雷帕霉素纳米粒滴眼液治疗兔高危角膜移植排斥反应

目的探讨雷帕霉素(RAPA)纳米粒滴眼液抑制高危角膜移植术后免疫排斥反应的疗效和机制。方法应用不同处理因素干预角膜移植术后免疫排斥反应,观察相关指标的变化。结果RAPA纳米粒滴眼液能明显延长角膜植片存活时间,并能使植片CD4 和CD8 T淋巴细胞表达时间延后。RAPA纳米粒滴眼液组植片中IL-2始终表达,而VEGF表达在14d开始被抑制。结论RAPA纳米粒滴眼液能延长高危角膜移植植片存活时间。     

雷帕霉素缓释膜治疗兔高危角膜移植排斥反应实验研究

目的 探讨局部应用雷帕霉素(RAPA)缓释膜抑制兔高危角膜移植免疫排斥反应的机制.方法 将角膜新生血管白兔68只分为4组,每组17只,行穿透性角膜移植术,供体为健康灰兔.A组为未治疗组;B组术中前房植入空白缓释膜;C组术后每天1%RAPA滴眼液滴眼3次共28天;D组术中前房植入RAPA缓释膜.术后隔天用裂隙灯显微镜观察植片免疫排斥反应发生的时间;用CD.和CD8单克隆抗体行免疫组织化学染色,观察各组T淋巴细胞的迁移和数量. 结果兔角膜植片存活时间:D组(44.89±8.95)d,较A组(11.11±1.69)d、B组(11.56±2.70)d、C组(17.78±8.41)d明显延长(P＜0.01).免疫组化检查:术后14 d,B、c组植片中聚积了大量CD4和CD8 T淋巴细胞,并随时间延长而增加.D组药物治疗后期,植片仅见少量CD4 和CD8 细胞浸润;当RAPA缓释膜消失后,CD4 和CD8 T淋巴细胞开始聚集.结论 RAPA缓释膜能显著延长兔高危角膜移植植片存活时间,减轻全身用药可能引起的并发症.     

雷帕霉素抑制大鼠角膜移植免疫排斥反应的实验研究

目的研究雷帕霉素对大鼠角膜移植免疫排斥反应的抑制作用。方法建立大鼠穿透性角膜移植动物模型,以SD大鼠为受体,Wistar大鼠为供体,分为4组,手术当日起分别腹腔注射0.5%羧甲基纤维素、雷帕霉素(2.5mg·kg-1·d-1)、环孢霉素A(10mg·kg-1·d-1)、雷帕霉素 环孢霉素A(RPM1.5mg·kg-1·d-1 CsA5mg·kg-1·d-1),共12d。对角膜植片进行临床观察,以混浊、水肿和新生血管3项指标作为临床评估标准。结果4组角膜植片的存活时间分别为11.29d±1.50d、20.57d±2.37d、20.86d±2·12d及22.57d±1.99d,各用药组与对照组比较差异有显著性(P<0.01)。结论雷帕霉素能显著延长角膜植片的存活时间,对大鼠穿透性角膜移植排斥反应具有抑制作用,联合用药具有协同作用。     

雷帕霉素滴眼液抑制大鼠角膜移植的免疫排斥反应

目的:研究雷帕霉素滴眼液眼局部应用对大鼠角膜移植免疫排斥反应的作用。方法:建立大鼠穿透性角膜移植动物模型,以SD大鼠为受体,Wistar大鼠为供体,分为A,B,C,D,E5组。手术后2d起分别以滴眼液基质、0.5g/L雷帕霉素滴眼液、1g/L雷帕霉素滴眼液、2g/L雷帕霉素滴眼液、10g/L环孢霉素A滴眼液滴眼,每次100μL,4次/d,用药至排斥反应发生。对角膜植片进行临床观察,以混浊、水肿和新生血管3项指标作为临床评估标准,并进行组织学检查。结果:角膜植片5组的存活时间分别为11.00±1.85d,19.71±3.86d,22.17±4.07d,31.71±8.44d及25.00±8.85d,各用药组与对照组比较差异有显著性(P<0.01);各浓度雷帕霉素组与环孢霉素A组比较差异无显著性(P>0.01)。组织学发现,术后14d各药物治疗组炎细胞浸润、新生血管形成及水肿程度均明显轻于对照组。结论:0.5,1及2g/L的雷帕霉素滴眼液均能显著延长角膜植片的存活时间,对大鼠穿透性角膜移植排斥反应具有抑制作用,随着药物浓度的增大抗排斥作用增强。     

