核因子-κB反义核苷酸对大鼠移植角膜存活时间的影响

目的研究核因子κB(NFκB)反义寡核苷酸对大鼠角膜移植排斥反应的影响。方法使用大鼠穿透角膜移植排斥反应动物模型,观察脂质体包裹的NFκB反义寡核苷酸对排斥反应指数(RI)及植片存活时间的影响,并测定受体鼠脾细胞混合淋巴细胞培养(MLC)和细胞毒T细胞(CTL)活性。结果反义寡核苷酸组角膜植片存活时间较对照组明显延长(P<0.01);MLC显示该组大鼠脾细胞对供体抗原刺激的增殖反应受到抑制(P<0.05),CTL活性明显减弱(P<0.05)。结论脂质体包裹的NFκB反义寡核苷酸能有效抑制受体鼠对移植物的排斥反应,使角膜植片存活时间延长。     

IL-1受体拮抗剂防治大鼠角膜移植排斥反应的实验研究

目的　从免疫病理学角度研究白细胞介素 1受体拮抗剂 (IL 1ra)防治角膜移植排斥反应。方法　将受体大鼠随机分为 4组 ,I、II、III组自手术之日起每日结膜下分别注射IL 1ra 5 0、10 0、2 0 0 μg。IV组 (对照组 )结膜下注射等体积生理盐水。观察各实验组IL 1ra和对照组的植片存活时间 ,用免疫组织化学染色方法检测IL 1ra及对照组角膜移植片中CD4 细胞、CD8 细胞的表达。结果　I、II、III组植片存活时间分别为 (11.0 0±1 4 6 )、(12 2 3± 1.13)、(13.5 8± 1.0 7)d ,IV组植片存活时间为 (7.85± 1.5 8)d。各用药组与对照组比较存活时间显著延长 (P <0 .0 1)。排斥的角膜植片大量表达CD4 、CD8 细胞 ,IL 1ra明显抑制这 2种细胞的表达。结论　结膜下注射IL 1ra可抑制角膜移植术后免疫排斥反应 ,减轻免疫性炎症反应 ,延长植片存活时间。IL 1ra剂量越大 ,效果越好。     

白介素-1受体拮抗剂滴眼液治疗大鼠高危角膜移植免疫排斥反应

目的　观察白介素 1受体拮抗剂 (IL 1ra)滴眼液对高危角膜移植排斥反应的治疗作用。方法　SD大鼠为供体 ,Wistar大鼠为受体。缝线法诱导角膜新生血管后行角膜移植。对照组用生理盐水点眼 ,实验组分别用IL 1ra 1、3、5mg/mL点眼。同时设IL 1ra 1mg/mL结膜下注射组。观察植片的存活情况并检测植片中CD1阳性细胞。结果　与对照组相比 ,IL 1ra 3、5mg/mL组和结膜下注射组均可明显延长植片的存活时间 (P <0 0 5 )并减少植片中CD1阳性细胞的浸润。结论　IL 1ra滴眼液可以用于治疗高危角膜移植免疫排斥反应     

联合自-异体穿透性角膜移植减少排斥反应研究

目的：观察联合自－异体穿透性角膜移植术后植片排斥反应的发生率和疗效。方法：对25例角膜病灶靠近边缘的患者,采用周边部新月形自体植片及中央部异体植片作穿透性移植术,另以植床靠边的旁中央穿透性移植术28例为对照组,比较其术后排斥反应的发生率和角膜散光。结果：两组植片的上皮排斥反应发生率分别为8．0％和32．1％,实质层和内皮排斥反应为28．0％及64．3％,角膜散光为3．82D和6．82D。结论：联合自－异体穿透性角膜移植术可降低术后植片排斥反应发生率及减少术后角膜散光。     

Leflunomide抑制大鼠角膜移植免疫排斥反应的研究

目的研究Leflunomide对大鼠角膜移植排斥反应的防治作用。方法建立大鼠穿透性角膜移植排斥反应的动物模型,观察Leflunomide对大鼠角膜植片存活和排斥反应指数（RI）的影响,并与阴性对照组和CsA治疗组相比较。结果阴性对照组角膜植片存活时间为12.375d±1.768d,而CsA组为17.375d±1.408d,Leflunomide组为18.250d±1.356d,均比阴性对照组显著延长（P<0.01）。结论Leflunomide能抑制大鼠穿透性角膜移植免疫排斥反应,显著延长角膜植片的存活时间。     

