重视角膜移植植片内皮细胞慢性失功的研究

随着配型技术及新型免疫抑制剂的临床应用,器官或组织移植术后早期急性免疫排斥反应发生率明显下降,慢性移植物失功则成为导致远期移植失败的主要原因之一。研究表明角膜移植术后和全身器官移植术后同样存在角膜植片慢性失功的过程,且角膜植片慢性失功已经成为再次角膜移植的主要原因之一。因此,重视角膜植片慢性失功对其临床防治及延长角膜植片的存活率有着重要的意义。     

Allograft rejection after living-related simple limbal epithelial transplantation

A 23-year-old man presented with congestion, peripheral corneal vascularization, an elevated ridge-like epithelial line and cellular infiltration around limbal transplants, 15 months after undergoing living-related simple limbal epithelial transplantation (SLET) for total limbal stem cell deficiency. A diagnosis of acute allograft rejection was made and he was treated with intravenous methylprednisolone, topical and oral prednisolone as well as systemic cyclosporine and azathioprine, leading to reversal of the signs. Similar findings were noted during a later rejection episode. An epithelial rejection line and cellular infiltration of limbal transplants are easily identifiable clinical signs of allograft rejection post SLET.     

Corneal allograft rejection. The role of the major histocompatibility complex

The greater success of corneal transplantation compared to other organ transplants has led to the concept that the cornea is a site of "immunological privilege." Corneal cells possess the antigens of the major histocompatibility complex responsible for allograft rejection in other tissues (i.e., HLA antigens). The avascularity of the cornea accounts for the relative protection of the donor cornea from the immunological surveillance of the recipient. As the roles and functions of the major histocompatibility complex are unravelled, the mechanisms responsible for host sensitization, lymphocyte activation and allograft rejection are becoming better understood. In particular, the HLA-DR antigen in humans is believed to play an integral part in allograft rejection. Langerhans cells in human corneal epithelium have been shown to bear this antigen. Evidence suggests that these cells or similar HLA-DR-bearing cells in the cornea play a major role in corneal allograft rejection. In light of these advances in transplantation immunobiology, new methods of suppressing and possibly preventing allograft rejection in corneal transplantation are presented.     

种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究

目的研究种植人骨髓间充质干细胞（MSCs）的猪角膜基质治疗兔角膜损伤的可能性。方法用全骨髓贴壁法分离纯化人MSCs并传代,流式细胞仪检测免疫表型及诱导成脂、成骨分化鉴定。12只新西兰白兔随机分为2组,实验组取第3代MSCs接种于去上皮的猪角膜基质上,培养4 d后移植到广泛损伤的兔角膜上,对照组单纯移植去上皮猪角膜基质。术后2、4、8周,取各实验眼行组织学检查,观察移植的MSCs及猪角膜基质的存活、转归及移植局部的反应。免疫组织化学、免疫荧光染色检测移植后角膜上皮细胞角蛋白12的表达。结果培养获得的MSCs中CD29阳性者占95.97%,CD44阳性者占96.49%,CD90阳性者占92.79%,CD105阳性者占94.66%,CD34阳性者占0.59%,CD45阳性者占0.36%,符合MCSs的免疫表型,并可以诱导成脂及成骨分化。实验组MSCs接种到去上皮猪角膜基质后贴附、生长迅速,术后植片在植床上存活良好,无排斥反应,角膜较对照组透明,新生血管少,而对照组在移植后发生排斥反应。实验组角膜免疫组织化学及免疫荧光染色均检测出CK12阳性细胞。结论种植MSCs的猪角膜基质移植到损伤兔角膜后可以存活,MSCs可以分化为角膜上皮样细胞,具有构建组织工程角膜的潜能。     

Mechanisms of corneal graft rejection: the sixth annual Thygeson Lecture, presented at the Ocular Microbiology and Immunology Group meeting, October 21, 2000