雷帕霉素抑制大鼠角膜移植免疫排斥反应的实验研究

目的探讨雷帕霉素(RAPA)对大鼠角膜移植排斥反应的防治效果。方法以SD大鼠为供体,Wistar大鼠为受体建立角膜移植实验模型。采用完全随机分组设计方案,将68只Wistar鼠(68只眼)随机分为4组,A组为Wistar鼠自体原位角膜移植,B、C、D组为SDWistar鼠之间进行同种异体角膜移植。术后灌胃给药,A组和B组给予空白液,C组和D组分别给予RAPA(3mg·kg-1·d-1)和环孢霉素A(CsA)(10mg·kg-1·d-1),连续用药12d。根据Holland排斥反应评分系统,判断术后植片排斥情况。比较各组角膜植片的平均存活时间和植片存活率。采用广义估计方程对角膜的新生血管评分进行统计学分析。术后14d,对各组角膜进行组织学检查。结果RAPA组发生排斥反应的时间明显延迟,同种异体移植对照组植片平均存活时间(MST)为(11.0±1.5)d,RAPA组MST为(36.1±14.9)d,两组比较差异有统计学意义(P<0.05)。RAPA组角膜植片存活情况较CsA组好,但两组植片存活率比较差异无统计学意义(P>0.05)。RAPA组术后角膜新生血管的生长明显低于异体移植对照组(P<0.05)和CsA组(P<0.05)。组织学检查证实,术后14d,RAPA组角膜植片未见明显淋巴细胞浸润。结论口服RAPA能够延缓大鼠角膜移植排斥反应的发生,并能够抑制术后新生血管的生成。     

雷帕霉素缓释片防治兔高危角膜移植免疫排斥反应和新生血管增殖的研究

目的探讨雷帕霉素(RAPA)缓释片防治兔高危角膜移植术后免疫排斥反应和角膜新生血管增殖的疗效及其作用机制。方法RAPA缓释片制作:RAPA与载体乙交酯-丙交酯-己内酯三元共聚物(PGLC)制成RAPA缓释片(W/W=50/50),每粒缓释片含RAPA0·5mg。65只健康新西兰大白兔,其中45只兔(45只眼)采用缝线法诱导角膜新生血管生成,选择大于3个象限、新生血管长入角膜超过3mm的40只兔按照随机数字表法分为对照组(A组)、1mgPGLC载体前房植入组(B组)、1%RAPA滴眼液组(C组)及0·5mgRAPA缓释片前房植入组(D组),每组10只兔;余20只兔为供体。对4组兔行右眼同种异体穿透性角膜移植术,术后观察90d,记录排斥指数(RI,为植片水肿、混浊及血管长入评分合计)和新生血管指数(NI,为血管长入植片评分)。定期检测C、D组兔房水中RAPA浓度;术后3周,原位杂交法对4组兔角膜植片中白细胞介素(IL)-2R、单核细胞化学吸引蛋白质(MCP)-1、Fas/FasL进行检测,采用免疫组化法对肿瘤坏死因子(TNF)α和血管内皮细胞生长因子(VEGF)进行检测。术后90d,病理组织学检查视网膜、肝肾结构变化。结果(1)免疫排斥:A、B、C及D组兔发生免疫排斥反应的平均时间分别为(16·5±2·5)、(16·0±2·6)、(47·1±13·2)、(87·6±5·8)d(P=0·000)。术后2周,4组兔植片RI分别为4·9±2·2、3·9±0·9、0·8±0·4、0·3±0·6(P=0·000)。术后12周分别为10·4±0·8、10·0±0·0、7·2±2·2、2·0±3·3(P=0·000)。(2)角膜新生血管:术后2周,4组NI数分别为2·4±0·7、2·1±0·5、0·6±0·5、0·3±0·5(P=0·000)。术后12周分别为3·8±0·5、3·8±0·4、0·8±0·7、0·4±0·8(P=0·000)。(3)房水中RAPA浓度:术后第2、4、8、12周,D组房水中RAPA浓度分别10·7、12·0、9·2、7·0ng/ml,C组房水中RAPA浓度检测不出。(4)细胞因子表达:A、B组植片大量表达IL-2R、MCP-1、TNF-α、VEGF细胞因子,C、D组不表达上述细胞因子,4组植片中Fas/FasL均不表达。(5)组织病理:4组兔视网膜、肝肾组织结构正常。结论局部应用RAPA可以防治兔高危角膜移植术后免疫排斥反应和角膜新生血管增殖,RAPA缓释片疗效优于滴眼液。     

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.     