CD4＋ NK T细胞在超抗原SEB诱导大鼠高危角膜移植免疫耐受中的作用

目的研究超抗原金黄色葡萄球菌肠毒素B亚单位（SEB）治疗高危角膜移植免疫排斥反应与CD4＋自然杀伤（NK）T细胞的相关性。方法Fisher 344大鼠21只作为供体,Lewis大鼠42只作为受体。缝线法诱导角膜新生血管,建立高危角膜移植动物模型。将受体大鼠随机分为3组,每组14只。SEB组在角膜移植术前腹腔内注射75μg/kgSEB 0.2 mL,每4日1次,共3次;SEB联合地塞米松组除按上述方法注射SEB外,在角膜移植术后每日结膜下注射5 g/L地塞米松0.1 mL,每日1次,共3次;生理盐水组按SEB组的方法腹腔内注射等体积生理盐水。术后观察植片混浊、水肿和新生血管指标并进行评分。术后第10天,每组取3只受体大鼠进行组织病理学和免疫学检测。结果SEB组植片平均存活时间为（12.50±1.41）d,较SEB联合地塞米松组（10.38±3.07）d和生理盐水组（7.30±0.67）d明显延长（P〈0.05）。SEB组淋巴细胞的增生能力明显降低,脾脏和颌下淋巴结中的CD4＋NK T细胞百分数明显升高。SEB联合地塞米松组脾脏和颌下淋巴结中的CD4＋和CD8＋细胞百分数均明显降低。SEB组房水和血清中IL-2质量浓度均明显降低,而IL-10质量浓度均明显升高。结论超抗原SEB能够明显延长大鼠高危角膜移植手术植片的存活时间,其作用机制与上调了CD4＋NK T细胞有关,CD4＋NK T细胞对于免疫耐受的形成有重要作用。     

α-GalCer活化的自然杀伤T细胞对高危角膜移植后排斥反应的防治作用

背景 角膜移植排斥反应是导致角膜移植手术失败的主要原因,抑制角膜移植排斥反应的各种药物均有不良反应.研究发现自然杀伤T(NKT)细胞可致器官移植患者免疫耐受,但目前有关NKT细胞用于治疗高危角膜移植免疫反应的研究较少. 目的 探讨体外α-GalCer活化的NKT细胞在防治大鼠高危角膜移植免疫排斥反应中的作用. 方法 无菌条件下取Lewis大鼠脾脏淋巴细胞,加入质量浓度为100 mg/L的α-GalCer,在RPMI 1640培养基培养1周后,流式细胞仪分选出NKT细胞(密度为5×106个/ml).取10只Fisher 344大鼠为供体,20只Lewis大鼠为受体,受体角膜移植前1周角膜缝线诱导角膜新生血管(CNV).将受体大鼠按照随机数字表法随机分为NKT细胞组和生理盐水组,每组各10只.Lewis大鼠行穿透角膜移植.NKT细胞组在手术结束时球后注射0.1 ml NKT细胞液,生理盐水组注射相同体积的生理盐水.术后裂隙灯下观察记录角膜植片的反应情况并按照Holland的标准进行评分.术后第14天,两组各获取3只大鼠角膜植片行组织病理学检测,采用免疫组织化学法检测角膜植片中CD4+和CD8+T淋巴细胞的浸润情况.结果 生理盐水组角膜植片平均存活时间为(7.90±1.37)d,NKT细胞组为(14.70±1.49)d,差异有统计学意义(t=10.61,P=0.00).术后2周,生理盐水组角膜植片重度混浊水肿,大量炎性细胞浸润,新生血管长入植片,而NKT细胞组角膜植片仅轻度混浊、水肿,炎性细胞明显少于生理盐水组.免疫组织化学检测可见,生理盐水组角膜植片中大量CD4+和CD8+T淋巴细胞浸润,NKT细胞组中CD4+和CD8+T淋巴细胞明显减少.流式细胞仪检查结果表明,NKT细胞组大鼠脾脏中NKT细胞百分数为(5.67±0.25)％,明显高于生理盐水组的(1.21±0.19)％,差异有统计学意义(t=8.43,P=0.00).结论 α-GalCer活化的NKT细胞球后注射可以明显延长大鼠高危角膜移植植片的存活时间,为角膜移植排斥反应的防治提供了新的手段.     

