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.     

MHC class I and II antigens as targets of rejection in penetrating keratoplasty in low- and high-risk mouse eyes

PURPOSE: To determine the extent to which expression of major histocompatibility complex (MHC) class I and II molecules contributes to rejection of orthotopic corneal transplants in mice. METHODS: Full-thickness corneas, prepared from eyes of normal C57BL/6 (B6) and BALB/c mice and from B6 mice in which the class II gene I-A or the beta2-microglobulin (beta2mu) gene was disrupted, were placed orthotopically in low- or high-risk eyes of BALB/c (fully incompatible), BALB.B [minor histoincompatible (H) only], and bm12 (class II only disparate) recipients. BALB/c grafts were placed in low-risk eyes of normal B6 and B6 mice with disrupted H-2 DMalpha genes. Graft survival was judged by clinical examination. RESULTS: Recipient-identical class II, but not class I, molecules on fully allogeneic corneas grafted to low-risk beds promoted graft rejection. Allogeneic class II molecules on fully allogeneic corneas placed in high-risk beds promoted graft rejection more strongly than did allogeneic class I molecules. Neither allogeneic class I molecules nor recipient-identical class II molecules on grafts placed in high-risk beds contributed to graft outcome. Mice deficient in H-2 DMalpha failed to reject fully incompatible cornea grafts. CONCLUSIONS: On corneal allografts, where minor H antigens are the major barriers to acceptance, allogeneic class II molecules promote rejection if the graft is placed in high-risk eyes, whereas recipient-type class II molecules promote rejection if the graft is placed in low-risk eyes. Allogeneic class I molecules make a minor contribution to rejection only if the grafts are placed in high-risk eyes.     

Effect of metalloprotease inhibitors on corneal allograft survival

PURPOSE: The expression of Fas ligand (FasL) in the cornea is essential for corneal allograft acceptance in mice. Because the expression of FasL on the surface of cells is sensitive to cleavage with matrix metalloproteases (MMPs), this study examined whether inhibitors of MMPs would lead to increased FasL expression and improved corneal allograft survival. METHODS: Corneal endothelia derived from mice and humans were treated with MMP inhibitors, and FasL expression was examined. BALB/c mice were engrafted with C57BL/6 or C57BL/6-gld corneas and treated with an ointment containing the MMP inhibitor, doxycycline. Corneal allograft survival was monitored for 50 days. RESULTS: Corneal endothelial cells from both mice and humans displayed increased surface expression of FasL after treatment with MMP inhibitors. The increase in surface expression was further evidenced by the ability of these cells to kill Fas-expressing target cells. Mice treated with doxycycline after corneal allograft transplantation showed significantly prolonged allograft survival and an increase in the overall acceptance rate. CONCLUSIONS: MMP inhibitor treatment of cornea-derived endothelial cells results in increased FasL expression and function. MMP inhibitor treatment prolongs corneal allograft survival and results in a modest increase in corneal allograft acceptance.     

Experimental study on cervical lymph nodes removal enhance allograft survival in alkali-burned cornea

AIM: To explore the inhibitive effects of cervical lympha- denectomy on keratoplasy after alkaline burns. METHODS: The Wistar rats' corneas were transplanted into Sprague-Dawley (SD) rats' eyes which were randomly divided into 4 groups: group A (control group); group B, the cervical lymphadenectomy group; group C, corneal transplantation after the alkali burn injury; group D, cervical lymphaden- ectomy following group C. Out of 6 rats in each group, the cornea of one rat was used for macrophage immuno- histochemistry at day 14 after the transplantation, and the remaining 5 rats were used for studying corneal immune rejection with a slit lamp. The time when allograft rejection occurred was recorded and mean survival times (MST) were compared among the groups. RESULTS: Compared with the MST of group A (10.40±1.14 days),the MST of group B(46.30±9.46 days) was significantly longer (P <0.05). MST of grafts between group C (7.00±1.58 days) and group D (15.00±3.39 days) was also significant (P < 0.05). At 14^th day after the transplantation, there was no CD₆₈immunoreactivity in the graft of group B, and CD₆₈ proteins were expressed to some extent in the grafts of group A and D. However, in the graft of group C, the expression of proteins was dramatically up-regulated. CONCLUSION: Cervical lymphadenectomy therapy has a significant effect in preventing corneal allograft rejection in normal and alkali burned corneal beds.     

