FK506对大鼠正位穿透性角膜移植排斥反应发生过程中外周血淋巴细胞免疫功能的影响

目的研究ＦＫ５０６对角膜移植排斥反应的防治作用机制。方法纯系大鼠正位穿透性角膜移植后球周注射ＦＫ５０６，以后每周注射二次，术后不同时间尾部取血，行双标激光流式细胞分析。结果术后不同时间对照组ＣＤ４、ＣＤ８及ＣＤ４／ＣＤ８比值的差别无明显的统计学意义，但ＣＤ８＋／ＣＤ２５＋及ＣＤ８＋／Ｉａ＋Ｔ细胞逐渐增加，直至角膜移植排斥反应出现。ＦＫ５０６治疗组动物ＣＤ４细胞逐渐降低，ＣＤ８细胞增加，ＣＤ４／ＣＤ８比值降低。ＣＤ８＋／ＣＤ２５＋Ｔ细胞在各时间点无统计学差异，ＣＤ８＋／Ｉａ＋Ｔ细胞在术后第６天升高具有明显统计学差异，但升高值低于对照组。结论ＦＫ５０６球周注射后通过局部作用及吸收作用抑制淋巴细胞活化，延长角膜移植片存活时间。     

鼠角膜碱烧伤的免疫学研究

目的 探讨角膜碱烧伤后眼局部的免疫反应机制。方法在大鼠角 制作碱烧伤模型。在烧伤后的不同阶段,制备角膜、虹膜组织平片,采用标准的卵白素－生物素过化物酶复合物免疫组方法。观察眼局部Ｔ淋巴细胞亚群,巨噬细胞、树突细胞、主要组织相容性复合物－Ⅱ类怕阳性细胞的动态变化。结果 碱烧伤后早期,角膜及虹膜即有Ｔ淋巴细胞浸润,以ＣＤ３淋巴细胞为主,ＭＨＣ－Ⅱ怕阳性细胞也轻度增多。在角膜溶解穿孔阶段,Ｔ淋巴细胞浸润     

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。     

异体角膜对人外周血T细胞及其亚群CD28分子表达的体外调节

目的观察异体角膜体外对人外周血Ｔ细胞及其亚群ＣＤ２８分子表达的调节。方法应用异体角膜与外周血淋巴细胞体外共同培养模型和流式细胞分析技术（ｆｌｕｏｒｅｓｃｅｉｎａｃｔｉｖａｔｅｄｃｅｌｓｏｒｔｅｒ，ＦＡＣＳ）。结果对照组Ｔ细胞ＣＤ２８表达为２１．３％；受角膜或沸波醇酯（ｐｈｏｒｂｏｌ１２，１３—ｄｉｂｕｔｙｒａｔｅ，ＰＤＢ）激活后，Ｔ细胞ＣＤ２８表达分别为５２．４％和８１．４％；受角膜激活后，ＣＤ４和ＣＤ８Ｔ淋巴细胞ＣＤ２８表达分别为６３．１％和５２．３％。结论角膜组织体外能够刺激人外周血Ｔ细胞ＣＤ２８表达和Ｔ细胞活化，ＣＤ４Ｔ细胞比ＣＤ８Ｔ细胞活化更明显。     

蚕蚀性角膜溃疡角膜及邻近球结膜的组织病理和免疫组化研究

目的：研究蚕蚀性角膜溃疡角膜和邻近球结膜的组织病理和免疫病理变化,探讨巨噬细胞、Ｔ细胞、ＶＣＡＭ－１等与其发病的关系。方法：接受球结膜切除术及板层角膜移植术的１８进行性蚕蚀性角膜溃疡患者,取病变角膜及邻近球结膜作组织病理研究,并采用链酶生物系（ｌａｂｅｌｌｅｄ ｓｔｒｅｐｔａｖｉｄｉｎ ｂｉｏｔｉｎ,ＬＳＡＢ）方法作免疫组化研究。检测的抗体包括：ＣＤ６８、ＣＤ３、ＣＤ４、ＣＤ８、ＶＣＡＭ－１、ＣＤ     