来氟米特活性代谢物A771726对大鼠角膜移植排斥反应的影响

目的探讨来氟米特活性代谢物A771726对大鼠角膜移植排斥反应的抑制作用。方法建立SD-Wistar大鼠同种异体穿透角膜移植模型,随机分组。A组为空白对照组;B、C、D组分别为0．5％、1．0％及2．0％A771726滴眼液组;E组为Wistar大鼠自体移植对照组。术后比较各组角膜植片排斥指数（RI）及植片存活时间,并对植片进行组织学及免疫组织化学染色观察。结果A组角膜植片存活时间为（9．38±2．26）d,B组（10．13±2．41）d,C组（17．57±1．72）d,D组（17．50±2．14）d,E组（〉28．00）d。A组、B组分别与C组、D组植片存活时间差异有统计学意义（P〈0．01）。移植术后10d,A组、B组角膜植片发生排斥反应,植片高表达IFN-γ及ICAM-1;术后20d,C组、D组植片发生排斥反应,植片IFN-γ及ICAM-1表达增高。结论局部应用A771726滴眼液能有效抑制角膜移植排斥反应。     

共刺激分子Tim-1在角膜移植排斥反应中的作用

背景 角膜移植是目前临床上治疗角膜盲可靠且有效的复明手段,角膜移植术后的免疫排斥反应是角膜移植失败的主要原因. 目的 研究共刺激分子Tim-1在大鼠角膜组织中的表达及其在角膜移植排斥反应发生和发展过程中的作用.方法 40只清洁级成年雌性Wistar大鼠随机分为正常对照组、自体角膜移植组和同种异体角膜移植组.正常对照组大鼠未行任何手术,自体角膜移植组分别以Wistar大鼠作为供体和受体施行穿透角膜移植术,而同种异体角膜移植组以SD大鼠角膜作为供体,Wistar大鼠作为受体行穿透角膜移植术,各组供体角膜植片均为3.5mm,植床直径为3.0 mm.分别于术后7d、14d在裂隙灯显微镜下观察术眼角膜炎症反应情况,按照Larkin的标准进行排斥反应评分,计算排斥反应指数（RI）,观察各组角膜植片的平均存活时间和植片存活率.术后7d、14d,3个组各取3只大鼠眼球制备角膜组织切片,分别行组织病理学检查和免疫组织化学检测,观察角膜组织的炎症反应和共刺激分子Tim-1蛋白在角膜组织中的表达;此外分别于术后7d、14d各组取5只大鼠角膜制备组织匀浆,采用实时荧光定量PCR法检测共刺激分子Tim-1 mRNA在大鼠角膜植片中表达量（吸光度,A）的变化.结果 术后7d,自体角膜移植组和同种异体角膜移植组大鼠角膜植片均出现轻度水肿;术后14 d,自体角膜移植组角膜植片水肿消失,植片透明,而同种异体角膜移植组大鼠角膜植片水肿增厚,呈灰白色混浊,可见新生血管形成.自体角膜移植组植片的存活率为100％,同种异体角膜移植组植片存活率为0,平均生存时间为（9.8±1.2）d.组织病理学检查表明,术后7d自体角膜移植组植片基质层可见炎性细胞浸润,但术后14d炎性细胞明显减少;同种异体角膜移植组术后7d植片水肿,基质层可见炎性细胞浸润,术后14 d阳性细胞大量增加,可见新生血管.免疫?     