The history of corneal transplantation reaches back over 150 years. Kissam performed one of the first penetrating keratoplasties when he transplanted a pig cornea onto a human in 1838. Only two interrupted sutures were used, and the surgery was performed without anesthesia! In retrospect, no one would be surprised to learn that the porcine corneal xenograft was rejected. Thirty years later, May transplanted rabbit corneal grafts to humans, but concluded that the failures in the first 24 attempts were the result of "imperfect technique and the inability to keep the eyes properly bandaged." The first documented report of a successful penetrating keratoplasty in a human subject was performed by Zirm in 1905. As we enter the new millennium, corneal transplantation remains the oldest, most common, and, arguably, the most successful form of solid tissue transplantation. In the United States alone, approximately 36,000 corneal transplants are performed each year. The success rate for corneal transplants is in excess of 90% in uncomplicated cases, even though HLA tissue typing is not performed and systemic immunosuppressive drugs are not administered. In spite of this extraordinary success, immune rejection remains the leading cause of corneal graft failure. Many inferences about the immunobiology of corneal graft rejection have been based on clinical observations; however, confirmation of these hypotheses requires prospective studies under controlled settings. The prudent use of animal models has fostered analytic studies on the immunobiology of corneal allografts without the complicating and confounding effects of topical steroids that are typically used on most keratoplasty patients. Although animal models of penetrating keratoplasty have been in use for almost a half-century, until recently, progress in understanding the immune mechanisms of corneal graft rejection has been slow. However, the widespread use of rodent models of orthotopic corneal transplantation has shed new light on the pathogenesis of corneal graft rejection.     

角膜移植排斥反应的共焦显微镜研究

目的：探讨角膜移植术后免疫排斥反应的共焦显微镜特征,在细胞水平为该症的基础和临床研究提供活体、三维与实时的科学依据。方法：应用NIDEK公司提供的共焦显微镜,对11例（11眼）发生角膜移植免疫排斥反应的患眼角膜在不同时期行三维实时扫描成像,并用计算机作定性与定量分析。结果：角膜移植免疫排斥反应的共焦显微镜呈现如下特征：①上皮排斥线是由较小的炎症细胞与受损的较大的上皮细胞混合形成的结构;②上皮下浸润表现为体积较小、折射率高的炎症细胞的聚集,上皮下神经纤维水肿呈串珠状改变,浅基层细胞水肿;③基质排斥反应可见角膜基质细胞水肿,胞体变形,数量减少,并见炎症细胞浸润,后者于缝线周围较明显;④内皮排斥线则是由较小的明亮的炎症细胞与核固缩胞体形态异常的内皮细胞混合而成,随着内皮排斥线的发展,内皮 细胞数量减少,体积变大呈伪足状;⑤以上排斥反应均见新生血管长入植片,并与血管壁可见游动的炎症细胞。结论：应用共焦显微镜可在活体上为角膜移植术后免疫排斥反应提供早期诊断与鉴别诊断的科学依据,并在细胞水平对排斥反应的触发与转归进行活体的动态观察。     

Systemic (serum) soluble interleukin-2 receptor levels in corneal transplant recipients

Elevated soluble interleukin-2 receptor levels (sIL-2R), a measure of T cell activation, have been used as a serum marker for early rejection in solid organ transplant patients. In this preliminary study, we measured sequential sIL-2R levels of 53 otherwise immunologically normal corneal transplant recipients. Eleven of these transplants rejected; sIL-2R was significantly elevated during the acute rejection episode, compared to pre-rejection and post-rejection levels (p = 0.01). Five patients' sIL-2R rose one to nine months prior to rejection. These data indicate that sIL-2R levels may correlate with corneal graft rejection and may be predictive of impending rejection. A larger prospective study with many immunologically normal patients and careful monitoring of sIL-2R levels prior to and during the acute rejection episode will be necessary to determine the value of sIL-2R monitoring as a predictive tool for corneal graft rejection.     

