颈淋巴结切除术抑制碱烧伤后角膜移植免疫排斥反应的实验研究

目的：探讨颈淋巴结切除术对碱烧伤后角膜移植免疫排斥反应的抑制作用． 方法：建立大鼠同种异体角膜移植模型，SD鼠为受体，Wistar鼠为供体，受体鼠再随机分为A，B，C，D四组，A组为对照组，B组为颈淋巴结切除组，C组为碱烧伤后角膜移植组，D组为碱烧伤后角膜移植合并颈浅淋巴结切除组。每组均为6只，其中1只于术后14d行植片的巨噬细胞免疫组化染色。其余5只通过裂隙灯观察角膜免疫排斥的状况，检测并比较各组植片平均存活时间（mean survival time，MST）。 结果：各组MST分别是10．40±1．14d；46．30±9．46d；7．00±1．58d和15．00±3．39d。B组MST较A组明显延长（P〈0．05），而D组MST较C组明显延长，差异具有显著性意义（P〈0．05）。角膜移植后14d，B组植片中无CD68阳性细胞出现，A组和D组植片中有不同程度的CD68阳性的巨噬细胞浸润，而C组植片中CD68呈强阳性表达。 结论：颈淋巴结切除术能有效抑制正常及碱烧伤后角膜移植术后的免疫排斥反应。     

Allograft survival enhancement using doxycycline in alkali‐burned mouse corneas

Purpose: To explore the inhibitory effects of doxycycline on allograft rejection in alkali‐burned cornea beds. Methods: The corneas of BALB/c mice were injured using a 1 mol/l NaOH solution. Following the injury, the corneas from C57BL/6 mice were transplanted into the eyes of BALB/c mice after being randomized into three groups: allogeneic corneal transplantation (group A), topical use of doxycycline after allogeneic corneal transplantation (group B) and syngeneic corneal transplantation (group C). Corneal angiogenesis was examined using whole‐mount immunofluorescence, and corneal inflammation was evaluated using inflammation index scoring. The immune rejection of the grafts was examined using a slit lamp. In addition, the expression of vascular endothelial growth factor A and interleukin‐1β in the transplanted corneas was examined using a real‐time polymerase chain reaction, immunohistochemistry and an enzyme‐linked immunosorbent assay. Results: The outgrowth of the corneal blood vessels in the group A mice was faster than that in the group B and group C mice. The inflammation index levels were highest in the group A mice, intermediate in the group B mice and lowest in the group C mice. Vascular endothelial growth factor and the interleukin‐1β protein and mRNA levels decreased dramatically in the group B mice compared with the group A mice (all p‐values < 0.01). In addition, the mean survival time in the group B mice (27.00 ± 2.00 days) was significantly longer than that in the group A mice (11.67 ± 1.51 days; p < 0.05). Conclusions: Doxycycline may have had a significant role in preventing corneal angiogenesis and inflammation in alkali‐burned corneal beds, which resulted in higher allograft survival rates.     

Crucial role of corneal lymphangiogenesis for allograft rejection in alkali‐burned cornea bed

Backgroud: To examine the time course of hemangiogenesis, lymphangiogenesis, inflammation after corneal alkaline burns and compare with the importance of corneal hemangiogenesis, lymphangiogenesis and inflammation in allograft rejection on alkali‐burned cornea bed, respectively. Methods: Rat corneal hemangiogenesis and lymphangiogenesis were examined by whole mount immunofluorescence and double enzyme‐histochemistry, and the state of corneal inflammation was evaluated by inflammation index scoring and histopathology. Then, corneal transplantations were divided into six groups and performed before the burn (group A) and on day 3 (group B), 2 weeks (group C), 5 weeks (group D), 6 weeks (group E) and 8 weeks (group F) after alkaline burns, respectively. The immune rejection of grafts was evaluated by interferon‐γ, interleukin‐2 enzyme‐linked immunosorbent assay and slit‐lamp examination. Results: Both corneal lymphatic and blood vessels reached the top 2 weeks after the burn. Corneal lymphangiogenesis disappeared 5 weeks after the burn, and corneal hemangiogenesis regressed completely 3 weeks later. Corneal inflammation was strong on day 3, but resolved 6 weeks after the burn. Compared with other groups, the mean survival time of groups B (4.67 ± 1.03 days) and C (5.00 ± 0.63 days) was significantly shorter (P < 0.05). The difference of mean survival time of grafts between group D (9.50 ± 1.05 days) and group E (9.83 ± 0.75 days), between group D and group F (10.00 ± 0.89 days) was not significant (P > 0.05). Conclusions: Corneal lymphangiogenesis presents for a shorter duration than corneal hemangiogenesis or corneal inflammation but plays a crucial role in allograft rejection on alkali‐burned cornea bed.     