CTLA4-Ig抑制兔角膜移植排斥反应的作用

目的:建立兔角膜移植高危及非高危模型,通过阻断CD28,探讨CTLA4-Ig对高危角膜移植排斥反应的影响。方法:实验分为新生血管化模型组及非新生血管化组,每组随机分成:空白对照组(空白保存液)、实验组(浸入含有CTLA4-Ig10mg/L保存液4℃孵育18h),每组10只兔。观察术后受体植片角膜混浊情况和植片病理改变,原位杂交方法检测角膜植片TNF mRNA的表达情况,比较植片生存时间。结果:非新生血管化角膜移植组:对照组和实验各组的植片排斥时间或平均植片存活时间上无统计学意义,超过半数植片(16/30,53%)存活时间超过100d。新生血管化角膜移植组:实验组生存时间较长69±34d,对照组26±4d,原位杂交检测移植术后4wk或排斥反应发生时对照组角膜植片上皮下基质层浸润细胞有明显的TNF mRNA的表达,实验组未见TNF mRNA表达。结论:在兔角膜移植排斥反应中应用CTLA4-Ig阻断CD28,可以明显抑制兔高危角膜移植排斥反应,提高移植的存活率。     

贝伐单抗抑制角膜移植术后新生血管及免疫排斥反应的作用

目的 探讨贝伐单抗对同种异体角膜移植术后免疫排斥反应的影响,评价其对角膜植片存活的疗效及作用机制。设计 实验研究。研究对象 近交系F344大鼠15只为供体,近交系Lewis大鼠30只为受体。方法 受体大鼠右眼行穿透性角膜移植术后随机数字法分为3组：对照组（A组）、贝伐单抗组（B组）、地塞米松组（C组）,每组10只。B、C组分别于术后0、3、6、9天结膜下注射40 μl贝伐单抗注射液及20 μl地塞米松磷酸钠注射液;A组不做处理。术后裂隙灯下观察14天,记录排斥指数（RI,为植片水肿、混浊、新生血管长入评分合计）和新生血管侵袭面积（IA）。术后14天,采用免疫荧光法检测角膜植片中CD4+、CD8+免疫细胞的表达。主要指标 角膜新生血管面积,免疫排斥时间,免疫细胞的数量。结果 术后7天IA分别为：A组（22.50±3.67）mm2,B组（14.21±2.79）mm2,C组（15.38±0.84）mm2;A组明显多于B组及C组（P=0.00和0.01）,B组与C组无显著差异（P=0.059）。术后14天IA分别为：A组（27.96±0.50）mm2,B组（18.76±2.73）mm2,C组（23.74±2.14）mm2。A组及C组多于B组（P=0.000）。免疫排斥时间A组8天,B组11天,C组13天。B组及C组角膜植片存活时间均长于A组（P均=0.000）。免疫细胞： CD4+阳性细胞数：A组（13.2±2.94）个,B组（6.14±1.07）个,C组（3.5±1.78）个;CD8+阳性细胞数：A组（14.4±2.44）个,B组（4.5±1.51）个,C组（3.38±1.68）个。术后14天,B组、C组角膜植片中CD4+、CD8+细胞亦明显少于A组（P均=0.000）。结论 结膜下注射贝伐单抗可有效抑制大鼠同种异体穿透性角膜移植术后新生血管的生长,并可能通过此作用延缓移植排斥反应,延长角膜植片存活时间。但贝伐单抗抗移植排斥反应疗效略逊于地塞米松。（眼科,2017, 26： 101-105）     

FTY720 prolongs clear corneal allograft survival with a differential effect on different lymphocyte populations

BACKGROUND: FTY720 is a potent immunomodulator with unique effects on lymphocyte homing and has recently proved to be safe and effective in renal transplantation in man. The authors investigated the potency of FTY720 in inhibiting allograft rejection in the rat model of orthotopic allogeneic penetrating keratoplasty. METHODS: Penetrating keratoplasties were performed using Fisher rats as donors and Lewis rats as recipients or donors: group 1 (n = 10), allogeneic control; group 2 (n = 10), Lewis/Lewis syngeneic control; group 3 (n = 9), mycophenolate mofetile (MMF) 40 mg/kg; group 4 (n = 10), FTY720 1.2 mg/kg; group 5 (n = 8), FTY720 0.3 mg/kg. Four animals from each group were sacrificed for immunohistological evaluation on day 14. Medication in the therapy groups was given for 18 days. RESULTS: The mean (SD) rejection free graft survival time was 11.3 (0.8) days for the allogeneic control (group 1), 24.6 (2.5) days for group 3 (MMF), 44.5 (5.7) days for group 4 (FTY720 1.2 mg/kg), and 35.3 (5.7) days for group 5 (FTY720 0.3 mg/kg) (p<0.05). The allogeneic control showed a dense infiltration with CD4+, CD8+, CD161+ (NK-cells), CD25+ (IL2 receptor), and macrophages. In the therapy groups the density of infiltrating CD4+, CD8+, CD161+ (NK-cells), and CD25+ (IL2 receptor) cells was notably reduced compared with the allogeneic control (p<0.05). In group 5 however, the reduction of infiltration by CD4+ cells was higher than the reduction of infiltration by CD8+ (p<0.05) and CD161+ (NK) cells. DISCUSSION: Oral immunosuppression with FTY720 significantly prolongs corneal allograft survival in this transplant model. The results suggest that FTY720 has a different effect on certain lymphocyte populations. CD4+ cells seem to be more affected than CD8+ cells and NK-cells.     

