角膜移植后患者角膜新生淋巴管与新生血管和炎症的关联

目的:研究角膜移植后角膜新生淋巴管与新生血管和炎症的关联。方法:人角膜取自行二次角膜移植的患者19例。淋巴内皮细胞受体(lymphatic vessel endothelial hyaluronan receptor,LYVE-1)和内皮细胞黏附因子-1(platelet endothelial celladhesion modecule-1,PECAM-1)双重免疫组化法标记角膜中的新生血管和淋巴管,进行淋巴管计数(lymphatic ves-sels counting,LVC)和血管计数(blood vessels counting,BVC),比较BVC、炎症指数(inflammation index,IF)、移植历史(transplantation history,TH)与LVC之间的关联。结果:角膜移植后BVC,IF与LVC间均呈显著性正相关,而TH与LVC间呈显著性负相关。角膜移植后新生淋巴管、血管、眼表炎症间大致成平行发展,新生淋巴管最先退化,其次是眼表炎症,新生血管最后消退。结论:人角膜移植后角膜新生淋巴管与新生血管、眼表炎症之间存在着极为密切的关联。     

Development of new lymphatic vessels in alkali‐burned corneas

Purpose: Corneal lymphangiogenesis provides an exit route for antigen‐presenting cells to regional lymph nodes, inducing immune response. The purpose of this study was to examine the development of corneal lymphatic vessels in alkali‐burned corneas. Methods: Corneal lymphatic vessels were examined by electron microscopy, 5′‐nase‐alkaline phosphatase (5′‐NA‐ALP) double enzyme‐histochemistry and whole mount immunofluorescence at 6 hr, 1 day, 3 days, and 1, 2, 3, 4, 5, 6, 7 and 8 weeks after rat corneal alkali injury. The expression of vascular endothelial growth factor‐C (VEGF‐C) protein and mRNA was examined 1, 3, 5, 7, 9, 11 and 14 days after the injury. Results: Corneal lymphangiogenesis developed 3 days after alkaline burns, reached its peak 2 weeks after the injury, decreased gradually and disappeared at the end of the fifth week. The expression of VEGF‐C in burned corneas increased dramatically on the third day but disappeared the 14th day after the injury. Conclusion: Corneal lymphatic vessels develop after alkaline burns and VEGF‐C may play an important role in corneal lymphangiogensis.     

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。     

Topical Ranibizumab inhibits inflammatory corneal hem‐ and lymphangiogenesis

Purpose: Ranibizumab (Lucentis^®) is a Fab‐Fragment of a recombinant, humanized, monoclonal VEGF (anti‐vascular endothelial growth factor) antibody. This study analyzed the ability of topical Ranibizumab to inhibit lymphangiogenesis in addition to hemangiogenesis after acute corneal inflammation in vivo. In addition, the effect of Ranibizumab on the proliferation of human lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) in vitro was studied. Methods: The inhibitory effect of Ranibizumab on LECs and BECs was studied in vitro using a proliferation enzyme‐linked immunosorbent assay (ELISA) assay. To study the in vivo effects of Ranibizumab, the mouse model of suture induced inflammatory corneal neovascularization was used. Study mice received topical Ranibizumab as eye drops. After 1 week excised corneas were stained with LYVE‐1 and CD31. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically by using a semiautomatic method based on the image analyzing program Cell^F. Results: An antiproliferative effect of Ranibizumab was seen in vitro on both human BECs and LECs with a significance of p < 0.0001 and p < 0.0004, respectively. In vivo experiments showed that topical application of Ranibizumab significantly inhibits both hemangiogenesis (p = 0.0026) and lymphangiogenesis (p = 0.0026) in the cornea. Conclusion: Ranibizumab is a potent inhibitor of inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo with a direct inhibitory effect on both endothelial cell types in vitro. This study for the first time demonstrates an inhibitory effect of Ranibizumab on lymphatic vessels which could have a wider range of clinical applications.     

