诱导受体产生对供体角膜的免疫耐受是预防角膜移植排斥反应的理想方法。新发现的一种细胞外膜性小囊泡“外泌体”具有诱导免疫耐受的潜能有望用于抗移植排斥治疗,但作用机制尚不清楚。我们前期研究证实未成熟树突状细胞的过继转移可有效延长小鼠角膜植片存活时间。我们假设未成熟树突状细胞分泌的外泌体（immature Dendritic Cells derived exosomes，imDex）可诱导小鼠角膜移植免疫耐受，使角膜植片长期存活。本研究获取供体imDex，体外验证其对效应T细胞的抑制作用；并在小鼠穿透性角膜移植模型上经结膜下或全身注射，观察移植术后角膜植片的存活状态，评价imDex对受体树突状细胞成熟的抑制作用、对全身和眼局部组织中的CD4+T细胞、调节性T细胞及相关细胞因子等的影响，阐明imDex诱导小鼠角膜移植免疫耐受的机制。从而为imDex用于抗角膜移植排斥反应的治疗提供新的免疫调节策略。

Penetrating keratoplasty(PKP) is often the unique solution for corneal disease. However, rejection is still an unsolved problem. Donor-derived immature Dendritic Cells (imDC) is an effective treatment in inducing immunosuppression in PKP. Exosomes are natural products released from many cells of the body and play a role in antigen presentation, immunoregulation, and signal transduction. The observation that exosomes isolated from immune cells such as DCs modulate the immune response has paved the way for these structures to be considered as potential immunotherapeutic reagents. In order to determine whether exosomes derived from imDC participate in transplantation tolerance, we isolated and purified imDC-derived exosomes (imDex) and established a murine model of PKP. Corneal recipients will receive the imDex by caudal vein injection or subconjunctival injection to observe the effect of transplantation tolerance in vivo and in vitro. From in vivo study, serum analysis and histology will show the function of exosomes in postpone allograft rejection and prolong the survival time of transplanted cornea. From in vitro study, imDex possessed the capacity to suppress T cells proliferation. Taken together, these results will suggest whether the imDex have a suppressive role on acute rejection and inhibit T cells proliferation. Thus, exosomes released from imDC could be considered as a possible immunosuppressive reagent for the treatment of transplant rejection.

目的：本研究探讨未成熟树突状细胞分泌的外泌体（imDex）对角膜植片存活时间的影响及诱导小鼠角膜移植免疫耐受的作用机制。方法：取供体C57BL/6小鼠骨髓细胞诱导获取未成熟树突状细胞（imDC），流式细胞检测鉴定imDC表面标志。采用EXO-Quick TC免疫吸附法分离提取imDex，并进行电镜观察及流式细胞检测鉴定表面标志物，以BCA蛋白测定法对imDex定量分析。建立小鼠异体穿透性角膜移植模型，供体C57BL/6小鼠受体BALB/c小鼠。将受体分为4组，角膜移植术前7天分别球后静脉注射imDex60μg/0.1ml、imDex30μg/0.1ml、imDC1*106细胞/0.1ml和PBS/0.1ml，术后观察角膜植片存活状态并评分。采用流式细胞技术分别检测角膜移植当天、移植后的1周、2周受体小鼠的同侧颌下淋巴结、外周血、脾脏中各淋巴细胞亚群CD4+T、CD8+T、Treg（CD4+CD25+FoxP3+）和树突状细胞的比率。结果：各组角膜植片平均存活时间分别为imDex60μ组9.2±0.37天，imDex30μg组13.67±1.05天、imDC组15.33± 1.76天、PBS组8.20± 0.58天，各组间存活时间比较imDEX30μg组明显长于PBS组（P<0.01）和imDex60μg组（P<0.01）。在球后注射后7天imDex30μg组局部淋巴结和脾脏中较PBS组CD4+T降低 、imDC增高、mDC降低，局部淋巴结Treg高于PBS组，差异均有统计意义(P<0.01)。结论：低剂量供体imDex可以延长小鼠角膜植片存活时间，其诱导小鼠角膜移植免疫耐受的机制与抑制DC成熟、阻止效应性CD4+T应答和上调Treg的表达有关。