高危角膜移植大鼠排斥反应及VEGF-C/D表达的研究

目的：探讨鼠角膜碱烧伤后VEGF-C/D的表达和意义,以及新生淋巴管在高危角膜移植后排斥反应中的作用。
　　方法：制作角膜碱烧伤模型,取不同时间段角膜进行电镜观察,观察角膜血管化情况;采用免疫组织化学方法检测l、3、5、7、14、28 d角膜组织VEGF-C/D及VEGFR-3的表达;并在角膜中仅有血管(A组),同时存在新生血管及新生淋巴管(B组),新生淋巴管消退期(C组),角膜新生血管消退期(D组)以及正常组(N 组)进行穿透性角膜移植,比较不同角膜植片的排斥反应指数( rejection index, RI)值及存活时间。
　　结果：电镜观察发现,在碱烧伤后第7d时鼠角膜出现新生血管,未出现新生淋巴管,在碱烧伤2 wk时出现新生血管的同时出现淋巴管,5wk时无明显的新生淋巴管,8wk时新生血管逐渐消退;大鼠角膜组织中 VEGF-C/D 及VEGFR-3的表达从第3 d开始明显上升,并于第5 d达到最高峰。角膜移植后N、A、B、C、D组的植片平均存活时间分别为14.25±0.62、9.35±1.02、5.06±1.13、8.71±0.83、9.44±1.05d。组间比较发现,B组植片平均存活时间显著性缩短(P<0.05),A、C、D的存活时间均显著性延长(P<0.05)。
　　结论：角膜碱烧伤后存在VEGF-C/D及VEGFR-3的高表达,而且新生淋巴管能加速高危角膜移植后的免疫排斥反应。     

Modulation of costimulation by CD28 and CD154 alters the kinetics and cellular characteristics of corneal allograft rejection

PURPOSE: To examine the effect of modulating the lymphocyte costimulation pathways through CD28 and CD154 (CD40 ligand) in a model of corneal allograft rejection, with particular interest in changes in the observed features of rejection. METHODS: CD28 knock-out (CD28KO) and wild-type BALB/c control mice received corneal grafts from fully major histocompatibility complex (MHC)-mismatched C3H donors and were treated with CTLA4-Ig and/or anti-CD154 Ab on days 0, 2, and 4 after transplantation. Proliferation of BALB/c and CD28KO T cells in response to C3H stimulators was examined in a mixed lymphocyte reaction (MLR) in the presence of CTLA4-Ig or anti-CD154 Ab. RESULTS: Corneal allograft survival in wild-type BALB/c mice (median survival time [MST] 14 days) was significantly prolonged by blockade of the costimulatory pathways with CTLA4-Ig or anti-CD154 Ab (MST 21 days and 25 days respectively). MST in recipients treated with CTLA4-Ig and anti-CD154 Ab in combination was 29 days, not significantly longer than graft survival in single-treatment groups. MST in CD28KO recipients was 46 days and was not prolonged after treatment with anti-CD154 Ab (MST, 43 days). A similar result was found in the MLR, in which anti-CD154 Ab had no effect on proliferation of CD28KO compared with wild-type T cells. In CTLA4-Ig-treated CD28KO, grafts were rejected at an accelerated tempo, similar to that in wild-type BALB/c recipients (MST 16 days). More severe graft injury after the onset of rejection in untreated allograft recipients was accompanied by a higher number of graft-infiltrating CD45(+) cells, but similar proportions of CD4(+) and CD8(+) cells. CONCLUSIONS: CD28- and CD154-mediated costimulation have significant functional roles in corneal allograft rejection. Agents that modulate CD28 and CD154 pathways delay onset and reduce the severity of observed allograft rejection. However, their use in combination did not have an additive effect, MLR data indicating that the CD40-CD154 system depends on a functioning CD28 costimulatory pathway.     