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

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.     

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

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.     

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

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.     

拮抗CD4小分子化合物J2抗小鼠角膜移植排斥机制的初步研究

目的 探讨拮抗CD4小分子化合物J2抗小鼠角膜移植排斥的机制.方法 实验研究.采用随机数字表法将53只Balb/c小鼠分成A、B、C、D及E组,A、C、E组各13只,B、D组各7只,A组为不做任何处理的正常对照,B组为自体角膜原位移植组,C、D、E组为Balb/c-C57BL/6同种移植组,其中B、C组给予不含药物的空白液灌胃,D组给予环孢菌素A(CsA)10 mg/kg体重,E组给予J2 15 mg/kg体重.在角膜移植后第1至4周,每周行外周血流式细胞学检查,观察CD3~+CD4~+T淋巴细胞、CD3~+CD8~+T淋巴细胞的动态变化趋势,在角膜移植后3周行迟发性超敏反应实验,在角膜移植后18 d行免疫酶联斑点实验,检测脾脏白细胞介素2(IL-2)、IL-10、γ干扰素(IFN-γ)的变化.实验数据通过单因素方差分析以及重复测量资料的单因素方差分析进行统计学处理.结果 流式细胞学检查显示:E组小鼠外周血总CD3~+T淋巴细胞、CD3~+CD4~+T淋巴细胞、CD3~+CD8~+T淋巴细胞较B、D组未发生特异性增殖(CD3~+CD4~+T淋巴细胞的E与B组间比较,P=0.776;E与D组间比较,P=0.606.CD3~+CD8~+T淋巴细胞的E与B组间比较,P=0.941;E与D组间比较,P=0.482).迟发性超敏反应结果显示:在移植后3周,E组小鼠对供体脾细胞和第3鼠系脾细胞均呈低反应,与A组小鼠比较差异无统计学意义(F=1.00,P=0.422).免疫酶联斑点试验结果显示:E组分泌IL-2较A组增加(36.0±7.6),分泌IFN-γ较A组增加(56.0+42.2),但与B组比较,差异无统计学意义(IL-2比较,P=0.832;IFN-γ比较,P=0.356).IL-10各组之间差异无统计学意义(F=2.911,P=0.240).结论 J2阻断了CD4与主要组织相容性抗原复合物Ⅱ(MHC-Ⅱ)类分子的抗原提呈过程,特异性抑制了抗原特异性迟发性超敏反应的发生;在J2阻断CD4/MHC-Ⅱ类分子的抗原提呈过程后,IL-2、IFN-γ及IL-10分泌未发生特异性变化.     

小分子化合物J2在抑制小鼠角膜移植排斥反应中作用的初步研究

目的 探讨小分子化合物J2在抑制小鼠角膜移植排斥反应中的作用。方法以23只C57BL／6小鼠作为供体,76只BALB／c小鼠作为受体建立角膜移植实验模型,随机数字法分为A、B、C及D组,A组为BALB／c小鼠自体原位角膜移植,B、C及D组为C57BL／6-BALB／c小鼠间同种异体角膜移植。术后灌胃给药,A组和B组给予不含药物的空白液,C组和D组分别给予环孢素A（CsA）和小分子化合物J2,连续灌胃12d,比较各组小鼠角膜植片的存活时间和存活率,术后21d对各组小鼠行外周血单核细胞行流式细胞学检查,并做角膜植片的组织学检查。结果A组观察期内角膜植片未发生排斥,B组角膜植片平均存活时间为（17．8±2．1）d,C组为（38．1±9．9）d,D组角膜植片存活时间为（40．6±8．3）d,D组与A组（P=0．04）及B组（P=0．00）比较存活时间差异均有统计学意义,与C组比较差异无统计学意义（P：0．99）。流式细胞学检查显示J2给药小鼠外周血CD^＋细胞、CD8^＋细胞未发生增殖,组织学检查证实术后21dD组角膜植片未见明显的淋巴细胞浸润。结论小分子化合物J2能够抑制排斥的发生,延长小鼠角膜植片存活时间。（中华胺群条峦,2007,43：608-612）     