荧光标记大鼠全角膜新生血管和淋巴管的方法研究

目的探讨标记大鼠全角膜新生血管和淋巴管的方法。方法应用心脏灌注荧光联合全角膜免疫荧光法．共焦显微镜下区分并标记全角膜的新生血管和淋巴管。结果适度的抗体浓度和灌注时间下可标记出全角膜新生血管和淋巴管。血管呈黄绿色，淋巴管呈红色。结论心脏灌注荧光联合全角膜免疫荧光法可用于标记大鼠全角膜新生血管和淋巴管。     

Age-related changes in murine limbal lymphatic vessels and corneal lymphangiogenesis

Important risk factors for graft rejection after corneal transplantation are pathologic corneal lymphangiogenesis and young recipient age. Purpose of this study was to investigate whether there are age-related differences in normal murine limbal and pathologic corneal lymphatic vessels, which could partly explain the unequal outcome of corneal transplantation in young versus old recipients. Furthermore, we investigated whether these observed differences correlate with changes in allograft survival in the murine model of corneal transplantation. Corneal whole mounts from untreated young (aged 6-8 weeks), untreated old (aged 9-15 months) and young and old mice after suture-induced, inflammatory corneal neovascularization were prepared and stained with LYVE-1 as a lymphendothelial marker. Angles of corneal parts with and without a main circumferential limbal lymphatic vessel were measured and then related to the total 360° of corneal circumference. Centrally directed vascular extensions from the main limbal lymphatic vessel (“sprouts”) of previously untreated old mice were counted. Concerning the outgrowth of pathologic lymphatic vessels after inflammatory corneal neovascularization, the area covered with pathologic lymphatic vessels was detected by an algorithm on digitized whole mounts using cell^F^® software. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed with one recipient group being young, the other group being old mice. In young, untreated mice, 70.5% of the total corneal circumference was covered by a main circumferential limbal lymphatic vessel versus 60.8% in old, untreated mice. Comparing the number of centripedal vascular extensions from the main limbal lymphatic vessel (“sprouts”), untreated old mice had significantly less extensions than young, untreated mice (p < 0.001). After an inflammatory stimulus, old mice had significantly less pathologic corneal lymphatic vessels than young mice (42% less, p < 0.001). Comparing the survival proportions after corneal transplantation, old recipient mice showed a significantly better graft survival 6 weeks after transplantation (65% versus 33%, p < 0.05). Thus, limbal lymphatic vascular sprouts and inflammation-induced pathologic corneal lymphangiogenesis decrease with age. The lower lymphangiogenic potency of older mice may explain the better outcome of corneal transplantations in old recipients, supporting the concept that lymphangiogenesis is an important risk-factor for corneal transplant rejection.     

羊膜移植对碱烧伤大鼠角膜缘干细胞的影响

目的观察羊膜移植对碱烧伤大鼠角膜缘干细胞的影响。方法SD大鼠40只制作眼碱烧伤模型,单眼行眼碱烧伤;随机选取20只大鼠（20眼）为实验组,20只大鼠（20眼）为对照组;实验组行新鲜羊膜移植,对照组不处理,观察大鼠眼表情况。并于术后1周、2周、3周、4周取角膜缘组织,采用免疫组化技术观察p63蛋白在角膜缘干细胞的表达情况。结果p63在实验组和对照组的角膜缘干细胞中均有表达,位于角膜缘上皮细胞基底层的细胞核内。但实验组明显高于对照组,两者比较有统计学意义（P〈0．05）。且随着烧伤时间的延长,p63的表达呈线性下降趋势。结论眼碱烧伤后行羊膜移植,能促进角膜缘干细胞的增生、分化,利于角膜上皮的修复。     

组织工程角膜上皮移植治疗兔角膜碱烧伤的形态学观察

目的:探讨组织工程角膜上皮移植治疗角膜碱烧伤的疗效和时机。方法:1mol/LNaOH制作改良兔角膜碱烧伤模型21只42眼,分对照组和移植组,移植组分别在碱烧伤后1,3,6,9d(早)和14d(中)行自体或同种异体组织工程角膜上皮移植术,比较移植组及对照组烧伤后28d内眼表和组织病理学变化。结果:角膜碱烧伤后7d开始出现角膜上皮的大片脱落,14d角膜上皮大片脱落或溃疡发生率达72%,持续至28d,而移植组在28d时发生率仅为25%,大多获得完整的角膜上皮;烧伤后早期移植组角膜基质深层炎性细胞浸润和新生血管生长较对照组明显受到抑制,而中期移植组角膜基质层较对照组并无明显差异;28d内异体组织工程角膜上皮移植的免疫排斥反应并不大于自体移植。结论:自体或同种异体组织工程角膜上皮移植均可尽快恢复眼表完整性,且烧伤后早期移植效果明显优于中期移植。     