蚕蚀性角膜溃疡角膜及邻近球结膜的免疫组织化学研究

用免疫组化技术对１４例蚕蚀性角膜溃疡的病变角膜及邻近球结膜标本的人类白细胞ＤＲ抗原（ＨＬＡ－ＤＲ）表达及Ｔ淋巴细胞亚群进行检测，发现有大量的角膜和球结膜的上皮细胞、角膜基质细胞异常地表达ＨＬＡ－ＤＲ抗原，同时，辅助性Ｔ细胞／抑制性Ｔ细胞（Ｔ＿Ｈ／Ｔ＿Ｓ）比例较正常对照组明显增高。结果提示，周边部角膜和球结膜主要组织相容性Ⅱ类抗原（ＭＨＣ－Ⅱ）的异常表达以及Ｔ＿Ｈ／Ｔ＿Ｓ升高导致过强的自身免疫反应，可能是本病发病的直接原因。     

Leflunomide抑制大鼠角膜移植排斥反应的免疫病理学研究

目的：研究角膜移植排斥反应的免疫病理学变化，阐明Ｌｅｆｌｕｎｏｍｉｄｅ的免疫抑制机理。方法：应用免疫组织化学染色方法检测环孢霉素Ａ、Ｌｅｆｌｕｎｏｍｉｄｅ及对照组角膜移植片中ＣＤ４＾＋细胞、ＣＤ８＾＋细胞、白细胞介素－２受体及主要组织相容性Ⅱ类抗原的表达。结果：排斥的角膜组织大量表达上述免疫细胞和分子，Ｌｅｆｌｕｎｏｍｉｄｅ可以对这些细胞和分子产生明显的抑制作用。结论：迟发型超敏反应和细胞毒性Ｔ细     

转化生长因子-β1对角膜上皮移植排斥反应的影响

为研究ＴＧＦ－β１对角膜上皮所致的外周淋巴细胞亚群ＣＤ４及ＣＤ８活化的影响。我们采用ＣＤ４、ＣＤ８免疫荧光标记及流式细胞仪检测技术,对ＢＡＬＢ／ｃ小鼠角膜上皮移植术后１２ｄ的外周血中ＣＤ４＾＋、ＣＤ８＾＋的表达进行分析。结果发现ＢＡＬＢ／ｃ小鼠角膜上皮移植术后１２ｄ的外周血中ＣＤ４＾＋、ＣＤ８＾＋的表达均有显著升高;术后经ＴＧＦ－β１治疗,ＣＤ４＾＋、ＣＤ８＾＋以及ＣＤ４＾＋／ＣＤ８＾＋明显受到抑     

Graves眼病患者球后组织免疫组化研究

目的：为了探讨Ｇｒａｖｅｓ眼病的确切发病机制。方法：应用单克降抗体人类白细胞ＤＲ抗原（ＨＬＡ－ＤＲ）、巨噬细胞（ＣＤ６８）、第八因子相关性抗原（Ｆ８／８６）、Ｔ细胞（ＣＤ３）、辅助性／诱导Ｔ细胞（ＣＤ４）、抑制性／细胞毒Ｔ细胞（ＣＤ８）、Ｂ细胞（ＣＤ２０）对１４例Ｇｒａｖｅｓ眼病患者球后组织主要为眼餐肌标本冰并或石蜡切用碱性磷酸酶－抗碱磷酸酶（ＡＰＡＡＰ）法进行免疫组织化学研究，５例正常人眼外肌作     