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。     

Role of recipient epithelium in promoting survival of orthotopic corneal allografts in mice

PURPOSE. To determine whether epithelium-deprived corneal allografts covered with syngeneic epithelium display immune privilege in orthotopic transplantation and whether syngeneic epithelium containing antigen-presenting cells nullifies this effect. METHODS. Epithelium-deprived allogeneic corneas (C57BL/6) and epithelium-deprived allogeneic corneas reconstituted with syngeneic (BALB/c) epithelium (containing or deprived of Langerhans' cells) were transplanted orthotopically into normal eyes of BALB/c mice. Graft survival was assessed clinically and evaluated histologically. RESULTS. Epithelium-deprived corneal grafts survived in syngeneic recipients but were swiftly rejected in allogeneic recipients. These allografts incited intense stromal inflammation and neovascularization. Epithelium-deprived allografts that were resurfaced in vivo by syngeneic epithelium derived from immune-incompetent SCID mice also underwent intense acute rejection when placed in normal eyes of BALB/c mice. The epithelium of in vivo resurfaced grafts was replete with Langerhans' cells. By contrast, most of the epithelium-deprived allografts reconstituted in vitro with fresh, normal BALB/c corneal epithelium survived indefinitely when placed in eyes of BALB/c mice. Similar grafts reconstituted with BALB/c epithelium containing Langerhans' cells were swiftly rejected. CONCLUSIONS. Replacement of donor epithelium with Langerhans' cell-deficient syngeneic epithelium protects orthotopic allogeneic cornea grafts (stroma plus endothelium) from immune-mediated rejection. The presence of an intact, histocompatible layer of corneal epithelium has two important effects on orthotopic corneal allografts: It suppresses nonspecific inflammation and neovascularization within the graft, and it blunts the alloimmunogenicity of the histoincompatible stroma and endothelium.     

Epithelium-deficient corneal allografts display immune privilege beneath the kidney capsule

PURPOSE: To determine whether corneal tissue as an allograft displays immune privilege in a nonprivileged site and, if so, whether CD95 ligand expression contributes to the privileged status. METHODS:Syngenic and allogeneic corneal tissues deprived of epithelium were transplanted beneath the kidney capsule of normal mice. Syngeneic BALB/c, allogeneic C57BL/6, and allogeneic B6Smn.C3H-gld (CD95 ligand-deficient) mice were used as donors for BALB/c recipients, and syngeneic C3H/HeJ-gld (CD95 ligand-deficient) mice were used for normal C3H/HeJ recipients. Allogeneic conjunctival segments served as positive grafting controls. Graft fate was assessed by visual inspection at 4, 7, 14, and 21 days and was confirmed by histologic study. Viability of graft endothelium was assessed by immunocytochemical analysis. RESULTS. Syngeneic corneas and C57BL/6 corneas survived almost indefinitely beneath the kidney capsule. Both the stroma and the endothelial layers remained healthy and intact. Allogeneic conjunctiva evoked a strong inflammatory response attended by neovascularization. Similarly, B6-gld corneas deficient in CD95 ligand expression showed acute destruction beneath the kidney capsule. Circumstantial evidence implicates alloimmune rejection as the mechanism. CONCLUSIONS. Epithelium-deprived corneas from normal mice possess inherent immune privilege that protects them from alloimmune rejection even at nonprivileged sites. Constitutive expression of CD95 ligand contributes to the privileged status. It is inferred that the extraordinary success of orthotopic corneal allografts owes as much to the qualities of the graft as an immune-privileged tissue as to the qualities of the eye as an immune-privileged site.     

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.     

