雷帕霉素纳米粒滴眼液联合环孢素A缓释膜治疗兔高危角膜移植术后免疫排斥反应的研究

目的 探讨局部联合应用雷帕霉素(RAPA)纳米粒滴眼液与环孢素A(CsA)缓释膜治疗兔高危角膜移植术后免疫排斥反应的效果和协同机制.方法 实验研究.(1)制备0.5%聚乳酸/胆固醇改性壳聚糖RAPA纳米粒滴眼液和聚乳酸/聚乙二醇CsA缓释膜.(2)设A组为同源对照组(17只兔),供受体均为新西兰白兔.建立102只(102只眼)新西兰白兔角膜新生血管模型,采用随机数字表法将其分为B、C、D、E、F、G6组,每组17只,供体为青紫兰兔,各组行穿透性角膜移植术.A组同源对照组;B组未治疗组;C组空白纳米粒滴眼液滴眼,每天2次共28 d;D组术中前房植入空白缓释膜;E组0.5%RAPA纳米粒滴眼液滴眼,每天2次共28 d;F组术中前房植入CsA缓释膜;G组术中前房植入CsA缓释膜并0.5%RAPA纳米粒滴眼液滴眼,每天2次共28 d.(3)术后观察100 d,隔天用显微镜观察角膜植片情况,记录免疫排斥反应发生时间和程度.(4)术后3、14、28及35 d用CD4和CD8单克隆抗体行免疫组织化学检查;用逆转录聚合酶链反应(RT-PCR)检测植片白细胞介素2(IL-2)和血管内皮生长因子(VEGF)表达.应用单因素方差分析和q检验,比较各组角膜植片平均存活时间.结果 各组兔眼角膜植片存活时间分别为A组(100.00±0.00)d、B组(8.44±1.24)d、C组(8.89±2.57)d、D组(8.56±2.30)d、E组(43.11±5.58)d、F组(43.67±9.54)d、G组(72.00±15.34)d.G组较E、F组,E、F组较B、C、D组,能显著延长角膜植片存活(qGE=11.42,qGF=11.24,qEB=13.64,qEC=13.38,qED=13.46,qFB=13.82,qFC=13.56,qFD=13.64;均P<0.01).免疫组织化学检查显示,B、C、D组术后14 d左右角膜植片中CD4+和CD8+T淋巴细胞大量聚集,呈进行性加重;E、F组术后35 d左右角膜植床与植片中CIM+和CD8+T淋巴细胞数开始出现;G组术后60 d以后,植片中CD4+和CD8+T淋巴细胞浸润.角膜植片RT-PCR检查显示,A组IL-2和VEGF始终未表达;F、G组IL-2表达在3 d开始被抑制;E、G组VEGF表达在14 d开始被抑制.结论 局部联合应用药物缓释剂治疗高危角膜移植术后免疫排斥反应较单独用药效果好,联合用药能减少单独用药的剂量和毒副作用.

Combined rapamycin eye drop in nanometer vector and poly (lactic add) wafers of cyclosporine A effectively prevents high-risk corneal allograft rejection in rabbits

Objective To evaluate the combined effect of topical rapamycin (RAPA) eye drop in nanometer vector and poly (lactic acid) (PLA) wafers of cyclospoirne A(CsA) in the prevention of acute allograft rejection after rabbit corneal transplantation. Methods It was an experimental study. RAPA was incorporated into the nanometer particles and CsA was incorporated into PLA wafers. A was syngeneic control whose both donor and recipient are New Zealand rabbit. Gray donor corneas were implanted into the 102 recipients of New Zealand albino rabbits with corneal neovascularization who were randomly divided into B, C, D, E, F, G 6 groups to receive the different types of therapy: B was no therapy control; C was eye drop of nanometer vector but no RAPA twice a day,28 days; D was PLA wafers in the anterior chamber of rabbit eyes but no drugs; E was 0.5% RAPA eye drop of nanometer vector twice a day,28 days; F was PLA wafers of CsA in the anterior chamber of rabbit eyes; G was PLA wafers of CsA in the anterior chamber of rabbit eyes and 0.5% RAPA eye drop of nanometer vector eye drop twice a day for 28 days together. Postoperative evaluation included slit-lamp biomicroscopy, histopathology and immunohistoiogy, Cytokines related with neovascularization and immunosuppression in the corneal tissue by RT-PCR. The graft survival was assessed by One-Way ANOVA and q test. Results Corneal allograft survival time: A (100.00±0.00), B ( 8.44±1.24), C (8.89±2.57), D (8.56±2.30), E (43.11±5.58), F (43.67±9.54), G (72.00±15.34)d. Group G led to a statistically significant prolongation of transplant survival and was superior than group E and F which was a statistical prolongation compared with group B, C and D (qGE=11.42, qGF=11.24,qEB=13.64, qEC=13.38, q<=13.46, qFB=13. 82, qFC=13.56, qFD=13.64; P<0.01). Immunohistopathologically, the grafts were subjected to an immune response contained a dense infiltrate of neutorphils,CD4+ and CD8+ T lymphocytes in the group B ,C and D. This cellular infiltrate was a significant reduction in group E,F,G. RT-PCR showed that the gene expression of IL-2 was inhibited earlier (3 days) in group F, G and VEGF gene expression being suppressed later (14 days) in group E, G. Conclusions Combined therapy with topical application of RAPA eye drop of nanometer vector and CsA PLA wafers can significantly prolong the survival of allograft at high-risk. Moreover, topical combined treatment of them is more effective, lower dosage, less side-effects and cheaper than the treatment with topical individual immunosuppressive drug.