诱导供体特异性前房相关免疫偏离对高危角膜移植排斥反应的影响

目的:探讨诱导供体特异性的前房相关免疫偏离(anterior chamber-associated immune deviation,ACAID)对高危角膜移植排斥反应的影响。方法:采用新西兰白兔眼角膜建立碱烧伤眼模型,实验动物随机分为4组,A:正常角膜常规角膜移植组(正常对照组);B:碱烧伤常规角膜移植组(碱烧伤对照组);C:正常角膜诱导ACAID的角膜移植组(正常诱导组)。D:碱烧伤后诱导ACAID的角膜移植组(碱烧伤诱导组)。B,D组进行左眼碱烧伤,烧伤后1mo进行角膜移植。C,D组在角膜移植前2wk于右眼前房注入可溶性抗原以预先诱导供体特异性ACAID。A,B组右眼前房注等量平衡盐溶液。术后记录植片存活时间;对角膜新生血管(corneal neovascularization,CNV)生长情况进行评分;记录移植排斥指数(rejection index,RI);角膜固定、包埋后制作切片行HE染色。结果:A、C组角膜植片长期存活,碱烧伤对照组植片平均存活25.13±0.64d,碱烧伤诱导组角膜植片平均存活时间为38.25±1.28d。与碱烧伤对照组相比,碱烧伤诱导ACAID组角膜植片平均存活时间显著延长,两组植片存活时间的差异具有统计学意义(P=0.00)。结论:诱导供体特异性ACAID可以延长碱烧伤高危眼角膜植片的存活时间。     

Local overexpression of nerve growth factor in rat corneal transplants improves allograft survival

PURPOSE: To investigate the effect and mechanisms of nerve growth factor (NGF) gene therapy to promote allograft survival in experimental rat corneal transplantation. METHODS: A rat major histocompatibility complex (MHC) class I/II disparate corneal transplant model was used. Recipients were randomly assigned to receive either local Ad (Ad)-mediated gene transfer of NGF or a single intraperitoneal injection of AdNGF 1 day before transplantation. Moreover, immunosuppressive therapy was introduced by systemic coapplication of an Ad expressing CTLA4Ig. The efficacy of this treatment was examined by intracorneal mRNA expression analysis of cytokines and cytoprotective molecules by quantitative RT-PCR at day 12 after transplant. Further graft integrity and immune response against adenoviral vectors were investigated. RESULTS: Local AdNGF-gene transfer significantly prolonged the mean survival time (MST) of rat corneal grafts (16.8 +/- 1.4 days) compared with control grafts (MST, 13.1 +/- 0.3 days; P < 0.03). In contrast, systemic AdNGF gene transfer did not result in improved corneal graft survival (MST, 15.2 +/- 1.0 days). RT-PCR analysis of cornea explants revealed diminished expression of proinflammatory cytokines (IFN-gamma, TNF-alpha) and increased expression of antiapoptotic molecules. In addition, graft endothelial integrity was improved, as measured by the detection of apoptotic cells. Moreover, coapplication of CTLA4Ig further significantly improved graft survival and protective effects of local NGF gene therapy. CONCLUSIONS: This is the first report showing the successful application of a neurotrophin gene therapy to prolong corneal graft survival in an experimental rat transplantation model. Moreover, immunomodulatory therapy further improves graft survival and demonstrates that both anti-inflammatory and cytoprotective mechanisms are involved in the prevention of corneal allograft rejection.     

