巩膜干细胞对实验性近视巩膜重塑的作用及调控机制研究

Myopia is a developmental oculopathy. Scleral remodeling is an important breakthrough in the etiology of experimental myopia. Type I collagen and cartilage proteins of sclera may be involved in the regulation mechanism of scleral remodeling. Stem cells are considered as a hot topic in the field of ophthalmology. Our prophase research found that adult stem cells with the potential abilities could be isolated from scleral tissue in guinea pig. Therefore, we hypothesize that scleral stem cells can regulate the process of scleral remodeling by regulating the expression of type I collagen and cartilage proteins. To test the hypothesize, (1) we will establish guinea pig experimental myopia model and simulate micro-environment of sclera remolding, then investigate the role of scleral stem cells in scleral remodeling related with experimental myopia by immunofluorescence, western-blot, RT-PCR and other molecular biological means. (2) We will regulate Wnt/β-catenin signal pathway by bidirectional intervention in vivo and vitro, and record the change of refractive error and axial length of guinea pig. We will confirm the regulation mechanism of scleral stem cells in scleral remodeling in experimental myopia with transmission electron microscopy and molecular biological means. We hope these researches will get some new findings that would lay the foundation for revealing the mechanism of experimental myopia, and bring new ideas of prevention and treatment of myopia.

近视是一种发育性眼病，巩膜重塑是实验性近视病因学研究的重要突破，研究认为I型胶原蛋白以及软骨相关蛋白参与调控巩膜重塑。干细胞作为眼科领域的研究热点，课题组前期研究发现，豚鼠巩膜组织可以分离出具有多向分化潜能的成体干细胞。为此，我们提出假说：豚鼠实验性近视模型巩膜干细胞通过影响I型胶原蛋白及软骨相关蛋白的差异性表达，继而调控巩膜重塑的发生发展。为验证以上假说，（1）构建豚鼠实验性近视模型以及模拟近视巩膜微观环境，采用免疫荧光、Western-blot、定量PCR等分子生物学手段，探讨巩膜干细胞对实验性近视巩膜重塑的作用；（2）通过体内外双向干预Wnt/β-catenin信号通路，观察豚鼠近视屈光度及眼轴变化，采用透射电镜技术观察和分子生物学手段明确巩膜干细胞对实验性近视巩膜重塑的调控机制。本项目将以巩膜干细胞为新视角，为揭示实验性近视发生机制奠定基础，为近视防治提供新的思路。

近视是目前全球发生率最高的屈光不正，在亚洲人群中其发生率显著增高，并呈现低龄化和高度化的趋势，已经成为严重的公共卫生问题。深入研究近视的发病机制和病理过程已经成为国内外学者和眼科医生密切关注的重点和难点。“巩膜重塑机制”作为实验性近视病因学研究的重要突破，其具体作用机制尚未完全阐明。干细胞是目前包括眼科在内的各种学科研究领域的热点。然而鲜有报道将干细胞应用于近视相关的基础和临床研究。本课题在前期研究工作的基础上，完成原代分离培养豚鼠巩膜干细胞及鉴定，通过诱导分化验证培养细胞的多向分化潜能；研究实验性近视豚鼠巩膜干细胞 I型胶原蛋白以及软骨相关蛋白的表达；通过体内外实验双向干预调控Wnt/β-catenin信号通路，研究巩膜干细胞对实验性近视巩膜重塑的调控机制。研究发现：豚鼠巩膜组织可以分离培养出具有多项分化潜能的巩膜干细胞;进一步研究培养的巩膜干细胞对于巩膜胶原蛋白以及软骨相关蛋白的调控机制，发现豚鼠实验性近视模型中巩膜干细胞软骨相关蛋白（II 型胶原蛋白、α-SMA）表达增加，而I型胶原蛋白表达降低；双向干预Wnt/β-catenin信号通路可以调控巩膜干细胞I型胶原蛋白以及软骨相关蛋白的表达，继而调节巩膜基质重塑，最终影响近视的进程，为近视的防治提供了新的干预靶点以及研究思路。