角膜新生淋巴管的分子免疫学研究进展

角膜新生淋巴管构成了角膜免疫反应的传入弧,它与角膜新生血管的协同作用是破坏角膜免疫赦免机制的关键因素.近年来,随着淋巴内皮细胞特异性标志物的相继发现,有关角膜淋巴管生成因子的生物学特性、调节机制及临床意义等方面的研究有很大进展.角膜新生淋巴管的免疫调节作用及与移植排斥反应的关系成为研究热点.为此,本文结合角膜免疫赦免特点对角膜新生淋巴管生成的调控机制及其与角膜移植排斥反应之间的关系作一综述.     

角膜新生淋巴管的研究进展

角膜新生淋巴管多见于感染、炎症、外伤或角膜移植术后.临床上,如何有效的防治角膜移植术后排斥反应是角膜移植手术成功的关键.随着近年来角膜新生淋巴管内皮特异性标记物及重要淋巴管生长因子的相继发现,人们对角膜新生淋巴管的生成机制及与角膜移植排斥反应的关系有了深入的研究,此文就新生淋巴管的生成及其在角膜移植排斥中的作用作一综述.     

角膜新生淋巴管的研究进展

近年来随着淋巴管内皮细胞特异性标记物VEGFR-3、LYVE-1、Podoplanin和Prox 1等的发现,对于角膜新生淋巴管的研究有了较大进展,成为近几年的研究热点。本文就角膜新生淋巴管是如何生成,其与新生血管之间的关系,新生淋巴管在角膜移植免疫排斥反应发生过程中的作用作一综述。     

角膜淋巴管生成的内源性调控因素及新生淋巴管相关眼表疾病研究进展

正常角膜无血管和淋巴管。角膜感染、化学烧伤、移植排斥反应等病理性刺激可破坏促淋巴管生成因素与抗淋巴管生成因素间的平衡,致使淋巴管从角膜缘向角膜中央延伸。角膜新生淋巴管的形成与多种调控因素及细胞信号通路密切相关,深入研究并阐明角膜淋巴管生成的机制将为角膜移植排斥反应、炎症性疾病、干眼症和肿瘤转移等相关领域的研究开辟新的方向。本文综述了角膜淋巴管生成的内源性调控因素及新生淋巴管相关眼表疾病的治疗策略。     

角膜新生淋巴管的研究进展

近年来随着淋巴管内皮细胞特异性标记物VEGFR-3、LYVE-1、Podoplanin和Prox 1等的发现，对于角膜新生淋巴管的研究有了较大进展，成为近几年的研究热点。本文就角膜新生淋巴管是如何生成，其与新生血管之间的关系，新生淋巴管在角膜移植免疫排斥反应发生过程中的作用作一综述。     

角膜新生淋巴管的作用研究进展

角膜新生淋巴管作为角膜免疫反应的传入弧,与许多疾病的发生密切相关,角膜新生淋巴管与角膜新生血管的关系一直是研究的热点,他们共同破坏了角膜的免疫赦免机制.因此,深入研究角膜新生淋巴管的作用机制,探讨相关眼科疾病的治疗方法,是目前亟待解决的问题.就角膜新生淋巴管与感染、角膜移植术后的并发症、干眼、翼状胬肉等疾病的关系展开综述,以期为临床治疗提供理论依据.     

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。     

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.     

细胞间黏附分子与角膜移植免疫

角膜移植免疫是多种免疫细胞和免疫分子参与的复杂的免疫反应过程。近来研究表明ICAM1在角膜移植免疫排斥反应的发生机制中起重要作用。ICAM1协同抗原递呈,促使免疫细胞在植片聚集,触发和促进移植排斥反应的发生和发展,引发粒细胞聚集于角膜基质层,导致组织损伤、破坏、角膜新生血管化使移植失败。目前研究抗ICAM1药物抑制角膜移植排斥反应已进入实验阶段。就细胞间黏附分子与角膜移植免疫的研究现状进行综述。     

Toll样受体对角膜免疫调节的研究进展

Toll样受体(TLRs)是先天性免疫的病原模式识别受体,在病原体的识别、获得性免疫反应的调节、信号转导等方面起重要作用.随着对感染性角膜炎和角膜移植术后急性、慢性排斥反应发生机制研究的深入,TLRs在角膜免疫应答中的诱导和调控作用日益受到研究者的关注.目前对TLRs激活细菌性角膜疾病、真菌性角膜疾病、病毒性角膜疾病、角膜移植后植片排斥反应等炎症过程中的免疫信号通路已有较多研究,对TLRs及其信号系统表达的调控有助于减轻或阻止上述炎症反应过程.就TLRs、TLRs信号转导途径及其对角膜炎症反应的免疫调控机制进行综述,探讨其关键作用及可能的治疗靶点.     