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。     

抗肿瘤坏死因子-α单克隆抗体抑制大鼠角膜移植排斥反应

目的:探讨肿瘤坏死因子-α(TNF-α)在角膜移植免疫排斥反应中的作用以及抗TNF-α单克隆抗体(TNF-α mAb)对排斥反应的抑制作用。方法:以近交系大鼠30只制作穿透性角膜移植模型(F344→Lou),随机等分入移植治疗组(每日结膜下注射0.5g/L抗TNF-α mAb 0.1mL)和移植模型组(结膜下注射等量生理盐水)。利用酶联免疫吸附实验(ELISA)技术分别检测术后3,7d及发生排斥反应时大鼠外周血清中TNF-α的水平,并观察移植角膜的组织病理学变化及植片存活情况。结果:角膜移植术后早期各组大鼠血清TNF-α浓度均有升高,与移植治疗组相比,移植模型组升高速度更快,水平更高(P<0.05);但当发生排斥反应时,二者已无显著性差异(P>0.05)。移植模型组大鼠植片存活时间为10.2±1.9d,而抗体治疗组平均13.8±2.2d,二者相比差异有显著性(P<0.01)。结论:TNF-α在角膜移植免疫排斥反应中发挥一定作用。动态检测外周血TNF-α浓度有助于预测、诊断排斥反应。应用抗TNF-αmAb治疗可部分延长植片存活时间。     

环孢素A缓释系统植入前房抑制鼠角膜移植免疫排斥反应机制的研究

目的　探讨前房植入环孢素A(cyclosporineA ,CsA)缓释系统抑制鼠角膜移植免疫排斥反应的机制。方法　 (1)环孢素A缓释系统的制备 :为CsA粉剂与已交酯 丙交酯 已内酯的三元共聚物混合体 ,每粒含环孢素A 0 5mg。 (2 )对 90只 (90只眼 )BALB c鼠 (受体 )行穿透性角膜移植术 ,将其分为A、B、C组 ,每组 30只。供体为C5 7BL 6鼠。A组术中鼠前房植入CsA缓释系统 ;B组术中鼠前房植入不含CsA的空白缓释系统 ;C组术后不作任何处理作为正常对照组。术后 3d用裂隙灯显微镜观察角膜植片情况 ,记录角膜植片免疫排斥反应发生的时间和程度。各组分别于术后 1、2、4及 6周随机取 2只鼠眼行组织病理学检查 ,并用CD4、CD8及CD11B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　A组鼠角膜植片排斥时间平均 (35± 3)d ,较B、C组 (14± 3)d明显延长 (P <0 0 0 1)。前房植入的CsA缓释系统体积缩小前 ,A组角膜植片均保持透明 ;当前房植入的CsA缓释系统消失后 ,角膜出现免疫排斥反应 ,植片逐渐混浊、增厚、血管化。B、C组免疫排斥反应均在术后 2周发生。组织病理学和免疫组织化学检查 :A组在术后 14d仅于植床角膜可见少量CD+ 4 　和CD+ 8　细胞浸润 ,在虹膜和睫状体中未见CD+ 11B、CD+ 4 、CD+ 8　T淋     