碱烧伤后角膜新生淋巴管与炎症反应指数间的关联

目的 探讨角膜碱烧伤后的角膜新生淋巴管与炎症反应指数间的关联.方法 实验研究.制备大鼠角膜碱烧伤模型.采用5'核苷酸酶-碱性磷酸酶(5'-NA-ALP)双重酶组织化学染色及全角膜免疫荧光法分别检测碱烧伤后1、3 d,1、2、3、4、5、6、7及8周的角膜新生淋巴管和血管的动态变化,并进行淋巴管计数(LVC)和血管计数(BVC).同时,于裂隙灯显微镜下观察角膜炎症反应的变化,记录炎症反应指数(IF),并比较LVC和IF之间的关联.11例人角膜取自碱烧伤后行角膜移植的11例患者.淋巴管内皮细胞受体(LYVE-1)免疫组织化学染色法标记人角膜中的新生淋巴管,LVC和IF之间的关联运用Pearson's相关分析,采用配对t检验比较角膜中存在淋巴管和不存在淋巴管的患者之间IF、炎性细胞计数、碱烧伤病史、年龄的差异.结果 碱烧伤后,角膜基质层存在着新生淋巴管.碱烧伤后3 d时出现角膜新生淋巴管,2周末达到高峰,5周末消退.新生淋巴管的出现滞后于炎症反应,但先于炎症反应和新生血管而消退.LVC与IF之间呈正相关(r=0.572,P＜0.01).11例患者中3例存在着角膜新生淋巴管.与另8例角膜中无新生淋巴管的患者相比,前者IF显著性升高(t=3.28,P＜0.05)、炎性细胞计数显著性增加(t=2.42,P＜0.05),年龄显著性下降(t=2.62,P＜0.05),而碱烧伤病史无显著性差异(t=1.28,P＞0.05).结论 角膜碱烧伤后有淋巴管生成,角膜新生淋巴管和炎症反应指数之间存在着密切的关联.     

不同浓度重组Canstatin蛋白对碱烧伤后角膜MMP-2及TIMP-2表达的影响

目的：探讨不同浓度重组Canstatin蛋白对碱烧伤后小鼠角膜基质金属蛋白酶－2（ matrix metalloproteinase－2,MMP－2）及其组织抑制剂－2（ tissue inhibitor of metalloproteinase－2, TIMP－2）表达的影响及其调节作用。方法：BALB／c小鼠60只随机分为实验组A、实验B及对照组C,每组20只。采用1mol／L氢氧化钠溶液烧伤小鼠右眼角膜,建立炎症性角膜碱烧伤动物模型,分别予以A组、B 组重组 Canstatin 蛋白3μg／mL、5μg／mL 点右眼,4次／d,对照组C组予以生理眼水点右眼。在碱烧伤后第1、3、7、14d以形态学分析评价角膜上皮损伤面积及新生血管生长的情况,并于碱烧伤后第1、3、7、14 d 应用Western－blot检测角膜MMP－2和TIMP－2的表达,增强化学发光法（ ECL）对结果进行分析。结果：形态学分析显示,A组和B组小鼠在碱烧伤后第3d起各时间点角膜上皮缺损面积均小于对照组（P＜0．01）,角膜新生血管均得到抑制,CNV面积明显小于对照组（ P＜0．01）。 Western－blot结果显示,碱烧伤后各时间点MMP－2的表达,A组和B组均明显低于对照组（P＜0．01）,TIMP－2的表达高于对照组（P＜0．01）,且A组和B组间MMP－2的表达在第14d比较差异有统计学意义（P＜0．05）,TIMP－2的表达在第7d及第14d比较差异有统计学意义（P＜0．05）。结论：重组Canstatin蛋白可通过抑制角膜细胞及浸润的炎性细胞产生MMP－2,促进TIMP－2表达,从而抑制和延迟碱烧伤后角膜融解的发生和发展,对碱烧伤后角膜的重塑起着重要作用。     

Preservation of donor cornea prevents corneal allograft rejection by inhibiting induction of alloimmunity