单疱病毒性角膜基质炎外周血T淋巴细胞体外活化CD71的表达

目的探讨单疱病毒性角膜基质炎的分子免疫发病机制。方法应用流式细胞技术对单疱病毒性角膜基质炎患者（７例）外周血Ｔ细胞体外活化ＣＤ７１表达进行研究，并与正常人进行对照。结果单疱病毒性角膜基质炎患者外周血ＣＤ３＋ＣＤ７１＋细胞表达、外周血培养７２ｈ组ＣＤ８＋ＣＤ７１＋细胞表达、外周血加ＰＨＡ培养７２ｈ组ＣＤ３＋ＣＤ７１＋及ＣＤ８＋ＣＤ７１＋细胞表达、外周血加ＰＤＢ培养７２ｈ组ＣＤ３＋ＣＤ７１＋细胞表达均明显低于正常人组。结论单疱病毒性角膜基质炎患者Ｔ细胞ＣＤ７１表达不足与其发病有明确关系，提示单疱病毒可能抑制了ＣＤ７１的表达。     

大鼠角膜热烧伤后眼组织平片的免疫组织化学研究

目的:探讨角膜热烧伤后角膜溃烂溶解穿孔以及眼内炎症的免疫学机制。方法:在大鼠角膜上制作热烧伤模型,在烧伤后的不同阶段,制备角膜,虹膜、脉络膜巩膜复合体以及视网膜平片,采用标准的ABC免疫组化方法,观察眼局部T淋巴细胞亚群,巨噬细胞、树突细胞,MHC Ⅱ类抗原阳性细胞的动态变化。结果:烧伤后早期,角膜及虹膜即有T淋巴细胞浸润,以CD3阳性细胞为主,MHCⅡ类抗原阳性细胞也轻度增多;当角膜溶解穿孔阶段,T淋巴细胞浸润达到高峰,在T淋巴细胞亚群中,CD4明显多于CD8阳性细胞;同时,巨噬细胞、树突细胞、MHCⅡ类抗原阳性细胞也大量出现。细胞密集分布于角膜缘,在溃疡溶解处,可见MHCⅡ类抗原阳性细胞及少至中等量CD3阳性淋巴细胞。Ⅱ类抗原阳性细胞在形态学上也发生了些变化,由初期的园形变为多形性,且大小不一。当烧伤恢复期病变稳定时,各种阳性细胞逐渐减少。结论:免疫反应参与了严重角膜烧伤的发病过程,在角膜溶解穿孔的发生中起重要作用。眼科学报 1997;13:70～74。     

Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells

PURPOSE: The presence of antigen-presenting cells (APCs) such as Langerhans cells (LCs), an epithelial form of dendritic cells (DCs), in corneal tissue is critical for generation of immune responses, including graft rejection and herpetic keratitis. The purpose of this study was to characterize the distribution and major histocompatibility complex (MHC) antigen expression of corneal LCs. METHODS: Normal and inflamed corneas were excised from BALB/c mice, and immunofluorescence staining for CD11c, CD11b, CD45, CD80 (B7.1), CD86 (B7.2), CD3, and MHC class II (Ia) was performed by confocal microscopy on wholemount corneal epithelium. RESULTS: CD11c(+) MHC class II-positive LCs were found in the limbus and corneal periphery, but not in the center of the normal cornea. These cells were CD45 positive, exhibiting bone marrow derivation, and CD3 and CD11b negative, confirming a DC lineage. Additionally, these cells were CD80 and CD86 negative, reflecting an immature phenotype. In the central and paracentral areas, however, significant numbers of CD11c(+) LCs were detected that expressed no MHC class II. It is important to note that although the density of the LCs declined from the limbus toward the center of the cornea, they were always present. In the inflamed cornea, the expression of MHC class II and costimulatory molecules CD80 and CD86 was significantly enhanced, and present in all parts of the cornea, in contrast to the normal cornea. CONCLUSIONS: The present study demonstrates for the first time the phenotype and distribution of MHC class II-negative LCs in the murine corneal epithelium. In the inflamed cornea, the LCs become activated as reflected by expression of B7 costimulatory markers. These changes in activation markers may provide additional information for devising novel immunomodulatory strategies.     