The impact of corneal allograft rejection on the long-term outcome of corneal transplantation

PURPOSE: To examine the influence of corneal allograft rejection on the survival of penetrating corneal transplantation, to review the status of conventional therapies to improve graft survival, and to consider prospects for alternative approaches to reduce the impact of rejection. DESIGN: Perspective, including prospective, observational cohort study. METHODS: An examination of the literature on human corneal graft rejection and data from the Australian Corneal Graft Registry, reviewed in the context of clinical experience. RESULTS: Corneal graft outcome is not improving with era. The sequelae of inflammation, whether occurring before corneal transplantation or subsequently, exert a profound influence by predisposing the graft to rejection. Of the developments that have been instrumental in reducing rejection in vascularized organ transplantation, living-related donation is not an option for corneal transplantation. However, HLA matching may be beneficial and requires reassessment. The evidence base to support the use of systemic immunosuppressive agents in corneal transplantation is thin, and topical glucocorticosteroids remain the drugs of choice to prevent or reverse rejection episodes. Experimental approaches to local allospecific immunosuppression, including the use of antibody-based reagents and gene therapy, are being developed but may be difficult to translate from the laboratory bench to the clinic. CONCLUSIONS: Corneal allograft rejection remains a major cause of graft failure. High-level evidence to vindicate the use of a particular approach or treatment to prevent or treat corneal graft rejection is lacking. In the absence of extensive data from randomized, controlled clinical trials, corneal graft registers and extrapolation from experimental models provide some clinically useful information.     

Angiogenesis and lymphangiogenesis in corneal transplantation–A review

Corneal transplantation has been proven effective for returning the gift of sight to those affected by corneal disorders such as opacity, injury, and infections that are a leading cause of blindness. Immune privilege plays an important role in the success of corneal transplantation procedures; however, immune rejection reactions do occur, and they, in conjunction with a shortage of corneal donor tissue, continue to pose major challenges. Corneal immune privilege is important to the success of corneal transplantation and closely related to the avascular nature of the cornea. Corneal avascularity may be disrupted by the processes of angiogenesis and lymphangiogenesis, and for this reason, these phenomena have been a focus of research in recent years. Through this research, therapies addressing certain rejection reactions related to angiogenesis have been developed and implemented. Corneal donor tissue shortages also have been addressed by the development of new materials to replace the human donor cornea. These advancements, along with other improvements in the corneal transplantation procedure, have contributed to an improved success rate for corneal transplantation. We summarize recent developments and improvements in corneal transplantation, including the current understanding of angiogenesis mechanisms, the anti-angiogenic and anti-lymphangiogenic factors identified to date, and the new materials being used. Additionally, we discuss future directions for research in corneal transplantation.     

异种角膜移植前景展望

角膜移植供体缺乏是一个亟待解决的难题.研究表明,与其他系统的器官移植相比较,角膜组织的免疫赦免特性使角膜移植不易发生免疫排斥反应,猪角膜的结构和生物力学与人角膜相似性较高,经过基因改造,降低免疫排斥反应使猪角膜植片具有作为人类角膜移植片替代品的潜力.就目前所了解的异种角膜移植的排斥机制,基因工程猪的最新研究进展以及猪角膜作为异种移植供体的可行性进行综述.     

The taming of the shrew? The immunology of corneal transplantation

Corneal transplantation, first reported a century ago, is the oldest and most frequent form of solid tissue transplantation. Although keratoplasty is also considered as the most successful transplant procedure, several studies indicate that the long term survival of corneal grafts is even lower than that of transplanted parenchymatous organs. Despite the immune privilege enjoyed by the cornea and anterior segment of the eye, immunologic graft rejection is a major limitation to corneal transplantation. This review gives an update on corneal immunobiology and the mechanisms of corneal graft rejection, focusing on antigen presentation, as well as on the molecular and cellular mediators of this particular immune response.     

