螺旋藻多糖对小鼠角膜血管新生的治疗作用及炎症因子的表达情况

目的 观察螺旋藻多糖对小鼠角膜碱烧伤后血管新生的抑制作用并探讨其机制.方法 将不同浓度的螺旋藻多糖滴眼液用于小鼠角膜碱烧伤模型,观察角膜浑浊度,ELISA法和免疫组织化学法分别检测MCP-1和IL-8及其受体CXCR1和CCR2 B在角膜组织中的表达水平.实验数据用SPSS 13.0统计软件分析.结果 0.5％螺旋藻多糖组角膜浑浊度、MCP-1和IL-8及其受体CXCR1和CCR 2 B表达水平明显比生理盐水对照组低(P＜0.05).结论 螺旋藻多糖通过减轻炎症,起抑制角膜血管新生的作用.     

TLR-2、TLR-4与大鼠角膜碱烧伤早期炎症反应相关性研究

目的 动态观察大鼠角膜碱烧伤后Toll样受体-2(Toll like-receptor 2,TLR-2)、Toll样受体-4(Toll like-receptor4,TLR-4)与炎性因子白细胞介素-1β(interleukin-1,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达情况及变化规律,分析Toll样受体家族(Toll like-receptor familys,TLRs)与炎性因子IL-1β、TNF-α的表达是否存在相关关系.方法 选用健康SPF级SD大鼠40只,右眼为烧伤实验眼,左眼为正常对照眼,用浓度为1 mol·L-1 NaOH建立Ⅲ级角膜碱烧伤模型(模型建立时及模型建立后出现大鼠角膜烧伤程度不是Ⅲ级、大鼠角膜穿孔或前房积血等情况,均予以剔除,最后随机选取32只符合条件的大鼠进行后续实验),随机分为碱烧伤后3d组、7d组、14 d组、21 d组,每组8眼.分别在模型制作后观察大鼠眼前节,评估其角膜炎症指数,然后摘取大鼠眼球,行HE染色,计算炎症细胞数,同时用Western blot蛋白检测法检测大鼠角膜中TLR-2、TLR-4、IL-1β、TNF-α表达情况,分析各组差异,评估TLRs与炎性因子的相关性.结果 在正常大鼠角膜中可检测到TLR-2、TLR-4、IL-1β、TNF-α蛋白少量表达,碱烧伤后其表达量增加,并于碱烧伤后7d达到峰值,以后逐渐递减,各组间(3d组、7d组、14 d组、21 d组)比较差异均有统计学意义(均为P＜0.05).相关性分析:TLR-2与IL-lβ(r=0.986,P＜0.05)、TNF-α(r=0.986,P＜0.05)的表达量呈正相关,TLR-4与IL-1β(r =0.975,P＜0.05)、TNF-α(r =0.990,P＜0.05)的表达量亦呈正相关.结论 TLRs参与并可能启动角膜碱烧伤的免疫炎症反应,介导炎性因子的产生.     

