基于角膜上皮细胞NF-κB促炎信号通路与外泌体的双重活化作用探讨IFN-γ促进角膜移植排斥的机制研究

Corneal graft rejection is the main cause of corneal graft failure and IFN-γ promotes the rejected progress. Recently, the role of NFκB pathway and exosomes has received extensive attention in immune rejection, but what is its role in corneal graft rejection? We confirmed that IFN-γ promoted apoptosis of human corneal epithelial cells (HCEC), and activated its NFκB pathway and downstream MICA molecules. Furthermore, HCEC exosomes were up-regulated. We speculated that NFκB pathway and exosomes play an important role in corneal immunity. Combining with the previous study, we hypothesized that on one hand, IFN-γ enhances the killing of NK cells and CD8+T cells through the NFκB/MICA pathway; simultaneously up-regulates the CCL5, which promotes lymphocyte aggregation, and regulates corneal immunity by cells direct contact; on the other hand, IFN-γ promotes HCEC exosomes secretion, regulates lymphocytes in a non-contact manner, which promotes corneal rejection. We plan to evaluate the expression of NFκB pathways and the effect of HCEC exosomes on lymphocyte function and their interactions after IFN-γ treatment by PCR, immunofluorescence, exosome protein chip and so on. To clarify the role of NFκB pathway and exosomes in corneal rejection and provides a new target for the regulation of corneal immunity.

角膜移植排斥是角移手术失败的主要原因，文献报道IFN-γ参与角移排斥。近期NFκB通路及外泌体在免疫中的作用得到广泛关注，但其在角移排斥中作用如何？课题组前期发现IFN-γ促进人角膜上皮细胞（HCEC）凋亡，其NFκB通路及下游MICA分子激活，外泌体分泌增多，推测NFκB及外泌体在角膜免疫中发挥重要作用。结合现有文献我们假设：IFN-γ一方面通过NFκB/MICA通路，增强NK细胞、CD8+T细胞杀伤性；同时上调趋化因子CCL5，促进淋巴细胞聚集，以细胞直接接触调节角膜免疫；另一方面IFN-γ促进HCEC外泌体分泌，以非接触方式调节淋巴细胞，促进角膜排斥。课题组拟通过PCR、免疫荧光、外泌体蛋白芯片等方法研究IFN-γ处理后HCEC中NFκB促炎信号通路、外泌体蛋白的表达规律，两者对淋巴细胞功能的影响及其交互作用，明确NFκB通路及外泌体在角膜排斥中的作用，为调控角膜免疫提供新的靶点。

本研究探讨外源性干扰素-γ（Interferon-γ，IFN-γ）对人角膜上皮细胞中MHC-I类相关蛋白 A （Major Histocompatibility Complex Class I Chain-related A，MICA）分子表达的影响及其与miRNA的相关性。明确miRNA-4448在IFN-γ上调人角膜上皮细胞MICA分子中的作用机，证实MICA分子对NK细胞及CD8+T细胞介导的人角膜上皮细胞具有细胞毒性影响。本研究将角膜上皮细胞作为研究对象，证实IFN-γ在角膜免疫中的相关作用，包含miRNA参与的细胞间信号转导，靶细胞功能的调控及外泌体在角膜免疫排斥中的作用。本研究首次发现IFN-γ通过抑制miRNA-4448进而上调转录因子NFkB的表达，NFkB通过作用于MICA启动子区，增强其基因表达。同时促进MICA介导的NK细胞、CD8+T细胞对角膜上皮细胞的杀伤作用，从而产生移植物损伤。同时，本课题证实，IFN-γ作用后HCEC-exo分泌增多，可促进T细胞增殖及NK、T细胞中IL-6，IL-2，TNF-α等炎症因子表达，说明 IFN-γ可通过外泌体促进淋巴细胞的免疫炎性作用。此外，IFN-γ刺激后HCEC-exo对血管新生具有促进作用，这是引发角膜免疫排斥的关键。为IFN-γ参与角膜移植排斥反应提供新的思路，为HCEC-exo治疗角膜移植术后排斥提供可靠的实验依据。