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。     

抗ICAM-1单克隆抗体抑制大鼠角膜移植排斥反应的实验研究

目的 :探讨应用抗细胞间粘附分子 (Intercellularadhesionmolecule 1,ICAM 1)单克隆抗体中和治疗大鼠角膜移植排斥反应的效果。方法 :应用两种主要组织相容抗原完全不同的大鼠Louis和RCS鼠 ,建立近交系大鼠穿透性角膜移植动物模型 ,分三组给药 ,其分别是抗细胞间粘附分子单克隆抗体组 (抗ICAM 1抗体组 )、生理盐水组和环胞霉素A(CsA)组。观察角膜移植排斥发生的时间及治疗效果。标本作HE染色及免疫组化染色 ,观察免疫排斥反应的程度及炎症程度。结果 :三组角膜植片平均存活时间为 :生理盐水组角膜移植排斥反应平均发生时间为术后 12 .1天 ,CsA组为18.3天 ,抗ICAM 1抗体组为 2 0 .2天。HE染色显示抗ICAM 1抗体组及CsA组的炎症反应较生理盐水组明显减轻 ,免疫组化染色显示抗ICAM 1抗体组和CsA组的ICAM 1表达较生理盐水组明显减少。结论 :抗ICAM 1单克隆抗体中和治疗可减少移植角膜ICAM 1表达及免疫炎性细胞的浸润 ,减轻角膜移植免疫排斥反应 ,延长移植片的存活时间 ,是一种有效的新型免疫抑制剂     

Trends in the distribution of donor corneal tissue and indications for corneal transplantation: the New Zealand National Eye Bank Study 2000-2009

Background: To investigate the indications for corneal transplantation and the distribution of donor corneal tissue in New Zealand. Design: Analysis of the prospective database of the New Zealand National Eye Bank. Participants: A total of 2205 corneal transplants were assessed. Methods: New Zealand National Eye Bank records were analysed for the decade 2000–2009. Main Outcome Measures: Variables analysed included donor corneal tissue distribution (including public and private sectors), indications for transplantation, donor corneal tissue recipient demographics (age and gender) and corneal transplantation type. Results: An average of 220 corneal transplants were performed each year over the 10‐year period (n = 2205). The median recipient age was 45 years (range 3 to 102 years) and 54.0% of recipients were male. In total 71.8% of transplants were performed in the public health sector. Surgeons in the Auckland metropolitan area performed 47.2% of all corneal transplants. The most common indications for corneal transplantation were: keratoconus (41.1%), repeat transplant (17.0%), aphakic/pseudophakic bullous keratopathy (13.9%), corneal dystrophy (10.7%), keratitis (7.9%) and trauma (3.7%). Overall, penetrating keratoplasty accounted for 90.7% of all corneal transplants, however, during the latter half of the study there was a progressive shift in transplantation type, with deep anterior lamellar keratoplasty and Descemet's stripping endothelial keratoplasty combined accounting for 32.3% of all transplants in the final year of the study period. Conclusions: This New Zealand National Eye Bank study provides valuable data regarding the indications for corneal transplantation, transplant recipient demographics and changes in transplantation type in New Zealand over the past decade.     

加强我国角膜移植免疫研究

通过角膜移植更换混浊或病变的角膜,是帮助角膜盲患者恢复视力的有效措施,但植片排斥反应的发生是导致角膜移植手术远期失败的主要原因,如何减少因植片排斥引起的再次致盲是角膜病研究的重点。本文分析了我国角膜移植免疫研究存在的问题：（1）没有建立标准的抗排斥治疗方案;（2）缺乏安全、有效的抗排斥反应治疗药物;（3）忽视围手术期的并发症处理;（4）角膜移植免疫基础研究薄弱。针对上述问题产生的原因,提出了相应的处理措施和建议,以增强角膜病专科医生对角膜移植免疫临床和基础研究的重视,促进我国角膜盲的防治工作取得更大的发展。     

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。     

Management of high-risk corneal grafts

Corneal transplantation is not invariably successful despite the anterior chamber of the eye being an immunologically privileged site. Inflammation erodes privilege. Other than by reducing inflammation through meticulous surgery, careful postoperative surveillance, and effective topical corticosteroids in the postoperative phase, there is little that a surgeon can do to improve the outlook for the majority of patients receiving corneal transplants. For patients at appreciable risk, HLA Class I matching may help where it is available. So too will systemic immunosuppression where it can be justified. Despite these measures, the results of corneal transplantation have not shown the improvement seen in solid organ transplantation over the last 30 years. New approaches applicable to corneal transplantation are required.     