To determine whether preservation of the donor cornea prevents allograft rejection, orthotopic corneal transplantation was performed using corneas preserved in storage medium (Optisol-GS((R))). Donor corneas harvested from C3H/He (H-2(k)) mice and B10.D2 (H-2(d)) mice were preserved in storage medium for 0, 3, 7 and 14 days, and then transplanted into the corneal beds of the recipient BALB/c (H-2(d)) mice. Graft survival was determined clinically and histologically. The expression of major histocompatibility complex (MHC) molecules in the preserved corneas was analysed by immunohistochemistry and Western blotting. Donor-specific cytotoxic T lymphocyte (CTL) and delayed-type hypersensitivity (DTH) responses were assessed 3 weeks after grafting. Active suppression of DTH in the recipient mice was also examined 3 weeks after grafting. The survival of 14 day preserved allografts was significantly higher than that of the non-preserved allografts in both MHC and minor histocompatibility (H) antigens, and minor H only disparate combination. The recipients of the preserved allografts failed to induce both CTL and DTH. The active suppression of DTH was not acquired in these recipients. The expression of donor-derived MHC class I antigens was markedly reduced in the corneas after preservation. Preservation of the donor cornea had a remarkable effect on the prevention of corneal allograft rejection. Since the preserved allografts failed to induce donor-specific CTL and DTH, and active suppression of DTH was not acquired in the recipients, the prevention of allo-rejection is due to a failure of allo-sensitization. These results indicate that the reduction of MHC class I antigens and minor H antigens expression in the preserved grafts induces a failure of allo-sensitization and leads to the high rate of acceptance in corneal allografts.     

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。     

细胞毒T淋巴细胞相关抗原4免疫球蛋白抑制鼠高危角膜移植免疫排斥反应的实验研究

目的　探讨细胞毒T淋巴细胞相关抗原 4免疫球蛋白 (CTLA4 Ig)对小鼠高危角膜移植免疫排斥反应的抑制作用及局部抗免疫排斥反应机制。方法　建立 5 0只BALB/c小鼠穿透性角膜移植动物模型。治疗组 (2 5只 ) :取C5 7BL/ 6小鼠角膜片 ,放置于 10 μg/mlCTLA4 Ig保存液中浸泡 2 4h后 ,移植到BALB/c小鼠。对照组 (2 5只 ) :植片不行任何处理。术后每 3d用裂隙灯显微镜检查植片情况 ,每周应用组织学和免疫组织化学方法检测植片中各种炎性细胞和淋巴细胞的变化。对出现排斥反应的角膜植片应用逆转录PCR(RT PCR)方法检测部分细胞因子的表达。另外 ,选择经CTLA4 Ig治疗、植片保持透明 6周以上的小鼠作为受体 ,接受来自C5 7BL/ 6小鼠皮肤的移植 ,当移植皮肤发生排斥时 ,进行迟发性超敏反应 (DTH)分析。结果　CTLA4 Ig治疗组角膜植片保持透明 >10 0d。对照组小鼠术后 14d内均发生免疫排斥反应。组织病理学检查显示 ,角膜移植术后 2周 ,CTLA4 Ig治疗组植片保持正常细胞结构 ,无明显炎性细胞和T淋巴细胞浸润 ;对照组的排斥植片有大量炎性细胞和T淋巴细胞浸润 (包括CD 4 ,CD 8及CD 11细胞 )。术后 2周发生免疫排斥的角膜植片中检测到白细胞介素 10 (IL 10 ) ,肿瘤坏死因子α(TNF α) ,γ干扰素 (IFN γ) ,B7及C     

Ballistic transfer of minimalistic immunologically defined expression constructs for IL4 and CTLA4 into the corneal epithelium in mice after orthotopic corneal allograft transplantation