趋化因子CX3CL1和CCL2对人单核细胞来源巨噬细胞的作用及意义

背景 研究发现巨噬细胞及其分泌的趋化因子与角膜新生血管（CNV）的发生相关.巨噬细胞具有异质性,在不同的微环境以及不同的刺激条件下发挥不同的作用. 目的 检测C-X3-C趋化因子配体1（CX3CL1）和C-C趋化因子配体2（CCL2）对巨噬细胞增生的作用,并探讨其意义. 方法 7～8周龄雄性BALB/c小鼠20只,用角膜碱烧伤的方法构建左眼CNV模型,术后4d在裂隙灯显微镜下检查小鼠CNV情况,颈椎脱臼法处死小鼠并制备角膜切片.用荧光免疫组织化学法检测小鼠角膜组织中巨噬细胞表面两种趋化因子受体C-C趋化因子受体2（CCR2）和CX3CR1的表达.用密度梯度离心法分离人外周血单核细胞,在含质量分数10％胎牛血清（FBS）的RPMI-1640培养液中加入30 μg/L粒细胞巨噬细胞刺激因子（GM-CSF）,诱导和培养人外周血单核细胞来源的巨噬细胞.将培养的细胞分为CD68＋CCR2组和CD68＋CX3CR1组,分别加入CD68＋CCR2抗体和CD68＋CX3CR1抗体,每组2管,其中一管作为IgG同型对照.采用流式细胞仪分别检测小鼠碱烧伤角膜组织中巨噬细胞和人外周血巨噬细胞中CX3CR1以及CCR2的表达.分别用人重组CX3CL1和CCL2蛋白刺激巨噬细胞,实时定量PCR（real-time PCR）法检测干预后巨噬细胞中血管内皮生长因子（VEGF）、血小板凝血酶敏感蛋白样模体的解整链蛋白金属蛋白酶1（ADAMTS-1）、凝血酶敏感蛋白1（TSP-1）等血管生成相关因子的表达.结果 角膜碱烧伤后4d,裂隙灯显微镜下可见角膜局部有新生血管发生,CNV沿角膜缘向角膜中心区域延伸.荧光免疫组织化学法检测发现,角膜组织内有F4/80阳性的巨噬细胞浸润,其表面可见CCR2和CX3CR1分子的表达,呈绿色荧光.未受GM-CSF诱导培养的巨噬细胞能维持生长约2周,但死亡细胞较多,而经GM-CSF诱导培养的细胞生长稳定,细胞数量增加,无明显悬浮死亡现象.培养的巨噬细胞中CX3CR1表达率平均约为75％,CCR2表达率为45％.Real-time PCR法检测发现,CCL2干预后巨噬细胞中VEGF mRNA的相对表达量明显增加,其中150 mg/L CCL2组VEGF mRNA的相对表达量明显高于对照组,差异有统计学意义（t=-5.09,P=0.03）,而ADAMTS-1 mRNA、TSP-1 mRNA表达下降,其中150 mg/L CCL2组ADAMTS-1 mRNA、TSP-1 mRNA表达明显低于对照组,差异有统计学意义（t=3.01,P=0.04;t=4.27,P=0.02）.CX3CL1干预后巨噬细胞中VEGF mRNA表达量明显下降,而ADAMTS-1mRNA、TSP-1 mRNA的表达水平升高,与对照组相比,150 mg/L CX3CL1组VEGF mRNA表达量下降,ADAMTS-1 mRNA、TSP-1mRNA表达量下降,差异均有统计学意义（t=6.35,P=0.02;t=-2.92,P=0.04;t=-3.81,P=0.03）. 结论 CCL2、CX3CL1能通过其对应受体影响巨噬细胞VEGF、ADAMTS-1、TSP-1等血管新生相关因子的表达,提示通过CCL2、CX3CL1等干预靶点治疗新生血管性眼病的可能.     

MMP-9及TIMP-1在碱烧伤小鼠角膜中的表达

目的探讨角膜烧伤后基质金属蛋白酶9(MMP9)及其组织型抑制剂(tissueinhibitorofmetallopoteinase1,TIMP1)在小鼠角膜中的表达及意义。方法采用1mol·L-1氢氧化钠溶液烧伤昆明小鼠角膜,建立角膜碱烧伤动物模型;用免疫组织化学染色方法和计算机图像分析系统检测小鼠角膜烧伤后不同时间点MMP9及TIMP1在角膜中的分布及其积分吸光度(A)值。结果小鼠角膜碱烧伤后第2d炎性细胞增多,第7d炎症达到高峰,21d后基本消失。小鼠角膜上皮层、基底膜、以及基质层中大量炎性细胞和新生血管内皮细胞均有MMP及TIMP1表达。角膜中MMP9在第2d出现表达,第7d达高峰,以后逐渐降低;TIMP1开始表达不明显,第7d出现表达,14d达高峰,以后逐渐降低。结论小鼠碱烧伤模型炎症早期MMP9活性增高,继而TIMP1分泌增多,MMP9活性受抑,炎症减轻。提示MMP9可能是参与碱烧伤角膜溃疡形成、角膜融解及纤维化的重要调控因子,而TIMP1则在炎症的抑制过程中发挥了重要作用。     