Non-specific tolerance induced by staphylococcal enterotoxin B in treating high risk corneal transplantation in rats

AIMS: To explore the role of staphylococcal enterotoxin B (SEB) in treating high risk corneal keratoplasty in rats. METHODS: Rat corneal high risk transplantation rejection models were set up using Fisher 344 and Lewis rats. The experimental rats were injected intraperitoneally with 0.2 ml SEB at different concentrations before keratoplasty. The rejection indexes of the allograft were recorded and the lymphocyte infiltration in the allograft and the percentage of the lymphocyte subpopulation in the lymphatic organs were also examined. Lymphocyte proliferation ability and the concentration of IL-2 and IL-10 in the serum were also evaluated. RESULTS: Compared with the control group, SEB prolonged the survival time of the allograft significantly from 7 to 12 days. It could also reduce CD4(+) and CD8(+) lymphocyte infiltration in the allograft and minimise the percentage of CD4(+) and CD8(+) lymphocytes in the lymphatic organs. The lymphocyte proliferation ability was also weakened. However, the percentage of CD4(+) NK T lymphocytes in the lymphatic organs was raised. The serum concentration of IL-10 was higher but IL-2 was lower in the SEB treated groups. CONCLUSIONS: SEB prolonged the survival time of the allograft in high risk rat corneal allo-transplantation, which may be caused by T cell deletion and acquisition of non-specific tolerance.     

Rat corneal allograft survival prolonged by the superantigen staphylococcal enterotoxin B

PURPOSE: The purpose of this study was to determine the optimal conditions for prolonging corneal allograft survival by inducing anergy with the superantigen staphylococcal enterotoxin B (SEB). METHODS: A rat model of penetrating keratoplasty, whereby Fisher344 donor corneas are implanted into Lewis recipients, was used to evaluate the effects of SEB on inhibiting immune-mediated allograft rejection. To induce anergy, SEB was injected into the peribulbar space of Lewis rats. Furthermore, histopathology and immunofluorescent staining were used to examine the levels of infiltrating CD4(+) and CD8(+) T lymphocytes and NK1.1(+) lymphocytes. RESULTS: By administering SEB, at doses of 90 or 120 micro g/kg 7 days before and after keratoplasty, we suppressed the episode of corneal graft rejection for a median of 12 and 30 days, respectively. In contrast, rejection was observed when 30 or 60 micro g/kg of SEB was administered. After SEB injections, lymphocyte infiltration into the corneal grafts was reduced, and the expression of NK1.1(+) lymphocytes was enhanced, suggesting that anergy may be occurring. Also, there were no differences in the number of infiltrating CD4(+) and CD8(+) T lymphocytes cells between the control group and groups injected with 30 and 120 micro g/kg SEB on postoperative days 10 and 30. CONCLUSIONS: Inducing anergy with the superantigen SEB prolonged corneal graft survival in a rat model of penetrating keratoplasty. Therefore, these results support the possibility of prolonging corneal allograft survival in a clinical setting by preventing immune-mediated rejection through the administration of the superantigen SEB.     