Background: Experiments were performed to determine whether corneal epithelium transfected with minimalistic immunologically defined expression constructs for the extracellular fragment of CTLA4 and for interleukin-4 (IL-4) or interleukin-10 (IL-10) is able to modulate an allospecific immune response after orthotopic corneal grafting in mice. Methods: Six groups of BALB/c (H-2d) mice received a C3H (H-2k) corneal graft and dexamethasone eye drops until day 11. Five groups of BALB/c mice had gold particles delivered into the corneal epithelium by Gene Gun on day 10 after transplantation. In four groups, minimalistic immunologic-ally defined gene expression (MIDGE) vectors were delivered into the corneal epithelium by ballistic transfer. The levels of expressed IL-4 and IL-10 were determined by an enzyme-linked immunosorbent assay (ELISA) in shock-frozen homogenized corneas. The expression kinetics of Gene-Gun-transfected corneas were determined by measuring luciferase in lysed whole corneas at different time intervals. Results: Luciferase expression was detectable during the first 5 days following transfection. ELISA was used to determine IL-4 and IL-10 expression in corneal tissue 36 h after transfection. Ballistic IL-4 and CTLA4 gene transfer significantly prolonged corneal graft survival in comparison with the gold-treated control group and the IL-10-treated group. Conclusion: The beneficial effect of IL-4 and CTLA4, but not IL-10 gene transfer into the corneal epithelium by MIDGE vectors was demonstrated for the first time in corneal transplantation. Received: 8 November 1999 Revised: 7 February 2000 Accepted: 9 February 2000     

Promotion of corneal allograft survival by the induction of oxidative macrophages

PURPOSE: A Th1-type immune response was detected in allotransplanted, rejected corneas. Because the intracellular thiol redox status of antigen-presenting cells (APCs) reportedly regulates the Th1/Th2 balance through distinctive cytokine production by APCs, this study was conducted to investigate the effect of the intracellular thiol redox status of macrophages (Mps) on corneal allograft survival. METHODS: N,N'-diacetyl-L-cystine dimethylester (NACOMe)(2) was injected intraperitoneally into BALB/c (H-2(d)) mice to induce Mps with a low intracellular glutathione content (icGSH). Corneal grafts from C57BL/10 (H-2(b)), B10.D2 (H-2(d)), and DBA/2 (H-2(d)) donor mice were placed on neovascularized BALB/c graft beds for assessment. B10.D2-grafted recipients were evaluated for donor-specific delayed-type hypersensitivity (DTH), and the cytokines produced by their lymphocytes were examined (IFN-gamma, IL-4, and IL-10). In other experiments, na?ve BALB/c mice, injected intravenously with Mps of low icGSH content, received B10.D2 corneal grafts. RESULTS: In (NACOMe)(2)-treated mice, 13 of 20 B10.D2 grafts and 6 of 10 DBA/2 grafts survived indefinitely. No grafts survived in the control mice (P < 0.0001). (NACOMe)(2) treatment did not enhance C57BL/10 graft survival. At 2 weeks after B10.D2 grafting, control mice exhibited DTH, but (NACOMe)(2)-treated mice did not (P < 0.01). Lymphocytes from (NACOMe)(2)-treated mice did not respond to donor splenocytes. Those of control mice showed Th1-type cytokine secretion. The intravenous transfer of peritoneal Mps from (NACOMe)(2)-treated mice prolonged corneal allograft survival (P < 0.003). CONCLUSIONS: The observed enhanced graft acceptance may be due to the suppression of alloantigen-induced Th1 polarization through the induction of Mps with reduced icGSH levels.     

人羊膜作为载体体外培养兔角膜上皮细胞移植治疗碱烧伤动物的实验研究

目的观察培养生长于人羊膜的兔角膜上皮细胞使其扩增并移植治疗角膜碱烧伤的效果。为培养角膜上皮细胞移植技术及应用于临床实践提供最佳的理论和实验依据。方法新西兰白兔30只（30眼）,随机分为3组（n=10）,右眼制成碱烧伤模型。A组：角膜上皮细胞羊膜移植组;B组单纯羊膜移植组;C组为对照组（碱烧伤后不作任何移植）。术后观察角膜透明度、角膜新生血管及上皮修复情况,裂隙灯显微镜照相记录。3组均于术后1周、2周、1个月及3个月时各取1眼角膜标本行病理组织检查。结果A组除1眼移植片在第7天脱落外,所有移植片在术后3d水肿消退,角膜逐渐透明。B组移植片持续水肿,眼前段炎性反应稍重,但较碱烧伤对照组轻。C组角结膜高度水肿浑浊,烧伤后观察3个月未发现角膜恢复透明现象。病理组织检查显示：A组角膜及周边上皮细胞为多层结构,角膜新生血管消失,基质的炎性细胞浸润减退;B组覆盖上皮细胞表现为完整上皮细胞型,C组角膜浑浊,新生血管及肉芽组织形成。结论人羊膜作载体体外培养兔角膜上皮细胞移植重建角膜基底膜和角膜上皮结构治疗兔碱烧伤后的角结膜损伤是一种合理有效的方法。     

