and development of therapeutic method for myopia aiming to HSP47.

巩膜重塑在近视眼发生发展中起关键作用，其与巩膜胶原合成减少、降解增加等有关。HSP47是胶原特异性分子伴侣，其缺乏可使胶原合成减少且易降解，因而推测HSP47可能参与近视巩膜重塑。课题组前期研究已发现形觉剥夺性近视眼巩膜HSP47表达降低。因此本项目拟构建HSP47重组质粒转染在体豚鼠近视模型眼巩膜和离体巩膜成纤维细胞，拟研究：（1）HSP47在豚鼠形觉剥夺性近视眼视网膜、脉络膜、巩膜中的表达和分布特点；（2）近视眼巩膜成纤维细胞生物力学特性的变化以及HSP47对近视眼巩膜成纤维细胞生物力学特性及其合成和分泌胶原的影响；3）HSP47对正常和形觉剥夺环境中眼屈光、胶原合成的影响。以期阐明HSP47在近视眼巩膜塑形中的作用和机制，为后续研究通过调节巩膜组织HSP47表达水平干预近视发生发展提供理论依据。

近视是一种世界性的常见眼病，其发病机制、自然病程和最佳治疗方法至今仍不清楚。.近视往往伴有眼轴延长， 95%以上的近视是由眼轴的延长所致。而眼轴的延长与巩膜重塑密切相关。巩膜是近视形成过程中的主要效应器和靶器官，由细胞外基质与少量的成纤维细胞组成。在近视发生早期，胶原合成快速减少。胶原合成与降解功能的平衡是影响巩膜重塑的重要因素。.胶原分子加工始于细胞内质网，然后转运至高尔基体后分泌到细胞外。在此过程中，需要HSP47协助。它参与了胶原的加工、合成、成熟和分泌，是调节胶原含量变化的关键因素。.本课题通过造豚鼠近视模型完成分析豚鼠巩膜 HSP47表达的基础水平；明确HSP47在近视发生发展中和诱导性近视恢复中的表达特征和分布特点：HSP47在豚鼠巩膜中有表达；近视眼巩膜中HSP47与I型胶原表达的下降，呈高度正相关；近视形成过程中胶原蛋白降解增加不仅与MMPs与TIMPs体系失衡有关，还可能与HSP47减少导致新合成的胶原对蛋白酶敏感，更易被水解有关。.通过完成质粒构建及检测；完成质粒转染巩膜成纤维细胞，并分析分别上调和下调HSP47表达对巩膜成纤维细胞生物力学及其合成和分泌的胶原的影响：（1）上调HSP47，可促进巩膜成纤维细胞合成和分泌胶原，同时减少胶原降解；下调HSP47，可抑制细胞合成和分泌，促进胶原的降解；（2）HSP47上调早期细胞增殖不明显，后期促进细胞的增殖；而下调HSP47则抑制细胞的增殖；（3）HSP47可以促进巩膜成纤维细胞转分化为肌成纤维细胞。.通过细胞培养完成对不同血清型腺相关病毒转染巩膜成纤维细胞的转染率及安全性的评估：（1）四种腺相关病毒转染豚鼠巩膜成纤维细胞能力：AAV8>AAV2>AAV5>AAV9（2）四种病毒对细胞调亡及细胞活力无明显影响，是安全可行的。.为近视的发生发展提供了新的可能机制，并可能为将来近视的防控找到了新的靶点与理论依据。