Lymphocyte infiltration and activation in iris-ciliary body and anterior chamber of mice in corneal allograft rejection

AIM: To investigate the infiltration and activation of lymphocyte in iris-ciliary body and anterior chamber after allogenic penetrating keratoplasty (PK), for further revealing the role of iris-ciliary body in corneal allograft immune rejection. METHODS: In the mice models of PK, BALB/C mice received orthotopic isografts (n =35) or C57BL/6 donor allografts (n =25). Grafts were examined daily for 3 weeks by slit-lamp microscopy and scored for opacity. The infiltration of CD4+ T lymphocyte in iris-ciliary body and anterior chamber was examined by immunohistology and the mRNA of CD80 and CD86 in both cornea graft and iris-ciliary body by RT-PCR was analyzed in allograft recipient at days 3, 6, 10 and the day when graft rejection occurred. Isograft recipients were examined as control at the corresponding time points. Transmission electron microscope was used to study the ultrastructure, especially cell infiltration, of iris-cilary body and corneal graft at day 3, 7 and the day when rejection occurred after allogenic PK. RESULTS: Rejection was observed in all the allograft recipients followed more than 10 days, at a median time of 15 days (range 12-18 days), but not in any of isografts. CD4+ T cells were first detected at day 6 after transplantation in limbus and Ciliary body, and then in the stroma of recipient, iris, anterior chamber and corneal allograft with an increased number until graft rejection occurred. CD80 and CD86 mRNA were detected under RT-PCR examination in both graft and iris-ciliary body of allograft recipient, but not in any of isograft recipient. Three days after operation, lymphocytes and monocytes macrophages were visible in iris blood vessels and the anterior chamber, and vascular endothelial cell proliferation and activation were significant under transmission electron microscopy examination. At day 7, corneal endothelial cells became thinner. Lymphocytes and mononuclear macrophages were found with great number in the anterior chamber and adhered to the corneal endothelium. Blood vessels in iris increased and were filled with lymphocytes. And lymphocytes were detected to migrate through endothelial cell gap out of vessels. When allograft rejection occurred, macrophages attached to endothelial cells with large number of lymphocytes and macrophages infiltrating in iris. CONCLUSION: Lymphocyte infiltration and activation occurred in iris-ciliary body after allogenic PK, and the lymphocytes could migrate from iris blood vessel to the anterior chamber, which might play an important role in corneal allograft immune rejection.     

Macrophages and MHC class II positive dendritiform cells in the iris and choroid of the pig

PURPOSE: Macrophages and dendritic cells (DC) are considered to play an important role in the initiation and propagation of uveitis. Little is known about these cells in the normal pig uveal tract, despite the fact that the pig eye shares many similarities with the human eye and is considered as a suitable species to investigate the pathogenesis of human ocular disease. The aim of this study was to investigate the presence of immunocompetent cells in the uveal tract of the normal pig. METHODS: Iris and choroid wholemounts and cryostat sections were obtained from normal pig eyes. Single and double immunohistochemistry was performed by using anti-porcine leukocyte (CD45), anti-porcine macrophage (CD163, CD14), anti-porcine MHC class II (MCA1335), anti-human MHC class II (MCA379G), anti-porcine B lymphocyte (IgM), anti-porcine T lymphocyte (CD6) and anti-porcine granulocyte (MCA1219) monoclonal antibodies. RESULTS: A rich network of dendritiform CD163 positive tissue macrophages was observed in normal pig iris and choroid wholemounts (368 + 84/mm(2), 402 + 97/mm(2), respectively). Approximately 50% of the CD163 positive tissue macrophages in the iris coexpressed MHC class II. Double immunostaining revealed that a small population of the MHC class II positive cells, did not express macrophage markers, and probably represent classical DCs. B lymphocytes and granulocytes were not detected in iris and choroid wholemounts. An occasional T cell could be observed per high power field in iris wholemounts but not in the choroid. CONCLUSIONS: The present study reveals that the normal pig uveal tract contains a rich network of tissue macrophages and MHC class II positive dendritiform cells. At least three populations could be distinguished: MHC class II positive and negative tissue macrophages and MHC class II+ dendritiform cells lacking tissue macrophage markers.     