新鲜羊膜移植对大鼠角膜碱烧伤后玫炎性细胞因子表达的影响

目的对SD大鼠角膜碱烧伤后行羊膜移植，分别检测碱烧伤及羊膜移植术后角膜上致炎性细胞因子的表达水平，探讨角膜碱烧伤的损伤机制及羊膜的抗炎和免疫调节机制。方法70只SD大鼠，以0．5mol／L的氢氧化钠碱烧伤右眼制作成碱烧伤模型，随机分组，A组（n=32只）为碱烧伤后未行羊膜移植的烧伤对照组，B组为烧伤后立即施行羊膜移植的羊膜移植组（n=32只），另有6只大鼠作为正常对照组即C组（n=6只），术后第1、4、7、10天处死动物，分别在裂隙灯显微镜下对角膜混浊度、角膜新生血管及角膜上皮缺损进行临床评分，取下角膜，酶联免疫吸附实验（ELISA）定量检测致炎性细胞因子IL-1d（interleukin-1α）、IL-6（interleukin-6）和IL-8（in—terleukin-8）的表达水平。结果羊膜移植组角膜上皮缺损、新生血管及角膜混浊度评分第4、7、10天明显低于对照组，在正常角膜中有IL-1α和IL-8的表达，而没有发现IL-6的表达，碱烧伤后IL-1d，IL-6和IL-8均显著表达，IL-1α于烧伤后第4天达到高峰，IL-6和IL-8于第7天达到高峰。羊膜移植组与烧伤对照组比较，羊膜移植组于术后第1、4天能显著降低IL-1α（P〈0．01）的表达水平，第4、7天能显著降低IL-6及IL-8的表达水平（P〈0．01）。结论角膜碱烧伤后早期致炎性细胞因子的过量表达直接关系到急性期角膜炎症，并可能通过激活免疫系统，参与随后的免疫反应，介导免疫损伤。新鲜羊膜移植可以抑制碱烧伤后角膜致炎性细胞因子的过量表达，可能通过调节致炎性细胞1子的表达而发挥抗炎和免疫调节机制。     

趋化因子受体4拮抗剂对实验性角膜新生血管的抑制作用及其机制

背景基质细胞源性细胞因子1α/趋化因子受体4（SDF-1αCXCR4）信号途径是机体内介导多种细胞趋化、黏附以及增生的关键分子,能通过促进血管内皮祖细胞向肿瘤组织的迁移而促进肿瘤新生血管的发生,同时也参与新生血管性眼病的病理过程。目的探讨阻断CXCR4信号通路对实验性角膜新生血管（CNV）发生发展的抑制作用及机制。方法收集8周龄BALB／c小鼠80只,将浸有1mol／LNaOH的滤纸贴附在左眼角膜中央40S以诱导小鼠CNV,按随机数字表法将动物分为透明质酸钠组（质量分数0．2％透明质酸钠点眼）及CXCR4拮抗剂组（0．2％透明质酸钠配制的CXCR4拮抗剂点眼）,自碱烧伤当日分别用相应的药物点眼共14d,于第14天裂隙灯下观察两组小鼠的CNV,然后制备角膜悬液,用流式细胞技术检测角膜悬液中CD31的表达值;制备角膜组织切片,以逆转录PCR（RT—PCR）和Western blot法检测CXCR4mRNA和蛋白在小鼠角膜组织中的表达,以免疫组织化学法检测角膜组织内CD31阳性标记的血管内皮细胞。以ELISA法检测角膜组织裂解液内血管内皮生长因子（VEGF）的表达。使用CXCR4拮抗剂干预小鼠腹腔来源的巨噬细胞,检测粒细胞巨噬细胞刺激因子（GM—CSF）刺激后培养上清液中VEGF的表达。结果小鼠角膜碱烧伤后2周,裂隙灯下可见CNV达高峰,与透明质酸钠组比较,CXCR4拮抗剂组CNV明显减少;角膜免疫组织化学检测显示,CXCR4拮抗剂组小鼠角膜中CD31阳性染色强度弱于透明质酸钠组,CD31阳性细胞数量明显减少;进一步流式细胞技术检测发现,CXCR4拮抗剂组CD31阳性表达率为（9．50±2．34）％,明显低于透明质酸钠组的（17．50±3．16）％,差异有统计学意义（t=-7．312,P〈0．05）;RT—PCR和Western blot法检测表明,碱烧伤后2、4、7d小鼠角膜组织中CXCR4mRNA和蛋白的表达均明显高于正常小鼠角膜组织,组间比较差异均有统计学意义（P〈0．01;P〈0．05）。ELISA检测结果显示,CXCR4拮抗剂点眼后4d、7d角膜组织内VEGF表达明显低于透明质酸钠组,差异均有统计学意义（t=10．927、5．151,P〈0．05）。体外实验发现,细胞培养后12、24和48h,GM—CSF＋CXCR4拮抗剂组上清液中VEGF的表达水平明显低于GM—CSF组,差异均有统计学意义（P〈0．05）。结论CXCR4拮抗剂能通过下调VEGF的表达抑制实验性CNV的形成。     