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）     

Immune Privilege and Immunogenicity Reside among Different Layers of the Mouse Cornea

Purpose: To determine the extent to which each layer of the mouse cornea displays alloimmuno-genicity or immune privilege. Methods: Intact corneas or individual or combined layers of corneas from normal or cauterized eyes of BALB/c, C57BL/6, and CD95L-deficient B6-gld mice were grafted beneath the kidney capsule of normal BALB/c, B10.D2, BALB.B mice or of BALB/c mice presensitized to donor antigens. Graft fate was assessed clinically and histologically and acquisition of donor-specific delayed hypersensitivity (DH) was assessed at selected intervals after grafting. Results: Full-thickness allogeneic corneas induced vigorous DH and were rejected acutely. Similar results were obtained with allografts of corneal epithelium alone (if supported by syngeneic viable stroma), allografts of epithelium from cauterized corneas (containing Langerhans' cells), and stromal allografts deprived of endothelium. Grafts comprised of stroma plus endothelium (without epithelium) were not rejected, nor did they induce DH unless the graft had no CD95L expression. If stroma-endothelium grafts had no CD95L expression, DH directed against major histocompatibility complex (MHC), but not minor histocompatibility, alloantigens was induced. Moreover, CD95L expressed on stroma-endothelium grafts protected endothelial cells, but not stromal cells, from rejection in presensitized recipients. Conclusions: When grafted to a heterotopic site, the alloimmunogenicity of the normal cornea resides within its epithelial and stromal layers, whereas immune privilege arises from the endothelium. In normal mice, CD95L-expressing endothelium can inhibit the stroma from inducing immunity directed at MHC alloantigens, but in presensitized mice the endothelium can protect itself only from immune rejection.     

Ex vivo adenovirus-mediated gene transfer to corneal graft endothelial cells in mice

PURPOSE: Genetic modulation of donor tissue before corneal transplantation may have the potential to modulate alloimmunity and/or to prevent corneal endothelial cell death. This study was conducted to optimize adenovirus-mediated gene transfer to donor corneal endothelium and to delineate the kinetics of marker gene expression in syngeneic and allogeneic corneal grafts. METHODS: BALB/c mouse corneas were incubated with replication-deficient adenovirus encoding green fluorescent protein (GFP) or empty vector ex vivo at a dose of 6 x 10(7) or 6 x 10(6) PFU at temperatures of 4 degrees C or 37 degrees C. After ex vivo infection, the donor corneas were transplanted orthotopically to BALB/c or C57BL/6 recipients. After transplantation, localization of GFP in the grafts was determined in cryosections of enucleated eyes, and GFP expression in the grafts was visualized in vivo by using epifluorescence microscopy over 12 weeks. All grafts were evaluated clinically by slit lamp biomicroscopy. RESULTS: GFP expression was found to be restricted to the corneal endothelium. In vivo expression of GFP in syngeneic corneal grafts was demonstrated for up to 12 weeks. Syngeneic grafts incubated with the vector at 4 degrees C exhibited a more extensive and longer duration of expression of green fluorescence than grafts incubated at 37 degrees C. Moreover, the syngeneic grafts infected at 4 degrees C maintained their transparency, whereas those infected at 37 degrees C displayed a high degree of opacity. Corneal allogeneic grafts infected with a low dose of the vector displayed longer GFP expression and graft survival than the allogeneic grafts infected with a high dose of the viral vector. CONCLUSIONS: Adenoviral vector can selectively and efficiently deliver exogenous gene(s) to the endothelium of corneal grafts during hypothermic organ preservation. Gene expression is retained in vivo in corneal syngeneic grafts for longer periods than are allogeneic grafts.