细胞毒性T淋巴细胞相关抗原4-凋亡相关蛋白配体双功能蛋白抑制小鼠角膜移植免疫排斥反应的研究

目的 探讨利用基因重组技术合成的细胞毒性T淋巴细胞相关抗原4(CTLA4)-凋亡相关蛋白配体(FasL)双功能蛋白预防小鼠角膜移植术后免疫排斥反应发生的疗效及其作用机制.方法 为实验研究.建立小鼠穿透性角膜移植动物模型,供体为C57BL/6小鼠(45只),受体为BALB/c小鼠(90只).利用随机分组法将实验动物分为3组,每组30只,A组即对照组(不进行任何治疗),B组即环孢素A缓释系统(CsA DDS)前房植入组,C组即10 μg/ml CTLA4-FasL双功能蛋白浸泡角膜植片组.术后比较3组间角膜植片的存活时间,并分别利用免疫组织化学检测CD4+T细胞,逆转录PCR(RT-PCR)检测CD80、CD86、白细胞介素10(IL-10)、肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)mRNA,DNA原位末端标记(TUNEL)检查凋亡的发生情况.结果 A、B、C组角膜植片的存活时间分别为(14.3±1.3)、(58.0±2.8)、(106.3±17.5)d.C与A组、C与B组、B与A组进行组间两两比较,差异均有统计学意义(均P=0.000).术后A组角膜植片中炎性细胞数量不断增加,以CD4+T细胞浸润为主;B组角膜植片中未见明显炎性细胞浸润;C组角膜植片中浸润的CD4+T细胞数量在术后7 d达到最多,之后发生骤减.RT-PCR检查可见在术后3 d A组和B组角膜和虹膜组织表达CD86 mRNA,术后7 d表达CD80 mRNA,而在相同时间点上C组均减弱表达CD86和CD80mRNA.术后14 d,A组发生排斥的角膜中可检测到IL-10、TNF-α、IFN-γ mRNA,B组和C组则检测不到上述细胞因子的表达.TUNEL检查显示术后7 d C组角膜和虹膜组织中分布着大量发生凋亡的单核细胞,而A、B组均未观察到细胞凋亡现象.结论 CTLA4-FasL双功能蛋白既能阻断T细胞活化所需的CD28-CD80/CD86共刺激信号途径,又能诱导T细胞凋亡,通过双重作用机制发挥免疫抑制作用,从而有效地延长角膜植片的存活时间.     

FK506缓释系统前房植入抑制兔高危角膜移植术后的免疫排斥反应

目的探讨前房植入FK506药物缓释系统（DDS）对兔高危角膜移植术后免疫排斥反应的抑制作用和FK506房水药物浓度与免疫排斥反应的关系。方法107只新西兰白兔中随机数字法选取73只兔进行角膜新生血管化模型的制作,其中68只兔作为受体成功建立高危角膜移植动物模型,随机数字法分为对照组、空白DDS前房植入组、环孢素A（CsA）DDS前房植入组（含CsA 1mg）、0．1％FK506眼液滴眼组及FK506 DDS前房植入组（含FK5060．5mg）。角膜移植术后观察各组角膜植片排斥发生的时间,移植术后1周取各组实验兔眼房水和静脉血进行FK506药物浓度检测。0．1％FKS06眼液滴眼组和FKS06 DDS前房植入组在移植术后的不同时间点抽取实验兔眼房水和静脉血,进行FK506药物浓度的检测。观察各组兔移植术后4周和观察期结束时角膜植片的病理变化,同时应用原位杂交的方法检测各组角膜植片内白细胞介素2受体a（IL-2Bot）、单核细胞趋化蛋白1（MCP-1）、Fas及FasL mRNA的表达。结果FK506 DDS前房植入组角膜植片存活时间超过180d,明显优于其他各组（F=926．37,P=0．0000）,其房水和角膜组织中的FK506药物浓度明显高于FKS06眼液滴眼组（T=21．00,P=0．0022）。FKS06 DDS前房植入组在术后24周内均能在房水中检测出FK506。术后4周对照组和空白DDS前房植入组有大量的炎性细胞浸润,并有明显的IL．2Bet和MCP-1mRNA的表达,而CsADDS前房植入组、FK506眼液滴眼组及FK506 DDS植入组角膜未见明显的炎性细胞浸润,未见IL-2Pux和MCP-1mRNA的表达。各组均未见明显的Fas和FasL mRNA的表达。结论前房植入FK506 DDS可有效地抑制高危角膜移植术后免疫排斥反应的发生,房水中较高的FK506药物浓度是防治术后发生免疫排斥反应的重要因素。