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.     

A quantitative electron microscopic study of growing corneal lymphatic vessels

Following silver nitrate injury to rabbit corneas, injection of carbon into the corneas showed the presence of new corneal lymphatic vessels in addition to the new corneal blood vessels.Quantitative measurements of several vessel parameters including endothelial thickness, area of vessel and lumen and circumference of vessel and lumen were made using electron micrographs of cross-sections of corneal lymphatic vessels of various ages. The thickness of the endothelium was found to decrease as the vessel matures, whereas both the area and the circumference of both vessel and lumen increase with age. This is in agreement with qualitative observations.Procedures for estimating the accurate age of vessels from their physical dimensions and for assessing the degree of tortuosity of the endothelial surfaces are presented and discussed.     

Corneal lymphangiogenesis: evidence,mechanisms, and implications for corneal transplant immunology

PURPOSE: The normal cornea is devoid of blood and lymphatic vessels but can become vascularized secondary to a variety of corneal diseases and surgical manipulations. Whereas corneal (hem)angiogenesis, i.e., the outgrowth of new blood vessels from preexisting limbal vessels, is obvious both clinically and histologically, proof of associated corneal lymphangiogenesis has long been hampered by invisibility and lack of specific markers. This has changed with the recent discovery of the lymphatic endothelial markers vascular endothelial growth factor receptor 3, LYVE-1 (a lymphatic endothelium-specific hyaluronan receptor), Prox 1, and Podoplanin. METHODS: We herein summarize the current evidence for lymphangiogenesis in the cornea and describe its molecular markers and mediators. Furthermore, the pathophysiologic implications of corneal lymphangiogenesis for corneal transplant immunology are discussed. RESULTS: Whereas corneal angiogenesis in vascularized high-risk beds provides a route of entry for immune effector cells to the graft, lymphangiogenesis enables the exit of antigen-presenting cells and antigenic material from the graft to regional lymph nodes, thus inducing alloimmunization and subsequent graft rejection. CONCLUSIONS: Antilymphangiogenic strategies may improve transplant survival both in the high- and low-risk setting of corneal transplantation.     

角膜移植免疫排斥反应防治的研究进展

角膜移植是众多器官和组织移植中成功率最高的,然而移植后的免疫排斥反应仍是导致角膜移植术失败的主要原因。本文综述角膜移植后免疫排斥反应发生的机制、排斥反应的预防及其治疗等几个方面的研究进展。     

Treatment strategy for rejection-free corneal transplantation--transition from full-thickness corneal transplantation to corneal endothelium transplantation

The avoidance of allograft rejection is the most critical factor for favorable surgical outcome after corneal transplantation. We report experimental data including distribution of white blood cells in human corneas for rejection-free corneal transplantation. We focused on leukocyte trafficking based on the immunological mechanism leading to allograft rejection in a mouse full-thickness corneal transplantation model. We identified two chemokine-receptors, CCR1 and CCR7 which are functionally relevant to the occurrence of allograft rejection. These chemokine receptors can be new targets for the suppression of allograft rejection after full-thickness corneal transplantation. In the human corneas, bone marrow-derived dendritic cells and monocyte-lineage cells reside constitutively in the normal epithelium and stroma, and may be associated with direct recognition of allo-antigen after corneal transplantation. We established a mouse model in which cultured allocorneal endothelium was transplanted onto a bullous keratopathy recipient cornea. During the follow-up period, the transplanted cultured allo-corneal endothelium did not show any sign of allograft rejection. Our findings demonstrated that a rejection-free mechanism is due not to suppression of immunity or to lack of response, but to failure to recognize the existence of resistance. Realization of the clinical application of cultured allo-corneal endothelium transplantation may be a shortcut to ideal rejection-free corneal transplantation.     