P-选择素对角膜上皮创伤修复及其中性粒细胞迁移的影响

目的观察P-选择素对角膜上皮创伤修复及其中性粒细胞迁移的影响。方法选用P-选择素基因敲除小鼠与野生型小鼠进行对比,观察角膜上皮创伤修复过程,同时用免疫组织化学方法观察中性粒细胞在角膜的迁移,并用酶联免疫吸附测定方法检测角膜中细胞因子和趋化因子的变化。结果与野生型小鼠相比,P-选择素基因敲除小鼠的角膜创伤修复延迟,向角膜迁移的中性粒细胞数量减少;中性粒细胞的迁移与趋化因子和细胞因子有密切的关系。结论P-选择素表达缺陷可导致角膜上皮创伤修复的延迟。这种延迟可能与创伤后中性粒细胞向创伤区迁移数量减少有关。     

新鲜羊膜移植对大鼠角膜碱烧伤后致炎性细胞因子表达的影响

目的对SD大鼠角膜碱烧伤后行羊膜移植,分别检测碱烧伤及羊膜移植术后角膜上致炎性细胞因子的表达水平,探讨角膜碱烧伤的损伤机制及羊膜的抗炎和免疫调节机制。方法70只SD大鼠,以0．5mol／L的氢氧化钠碱烧伤右眼制作成碱烧伤模型,随机分组,A组（n=32只）为碱烧伤后未行羊膜移植的烧伤对照组,B组为烧伤后立即施行羊膜移植的羊膜移植组（n=32只）,另有6只大鼠作为正常对照组即C组（n=6只）,术后第1、4、7、10天处死动物,分别在裂隙灯显微镜下对角膜混浊度、角膜新生血管及角膜上皮缺损进行临床评分,取下角膜,酶联免疫吸附实验（ELISA）定量检测致炎性细胞因子IL-1d（interleukin-1α）、IL-6（interleukin-6）和IL-8（in—terleukin-8）的表达水平。结果羊膜移植组角膜上皮缺损、新生血管及角膜混浊度评分第4、7、10天明显低于对照组,在正常角膜中有IL-1α和IL-8的表达,而没有发现IL-6的表达,碱烧伤后IL-1d,IL-6和IL-8均显著表达,IL-1α于烧伤后第4天达到高峰,IL-6和IL-8于第7天达到高峰。羊膜移植组与烧伤对照组比较,羊膜移植组于术后第1、4天能显著降低IL-1α（P〈0．01）的表达水平,第4、7天能显著降低IL-6及IL-8的表达水平（P〈0．01）。结论角膜碱烧伤后早期致炎性细胞因子的过量表达直接关系到急性期角膜炎症,并可能通过激活免疫系统,参与随后的免疫反应,介导免疫损伤。新鲜羊膜移植可以抑制碱烧伤后角膜致炎性细胞因子的过量表达,可能通过调节致炎性细胞1子的表达而发挥抗炎和免疫调节机制。     

趋化因子受体3及其配体在眼部疾病中的作用

趋化因子受体3 （CCR3）及其配体eotaxin是一种与哮喘和过敏相关的炎性因子,研究发现CCR3及其配体与多种眼科疾病相关,如脉络膜新生血管、变态反应性结膜炎、视网膜中央静脉阻塞、年龄相关性黄斑变性、视网膜变性等.CCR3的生物学功能研究认为,CCR3及其配体eotaxin不仅参与促进嗜酸性粒细胞与肥大细胞的运输,还参与炎症反应过程,了解其在眼部疾病发生及发展过程中的作用对于眼科相关疾病的认识和诊治有重要意义.就CCR3及其配体的结构和生物学功能以及其与一些眼部疾病的关系进行综述.     