肿瘤坏死因子相关凋亡诱导配体转基因治疗鼠角膜移植免疫排斥的研究

目的：探讨肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand,TRAIL）对角膜移植免疫排斥反应的作用。方法：选择受体BALB/c和供体C57BL/6小鼠,各32只。将其分为正常对照组、可溶性死亡受体5(soluble death receptor5,sDR5)浸泡组、TRAIL的重组腺病毒（Ad-TRAIL）转染组及绿荧光蛋白（green fluorescent protein,GFP）的重组腺病毒（Ad-GFP）转染组,每组8只。采用免疫组化技术分别对病毒接种后不同时间的体外角膜内皮细胞中TRAIL蛋白进行检测。将携带有Ad-TRAIL的供体C57BL/6小鼠角膜移植片移植到受体BALB/c小鼠眼上,观察术后角膜免疫排斥反应发生的时间及炎性反应强度,并检测免疫排斥的角膜移植片中CD4^ 及CD8^ T淋巴细胞的浸润情况。采用DNA原位缺口末端标记检测角膜移植片中的凋亡细胞。结果：Ad-TRAIL转染3d,50％以上内皮细胞TRAIL蛋白表达阳性;转染10-14d时,内皮细胞TRAIL蛋白表达程度最高;转染3周后,TRAIL蛋白表达阴性。正常对照组、sDR5浸泡组、Ad-TRAIL转染组及Ad-GFP转染组小鼠角膜移植术后发生免疫排斥反应的平均时间分别为17.1、12.3、22.0及17.4d;4个组间角膜免疫排斥反应时间比较,差异有显著意义（P=0.000）。排斥的角膜组织中可见CD4^ 及CD8^ T淋巴细胞浸润,凋亡细胞呈散在分布。结论：TRAIL能抑制小鼠角膜移植术后免疫排斥反应,延长免疫排斥反应发生的时间。     

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）     

Prolongation of corneal allograft survival by CTLA4-FasL in a murine model

Background To investigate the therapeutic effect of CTLA4-FasL—B7 costimulatory pathway blockage—on graft survival in a murine model of corneal transplantation. Methods Orthotopic penetrating keratoplasty was performed on BALB/c mice. The mice were randomized into four groups: the isograft group, untreated allograft group, cyclosporine A drug delivery system (CsA DDS)-anterior chamber implanted group, and 10 μg/mL CTLA4-FasL-treated group. Allografts were from C57BL/6 mice. Survival time of corneal grafts was evaluated. Immunohistological method and TdT-mediated dUTP Nick End Labeling (TUNEL) were applied for the detection of CD4+ T cells and apoptotic cells in corneal transplants. To assess whether peripheral immune tolerance appeared after the treatment of CTLA4-FasL, CsA DDS-implanted- and CTLA4-FasL-treated BALB/c mice with clear grafts received skin allografts at 4 weeks after keratoplasty, and the status of corneal transplants were observed when skin grafts were rejected. Results Allografts in the CTLA4-FasL group (median survival time [MST] = 106 days, p = 0.0042) and the CsA DDS group (MST = 60 days, p = 0.0037) revealed extending survival time, compared with that in the untreated allograft group (MST = 14 days). There were significantly fewer CD4-positive T cells in both the isograft group and the CsA DDS group. In the untreated allograft group, the number of CD4+ T cells gradually increased from day 1 until the final day of observation (day 21). By contrast, it reached a peak on day 7 and then absolutely reduced in the CTLA4-FasL group. Many apoptotic cells were detected on day 7 in the CTLA4-FasL group, but very few were seen in the other groups. Within 30 days of skin-graft rejection, previously healthy and long-standing corneal grafts became rejected in the CsA DDS group but remained clear in the CTLA4-FasL group. Conclusions CTLA4-FasL can prolong the survival time of corneal allografts in mice, exerting a negative regulation on T-cell activation simultaneously by blocking B7 costimulatory signals and inducing Fas-FasL apoptotic pathway. Due to the adjunctive role of FasL, it also appears to be a potential activity of tolerance induction through T-cell apoptotic pathways.     

