有氧糖酵解途径对高危角膜移植免疫排斥反应的影响及其机制研究

AS the second largest blind eye diseases, the corneal blindness needs keratoplasty for eyesight restoration. However, immune rejection is the main cause of corneal allograft failure. Emerging evidence indicates that activated antigen-presenting cells (APCs) and effector T cells（Teffs）recognize and attack allografts mainly through aerobic glycolysis. Our previous study have found that the proteins involved in aerobic glycolysis, such as glucose transporter GLUT1 and lactate dehydrogenase LDHA, were highly expressed in rejected corneal allografts. Blockade of glycolysis could significantly prevent corneal allograft rejection. Based on these observations, we hypothesize that aerobic glycolysis, as a key energy switch, plays an important role in the high-risk corneal allograft rejection by regulating APCs and Teffs. To verify this hypothesis, here we will investigate the mechanisms of aerobic glycolysis involved in the high-risk corneal allograft rejection through regulating the functions of APCs and Teffs. Our investigation will not only elucidate the effect and mechanism of anaerobic glycolysis on corneal allograft rejection, but also provide a theoretical basis for clinical prevention and treatment of corneal allograft rejection.

角膜盲是我国第二大致盲性眼病，一般需角膜移植手术复明，然而术后免疫排斥反应是移植失败的最主要原因。最新研究表明对移植物进行识别和攻击的免疫细胞群，如抗原递呈细胞和效应T细胞，主要借助有氧糖酵解途径获得能量从而发挥其免疫功能。本团队前期研究结果：有氧糖酵解代谢相关蛋白葡萄糖转运蛋白GLUT1、乳酸脱氢酶LDHA等在免疫排斥角膜组织中高表达，抑制糖酵解过程可显著延缓角膜移植免疫排斥反应的发生。因此，我们推测：有氧糖酵解途径是抗原递呈细胞以及效应T细胞的发挥其免疫识别和攻击功能的关键能量开关，在角膜移植免疫排斥反应中发挥重要作用。为验证上述假说，我们拟采用小鼠高危角膜移植模型，结合体内外实验，探讨有氧糖酵解途径通过调控抗原递呈细胞和效应T细胞功能参与角膜移植术后免疫排斥反应的机制。通过本研究不仅可以明确有氧糖酵解途径对免疫排斥反应的影响及其机制，还可以为临床防治角膜移植免疫排斥反应提供理论依据。

角膜移植是角膜盲复明的主要手段，但术后的免疫排斥反应是导致移植失败的最主要原因。本课题主要围绕有氧糖酵解途径在角膜移植免疫排斥反应的作用开展系列研究，并取得一定的进展：①利用小鼠穿透性角膜移植模型并结合转录组测序发现：糖酵解关键酶在免疫排斥角膜中显著高表达，糖酵解活性明显增强，mTOR/HIF-1信号通路参与糖酵解活性调控；阻断糖酵解活性有效延长角膜植片存活时间。②通过巨噬细胞（BMDM）模型发现，阻断乳酸转运体（MCT-4）能抑制脂多糖（LPS）引起的炎症反应；MCT-4抑制剂（CHCA）通过影响乳酸化修饰抑制炎症反应；体内实验证实，阻断乳酸化修饰酶p300活性可逆转MCT-4抑制剂抗免疫排斥效果。③在前期发现雷帕霉素纳米胶束滴眼液（RAPA-NM）有效抑制角膜移植免疫排斥的基础上，进一步评估RAPA-NM的理化特征、稳定性与生物相容性等；发现RAPA可以通过诱导自噬缓解角膜移植免疫排斥反应，在机制上，自噬通过诱导NLRP3蛋白降解抑制炎症反应进而发挥其抗免疫排斥效果。此外，我们还评估了整体基质/水凝胶复合物负载曲安奈德防治角膜新生血管的效果，并证明其有良好的抗角膜新生血管作用。截至目前，在本项目的资助下共发表学术论文36篇、授权专利2项，获山东省科技发明一等奖1项。