CD86在排斥反应和前房相关免疫偏离过程中的作用

目的检测大鼠角膜共刺激分子CD86（B7—2）的原位表达,探讨CD86分子与角膜移植排斥反应和前房相关免疫偏离（ACAID）过程的关系。方法制作穿透角膜移植排斥反应和同一供体前房内注射脾细胞诱导ACAID的大鼠模型;角膜移植组进行排斥反应指数（RI）评分;ACAID组进行迟发型超敏反应（DTH）评价;采用免疫组织化学的方法检测CD86在角膜中的原位表达（以脾脏的表达为阳性对照）。结果角膜移植组植片均出现不同程度的新生血管、角膜水肿、混浊、增厚;ACAID组角膜透明,房水清,DTH评价术后2周及4周诱导成功率100％;免疫组织化学检测CD86在正常大鼠角膜组织全层无阳性表达,在移植后出现急性排斥反应的大鼠角膜上皮层中有大量阳性细胞表达,在ACAID诱导成功的大鼠角膜中未见阳性细胞表达。结论共刺激分子CD86参与移植后的急性排斥反应,但可能不参与免疫赦免过程。     

Development of organised conjunctival leucocyte aggregates after corneal transplantation in rats

AIM: To investigate the development of lymphoid aggregates in the conjunctiva after corneal transplantation in rats. METHODS: LEW or PVG strain corneas were transplanted orthotopically to PVG rats. Cornea and conjunctiva were examined clinically for up to 42 days. Eyes were removed with attached conjunctiva on days 10 and 15 after transplantation (before and during rejection), together with normal eyes, fixed, paraffin embedded, and examined immunohistochemically. RESULTS: Clinically, the temporal half of the upper palpebral conjunctiva of recipients of 10/19 allografts and 1/10 isografts developed pronounced swelling, correlating with inflammation and rejection. Histologically, the swelling comprised leucocytic aggregates with an altered overlying epithelium. Aggregates contained granulocytes, macrophages, and cells expressing major histocompatibility complex (MHC) class II, CD4, and CD8, all more numerous in allograft associated conjunctiva. Class II+ cells were more abundant at the surface, whereas macrophages and T cells were more numerous in the deeper stroma. There were few B cells. There was greater CD54 expression by vascular endothelium in allograft associated aggregates. Cells expressing TNFalpha and IFNgamma but not IL1beta were present in stromal and superficial areas. CONCLUSIONS: Corneal transplantation in rats induces the development of organised conjunctival leucocytic aggregates in a fixed location that are significantly more pronounced in recipients of allografts compared with isografts and show characteristics of a Th1 type immune response. These aggregates have characteristics of conjunctiva associated lymphoid tissue and may be sites of presentation of graft antigens and lymphocyte proliferation at the ocular surface.     

Differences in leukocyte phenotype and interferon-gamma expression in stroma and endothelium during corneal graft rejection

Critical to the success of human corneal transplants is prevention of corneal endothelial rejection, yet little is known about the endothelial infiltrate. To examine the endothelium, a method for removal and processing this layer as a flat sheet was used and the infiltrate was compared with stroma and epithelium. LEW or PVG strain rat corneas were transplanted to PVG strain recipients. Clinical changes after transplantation were monitored by slit lamp and animals sacrificed at a range of time points during rejection. Clinically defined rejection, accompanied by an epithelial rejection line and endothelial cell infiltration, occurred between days 10 and 15. There was some infiltration of leukocytes in the stroma of isografts at these time points, but significantly more in allografts (p<0.003 for all subsets). There was no infiltration of isograft endothelium at any time and no infiltration of allograft endothelium on day 10. On day 15, there were similar numbers of all major subsets except B cells in the stroma, while on the endothelium macrophages, MHC class II(+) cells and CD8(+) cells predominated (p<0.001 CD4(+) vs CD8(+) cells). T cells and NK cells predominated in the epithelial rejection line. While TNF-alpha and IFN-gamma-producing cells were numerous in stroma and epithelium, no IFN-gamma-producing cells were found on endothelium. Distinct differences in infiltrative profile within layers of the cornea suggest that the mechanisms of rejection may also differ. The restricted endothelial cell profile and lack of IFN-gamma suggests that the anti-endothelial response may be modulated by the anterior chamber environment.     