Differential effects of costimulatory pathway modulation on corneal allograft survival

PURPOSE: T lymphocytes have a central role in allograft rejection. On engagement of the T cell receptor by antigenic peptide-major histocompatibility complex (MHC) complex, a second "costimulatory" signal is critical to full T-cell activation or downregulation. In this study, the effect on corneal allograft survival of modulation of the costimulatory molecules programmed death-1 (PD-1) and inducible costimulatory (ICOS) molecule was examined. These molecules are known to modulate, respectively, negative or positive T-cell activation signals. METHODS: A dimeric PD-L1 immunoglobulin (Ig) fusion protein was generated to stimulate the inhibitory receptor PD-1, and a monoclonal antibody was used to block ICOS. The effect of PD-1 engagement and ICOS blockade on lymphocyte activation by in vitro T-cell proliferation and the effect on orthotopic corneal allograft survival in BALB/c mice were determined. RESULTS: Both reagents demonstrated T-cell inhibition in vitro. PD-L1.Ig treatment of BALB/c mice prolonged fully MHC-mismatched C3H donor corneal allograft survival, with a median survival time (MST) of 21 days. This was significantly prolonged compared to isotype control protein-treated recipients (MST 13 days, P < 0.003). Allograft survival in BALB/c recipients treated with anti-ICOS antibody showed no prolongation of survival compared with the isotype control antibody (MST, 12 days in both groups). CONCLUSIONS: Augmented ligation of the PD-1 negative costimulatory molecule significantly prolongs corneal allograft survival. However, in contrast to findings in other allograft models, signaling through the positive costimulatory molecule ICOS appears to be less important in allogeneic rejection of cornea.     

Efficacy of subconjunctival cyclosporin-containing microspheres on keratoplasty rejection in the rabbit

· Background: The purpose of this study was to evaluate microspheres of PLGA containing cyclosporin (CsA) as a subconjunctival drug delivery system and to test their efficacy in the prevention of corneal allograft rejection in the rabbit. · Methods: Rabbits were injected subconjunctivally with a solution of CsA (CsA-AR) (20 animals) or a microsphere suspension of CsA (CsA-MP) (20 animals), with equivalent drug concentrations (15 mg/ml). The concentration of CsA in the aqueous, cornea and blood was measured by radioimmunoassay at different times thereafter. In other rabbits, 40 allogeneic grafts were performed. Animals were divided into four groups that received the following subconjunctival treatments: group 1: AR solution (solvents of CsA-AR solution); group 2: CsA-AR solution; group 3: MP suspension (empty microspheres); group 4: CsA-MP suspension. · Results: Mean corneal levels of CsA were 1174±830, 918±179, 972±580, 268±182 and 243±162 ng/ml at 12, 24 and 48 h and 7 and 14 days after the injection of CsA-AR. For the CsA-MP suspension, corneal concentrations were 1195±321, 234±147 and 88±77 ng/ml at 12, 24 and 48 h but subsequently dropped to undetectable levels. Blood and aqueous levels were undetectable. Treatment with CsA significantly improved the survival time and survival rate of grafts in the CsA-treated groups (2, 4) over grafts in non-CsA-treated groups (1, 3). There was no significant difference in the graft survival curve between groups 2 and 4. · Conclusion: CsA-containing microspheres might be a promising formulation in the prevention of corneal graft rejection. Since the levels of CsA in blood were undetectable, this treatment might avoid the problems associated with systemic side effects. Received: 28 October 1998 Revised version received: 5 February 1999 Accepted: 2 March 1999     

