纳米磁珠连接Y-27632促人脐血内皮祖细胞修复角膜内皮缺损的研究

Fuchs’ dystrophy and bullous keratopathy are two common corneal endothelial diseases that involve progressive corneal edema and the loss of vision, and these diseases require corneal transplantation. However, the global shortage of donor corneas limits the transplantation. There is a great need to find new therapies to restore corneal clarity that is lost due to endothelial dysfunction. Using stem cells to repair corneal endothelium might resolve this problem. We have found the human umbilical cord blood endothelial progenitor cells (UCB EPC) had the possibility to repair corneal endothelium defect, but in the follow-up time the cornea failed to regain totally transparent. It has been reported that the ROCK inhibitor Y-27632 can facilitate corneal endothelial cells proliferation. We also found that Y-27632 could facilitate UCB EPC to different to cornea endothelial cells. In this study, Fluorescent magnetic nanoparticles (FMNPs) are conjugated with Y-27632. The UCB EPCs labeled with Y27632-FMNPs are transplanted into the rabbit anterior chamber combined with magnetic attraction, to repair corneal endothelium defect. The feasibility of this method is investigated. This study will provide a promising cell therapy for corneal endothelial dysfunction.

角膜内皮失代偿导致角膜混浊、视力下降甚至失明，角膜内皮移植是主要治疗方法，但角膜供体来源缺乏严重限制了该技术的开展。为了突破这个瓶颈，研究者们一直在寻找自体来源的干细胞替代角膜内皮细胞治疗角膜内皮失代偿疾病。我们前期研究发现，人脐血内皮祖细胞（EPC）能初步修复角膜内皮缺损，但在随访期内角膜未能完全恢复透明，预实验中发现小分子化合物Y-27632在体外能促进人脐血EPC向角膜内皮细胞分化。为进一步提高人脐血EPC修复角膜内皮缺损的效果，本项目以纳米磁珠为载体连接Y-27632，通过人脐血EPC吞噬磁珠，将Y-27632导入细胞内，再将细胞注射至兔前房，在体外磁场作用下，使人脐血EPC定向移动至角膜内皮缺损处，促进人脐血EPC向角膜内皮细胞分化，修复角膜内皮缺损。本项目的实施有望为角膜内皮失代偿疾病提供新的细胞来源，解决临床角膜供体来源缺乏的难题。

角膜内皮失代偿疾病会导致角膜混浊、视力下降甚至失明，角膜内皮移植是主要治疗方法，但角膜供体来源缺乏，医生手术学习曲线长，严重限制了该技术的开展。本课题创新性使用Nd: YAG激光制备兔角膜内皮失代偿模型，效果可维持4周，无并发症，为临床药物及细胞治疗提供了理想的模型。本课题利用ROCK1/2的小分子抑制剂Y-27632联合角膜内皮细胞条件培养液，诱导人脐血内皮祖细胞，使其分化为角膜内皮样细胞，表达ZO-1，AQP1，N-Cadherin、ATP1A1和Pax6 COL8A2、COL4A2，且较单纯条件培养液诱导组表达明显升高，说明更好地建立了屏障功能和泵功能。进一步将Y-27632联合人脐血内皮祖细胞行前房注射，发现可修复兔角膜内皮缺损，使角膜恢复透明，疗效优于单纯Y-27632组或单纯内皮祖细胞注射组。本研究为临床角膜内皮失代偿疾病提供了新的治疗方法。随着脐带血库的兴起，自体脐血内皮祖细胞移植将成为可能。作为一种新技术，人脐血内皮祖细胞联合Y-27632前房注射治疗角膜内皮失代偿疾病，克服了临床角膜供体来源缺乏的难题，而且具有免疫排斥低、手术微创、操作简便、易于推广、治疗费用低的优点，具有很好的临床应用前景。如果能将这一技术应用于临床，那么在角膜病致盲居世界第一的我国，将产生重大的社会价值和深远的科学意义。