Time course of angiogenesis and lymphangiogenesis after brief corneal inflammation

PURPOSE: To study the time course of angiogenesis and lymphangiogenesis in the cornea after a short inflammatory insult. This might be helpful for the timing of corneal transplantation in high-risk eyes. METHODS: The mouse model of suture-induced inflammatory corneal neovascularization was used. After placement of 3 interrupted 11-0 sutures into the corneal stroma of BALB/c mice (left in place for 14 days), corneas were excised 2, 3, 5, 7, 14, and 21 days as well as 1, 2, 3, 6, and 8 months after surgery. Hem- and lymphangiogenesis were evaluated using double immunohistochemistry of corneas with CD31/PECAM1 as panendothelial and LYVE-1 as lymphatic endothelial marker. RESULTS: Both blood and lymphatic vessels grew into the cornea as early as day 2 after suture placement. The outgrowth was initially parallel. Hem- and lymphangiogenesis peaked around day 14. Thereafter, both vessel types started to regress. Regression of lymphatic vessels started earlier and was more pronounced than that of blood vessels. Whereas at 6 and 8 months (partly) perfused CD31+++/LYVE-1(-) blood vessels and (nonperfused) ghost vessels could still be observed, there were no CD31+/LYVE-1+++ lymphatic vessels detectable beyond 6 months after this short inflammation. CONCLUSIONS: After a temporary inflammatory insult to the cornea, there is initially parallel outgrowth of both blood and lymphatic vessels. But thereafter, lymphatic vessels regress earlier than blood vessels and are completely regressed by 6 months. Earlier regression of pathologic corneal lymph versus blood vessels suggests that corneal graft survival in high-risk eyes might best be delayed for a prolonged interval following an inflammatory insult.     

Standardized semiquantitative analysis of corneal neovascularization using projected corneal photographs--pilot study after perforating corneal keratoplasty before immune reaction

BACKGROUND: A semiquantitative scheme for analysis of corneal neovascularization using projected corneal photographs is demonstrated and tested in a pilot study to analyze occurrence of corneal neovascularization in patients after perforating keratoplasty which subsequently developed transplant rejection. METHODS: Corneal photographs on the slit lamp with diffuse frontal illumination were obtained in a standardized technique. Slides were projected with 100 x magnification and analyzed twice with a 2 months interval. Corneal vessels were graded by two independent observers in each of 12 corneal sectors in a standardized fashion (grade 0: no vessels beyond limbus, 1: vessels between limbus and outer end of a double-running diagonal suture; 2: vessels between outer suture end and graft-host junction; 3: vessels reaching graft-host junction; 4: vessels within donor cornea). All patients with endothelial graft rejection of the prospective Erlangen non-high-risk keratoplasty study were included in a pilot study (1/1997-6/2000: 13 of 325; 4%). One patient without photographs available was excluded. Corneal photographs taken prior to surgery (n = 10), at the last 3 monthly-routine control before (10), at rejection episode (12) and one year later (10) were evaluated for corneal neovascularization. RESULTS: Interobserver correlation at the two assessments was 0.79 and 0.86 (Kendall's Tau B). Correlation between the assessments at the two analyses 2 months apart was 0.8. New vessels with diameter up to 6 microns can be detected. 8 of 12 analyzed patients (67%) with immune reaction after keratoplasty developed corneal neovascularization within 1 year after operation prior to transplant rejection in at least one corneal sector (2.1 +/- 1.9 sectors; 1-6). At time of rejection, new vessels reached the graft-host junction in 2 patients, in 1 patient vessels grew into the donor cornea, whereas in 8 the vessels were seen beyond the outer suture end without reaching host-graft junction (grade I: 1 patient). New vessels usually pointed to the outer suture ends of the double-running suture. CONCLUSIONS: Development of corneal neovascularization e.g. after keratoplasty can be assessed reliably using projected slides of corneal photographs at 100 x magnification. This method has the advantage of being more objective, precise and available compared to simple evaluation at the slit lamp. Postkeratoplasty corneal neovascularization seems to be common in non-high-risk eyes later developing transplant rejection. However, new vessels usually do not reach the host-graft junction. Whether neovascularization after keratoplasty demonstrates a risk factor for subsequent transplant rejection remains to be analyzed in a greater study.     

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。