整合素在角膜上皮创伤愈合中的研究进展

整合素作为一类重要的细胞黏附分子,通过影响细胞的形态,介导细胞的黏附、迁移和增生,在角膜上皮创伤愈合中发挥了重要的作用。讨论α2β1、α3β1、α5β1、αvβ3、α6β4、α9β1和αvβ6这7种整合素在角膜上皮创伤愈合中的研究进展及其临床意义。α6β4整合素为半桥粒的主要组成部分,介导角膜上皮细胞在细胞外基质上的静态黏附,损伤后该黏附就转变为α2β1、α3β1整合素介导的动态黏附,细胞在黏附-去黏附的过程中实现迁移,从而修复创面。α6β4、α3β1整合素相互协调作用,实现上皮细胞的板层状运动。研究还发现α6β4、α3β1整合素的活化还能促进细胞的增生。损伤后上皮细胞表面α5β1、αvβ3整合素的表达上调,二者与黏着斑的形成密切相关。α9β1和αvβ6为近年来新发现的与角膜上皮创伤愈合有关的整合素,其具体作用尚有待进一步的研究。     

β1整合素过表达促进体外兔角膜上皮细胞黏附和迁移

目的:探讨β1整合素过表达对角膜上皮细胞黏附和迁移的影响机制。方法:将β1整合素-GFP融合蛋白真核细胞重组表达质粒转染兔角膜上皮细胞,观察转染细胞的融合基因表达以及细胞的黏附和迁移能力。检测β1整合素转染对角膜上皮细胞粘着斑激酶(FAK)磷酸化的影响。结果:成功将β1整合素-GFP融合蛋白转染至兔角膜上皮细胞并使其过表达;β1整合素过表达能够明显增加角膜上皮细胞的黏附和迁移能力(P<0.05)并促进FAK磷酸化(P<0.05)。结论:β1整合素过表达能够明显促进角膜上皮细胞的黏附和迁移。     

β1整合素过表达促进体外角膜上皮细胞黏附和迁移

目的:探讨β1整合素过表达对角膜上皮细胞黏附和迁移的影响机制。方法:将β1整合素-GFP融合蛋白真核细胞重组表达质粒转染兔角膜上皮细胞,观察转染细胞的融合基因表达以及细胞的黏附和迁移能力。检测β1整合素转染对角膜上皮细胞粘着斑激酶(FAK)磷酸化的影响。结果:成功将β1整合素-GFP融合蛋白转染至兔角膜上皮细胞并使其过表达;β1整合素过表达能够明显增加角膜上皮细胞的黏附和迁移能力(P<0.05)并促进FAK磷酸化(P<0.05)。结论:β1整合素过表达能够明显促进角膜上皮细胞的黏附和迁移。     

真菌性小鼠角膜炎中炎性因子的表达

目的 探讨4种炎性因子在真菌性小鼠角膜炎发病中的表达和作用.方法 实验研究.240只BALB/c小鼠采用随机数字表法分为4组,分别为茄病镰刀菌感染组、烟曲霉菌感染组、白色念珠菌感染组及损伤对照组,每组60只.建立小鼠真菌性角膜炎模型.分别于术后1、3、5及7 d,于裂隙灯显微镜下观察角膜疾病的发展,病理学检查病变角膜的炎症反应和菌丝生长情况,反转录聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测巨噬细胞炎性蛋白2(MIP-2)、细胞因子诱生性中性粒细胞趋化物(KC)、白细胞介素(IL)1β和IL-6炎性因子的mRNA和蛋白质表达水平.对临床评分、RT-PCR和ELISA结果比较应用参数检验中方差分析(one-way ANOVA),进一步两两比较采用LSD检验法.结果 随时间变化各感染组小鼠角膜病变和炎症反应出现先增强到术后7 d开始减弱的趋势.RT-PCR结果显示,MIP-2和IL-1β的表达在烟曲霉菌感染组最强,KC在白色念珠菌感染组最强,IL-6在茄病镰刀菌感染组最强.各感染组中IL-1β和KC的表达强于IL-6和MIP-2.MIP-2、KC及IL-1β在茄病镰刀菌感染组、烟曲霉菌感染组及白色念珠菌感染组的表达高峰分别为术后5、1及3 d,IL-6在茄病镰刀菌和烟曲霉菌感染组的高峰为术后1和7 d,在白色念珠菌感染组的高峰为术后1和5 d.对各实验组同一因子mRNA的表达强度的相对值进行相互比较分析显示,各组间差异均有统计学意义(MIP-2:F=1675.339,P<0.01;KC:F=730.267,P<0.01;IL-1β:F=297.106,P<0.01;IL-6:F=174.513,P<0.01).ELISA结果显示,各感染组的4种因子蛋白表达量从高到低依次为IL-1β、MIP-2、KC及IL-6.不同实验组间和相同实验组中每种因子的蛋白表达随时间变化规律与4种因子的mRNA表达规律吻合.对各实验组同一因子的蛋白表达水平进行相互比较分析表明,各组间差异均有统计学意义(MIP-2:F=2871.736,P<0.01;KC:F=886.598,P<0.01;IL-1β:F=3595.488,P<0.01;IL-6:F=89.225,P<0.01).结论 MIP-2、KC和IL-1β的mRNA和蛋白表达水平与真菌性角膜炎的病变程度和炎症反应密切相关,其中MIP-2和IL-1β的持续高水平表达可能在真菌性角膜炎病变损害中起着重要的作用.     

