巨噬细胞功能异常影响糖尿病角膜创伤修复的机制研究

Diabetes impairs corneal wound healing and acts as a high risk fctor for corneal surgery, however, the mechanism remains elusive. In our previous study, we found that: macrophage played a critical role in wound healing process after corneal graft, macrophage depletion significantly impaired wound healing after autologous corneal transplantation and macrophages transfusion recovered the defect of corneal wound healing. Likewise, we found that diabetic mice presented similar symptoms characterized by impaired wound healing and decrease of myofibroblast number. Moreover, in contrast with their counterparts, diabetic mice showed decreased number of infiltrated CD206+ M2 macrophage in the in the junction area. Based on the above findings, we will set up the model of mouse corneal transplantation and in vitro transwell co-culture, using the technology of molecular biology, immunology, flow cytometry, 1) study the discrepancy of infiltrated macrophage and myofibroblasts dynamics between diabetic and normal mice 2) explore the mechanism of how M1/M2 macrophage regulate corneal keratocyte function. The purpose of this study is to explain the cause of the delayed healing of corneal epithelium and stroma after eye surgery, and find the theoretical foundation to deal with the wound healing after keratoplasty in diabetic patients.

糖尿病影响角膜创伤后的正常愈合，是各类角膜手术的高危因素之一，但其机制不清楚。我们前期研究发现：巨噬细胞在角膜愈合过程中起关键作用，糖尿病小鼠与眼局部巨噬细胞清除后小鼠的角膜创伤愈合过程和病理特征相似，表现为植片-植床交界处肌成纤维细胞数量减少，愈合障碍，而巨噬细胞回输可显著恢复植片愈合；同时，正常小鼠角膜植床-植片交界处存在大量的M2型巨噬细胞，而糖尿病小鼠主要是M1型巨噬细胞。在此工作基础上，本项目拟采用角膜移植模型和体外细胞共培养模型，通过分子细胞生物学、免疫荧光、流式细胞术等技术研究糖尿病角膜移植愈合过程中巨噬细胞、肌成纤维细胞分化的动态变化差异；M1/M2巨噬细胞调控角膜基质细胞的功能及机制以及巨噬细胞回输对角膜移植愈合的影响。本项目的实施将有望解释糖尿病患者眼部手术后角膜上皮及基质愈合缓慢的原因，为糖尿病患者角膜移植术后切口愈合提供新的治疗靶点。

本项目计划通过糖尿病角膜移植模型，研究巨噬细胞功能异常影响糖尿病角膜创伤修复的机制，研究糖尿病条件下角膜创伤后上皮和角膜基质愈合缓慢的原因，为糖尿病患者角膜移植术后切口愈合提供新的治疗靶点。在项目执行过程中，一方面，我们按项目计划研究了不同时间点糖尿病角膜移植后巨噬细胞M1/M2极化与肌成纤维细胞形成、分化的相互关系；另一方面也研究了骨髓间充质干细胞促进糖尿病角膜上皮损伤修复的机制。经过研究，本课题在以下2个方面取得了进展：1、糖尿病小鼠角膜基质愈合显著延迟，植片存活时间显著下降，α-SMA阳性的肌成纤维细胞显著减少，M1型巨噬细胞数量和比例均显著上调，M2型巨噬细胞数量和比例显著下降，巨噬细胞异常极化是糖尿病小鼠角膜移植排斥的重要原因；2、 骨髓-间充质干细胞通过分泌重组肿瘤坏死因子α刺激基因6（TSG-6）促进角膜上皮细胞增殖，促进M2型巨噬细胞极化，降低M1型巨噬细胞比例进而促进糖尿病角膜上皮损伤愈合。通过本课题的研究，共发表SCI收录期刊论文3篇，在国内专业学术会议发言5次。