β趋化因子受体CCR1在遗传性视网膜变性小鼠视网膜中的表达

目的探讨B趋化因子主要的受体之一β趋化因子受体1（CCR1）在视网膜变性模型小鼠（rd小鼠）的视网膜变性过程中的表达及其在感光细胞凋亡中的病理作用。设计实验研究。研究对象出生后8、10、12、14、16及18天的rd小鼠各10只（共60只）及同龄C57BL／6N对照小鼠各10只（共60只）。方法小鼠断颈处死,取双眼眼球制作冰冻切片或分离新鲜视网膜组织。逆转录多聚酶链反应（RT．PCR）测定各鼠龄rd小鼠及对照鼠视网膜CCR1mRNA的表达水平。免疫组织化学法观察CCR1蛋白在各鼠龄rd小鼠视网膜的定位表达。CCR1在感光细胞及凋亡细胞中的表达由免疫荧光双标法确定。主要指标视网膜CCR1mRNA及蛋白的表达、CCR1的细胞定位及与凋亡细胞的关系。结果CCR1mRNA在对照组及各鼠龄rd小鼠视网膜中均有表达,但在出生后12、14天rd小鼠视网膜中表达明显升高（光密度值分别为0．986±0．17和1．152＋0．22,P=0．010和0．008）。CCR1阳性染色细胞开始出现于出生后8天的rd视网膜外核层,并于12及14天达到高峰。对照组视网膜外核层无CCRl阳性染色。CCR1与视紫红质、CD11b或TUNEL染色免疫荧光双标结果显示,CCR1表达于感光细胞而非小胶质细胞中,部分CCR1表达于凋亡的感光细胞中。结论在rd小鼠视网膜中CCR1表达于感光细胞并随其变性程度加重表达升高。CCR1的活化可能在rd小鼠感光细胞凋亡中发挥作用。     

Deletion of the chemokine receptor CCR1 prolongs corneal allograft survival

PURPOSE: Many corneal grafts undergo immune rejection, and current therapies are associated with many side effects. The purpose of this study was to identify critical chemokine pathways involved in generating the alloimmune response to corneal transplants. METHODS: Orthotopic corneal transplantation was performed in fully mismatched strains. Cytokine and chemokine receptor gene expression was determined by the RNase protection assay. Knockout (KO) strains for chemokine-chemokine receptors that are upregulated after transplantation underwent corneal transplantation. Results derived from KO murine hosts were compared with cyclosporine (Cy) therapy. In addition to graft survival, graft infiltration, allospecific delayed-type hypersensitivity (DTH), and cytokine expression were compared among the recipient groups. RESULTS: Initial experiments revealed gene upregulation of the chemokine receptors CCR1, -2, and -5 after corneal allorejection. Although CCR1 KO hosts showed a significant increase in graft survival compared with wild-type (WT) hosts, allografts in CCR5, CCR2/CCL3(MIP-1alpha), CXCR3, CXCL10/IP-10, and CCL3/MIP-1alpha KO mice did not show a significant improvement in graft survival. Further, CCR1 KO hosts showed a significantly higher survival rate than with systemic Cy therapy in WT hosts. Moreover, graft infiltration by leukocytes and gene expression of proinflammatory cytokines were reduced in CCR1 KO mice compared with both Cy treated and untreated WT mice, as was the induction of allospecific DTH. CONCLUSIONS: These studies provide, for the first time, evidence that targeting of specific chemokine pathways can significantly promote survival of corneal transplants, and suggest that select deletion or suppression of CCR1 can be a useful therapeutic target in corneal transplant immunity.     

The inflammatory milieu associated with conjunctivalized cornea and its alteration with IL-1 RA gene therapy

PURPOSE: This study was designed to gain an insight into the inflammatory milieu into which a donor limbal graft is routinely introduced. The objective of this study was to modulate this environment by gene therapy with the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1 RA). METHODS: In a mouse model, the ocular surface cytokine environment associated with a conjunctivalized cornea was assessed 4 weeks after injury. Total corneal epithelial and limbal debridement was performed with a combination of alkali and scrape injury. The cytokines and adhesion molecules measured included IL-1alpha, IL-1beta, IL-6, VEGF, intercellular adhesion molecule (ICAM)-1, and vascular adhesion molecule (VCAM)-1, by real-time PCR or ELISA. Injured corneas were transfected with IL-1 RA by injection of naked plasmid vector pIRES-EGFP-IL-1 RA immediately after injury. Corneas transfected with pIRES-EGFP served as the control. Expression of corneal IL-1 RA after transfection with pIRES-EGFP-IL1-RA was assessed over a 2-week period by real-time PCR and Western blot analysis. In addition, limbal stem cell grafts transfected with IL-1 RA were assessed for leukocyte influx. RESULTS: Conjunctivalized corneas showed increased expression of IL-1alpha, IL-1beta, IL-1 RA, IL-6, VEGF, ICAM-1, and VCAM-1, compared with normal cornea. Transfection-efficiency experiments indicated that corneal expression of IL-1 RA peaked between 12 and 24 hours and lasted up to 2 weeks after the initial transfection. IL-1 RA corneal gene therapy resulted in a downregulation of IL-1beta and VCAM-1 expression at 4 weeks after injury, whereas downregulation of IL-6 was evident only at 1 week after injury. Corneal neovascularization was also reduced. In addition, corneal limbal stem cell grafts transfected with IL-1 RA showed a decreased leukocyte influx compared with control grafts. CONCLUSIONS: Transfection of a cornea with IL-1 RA immediately after epithelial injury selectively altered the cytokine profile of the resultant conjunctivalized cornea and suppressed corneal neovascularization. Transfection of corneal limbal donor tissue with IL-1 RA before engraftment can reduce leukocyte influx into the graft. The findings demonstrate the feasibility of using transient cytokine gene expression, either in donor or recipient corneal tissue, to alter the ocular surface environment beneficially.     

Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea.

PURPOSE: To determine the effect of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha on cytokine, chemokine, and receptor expression in corneal stromal cells; the effect of corneal scrape injury on monocyte chemotactic and activating factor (MCAF) expression and monocyte-macrophage influx into the stroma; and the effect of MCAF and granulocyte colony-stimulating factor (G-CSF) microinjection on inflammatory cell infiltration into the stroma. METHODS: Gene array technology was used to evaluate changes in cytokine, chemokine, and receptor gene expression in stromal fibroblasts in response to IL-1alpha and TNFalpha. Expression of MCAF mRNA and protein was monitored with an RNase protection assay and Western blot analysis, respectively. Keratocyte MCAF protein expression in the rabbit cornea was detected with immunocytochemistry. After epithelial scrape injury, monocytes-macrophages were detected in rabbit corneas, by immunocytochemistry for monocyte-macrophage antigen. Inflammatory cell infiltration after MCAF and G-CSF microinjection into the stroma of mouse corneas was monitored with hematoxylin and eosin staining. RESULTS: IL-1alpha or TNFalpha upregulated the expression of several proinflammatory chemokines in stromal fibroblasts in culture. These included G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF). MCAF mRNA upregulation was confirmed by RNase protection assay, and MCAF protein was detected by Western blot analysis. MCAF protein was detected in keratocytes at 4 hours and 24 hours after epithelial injury, but not in keratocytes in the unwounded cornea. Corneal epithelial injury triggered the influx of monocytes-macrophages into the corneal stroma in the rabbit. Microinjection of MCAF and G-CSF into mouse cornea resulted in the influx of monocytes-macrophages and granulocytes, respectively, into the stroma. CONCLUSIONS: Proinflammatory chemokine induction in keratocytes is mediated by IL-1alpha and TNFalpha. The proinflammatory chemokines produced by the keratocytes probably trigger the influx of inflammatory cells into the stroma after epithelial injury associated with corneal surgery, contact lenses, or trauma.     

Cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis on murine photoreceptor cells

AIM: To investigate the cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis (EAU) as well as their secreted interferon (IFN)-γ and interleukin (IL)-17A on murine photoreceptor (661W) cells. METHODS: An EAU model was established in female mice by injection of interphotoreceptor retinoid binding protein (IRBP) emulsion supplemented with complete Freund’s adjuvant (CFA) and Mycobacterium tuberculosis (TB). On day 12 after induction of EAU, specific T cells from spleen and lymph node tissues were isolated and cultured for 4d and the levels of IFN-γ and IL-17A in the supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). T cells and their supernatants were added to 661W cells to observe the alteration of cell morphology; IFN-γ and IL-17A were separately added to 661W cells to observe the effect of IFN-γ and IL-17A on cell proliferation. RESULTS: The levels of IFN-γ and IL-17A in the T cell supernatants were 1568.64±38.79 pg/mL and 1456.57±46.98 pg/mL, respectively. The supernatants apparently inhibited 661W cell proliferation (P<0.05). T cells could also attach to the surface of 661W cells, and IFN-γ showed a more serious cytotoxic effect on 661W cells than IL-17A, inhibiting cell proliferation (P<0.01). CONCLUSION: IFN-γ and IL-17A from T cells of EAU mice model can exert cytotoxic effects on murine photoreceptor cell proliferation, and IFN-γ shows more serious cytotoxic effects on murine photoreceptor cells than IL-17A.     