同种异体角膜移植后角膜内皮细胞的特征表现及生物学意义

同种异体角膜移植目前仍是治疗角膜疾病最为有效的方法,但术后发生的免疫排斥所引起的角膜炎症反应和血管化,最终导致植片水肿,甚至功能丧失仍是造成手术失败的最主要原因.角膜内皮细胞作为免疫赦免及角膜正常生理功能维系的首要屏障,在眼部系统中占据着至关重要的位置.本文就同种异体角膜移植术后免疫的识别,应答和最终反应的各阶段角膜内皮所呈现的特征改变及生物学意义进行阐述.

Abstract：

Allograft corneal transplantation is currently the most effective and optimum method to treat corneal disease, however it is the immune rejection that the main reason for graft edema caused by vascularization and inflammation of the cornea and surgical failure after transplantation. Corneal endothelial cells occupy a crucial position as immune privilege and the primary barriers for maintaining normal physiological function of eye. In this paper, the various stages (recognition, response and reaction of immune system) of corneal endothelial changes in the morphological and biological characteristics had been presented after allograft corneal transplantation.     

Exacerbated corneal inflammation and neovascularization in the HO-2 null mice is ameliorated by biliverdin

Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. An 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827,600+/-129,000 vs. 294,500+/-57,510; p<0.05), neovessels measured by vital microscopy (21.91+/-1.05 vs. 12.77+/-1.55mm; p<0.001) 4days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.     

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。     

Immunology of corneal allograft rejection. Associated human leucocyte populations

Human serum samples and peripheral blood mononuclear leucocytes (PBML) were collected from ten corneal transplant recipients over a two-year period at intervals before and after surgery, and during and after episodes of immunologic (rejection) reaction. Serum was monitored for the development of antibodies to histocompatibility antigens and PBML were stained with a panel of monoclonal reagents specific for T cell and for monocyte populations using indirect immunofluorescence. Patients who demonstrated an elevation in the Leu 2a+ (suppressor/cytotoxic) T cell population following an immunologic reaction ultimately accepted their grafts, whereas patients whose Leu 2a+ population was unaffected developed irreversible graft clouding. Additionally, a direct positive correlation between the development of elevated levels of lymphocytotoxic antibodies and helper T cells was noted in some patients during immunologic reactions. Neither DR-positive nor monocyte population changes demonstrated any consistent correlation with rejection episodes or final graft outcome.     

前房相关免疫偏离对碱烧伤眼角膜移植的影响

目的观察诱导供体特异性的前房相关免疫偏离(ACAID)是否有助于角膜植片在碱烧伤高危眼中的存活。方法用1mol/L的NaOH烧灼新西兰白兔单眼角膜中央建立高危眼模型,然后将动物随机分为诱导组和对照组,1个月后进行角膜移植。诱导组在角膜移植前10d前房植入供体肌肉诱导供体特异性ACAID,对照组未处理。观察诱导组与对照组相比角膜植片存活时间是否延长。结果与对照组相比,诱导ACAID组角膜植片平均存活时间延长,两组植片存活率的差异具有显著统计学意义。结论诱导供体特异性ACAID有助于碱烧伤高危眼角膜植片的存活。     

高危角膜移植排斥反应的预防和治疗进展

高危角膜移植术后免疫排斥反应是影响角膜移植成败的首要因素,因此,免疫抑制剂在高危角膜移植排斥反应中的应用占有不可替代的地位。然而,在应用过程中出现许多局部和全身不良反应,为了提高角膜移植成功率,国内外专家、学者在剂型、用药途径以及联合用药方面都做了卓著的研究; 同时在基因工程方面也有可观的进步。本文就高危角膜移植排斥反应的预防和治疗方面做一综述。     