β1整合素过表达抑制角膜上皮细胞凋亡的研究

目的探讨β1整合素功能性过表达对角膜上皮细胞凋亡的影响及机制,为角膜细胞移植提供理论依据。方法构建β1整合素-绿色荧光蛋白(GFP)融合基因并转染兔角膜上皮细胞。观察融合基因在角膜上皮细胞的表达及对各细胞外基质蛋白的黏附能力。检测β1整合素功能性转染对角膜上皮细胞凋亡及丝裂素活化蛋白激酶(MAPK)磷酸化的影响。结果β1整合素-GFP融合基因成功转染至兔角膜上皮细胞并过表达;β1整合素过表达能够明显增加角膜上皮细胞对各细胞外基质蛋白的黏附力并抑制角膜上皮细胞凋亡及促使MAPK磷酸化。结论β1整合素过表达能有效抑制角膜上皮细胞凋亡,MAPK磷酸化可能在这一过程中起重要作用。     

β1整合素过表达抑制角膜上皮细胞凋亡的实验研究

目的:探讨β1整合素功能性过表达对角膜上皮细胞凋亡的影响及机制,为角膜细胞移植提供理论依据。方法:构建β1整合素-绿色荧光蛋白(GFP)融合基因并转染兔角膜上皮细胞。观察融合基因在角膜上皮细胞的表达及对各细胞外基质蛋白的黏附能力。检测β1整合素功能性转染对角膜上皮细胞凋亡及丝裂素活化蛋白激酶(mitogen-activated protein kinase,MAP激酶)磷酸化的影响。结果:β1整合素-GFP融合基因成功转染至兔角膜上皮细胞并过表达;β1整合素过表达能够明显增加角膜上皮细胞对各细胞外基质蛋白的黏附力并抑制角膜上皮细胞凋亡及促使MAP激酶磷酸化。结论:β1整合素过表达能有效抑制角膜上皮细胞凋亡,MAP激酶磷酸化可能在这一过程中起重要作用。     

整合素-α5及其配体在大鼠角膜创伤愈合中的表达

整合素属细胞黏附分子，是由α和β亚单位通过非共价键形式结合成为跨膜糖蛋白，主要介导细胞与细胞和细胞与细胞外基质间的黏附，并在角膜创伤愈合中起重要作用。α5主要与β1结合。文献报道，配体纤维连接蛋白(fibronectin，FN)是以二聚体形式存在的大分子糖蛋白，经典受体为整合素-α5，大鼠角膜移植术后角膜创缘上皮细胞、迁移上皮细胞膜基底面及基质切口边缘α5及FN蛋白表达量明显增加；当创伤愈合后，其α5及FN蛋白表达消失。为此我们对大鼠角膜创伤愈合中整合素-α5及FN蛋白表达量进行检测，探讨其在角膜创伤愈合中的作用，现将结果报告如下。     

主要致病真菌在角膜内生长方式的研究

目的 研究白色念珠菌、茄病镰刀菌、烟曲霉菌在角膜内的生长方式.方法 应用表层角膜植片术,在植床与植片间注射真菌菌液,建立浅层真菌性角膜感染的兔模型.28只健康成年家兔,分为白色念珠菌感染组、茄病镰刀菌感染组、烟曲霉菌感染组,每组随机取8只兔,并设立4只兔为对照组.进行组织病理学及透射电镜观察.结果 大体观察发现3种真菌感染的临床表现各有特点.组织病理学及透射电镜检测可见白色念珠菌垂直生长,茄病镰刀菌平行于角膜板层生长,烟曲霉菌斜行生长.结论 3种不同真菌在角膜内生长方式不同,可能存在不同的致病机制.     