小鼠角膜移植排斥反应中植片内细胞因子表达水平的变化

背景研究表明细胞因子在角膜移植免疫调节中起重要的作用,但关于角膜移植术后角膜植片内细胞因子表达的变化报道较少。目的探讨角膜移植术后不同时间点细胞因子的表达变化。方法采用雌性6～8周龄BALB／c小鼠及C57BL／6小鼠分别建立小鼠自体角膜移植和同种异体角膜移植模型,每组各10只,自体角膜移植术组供体和受体同为BALB／c小鼠,同种异体角膜移植术组供体为C57BL／6小鼠,受体为BALB／c小鼠。于术后1d开始临床观察角膜植片并对炎症程度进行评分,当植片评分总和≥5分或植片混浊≥2分时为发生植片排斥反应。将BALB／c小鼠随机分为正常对照组3只和同种异体角膜移植术组15只,后者分别于术后6h及1、3、7、14d对角膜植片行逆转录PCR（RT—PCR）检测植片中细胞因子白细胞介素4（IL-4）、γ-干扰素（IFN-γ）、IL-10、肿瘤坏死因子α（TNF—α）表达的变化。结果在60d的观察期中,自体角膜移植术后小鼠角膜植片混浊评分均〈2分,植片的炎症评分之和均〈5分,植片存活率为100％,但同种异体角膜移植术后角膜植片水肿、混浊,伴有角膜新生血管形成,至术后24d角膜植片排斥反应评分之和均≥5分,植片混浊评分≥2分,全部发生排斥反应,角膜植片平均存活时间为（17．80±4．66）d。RT—PCR检测结果表明,正常角膜中IL-4和IFN-γ呈阳性表达,IL-10和TNF-α表达阴性。同种异体角膜移植术后6h可检测到IL-4呈阳性表达,IFN-γ和IL-10表达呈强阳性,而未检测到TNF—α的表达。术后1～3d角膜植片中IL-4呈强阳性表达,IFN-γ阳性表达,TNF—α表达增强,IL-10表达逐渐消失。角膜移植术后7d,可见IL-4表达阴性,而IFN-γ、IL-10和TNF—α仍呈强阳性表达。至术后14d,角膜植片中IL-4、IFN-γ和TNF—α．均未见表达,仅检测到IL-10呈阳性表达。结论TNF-α是角膜移植术后局部参与调控的主要因子。     

复方倍他米松对异种角膜板层移植术后免疫排斥反应的影响

目的：观察结膜下注射复方倍他米松对鸵鸟-兔眼板层角膜移植术后免疫排斥反应的缓释治疗作用。

方法：16只6周龄健康新西兰白兔角膜(单眼)行板层角膜移植术,植片应用鸵鸟脱细胞的角膜基质,术后分成两组,实验组术毕及术后结膜下注射复方倍他米松注射液(每隔7d一次),对照组术毕及术后结膜下注射地塞米松磷酸钠注射液(每隔7d一次)。分别于术后1、2wk, 1、2mo取兔角膜组织做石蜡包埋切片,进行HE染色观察组织特点,同时进行间接免疫荧光法检测CD4⁺、CD8⁺ T淋巴细胞的变化。

结果：术后2mo,实验组角膜植片在位,并保持透明,新生血管极少,组织切片HE染色和间接免疫荧光染色结果显示,仅见少许中性粒细胞浸润,未见CD4⁺、CD8⁺ T淋巴细胞; 对照组炎症反应明显,角膜植片混浊,组织切片HE染色和间接免疫荧光染色结果显示,炎性反应以中性粒细胞浸润为主,可见CD4⁺、CD8⁺ T淋巴细胞。

结论：复方倍他米松作为长效缓释剂可有效抑制鸵鸟-兔板层角膜移植术后免疫排斥反应,延长植片的存活时间。     

Sustained intraocular rapamycin delivery effectively prevents high-risk corneal allograft rejection and neovascularization in rabbits

