泛素蛋白连接酶E3D在年龄相关性黄斑变性中的作用机制研究

ubiquitin-proteasome system plays a significant role in maintaining cellular homeostasis and it is one of the major mechanisms for clearing misfolded protein or toxic protein, playing an important role in aging, neurogenerative diseases and immune system.Age-related macular degeneration actually is a multifactorial neurodegenerative disease associated with aging.Inflammation and immune is closely related to its onset.Retinal pigment epithelium(RPE) oxidative damage play a crucial role in the development of AMD.In 2015, we first reported that the ubiquitin protein ligase E3D (UBE3D) gene polymorphism was significanlty associated with AMD, ablation of the UBE3D protein lead to an abnormal amount of pigment granules deposited in retinal pigment epithelium microvilli area and an abnormal response on electroretinography (ERG) in UBE3D+/- heterozygous mice. But the specific mechanism about the gene involved in angiogenesis, protein degradation and RPE cell function were not clear. Conditional UBE3D gene knock in /knock out mice, in vitro gene overexpression/gene knock down method were used to study the behavior changes of RPE;the alteration of pro-/anti-angiogenesis factors, inflammasome were evaluated to elucidate the role of UBE3D in the pathogenesis of AMD. This study will enrich our knowledge on the mechanisms of AMD and explore new strategies for AMD treatment.

泛素蛋白酶体系统对维持细胞稳态意义重大，是真核细胞清除错误折叠蛋白或毒性蛋白的主要途径，参与炎症、细胞信号转导、凋亡等病理生理过程，在衰老、神经退行性疾病、免疫等具有重要作用。年龄相关性黄斑变性（AMD）本质是一种与衰老相关的多因素神经退行性疾病。炎症免疫与其发病密切相关。视网膜色素上皮细胞（RPE）的氧化损伤在其发生发展中也具有关键作用。2015年我们首次在《自然通讯》报道UBE3D基因多态性与AMD显著相关，杂合子小鼠电镜下可见光感受器外节与RPE层之间色素颗粒增加，ERG检测显示a波和b波均下降。但其参与血管新生、蛋白降解、RPE细胞功能等具体机制并不明确。本课题拟通过构建UBE3D定向敲入/减小鼠、体外基因过表达/敲减方法，围绕氧化应激、炎症小体、血管生成促进/抑制因子表达及RPE细胞功能变化,明确UBE3D在AMD发生发展中的作用，该研究将为探索AMD发病机制提供新的理论依据。

2015年我们首次在《自然通讯》报道UBE3D基因多态性与AMD显著相关，杂合子 小鼠电镜下可见光感受器外节与RPE层之间色素颗粒增加，ERG检测显示a波和b波均下降。 但其 参与血管新生、蛋白降解、RPE细胞功能等具体机制并不明确。本课题拟通过构建UBE3D定向敲 入/减小鼠、体外基因过表达/敲减方法，围绕氧化应激、炎症小体、血管生成促进/抑制因子 表达及RPE细胞功能变化,明确UBE3D在AMD发生发展中的作用。以慢病毒为载体的基因转染技术使人RPE细胞过表达/敲减UBE3D，叔丁基过氧化氢诱导RPE细胞氧化损伤。过表达UBE3D基因的RPE在氧化损伤后增殖、迁移能力均高于空载组,敲减UBE3D基因的RPE在氧化损伤后增殖、迁移能力均低于阴对照组;过表达UBE3D基因的RPE在氧化损伤后下调Cleaved-caspase-3、磷酸化p38MARK、PERK、LC3、Beclin1的表达量，上调了P62的表达，敲减UBE3D基因的RPE在氧化损伤后上调Cleaved-caspase-3、磷酸化p38MARK、PERK、LC3、Beclin1的表达量，下调了P62的表达。 蓝光诱导光氧化损伤模型，UBE3D杂合子敲除小鼠与野生型相比，ERG结果显示a波和b波升高；OCT显示RPE细胞层略增厚。激光诱导CNV模型，UBE3D过表达小鼠中可检测到VEGF和自噬蛋白LC3表达增加。UBE3D基因参与了AMD病程中的氧化应激和炎性反应，UBE3D障碍可以加重氧化损伤和炎性反应。UBE3D基因通过介导自噬-内质网应激发挥抗氧化损伤的协同作用。