Hydrogen promotes the activation of Cu, Zn superoxide dismutase in a rat corneal alkali-burn model

AIM: To investigate the cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis (EAU) as well as their secreted IFN-γ and IL-17A on murine photoreceptor (661W) cells.METHODS: An EAU model was established in female mice by injection of interphotoreceptor retinoid binding protein (IRBP) emulsion supplemented with complete Freund’s adjuvant (CFA) and Mycobacterium tuberculosis (TB). On day 12 after induction of EAU, specific T cells from spleen and lymph node tissues were isolated and cultured for 4d and the levels of IFN-γ and IL-17A in the supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). T cells and their supernatants were added to 661W cells to observe the alteration of cell morphology; IFN-γ and IL-17A were separately added to 661W cells to observe the effect of IFN-γ and IL-17A on cell proliferation. RESULTS: The levels of IFN-γ and IL-17A in the T cell supernatants were 1568.64 pg/mL and 1456.57 pg/mL, respectively. The supernatants apparently inhibited 661W cell proliferation (P<0.05). T cells could also attach to the surface of 661W cells, and IFN-γ showed a more serious cytotoxic effect on 661W cells than IL-17A, inhibiting cell proliferation (P<0.01).CONCLUSION: IFN-γ and IL-17A from T cells of EAU mice model can exert cytotoxic effects on murine photoreceptor cell proliferation, and IFN-γ shows more serious cytotoxic effects on murine photoreceptor cells than IL-17A.     

α1抗胰蛋白酶抑制角膜碱烧伤的实验机制研究

目的研究α1抗胰蛋白酶(alpha-1 antitrypsin,AAT)抑制角膜碱烧伤新生血管的效应和可能机制。方法2018年1月~2018年6月应用NaOH构建小鼠碱烧伤模型,应用AAT制备的滴眼液干预碱烧伤模型。通过眼前段照相、CD31角膜铺片免疫荧光观察AAT对角膜碱烧伤模型新生血管的抑制作用。H&E切片观察角膜组织中炎症细胞浸润的情况。RT-PCR检测炎症血管因子的mRNA表达情况。结果裂隙灯眼前段照相发现AAT明显抑制小鼠碱烧伤角膜新生血管的生长,造模后7d、14d新生血管增生的面积较对照组减少24.6%、32.3%,差异具有统计学意义(P<0.05),CD31角膜组织免疫铺片检测取得相近的实验结果,7d、14d新生血管密度较对照组降低8.2%、15.1%。HE切片也提示AAT明显抑制了角膜组织中炎症细胞的浸润,造模14d对照组、实验组炎症细胞浸润个数为231.4±80.7、113.5±56.1,二者之间具有统计学差异。RT-PCR检测发现AAT下调角膜组织β-FGF,CCL2,IL-6,MMP9等血管炎症因子的mRNA表达水平。结论AAT可能通过抑制角膜组织的免疫炎症发挥抑制新生血管的作用,AAT应用治疗角膜化学性烧伤疾病具有广阔的临床使用前景。     

碱烧伤小鼠角膜组织中SDF-1／CXCR4的表达及意义

目的探讨基质细胞衍生因子-1（SDF-1）和CXCR4在小鼠角膜碱烧伤新生血管形成中的作用。方法应用在角膜中央放置NaOH滤纸片的方法构建C57／BL小鼠角膜碱烧伤角膜新生血管动物模型,通过免疫组织化学、RT—PCR、Western blot法检测碱烧伤后不同时间点角膜组织中SDF-1和CXCR4 mRNA及蛋白的表达情况。结果免疫组织化学检测显示,正常小鼠角膜基质层无明显SDF-1的阳性表达,CXCR4仅在角膜上皮层呈弱阳性表达。RT—PCR检测显示,与正常对照组相比,碱烧伤后各时间点SDF-1和CXCR4 mRNA表达均明显升高（P〈0．05）;Western blot检测显示SDF-1和CXCR4蛋白的表达也可见相似的变化趋势（P〈0．05）。SDF-1和CXCR4在角膜中的表达于第7天达高峰,第14天开始下降,但仍高于正常。结论SDF-1／CXCR4在碱烧伤后小鼠角膜组织中的表达增加,在角膜碱烧伤的炎症和新生血管的形成和发展过程中可能发挥重要作用。