PURPOSE: To evaluate the immunosuppressive and antiangiogenic activities of an intraocular rapamycin (RAPA) drug delivery system (DDS) in a rabbit model of high-risk penetrating keratoplasty. METHODS: Forty New Zealand White rabbits with corneal neovascularization underwent allograft cornea transplantation and were randomly divided into four groups: a control group, a glycolide-co-lactide-co-caprolactone copolymer (PGLC)-implanted group, a RAPA eye drop group, and a RAPA-PGLC DDS-implanted group. Graft survival, corneal neovascularization, and RAPA concentration in the aqueous humor were monitored for 90 days. Corneal grafts were also examined by in situ hybridization and immunohistochemistry for proinflammatory gene expression. RESULTS: In the control and PGLC groups, graft rejection occurred within 3 weeks of keratoplasty. In the RAPA eye drop and RAPA-PGLC groups, corneal rejection was significantly delayed, and neovascularization was markedly inhibited. Median graft survival times were 36 and >90 days in the eye drop and RAPA-PGLC groups, respectively. Mean RAPA concentrations in the aqueous humor were 10.7 ng/mL, 12.0 ng/mL, 9.2 ng/mL, and 7.0 ng/mL in the RAPA-PGLC group 2, 4, 8, and 12 weeks after surgery, respectively. By contrast, RAPA was undetectable in the aqueous humor in the eye drop group. High levels of IL-2R, MCP-1, TNF-alpha, and VEGF were detected in the corneal grafts of the control and PGLC groups but not in those of the RAPA-treated groups. CONCLUSIONS: RAPA-PGLC DDS and RAPA eye drops can significantly prolong the survival of allografts at high risk and inhibit corneal neovascularization. However, RAPA-PGLC DDS is far more effective than RAPA eye drops in preventing corneal graft rejection.     

二十碳五烯酸对大鼠角膜新生血管核转录因子KB及细胞因子表达的抑制作用

背景角膜新生血管（CNV）是炎症性角膜病变导致视力下降和角膜移植后发生排斥反应的重要原因,其发生机制和治疗一直是国内外角膜病研究的热点。目的探讨二十碳五烯酸（EPA）对碱烧伤大鼠CNV核转录因子KB（NF—KB）、白细胞介素1α（IL一1αt）、IL一6表达的影响及作用机制。方法用浸有1mol／LNaOH的3mm圆形滤纸贴附于72只SD大鼠的右眼角膜中央30s制作角膜碱烧伤模型,用随机数字表法将大鼠随机分为3个组,另6只匹配的正常大鼠作为正常对照组。碱烧伤0．02mgEPA治疗组和碱烧伤0．03mgEPA治疗组大鼠分别于碱烧伤后即刻结膜下注射0．04ml剂量为0．02mg或0．03mgEPA,碱烧伤模型组结膜下注射0．04ml生理盐水,正常对照组大鼠未行结膜下注射。大鼠干预后每天裂隙灯下观察CNV的生长面积和角膜水肿。分别于造模后24h,4、7、14d过量麻醉处死动物后制备角膜标本,采用苏木精一伊红染色法检测各组大鼠角膜的组织病理学变化,免疫组织化学方法检测各组大鼠角膜组织中CD34的表达以计数血管内皮细胞,用逆转录聚合酶链反应（RT—PCR）及蛋白印迹法分别检测各组大鼠角膜组织中IL-1α-mRNA和IL．6mRNA的表达及NF—KBp65的蛋白表达。结果裂隙灯下见造模后2～4d,CNV缓慢生长,角膜水肿,上皮缺损;造模后7～10d,CNV面积增加,角膜水肿减轻;造模后14d,CNV面积最大,血管变细。苏木精一伊红染色显示,碱烧伤后7d碱烧伤组角膜上皮部分缺损,基质层水肿明显,可见较多炎性细胞及大量CNV;碱烧伤0．03mgEPA治疗组与碱烧伤0．02mgEPA治疗组均可见角膜上皮修复,基质层水肿减轻,少量炎性细胞,未见CNV。碱烧伤后7d和14d,碱烧伤0．02mgEPA治疗组CNV相对面积分别为（15．80±6．43）％、（11．06±2．14）％,碱烧伤0．03mgEPA治疗组为（16．10±7．41）％、（11．06±2．51）％,均明显小于碱烧伤组的（84．74±7．77）％、（89．63±7．50）％,差异均有统计学意义（P〈0．05）。碱烧伤后7d,碱烧伤组CD34在CNV的内皮细胞中呈强阳性表达,而在碱烧伤0．02mgEPA治疗组、0．03mgEPA治疗组角膜中未见表达。RT—PCR检测结果表明,碱烧伤后4d,碱烧伤0．02mgEPA治疗组、0．03mgEPA治疗组IL一1α、IL一6mRNA在角膜组织中的表达量明显低于碱烧伤组,蛋白印迹检测证实NF—KBp65蛋白在角膜组织中的表达低于碱烧伤组,差异均有统计学意义（P〈0．05）。结论EPA能够抑制NF—KB、IL一1α、IL一6的表达,从而抑制碱烧伤后CNV的生长。     

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.