碱烧伤人角膜组织新生淋巴管的检测

目的 探讨人角膜碱烧伤后新生淋巴管的生长情况及其影响因素.方法 回顾性系列病例研究.分析2005年1至12月期间在中山大学中山眼科中心因角膜碱烧伤住院手术治疗的22例(22只眼)患者.记录患眼的烧伤时间(IT)和损伤等级(ID),并测量炎性反应指数(II)和角膜新生血管的相对面积(BVA);应用双重酶组织化学、免疫组织化学染色及透射电镜检测角膜标本的新生淋巴管(LVC)和血管(BVC);采用苏木素-伊红染色检测多形核白细胞(PMN)浸润情况.结果 采用配对Student t检验、Pearson相关检验及Stepwise回归进行统计学分析.结果 22例患者观测结果 IT为(57.62±31.72)个月、ID为(12.00±2.76)分、II为(2.32±2.63)分、BVA为29.79%±18.61%、BVC为(14.45±9.29)个、LVC为(2.73±4.57)个、PMN为(13.45±13.09)个,其中7例(占32%,IT<64个月)同时存在角膜新生淋巴管(8.6±3.8)个和血管(22.3±11.1)个,LVC总数为60个,占发生新生淋巴管的角膜中总管腔数(BVC和LVC,378个)的16%.LVC与IT、ID、BVA、PMN、II的相关系数分别为-0.673、0.604、0.755、0.806、0.873,均P<0.05;进一步回顾分析得出LVC近似等于II和BVA分别和特定的常数相乘后取和所得的淋巴管指数(LI).透射电镜从微观上证实了碱烧伤人角膜组织中存在具有典型结构特征的新生淋巴管和炎性细胞浸润.结论 碱烧伤后部分角膜组织存在新生淋巴管,通过II和BVA可间接估计其发生情况.LI是一项评估碱烧伤角膜新生淋巴管的有用临床参数.(中华眼科杂志,2009,45:115-121)     

调节性T细胞在角膜移植排斥反应中的研究进展

角膜移植术是治疗终末期角膜疾病的常用方式,虽然角膜移植的成功率较其它器官移植高,但是术后排斥仍然是手术失败的主要原因。器官移植后排斥反应高度依赖于免疫细胞向淋巴组织或炎症部位的定向迁移和归巢,并受粘附分子和趋化因子的调控。调节性T细胞在免疫调节中起着关键性作用,通过诱导免疫耐受维持内环境稳定,在器官移植排斥反应、自身免疫性疾病及肿瘤相关研究中发挥重要作用。本篇综述主要介绍调节性T细胞参与眼部免疫耐受的相关研究,着重阐述调节性T细胞在角膜移植排斥过程中的作用、机制和应用。     

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。     

Inhibitory effect of tetrandrine eye drops on corneal allograft rejection in rats

To observe the effect of tetrandrine (Tet) eye drops of different concentrations on corneal graft and on allograft rejection in rats. · METHODS: Models of allograft rejection were set up in 64 SD rats and they were then randomly divided into 3, 5, 10g/L tetrandrine eye drops-treated and control groups. At different times postoperatively, neovascularization and inflammation of corneal graft were observed using slit-lamp microscopy, HE staining, light microscopy and microphoto-analysis. · RESULTS: The graft was infiltrated mainly with lymphocytes and mononuclear-macrophages. Corneal neovascularization and inflammation were significantly inhibited in the 5g/L Tet-treated group (P <0.05), compared with control group on day 7, 14, 21, 28 postoperatively. · CONCLUSION: Corneal edema and corneal epithelial bubble appear when the graft is treated with tetrandrine of higher concentration (10g/L), but 5g/L Tet eye drops significantly inhibit corneal allograft rejection in rats without serious side-effects.     

雷公藤对异位角膜移植抗排斥反应的研究

目的：采用雷公藤对角膜移植排斥反应进行实验性治疗。 方法以 Ｗｉｓｔａｒ大鼠异种角膜异位移植为模型，将 ３６只鼠随机分为治疗组及对照组，并在术后ｄ２、ｄ５、ｄ７、ｄ１０、ｄ１４分别取出植片做病理切片，检测外周血Ｔ淋巴细胞亚群及Ｔ淋巴细胞集落形成数。 结果雷公藤可推迟排斥反应的发生时间（排斥时间 ７．５±１．０７ｄ，对照组 ５．４±０． ９７ｄ）；有明显减少外周血中辅助 Ｔ淋巴细胞百分率的作用（术后 ｄ５治疗组 ＣＤ４＋／ＣＤ８＋为 １． ４２±０． ２７，对照组为１， ９６±０． ２６）；对大鼠外周血 Ｔ淋巴细胞集落形成能力无明显影响。 结论雷公藤多甙是一种有效的免疫抑制剂，能明显抑制角膜移植排斥反应发生时间及 ＣＤ４＋／ＣＤ８＋比值的上升。