结缔组织生长因子对角膜成纤维细胞α5β1整合素表达的影响

目的 :观察结缔组织生长因子 (connectivetissuegrowthfactor,CTGF)对体外培养的角膜成纤维细胞α5β1整合素表达的影响。方法 :体外培养兔角膜成纤维细胞 ,经 0 .5、5、5 0和 5 0 0ng/mlCTGF处理 2 4h后 ,用免疫组化染色检测角膜成纤维细胞α5β1整合素的表达情况。结果 :与对照组相比 ,0 .5、5、5 0和 5 0 0ng/ml的CTGF能呈剂量依赖性地促进角膜成纤维细胞α5β1整合素的表达 (P <0 .0 1)。结论 :一定浓度的CTGF能促进角膜成纤维细胞α5β1整合素的表达 ,它可能参与角膜基质损伤修复过程中角膜成纤维细胞与细胞外基质以及角膜成纤维细胞之间的黏附和角膜成纤维细胞的移行过程。     

高糖和糖浓度波动对人角膜上皮细胞中MMP-9表达及真菌黏附的影响

目的 探讨不同浓度葡萄糖环境下茄病镰刀菌和尖端赛多孢子菌对人角膜上皮细胞(HCEC)的黏附能力和HCEC中MMP-9表达的影响.方法 将处于对数生长期的HCEC分为3组:低糖组(5.5 mmol·L-1葡萄糖培养基培养)、高糖组(25.0 mmol·L-1葡萄糖培养基培养)和糖波动组(5.5 mmol·L-1和50.0...     

Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha(2), alpha(3), alpha(6), beta(1)and beta(4)subunits, integrins alpha(6)and beta(4)being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha 3, beta 3 and gamma 2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in colocalization with beta(4)integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin beta(1)subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin alpha-subunits inhibited the adhesion. Integrin alpha(3)beta(1)complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex alpha(3)beta(1), as well as laminin alpha(3)chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha(3)beta(1)complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin alpha subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes.     

Roles of adherence and matrix metalloproteinases in growth patterns of fungal pathogens in cornea

PURPOSE: To investigate the roles of adherence and matrix metalloproteinases (MMPs) in growth patterns of major fungal pathogens in cornea. METHODS: Ninety-six eyes in 96 rabbits were equally divided into four groups receiving inoculation of fungal conidia of Aspergillus fumigatus, Candida albicans, Fusarium solani, and Penicillium citreo-viride, respectively, to induce fungal keratitis. Corneas in each group were obtained at 2, 8, 16 hr, and 1, 2, 3, 5, and 8 days after inoculation and were subjected to scanning electron microscopy, histopathological examination, and gelatin zymography. Eight saline-inoculated eyes in another eight rabbits served as controls. RESULTS: All eyes in the fungus-inoculated groups developed fungal keratitis. The binding of conidia to corneal epithelial basement membrane was initiated earlier in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. Destruction of basement membrane began at 1 to 3 days. Histopathologically, infiltration of inflammatory cells was more evident in the A. fumigatus and C. albicans groups than the F. solani and P. citreo-viride groups at 3 days. The hyphae of A. fumigatus and C. albicans traversed the cornea in a plane perpendicular to the stromal lamellae, whereas the hyphae of F. solani and P. citreo-viride lay parallel to the corneal lamellae. MMP-9 and MMP-2 were found in all infected corneas. At 3 days, proteolysis was most active; the level of MMP-9 was higher in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. There were positive correlations among the number of binding conidia, degree of inflammation, and level of MMP-9 (p < 0.05). CONCLUSIONS: The adherence ability, chemotaxis to neutrophils, and MMP-9 expression level differ in eyes with different fungal pathogens, which may contribute to the different growth patterns of fungi in cornea.     

Corneal biopsy in the diagnosis of keratomycosis

In two patients, a 55-year-old man and a 49-year-old man, who had fungal keratitis initially undiagnosed by corneal scrapings the condition was successfully diagnosed by corneal biopsy. We compared corneal biopsy specimens and corneal scraping in the diagnosis of keratomycosis in rabbits with experimental bilateral fungal keratitis caused by Fusarium solani, Aspergillus fumigatus, and Candida albicans. Corneal scrapings disclosed three specimens (30%) positive for Candida, five (50%) for Fusarium, and six (60%) for Aspergillus keratitis, whereas corneal biopsy specimens showed fungal elements of Fusarium, Aspergillus, and Candida in all inoculated eyes.     

Direct examination vs culture of biopsy specimens for the diagnosis of keratomycosis

In two patients with fungal keratitis, direct examination of corneal biopsy specimens showed positive fungal elements, but cultures of biopsy specimens failed to disclose fungal growth. We compared the value of direct examination and culture of biopsy specimens in the diagnosis of keratomycosis in rabbits with experimental fungal keratitis caused by Fusarium solani, Aspergillus fumigatus, and Candida albicans. Cultures disclosed seven specimens (70%) positive for Candida and eight (80%) for Fusarium and Aspergillus keratitis, whereas direct examination showed positive fungal elements of Fusarium, Aspergillus, and Candida in all specimens.     