兔自体骨髓间充质干细胞移植治疗角膜内皮损伤的研究

目的研究自体骨髓间充质干细胞（MSCs）移植到兔角膜内皮细胞（CECs）部位后的形态和功能分化。方法体外培养新西兰兔自体MSCs并用5-溴尿嘧啶脱氧核苷（Brdu）标记后接种在明胶载体膜上;利用α-氰基丙烯酸脘基酯作为组织黏合剂,将接种有兔自体MSCs的载体与机械去除了内皮细胞的自体兔角膜植片相黏合,原位缝合进行MSCs移植;对照分别移植体外培养的同种异体CECs和空白载体膜。59只新西兰白兔接受了移植实验,其中21只新西兰兔的右眼接受了自体MSCs移植,21只兔的右眼接受了CECs移植,另外17只兔的右眼移植了空白载体。术后不同时间观察兔眼角膜移植片的透明情况、角膜厚度及眼压,并利用共焦显微镜进行移植细胞的形态观察和精确计数。术后观察12周,取角膜标本分别进行组织切片、抗Brdu单克隆抗体免疫组织化学染色和电镜观察。结果MSCs和CECs在明胶载体膜上生长良好,体外培养3～5d后细胞汇合,细胞密度可达约MSCs3000个／mm^2、CECs2700个／mm^2。术后2周,57只植片均发生了水肿,并逐渐混浊。自体MSCs移植组术后8周植片水肿逐渐减轻,角膜透明度恢复;CECs移植组植片术后约4周水肿减退;而对照组角膜植片则持续水肿、混浊。术后12周时,移植细胞的平均密度为：MSCs组（2105±677）个／mm^2、CECs组（2023±330）个／mm^2。扫描电镜显示MSCs移植后逐渐分化成为类似多角形的细胞,但是细胞间的间隙较大,细胞表面有丰富的微绒毛。Brdu单克隆抗体免疫组织化学染色可将移植的细胞核着色。结论自体MSCs细胞替代CECs进行移植后,这些细胞在角膜内表面可以分化成为一种类似CECs形态和功能的细胞。（中华腠科杂志,2007,43：540-545）     

Mycophenolate mofetil versus cyclosporin A in high risk keratoplasty patients: a prospectively randomised clinical trial

BACKGROUND/AIMS: The requirement for an effective, minimally toxic immunosuppressive agent remains a major obstacle to performing high risk corneal transplantation. Although therapy with cyclosporin A (CSA) allows superior graft survival, its use is limited because of a wide range of side effects. Mycophenolate mofetil (MMF) has been shown to be a safe and effective immunosuppressive agent following renal transplantation. This prospective, randomised clinical trial was carried out to investigate the efficacy and safety of MMF in preventing corneal allograft rejection. METHODS: Recipients of corneal transplants who were at high risk for graft failure were randomly assigned to either CSA or MMF immunosuppressive therapy. CSA was given in doses to achieve whole blood trough levels of 120-150 ng/ml. MMF was given in a daily dose of 2 g. Both therapy groups additionally received oral corticosteroids (fluocortolone 1 mg/kg) which were tapered and discontinued within the first 3 postoperative weeks. Patients were monitored closely for evidence of corneal graft rejection and adverse side effects. Drug efficacy was measured, primarily, by the number of patients who experienced at least one episode of clinical graft rejection. Safety analysis focused on reported adverse side effects and laboratory measurements. RESULTS: 41 patients were enrolled in the study. There was no statistically significant difference between the two groups. 20 patients received CSA and 21 patients received MMF. Two patients in each group showed evidence of acute graft rejection which could be treated effectively by corticosteroids. All corneal grafts remained clear throughout the follow up. CONCLUSIONS: In this study it was shown that MMF is just as effective as CSA in preventing acute rejection following high risk corneal transplantation. Mycophenolate mofetil represents a promising alternative therapeutic option in patients who are at high risk for corneal graft failure.