小分子化合物J2抑制小鼠角膜移植术后排斥反应的研究

目的 探讨新型药物J2抗同种异基因小鼠角膜移植术后排斥反应的作用及其机制.方法 实验研究.采用随机分组设计的研究方法.建立以C57BL/6小鼠为供体,BALB/c小鼠为受体的角膜移植实验模型,其中供体38只,受体100只,随机分为A、B、C、D 四组,每组25只.A组为BALB/c小鼠白体原位角膜移植,B、C及D组均采用C57BL/6-BALB/c同种异体角膜移植,术后采用腹腔注射给药,自手术当日开始,A组、B组给予安慰剂,C组给予CsA 10 mg/kg体重,D组给予J215 mg/kg体重,每天1次,连续给药12 d.观察各组植片生存指数变化,苏木素-伊红染色及冰冻切片免疫组织化学观察角膜植片病理改变;分别在术后1、2、3及4周行逆转录聚合酶链反应(RT-PCR)检测角膜植片中细胞因子的表达.各组间植片存活时间差异采用One-Way ANOVO分析进行比较,两组之间比较采用SNK法.结果 B组角膜植片平均存活时间为(18.88±4.19)d,D组发生排斥时间明显延迟为(33.62±6.S0)d,两组比较差异有统计学意义(q=8.33,P=0.00),而D组与C组比较差异无统计学意义(q=0.85,P=0.60).组织学检查证实,术后21 d,B组见大量细胞浸润,而D组与C组植片未见明显的细胞浸润.免疫组织化学检测显示安慰剂组角膜植片大量CD4+和CD8+T淋巴细胞浸润,J2组与CsA组植片仅有少量的CD4+和CD8+T淋巴细胞浸润.RT-PCR结果显示,在正常小鼠角膜未见IL-10和IFN-γ基因表达;异基因移植组从第1周开始表达各种基因,第3周表达明显增强;而J2组与CsA组从第1周开始表达,第2、3周表达减弱,第4周表达强于前3周.结论 J2通过拮抗CD4与主要组织相容性抗原(MHC)Ⅱ分子结合抑制CD4+T淋巴细胞激活,从而具有抗小鼠角膜移植排斥作用.     

敲除TLR2基因对同种异体小鼠角膜移植排斥反应的影响

目的　探讨敲除Ｔｏｌｌ样受体２（ｔｏｌｌ-ｌｉｋｅｒｅｃｅｐｔｏｒ２,ＴＬＲ２）基因后是否抑制同种异体小鼠角膜排斥反应的发生。方法　以ＢＡＬＢ／ｃ为供体,各取３０只Ｃ５７ＢＬ／６和同背景ＴＬＲ２基因敲除鼠为受体行右眼角膜移植术,设为野生组（ＷＴ）和基因敲除组（ＫＯ）;取１９只Ｃ５７ＢＬ／６行右眼自体角膜移植术,为自体组（ＩＳＯ）;各取９只Ｃ５７ＢＬ／６和ＴＬＲ２基因敲除鼠分别设为野生对照组（ＷＴｃｏｎｔｒｏｌ）和基因敲除对照组（ＫＯｃｏｎｔｒｏｌ）。术后每周两次观察植片并记录免疫排斥的发生时间;术后１４ｄ,收集术眼同侧颈部淋巴结,行流式细胞术分析ＣＤ４＋Ｔ细胞百分比;收集术眼角膜,行免疫组织化学染色和实时荧光定量ＰＣＲ检测角膜干扰素（ｉｎｔｅｒ-ｆｅｒｏｎ,ＩＦＮ）-γ、ＴＬＲ２和ＭｙＤ８８的表达。结果　ＷＴ组和ＫＯ组小鼠角膜移植术后中位生存时间ＫＯ组（３５．０±３．８）ｄ长于ＷＴ组（２１．０±１．５）ｄ（Ｐ＜０．０５）。流式细胞术分析显示,ＷＴ组同侧颈部淋巴结ＣＤ４＋Ｔ细胞百分比较ＷＴｃｏｎｔｒｏｌ组明显增高（Ｐ＜０．０５）,而ＩＳＯ和ＫＯ组相对于其对应的ｃｏｎｔｒｏｌ组（ＷＴ／ＫＯｃｏｎｔｒｏｌ）差异均无统计学意义（均为Ｐ＞０．０５）;免疫组织化学结果显示,ＩＳＯ和ＫＯ组间角膜ＩＦＮ-γ和ＭｙＤ８８分子表达无差异,但两者均低于ＷＴ组。ＫＯ组角膜几乎不表达ＴＬＲ２分子,ＩＳＯ组有微量表达,ＷＴ组表达明显增高;ＰＣＲ检测显示,ＩＳＯ和ＫＯ组角膜ＩＦＮ-γ和ＴＬＲ２ｍＲＮＡ相对表达量差异均无统计学意义（均为Ｐ＞０．０５）,但两者均低于ＷＴ组（均为Ｐ＜０．０５）;ＫＯ组角膜ＭｙＤ８８ｍＲＮＡ相对表达量比ＩＳＯ组高（Ｐ＜０．０５）,但两者均低于ＷＴ组（均为Ｐ＜０．０５）。结论　敲除ＴＬＲ２基因可以一定程度抑制同种异体小鼠角膜排斥反应的发生。     