Modulation of corneal epithelial cell functions by the neutrophil-derived inflammatory mediator CAP37

PURPOSE: To investigate the effect of CAP37, an inflammatory mediator in neutrophils, on three important events in corneal wound healing: proliferation, migration, and adhesion. METHODS: Immortalized human corneal epithelial cells (HCEC) were treated with CAP37, and its effects on migration and proliferation were measured using the modified Boyden chemotaxis chamber and the proliferation assays (CyQUANT; Molecular Probes, Eugene, OR), respectively. Effects on adhesion were determined by measuring upregulation of adhesion molecules belonging to the selectin, integrin, and immunoglobulin superfamily using RT-PCR and flow cytometry. RESULTS: CAP37 promoted proliferation of HCEC in a time- and dose-dependent fashion. CAP37 was maximally chemotactic for HCEC over a range of 1.3 x 10(-8) to 5.2 x 10(-8) M. CAP37 upregulated intercellular adhesion molecule (ICAM)-1, platelet endothelial cell adhesion molecule (PECAM)-1, and integrin molecules alpha3 (CD49c) and beta1 (CD29). Data on migration and ICAM-1 and PECAM-1 upregulation were corroborated using primary human corneal epithelial cells. CONCLUSIONS: CAP37 modulated corneal epithelial cell proliferation and migration and upregulated adhesion molecules involved in leukocyte-epithelial and epithelial-extracellular matrix interactions.     

明胶酶在兔真菌性角膜炎病理改变中的作用研究

目的探讨明胶酶包括基质金属蛋白酶（MMP）2与MMP-9在兔真菌性角膜炎病理改变中的作用。方法80只新西兰白兔随机分为4组,每组20只。其中3组为实验组,兔右眼分别注入100μl茄病镰刀菌、烟曲霉菌及白色念珠菌的悬液;另1组为对照组,兔右眼注入等量生理盐水。免疫组织化学方法观察MMP-2与MMP-9的来源,明胶酶谱法检测其活性。组织病理学方法观察炎性细胞的浸润、角膜细胞外基质（ECMs）的降解以及真菌菌丝在角膜内的生长方式与入侵深度。结果MMP-2主要由角膜基质细胞产生,真菌感染后5d检测出活性,8d活性升高。MMP-9主要来源于嗜中性粒细胞,接种后1d即检测到活性,3d活性升高,之后逐渐下降。茄病镰刀菌感染后3d,角膜内散在嗜中性粒细胞,浅层ECMs被降解,菌丝平行于角膜基质纤维生长。烟曲霉菌和白色念珠菌感染后3d,角膜内可见大量嗜中性粒细胞,周围ECMs降解明显,菌丝表现为垂直生长。接种后8d,茄病镰刀菌和白色念珠菌感染的角膜内炎性细胞和菌丝明显减少,而烟曲霉菌感染的角膜变化不明显。结论茄病镰刀菌、烟曲霉菌及白色念珠菌感染兔角膜后,产生的明胶酶活性明显不同;明胶酶对降解角膜ECMs发挥了重要作用;随着ECMs降解程度的不同,菌丝在角膜内的生长方式、入侵深度等病理改变出现差异。     

Rapid visualization of three common fungi using fluorescein-conjugated lectins

The feasibility of using fluorescein-conjugated lectins to visualize and differentiate three fungi commonly involved in ophthalmic mycoses was evaluated. Using a panel of fluorescein-conjugated lectins, Candida albicans, Aspergillus fumigatus, and Fusarium solani were rapidly and reproducibly visualized in in vitro culture isolates, as well as in tissue samples and fixed histopathologic specimens taken from experimental mycoses. Additionally, Aspergillus and Fusarium were consistently differentiated from Candida. The binding affinities of the different lectins corresponded well with the individual sugar composition of the fungal cell walls.     

Calcofluor and ink-potassium hydroxide preparations for identifying fungi

Calcofluor and ink-potassium hydroxide preparations identified Fusarium solani, Aspergillus fumigatus, and Candida albicans, the three most common ocular fungal pathogens, in scrapings, biopsy specimens, and tissue sections of corneal mycotic infections in rabbits. These stains also identified fungal organisms in specimens from four human patients with keratomycoses. Neither procedure requires more than a few minutes to perform or extensive training or experience to interpret. The specimen stained with calcofluor can be examined immediately, but may not identify all fungi. The more sensitive ink-potassium hydroxide preparation should be examined after 18 to 24 hours, and is less likely to provide false-positive results than the calcofluor method.