WnT/β-catenin通路在实验性近视中的作用机制及调控研究

Myopia is an overdevelopment disease of the eye. WnT is an important signal molecule for the regulation of embryonic eye development. In mature retina, WnT still expresses and plays a role during the eye growth. Preliminary study of our research group founds that WnT2b/β-catenin and membrane frizzled protein(MFRP)5 is upregulated in the mice form deprivation myopia(FDM)and is positively correlated with myopia degree. However, the role of WnT/β-catenin/MFRP in the development of myopia still remains unclear. For this reason, our research project plan to use gene chip technology, immunofluorescence, RT-PCR and Western-blot method respectively to detect different isoforms of WnT/MFRP expression in FDM, as well as its expression sites and variation patterns, to observe its relationship with myopia development, to clarify its mechanism of the expression variation.Through DKK1( WnT blocker) and SB-2167631(WnT activator) intravitreal injection ,we compare the change of myopia with the control group, detect the expression of WnT/β-catenin/MFRP,T cell factor/lymphocyte enhancer factor, retinoic acid and transforming growth factor-beta, in order to clarify the effect and mechanism of myopia by regulation of the WnT pathway. We expect to provide a novel and effective target spot for myopia intervene and to lay the foundation for new anti-myopia drug. We hope it will possess scientific significance for the clinical prevention and treatment of myopia.

近视是一种眼球过度发育性眼病。WnT是胚胎时期调控眼球发育的重要信号分子，在成年视网膜仍有表达并对眼球的生长起调节作用。课题组前期研究发现，小鼠形觉剥夺性近视（FDM）的视网膜中WnT2b、β-catenin及膜型卷曲蛋白5（MFRP5）表达上调，且与近视的程度呈正相关，但其在近视发生中的作用不清。为此，本项目分别采用基因芯片技术、免疫荧光、定量PCR及Western-blot等方法，检测FDM中WnT/MFRP各亚型的表达、表达部位及变化规律，观察其与近视发生的关系，并阐明其表达变化的机制；分别实施玻璃体腔注射WnT抑制剂DKK1和激活剂SB-2167631进行双向干预，观察其对近视的作用，分析WnT/β-catenin/MFRP、T细胞因子/淋巴细胞增强因子、视黄酸和转化生长因子β的表达，阐明调控WnT通路对近视的作用及其机制，为发现近视干预的可能有效靶点及研制新型抗近视药物奠定基础。

本课题成功构建了豚鼠形觉剥夺性近视模型，基因芯片研究表明Wnt家族中Wnt3亚型在形觉剥夺近视组的表达高于对照组2.13倍，进一步采用Western-blot方法对巩膜和视网膜组织检测，发现：与对照组相比，近视组Wnt3在蛋白水平表达也显著增加，这与基因芯片结果一致，这为本课题后续的体内体外研究奠定了基础。而且，我们进一步关注Wnt通路在近视调控中的机制和对巩膜胶原重塑的影响。在离体培养的巩膜成纤维细胞研究中发现：①近视组Wnt3和β-catenin蛋白表达均显著增加，加入DKK-1（Wnt通路抑制剂）干预后Wnt3和β-catenin的表达降低；②近视组细胞上清液的TGF-β1较对照组显著降低，加入DKK-1后近视组TGF-β1的表达增加，加入TGF-β1中和抗体后近视组TGF-β1的表达进一步降低；③近视组I型胶原蛋白较对照组显著降低，加入DKK-1后表达增加。在加入TGF-β1中和抗体后，I型胶原蛋白表达进一步降低。在体的研究表明：豚鼠近视组Wnt3及β-catenin增加，I型胶原蛋白降低。DKK-1干预后Wnt3及β-catenin表达降低，而I型胶原蛋白有所增加。TGF-β1中和抗体干预后I型胶原蛋白表达进一步降低。以上离体和在体的研究均表明：实验性近视中Wnt3/β-catenin信号通路激活，通过调控巩膜的TGF-β1来影响I型胶原蛋白的表达，从而影响巩膜的重塑过程。本项目的研究成果可为寻求近视的新的干预靶点提供有价值的实验室依据。受本课题资助已发表论著4篇，其中SCI收录 3篇。培养博士3名，硕士1名。