脂肪干细胞来源外泌体对角膜基质损伤修复的作用和机制研究

Regeneration of corneal stroma plays important roles in maintaining corneal transparency and tenacity after corneal injury. Our previous study have found that exosomes derived from adipose derived stem cells(ADSCs) could inhibit keratocyte-fibroblast-myofibroblast transition of corneal stromal cells(CSCs) which increase the risk of corneal scar formation, with a underlying mechanism. Several signaling pathways are thought to be involved in tissue injury repair such as TGF-β/Smad, Notch and p53, all upregulated by homeodomain-interacting protein kinase 2(HIPK2). The 3'-UTR of HIPK2 mRNA is able to complement the miRNA encoded by miR-17-92 gene cluster, which has documented its existence in exosomes from stem cells. In the current study, we aim to demonstrate the role of miR-17-92 gene cluster encoding miRNA from ADSC-derived exosomes in regulating HIPK2 expression to regulate signaling pathways like TGF-β/Smad, Notch and p53，and subsequently inhibit CSCs fibrosis, and verify the regeneration mechanism of ADSCs-derived exosomes in CSCs on animal injury models to probe its clinical ophthalmic application value.

角膜基质非纤维化修复是角膜损伤修复、恢复透明度的核心内容。前期研究发现脂肪来源干细胞(ADSCs)分泌的外泌体促进角膜基质细胞生长并抑制其过度活化为成纤维细胞造成瘢痕化修复，但作用机制不明。组织损伤修复与多种纤维化信号通路如TGF-β/Smad、Notch、p53等有关，均受到同源结构区域相互作用蛋白激酶2（HIPK2）的正向调节。HIPK2 mRNA分子3'-UTR与miR-17-92基因簇编码miRNA序列互补，两者下游分子高度重合，而干细胞来源外泌体中存在miR-17-92基因簇。项目拟在角膜基质细胞上，研究ADSCs来源外泌体中miR-17-92基因簇编码miRNA通过调控HIPK2表达，来调控TGF-β/Smad、Notch、p53等信号通路，从而抑制角膜基质细胞成纤维化；并在兔角膜基质损伤动物模型上验证，揭示ADSCs来源外泌体修复角膜基质损伤的机制，探讨其在眼科临床应用前景。

角膜损伤引起的角膜盲是我国主要的致盲性眼病之一。脂肪干细胞（ADSCs）被发现有修复角膜损伤作用。项目前期研究发现ADSCs分泌的外泌体能促进角膜基质细胞（CSCs）生长并抑制其过度活化为成纤维细胞。研究期间按计划完成实验内容：分离ADSCs和CSCs并鉴定，10%血清激活CSCs为成纤维细胞。ADSCs来源外泌体处理纤维化的CSCs，观察对CSCs过度纤维化的影响及纤维化信号通路变化。筛选出外泌体中高表达miRNA-19a，合成激动剂，检测其对HIPK2水平和活性影响。miRNA-19a agomir转染ADSCs后提取外泌体，处理活化的CSCs；过表达 HIPK2并观察外泌体是否通过miRNA-19a抑制CSCs中纤维化信号通路活性。动物实验分别采用碱烧伤及角膜基质切除的方法制作角膜基质损伤模型。此外还尝试采用化学缺氧方法CoCl2模拟低氧环境，研究BMP4与VEGF联合诱导ADSCs向角膜内皮细胞分化。.成功分离高纯度ADSCs和CSCs，ADSCs及外泌体抑制活化的CSCs增殖。CSCs激活后，HIPK2表达和活性增加，smad3和p53途径激活，干扰HIPK2抑制HIPK2表达和活性，抑制smad3和p53激活。筛选得到外泌体中高表达miRNA-19a可以抑制HIPK2表达和活性。高表达miRNA-19a的外泌体进一步抑制CSCs中HIPK2含量和活性，抑制p-Smad3、p-p53蛋白的水平，过表达HIPK2会抑制该过程。说明ADSCs 来源的外泌体中miR-19a通过抑制CSCs中HIPK2的水平，抑制细胞中HIPK2含量和活性，抑制Smad3和p53途径，抑制细胞纤维化进程。构建了角膜基质损伤动物模型，研究眼部给药方式。对ADSCs诱导修复角膜内皮细胞的初探，摸索了诱导条件。