白细胞介素-34在大鼠角膜移植排斥反应中的作用机制

目的　探讨白细胞介素-34（interleukin-34,IL-34）在大鼠角膜移植术后的表达情况以及在免疫排斥反应中的作用。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。Wistar大鼠60只按照随机数字表法随机分为3组,B组为自体角膜移植组,C、D组行同种异体角膜移植术。另取10只为正常对照组（A组）。术后B、C组术眼滴泰利必妥眼液,D组术眼滴典必殊眼液。术后各组分别取10只大鼠判断四组角膜植片的存活情况并作生存分析。其余大鼠于术后14 d取术眼角膜植片,行组织病理学、免疫组织化学及RT-PCR检测。结果　生存分析结果提示,A组和B组角膜不发生排斥反应,D组角膜存活时间为（26.00±0.97）d,远高于C组（10.00±1.55）d（P<0.001）。HE染色结果显示,C组角膜组织各层有大量炎性细胞浸润以及新生血管形成,D组仅有少量炎性细胞、新生血管。免疫组织化学结果提示,IL-34蛋白在C组的表达量（0.089 4±0.005 6）明显高于A组（0.037 7±0.002 3）、B组（0.068 4±0.004 4）和D组（0.044 5±0.004 5）的表达量（F=145.21,P＜0.01）,且主要集中在上皮层和基质层。RT-PCR结果提示,IL-34、IL-1β、TNF-α、IL-17A的mRNA 在C组角膜组织中表达水平明显高于A组、B组和D组（均为P<0.05）。结论　IL-34参与了大鼠角膜移植术后的排斥反应,而典必殊可能通过抑制IL-34的表达及相关的信号通路,延缓排斥反应的进展。     

Effect of alloantibodies on corneal allograft survival

PURPOSE: The precise role of antibodies in corneal transplantation is ambiguous, with evidence to support as well as repudiate their involvement in graft rejection. Accordingly, this study was undertaken to investigate the direct contribution of donor-specific antibodies to corneal graft rejection. METHODS: Serum samples from CB6F1 rejecters of orthotopically grafted C3H/Hej corneas were tested by ELISA for elevated levels of donor-specific alloantibody. Orthotopic corneal allograft rejection was also examined in B-cell-deficient mice. In a prospective study, na?ve BALB/c T-cell-deficient nude mice and complement-depleted nude mice were passively infused with immune donor-specific serum and grafted with fully allogeneic C57BL/6J corneas. The incidence and speed of graft rejection were observed in each case. The susceptibility of corneal cells to antibody-mediated lysis was tested in vitro. RESULTS: Seventy percent of the CB6F1 hosts that rejected the C3H/Hej corneal allografts possessed significantly elevated levels of alloantibody in serum. Although BALB/c corneal allografts were rejected by B-cell-deficient mice at the same incidence as wild-type control mice, their mean survival time (MST) was significantly longer than that of their wild-type counterparts. Serum of BALB/c mice immunized against C57BL/6J alloantigens produced complement-dependent cytolytic activity against C57BL/6J corneal cells in vitro. Passive transfer of this alloantiserum to T-cell-deficient BALB/c nude mice produced complement-dependent corneal lesions, resulting in significantly increased opacity of C57BL/6J corneal grafts, compared with the relatively clear grafts in control hosts. CONCLUSIONS: Alloantibody, although not necessary for corneal graft rejection, can produce extensive injury to corneal allografts in a complement-dependent manner.