IL-10基因修饰的未成熟树突状细胞在大鼠角膜移植排斥反应中的作用

目的：通过建立大鼠角膜穿透性移植模型,探讨IL－10基因修饰的未成熟树突状细胞在大鼠角膜移植排斥反应中的作用及其作用机制。方法：建立大鼠同种异体穿透性角膜移植模型,将受体SD大鼠随机分为：阳性对照组、GFP－DC组、8－DC组及IL－10－GFP－DC组,分别于角膜移植术前3 d尾静脉注射等量的PBS、供体Wistar 大鼠骨髓源8－DC （培养8d的DC）、转染48h的GFP－DC及IL－10－GFP－DC。术后每天在裂隙灯下观察角膜植片情况,记录排斥反应指数及角膜植片存活时间,在移植术后第14 d行各组角膜组织的病理学检查及免疫组织化学检查。结果：IL－10－GFP－DC组角膜植片存活时间较GFP－DC组、8－DC组比较显著延长（ P＜0．01）。术后第14 d时IL－10－GFP－DC组角膜植片的混浊、水肿、新生血管及排斥指数均显著降低（P＜0．01）。病理组织学检查结果显示各实验组角膜植片的炎症反应较阳性对照组轻,植片中央未见明显新生血管。免疫组织化学结果显示：IL－10－GFP－DC组的CD4＋、CD8＋、CD25＋、IL－2＋、NK＋及NF－κB＋阳性细胞数量较阳性对照组、GFP－DC组、8－DC组减少,差异均具有显著统计学意义（P＜0．01）。结论：经过供体来源未成熟树突状细胞预处理的受体,角膜植片的存活时间显著延长,成功诱导角膜移植免疫耐受。 CD4＋、CD8＋、CD25＋、IL－2＋、NK＋及NF－κB＋阳性细胞参与了同种异体角膜移植排斥反应的调控,IL－10－GFP－DC可降低CD4＋、CD8＋、CD25＋、IL－2＋、NK＋及NF－κB＋阳性细胞的浸润,抑制角膜移植排斥反应的发生。     

Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival

PURPOSE: To examine the role of the CD28-CD80-CD86 pathway of T-lymphocyte costimulation in corneal allograft rejection and the effect of blockade of that pathway on graft survival. METHODS: Kinetics of CD80 and CD86 expression in the cornea and draining lymph nodes were examined by RT-PCR and immunohistochemistry in untreated allograft recipients in a high-responder rat model. The effect of blockade of CD28-mediated costimulation was first examined by ex vivo incubation of excised Brown Norway rat donor cornea with the inhibitory protein CTLA4-Ig or an adenovirus vector (AdCTLA) expressing CTLA4-Ig, before grafting into Lewis rat recipients. A second group of graft recipients received systemic posttransplantation treatment with either CTLA4-Ig or AdCTLA. RESULTS: Expression of CD80 mRNA was increased in both donor and recipient cornea 16 hours after transplantation, whereas CD86 was detected constitutively, with no significant early increase. Immunohistochemistry on day 5 after transplantation demonstrated major histocompatibility complex (MHC) class II expression, no CD80, and only a trace of CD86 in corneal allografts. In lymph nodes strong MHC class II, weak CD80, and moderate CD86 expression was noted. Both donor cornea and recipient treatment with CTLA4-Ig resulted in prolonged allograft survival. AdCTLA was found to induce sustained secretion of bioactive CTLA4-Ig from corneas infected ex vivo. Survival of corneal allografts incubated with AdCTLA was marginally prolonged, and systemic treatment with AdCTLA significantly prolonged survival. CONCLUSIONS: Protein- or gene-based administration of CTLA4-Ig prolongs allograft survival by treatment of either the recipient or the donor tissue ex vivo before grafting.