过表达白介素10的间充质干细胞外泌体修复NPDR血视网膜屏障的机制研究

Interleukin 10 (IL10) is a potential therapy for the chronic immune inflammation by regulating T cells and leukocyte, and suppressing inflammatory factors in NPDR. The exosome derived from mesenchymal stem cells (MSC) has been applied to diseases because it has the similar function as MSC and overcome the differentiation, integration and ethics of stem cell transplantation. At present, the treatment of NPDR is just to control the blood glucose level, and there is no effective treatment to suppress the development of NPDR. While few reports showed that exosome derived from MSC protect retinal vascular endothelial cells. Our previous study has shown that over expressed IL10 in stem cells could delay the progression of DR by inhibiting NF-Κb pathway. Therefore, we assume that the exosome derived from IL10-transfected MSC could repair BRB via improving the local inflammatory microenvironment and repairing the retinal vascular endothelial cells. Our previous study has established MSC cell lines transfected by IL10. And then, we will extract exosome from IL10-transfected MSC, identify its phenotypes, and evaluate the function for proliferation, tabulation, migration and apoptosis of vascular endothelial cells in vitro and BRB in vivo. Finally, we will investigate the exosome derived from IL10-transfected MSC to repair BRB via regulation T cells, inhibition of leukocyte adhesion and up-regulation the angiogenic factors, which could provide a new therapy and scientific basis for the treatment of NPDR.

白介素10（IL10）调控T细胞、白细胞及炎性因子等发挥免疫抑制作用是控制NPDR慢性免疫炎症的潜在治疗方式。间充质干细胞（MSC）外泌体因具有与MSC相似的功能且克服了干细胞移植后分化、整合及伦理等问题被广泛应用。目前对于DR早期的治疗以控制全身一般情况为主，且MSC外泌体对DR血管的保护作用研究较少。我们前期研究表明过表达IL10的干细胞通过抑制NF-κB通路及下游炎症因子延缓NPDR发展，推测过表达IL10的MSC外泌体通过改善视网膜炎性微环境和保护血管，双重机制修复血视网膜屏障(BRB)控制NPDR发展。本课题在已建立了过表达IL10的MSC基础上提取其外泌体，并利用细胞实验和DR大鼠模型，研究对血管内皮细胞增殖、成管、迁移及凋亡的作用和对BRB渗漏的保护作用；通过调节T细胞、抑制白细胞粘附和上调促血管生成因子表达，抗炎和保护双重机制修复BRB，为NPDR的治疗提供思路并奠定基础。

白介素10（IL10）调控T细胞、白细胞及炎性因子等发挥免疫抑制作用是控制NPDR慢性免疫炎症的潜在治疗方式。间充质干细胞（MSC）外泌体因具有与MSC相似的功能且克服了干细胞移植后分化、整合及伦理等问题被广泛应用。血-视网膜屏障（BRB）的破坏是DR形成的标志，DR早期（NPDR）的治疗主要以控制血糖、血压、血脂等全身一般情况为主，还没有有效的治疗方式控制视网膜局部慢性免疫炎症及修复血管内皮细胞损伤，进而修复BRB，逆转DR发展。本科题完成了过表达IL10的人脐带MSC外泌体的提取，成功获得了过表达IL10的MSC外泌体，拓宽了单一细胞外泌体的功能。并通过体内、体外实验，在分子、细胞及组织水平检测过表达IL10的MSC外泌体对视网膜血管内皮细胞（RMEC)和DR大鼠视网膜的影响并探讨其分子机制。结果显示：过表达IL10的MSC外泌体可以增强RMEC的迁移和成管能力，减少DR大鼠视网膜血管的渗漏和白细胞粘附。WB结果显示：过表达IL10的MSC外泌体可以显著降低DR大鼠视网膜中促血管生成因子VEGF、VCAM-1和ICAM-1表达降低，促炎因子IL-2、IFN-γ表达降低，而抗炎因子IL10及TGF-β表达水平上升。同时还发现了MSC外泌体可以增强高糖诱导的Müller细胞活力并减少细胞凋亡。因此，本科题的研究表明过表达IL10的MSC外泌体一方面通过调节T细胞分化和抑制白细胞的粘附从而改善局部炎性微环境；另一方面促进视网膜局部促血管生成因子表达从而修复视网膜血管。同时通过生物测序分析发现MSC外泌体通过携带miR-93-5p抑制TXNIP/NLRP3/ASC/Caspase-1引起的炎症小体激活，从而增强了高糖诱导的Müller细胞活力并减少细胞凋亡，保护DR患者Müller细胞。多角度实现控制DR病情发展的目标，为治疗NPDR提供新的思路和理论依据。