氧化应激诱导的RPE细胞源性外泌体在湿性AMD发生中的作用和机制

Exosomes are small membrane bounded vesicles that are released by almost every cell type to the extracellular environment. Exosomes promote the communication between cells by conveying biologically active cargo including proteins and RNA molecules. These can be delivered to the cells and taken up, so that exosomes play a crucial role in many physiological processes and in pathological processes in some diseases. Based on current research, exosomes could not only have protective effect on specific cells, but could also promote the infiltration and development of cancer. In addition, some kinds of exosomes could have important impact on some diseases by promoting angiogenesis. Retinal pigment epithelium (RPE) dysfunction is hypothesized to be fundamental in the pathogenesis of age-related macular degeneration (AMD). Because RPE cells are in a highly oxidative environment, we hypothesize that the secretion of the exosomes from the RPE will reflect their metabolic status and mediate communication between this cell layer and the retina and the choroid. Therefore, we plan to study the effect of exosomes originating from RPE cells that are cultured in an environment of oxidative stress on the development of choroidal neovascularization (CNV): 1) We will detect the level of exosomes and changes in proteins and miRNA expression in aqueous humor (AH) and serum of AMD patients;.2) We will detect the effect of AMD patients’ AH-originated exosomes; control patients’ AH-originated exosomes on human choroidal microvascular endothelial cell (HCMEC). In parallel we will obtain exosomes from human RPE cells grown under standard culture conditions and under oxidative conditions and study their impact on HCME cells. 3) We will use RNA sequencing (RNAseq) to identify miRNA and other small RNAs that are over-expressed or suppressed in RPE derived exosomes under various conditions to investigate the role of specific exosome miRNAs in the occurrence of CNV. 4) We will also compare the effect of the four kinds of exosomes mentioned above on the development of CNV in wet-AMD mouse model. We hypothesize that exosomes originating from RPE cells which are cultured in oxidative stress environment will promote the development of CNV in mice.

不同宿主细胞来源的外泌体在胞间信息传递和物质运输中具有不同的功能。RPE细胞是AMD 发病的主要靶点之一，近年来我们系统研究了氧化应激诱导RPE细胞死亡的分子机制,并在预实验中发现氧化应激的RPE可分泌更多的外泌体，且能促进培养的人脉络膜微血管内皮细胞增殖和迁移。因此我们拟进一步研究氧化应激诱导RPE细胞来源外泌体在CNV发生中的作用：1）检测湿性AMD患者房水及血清外泌体的水平，用miRNA芯片筛选出差异性表达的miRNAs；2）分别探讨不同来源的外泌体（AMD 患者房水、对照组患者房水、常规和氧化应激培养的RPE细胞）对人脉络膜微血管内皮细胞的增殖、迁移、小管形成的作用及机制；3）以筛选出的miRNA为靶点，对其过表达和抑制，探讨特定miRNA在外泌体介导CNV发生中的作用；4）比较以上四种来源的外泌体和特定miRNA对小鼠CNV模型发展的影响及机制。为AMD机理及治疗提供新靶点。

本项目探究了氧化应激诱导RPE细胞来源外泌体在CNV发生中的作用：检测了湿性老年患者房水及血清外泌体的水平，用miRNA芯片筛选出差异性表达的miRNAs；探讨了不同来源的外泌体（AMD患者房水、对照组患者房水、常规和氧化应激培养的RPE细胞)对人脉络膜微血管内皮细胞的增殖、迁移、小管形成的作用及机制;以筛选出的miRNA-3610为靶点，探讨miRNA-3610调控FOXA2在外泌体介导AMD血管新生的分子机制。