连接黏附分子-1和咬合蛋白在人角膜上皮中的表达

目的检测咬合蛋白（0ccludin）和连接黏附分子-l（junction adhesion mo1ecule-l，JAM-1）在正常人角膜上皮各层中的表达。方法培养人角膜上皮细胞提取细胞总RNA，以逆转录后获得的cDNA为模板，PCR扩增目的基因JAM-1及0ccludin。流式细胞仪检测JAM-l蛋白表达。双重免疫荧光观察JAM-l与0ccludin在正常人角膜上皮组织的原位表达。结果RT-PCR在培养人角膜上皮细胞中检测到JAM-l与0ccludin扩增片段；流式细胞仪检测到JAM-l蛋白表达；双重免疫荧光结果显示0ccludin染色主要位于表层上皮层细胞之间；而JAM-l荧光染色不仅见于上皮表层，在整个上皮层细胞之间均可见其荧光反应。结论 0ccludin主要位于正常人角膜上皮表层细胞之间，JAM-1在正常人角膜上皮的全层中均有表达。     

接合黏附分子-1在大鼠角膜中的表达研究

背景 接合黏附分子-1(JAM-1)是新发现的跨膜蛋白,参与细胞紧密连接的结构组成和功能发挥.在眼组织方面,紧密连接对维持角膜的透明性十分重要,但是目前就JAM-1在角膜紧密连接结构和功能方面的研究较少. 目的 确定JAM-1在大鼠角膜上皮层、基质层和内皮层的构成.方法 选取4只SPF级Wistar大鼠,2只用于JAM-1基因在角膜组织中表达的逆转录聚合酶链反应(RT-PCR)检测,另2只用于免疫组织化学检测.动物过量麻醉处死后获得角膜组织并制备角膜上皮、基质和内皮标本,RT-PCR法检测角膜标本中JAM-1、occludin和claudin-1 mRNA的表达.反应产物行质量分数1.5％琼脂糖凝胶电泳并用凝胶成像系统进行分析.用兔抗鼠JAM-1单克隆抗体对角膜石蜡切片、上皮及内皮铺片行免疫组织化学检测,评估JAM-1蛋白在大鼠角膜组织各层的表达部位和表达强度. 结果 在大鼠角膜各层均可检测到JAM-1、occludin和claudin-1 mRNA的表达,PCR熔解曲线为清晰的单峰.角膜组织各层中JAM-1 mRNA表达水平与occludin mRNA相似,均高于claudin-1 mRNA.3种黏附分子均在上皮层表达最强,角膜基质层表达较弱.免疫组织化学检测显示,JAM-1蛋白在角膜各层均有明确的阳性染色,角膜上皮基底层的表达强于基质层和内皮层.角膜上皮、内皮铺片检测显示,JAM-1蛋白主要表达于上皮细胞和内皮细胞的连接部位,而角膜内皮中JAM-1蛋白的阳性染色广泛而弥散.结论 JAM-1作为细胞连接的构成成分,在角膜上皮层、内皮层和基质层均有表达,但其表达的形态和水平因组织层次的不同而不同.     

Coordinated reassembly of the basement membrane and junctional proteins during corneal epithelial wound healing

PURPOSE: To characterize changes in the localizations of the basement membrane protein laminin-1 and of adhesion proteins of intercellular junctions during wound healing after epithelial ablation in the rat cornea. METHODS: Epithelial ablation was performed with an excimer laser. Rats were killed immediately, 12 hours, 24 hours, 3 days, or 4 weeks after ablation, and corneal cryosections were subjected to two-color immunofluorescence staining with antibodies to laminin-1 and antibodies to connexin43 for gap junctions, desmoglein 1 or 2 (desmoglein 1 + 2) for desmosomes, or E-cadherin for adherens junctions. Sections were also stained with antibodies to occludin for examination of tight junctions. RESULTS: Laminin-1 was detected in the basement membrane, connexin43 in the basal cell layer, desmoglein 1 + 2 in the wing cell layer, E-cadherin in all cell layers, and occludin in the wing and superficial cell layers of the intact corneal epithelium. Laminin-1 immunostaining was not detected at the leading edge of migrating epithelial cells until 24 hours after ablation. Expression of connexin43 and desmoglein 1 + 2 coincided with the reappearance of laminin-1, whereas that of E-cadherin and occludin was apparent regardless of the absence or presence of laminin-1. Epithelial remodeling was complete after 4 weeks. The basement membrane was re-established, and the expression patterns for all the adhesion proteins were identical with those characteristic of the intact cornea. CONCLUSIONS: Actively migrating epithelial cells no longer manifested gap junctions and desmosomes in the wounded area with no basement membrane. Re-establishment of the basement membrane coincided with reassembly of these intercellular junctions, suggesting that the presence of the basement membrane may be required for their reformation in the rat cornea.     

Expression of the 55-kD/64-kD corneal keratins in ocular surface epithelium.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells. Immunofluorescence microscopic analysis of frozen sections of guinea pig and bovine tissue revealed that the 55-kD antiserum labeled corneal and limbal epithelium. In addition, the antiserum stained a subpopulation of peripheral limbal cells that were distributed in both basal and suprabasal layers of the epithelium. The monoclonal antibody AE5 was used to investigate the distribution of the 64-kD polypeptide in guinea pig and bovine tissue. Immunoblotting analysis revealed that AE5 antibodies recognized a 64-kD polypeptide in guinea pig cornea, but recognized a 66-kD polypeptide in bovine cornea. Immunofluorescence microscopic analysis of guinea pig tissue revealed that AE5 antibodies labeled suprabasal layers of corneal epithelium, in suprabasal layers of limbal epithelium, and in groups of cells in the peripheral limbal epithelium. We discuss the possibility that the ocular epithelial cells recognized by either the 55-kD or the 64-kD antibodies in the peripheral limbus may play a role in the reepithelialization of the cornea after wounding.     

The spatial organization of apical junctional complex-associated proteins in feline and human corneal endothelium

PURPOSE: Previous studies suggest that proteins associated with the apical junctional complex (AJC) play essential roles in the development, maintenance and regulation of barrier function in transport epithelium and vascular endothelium. The goal of this study is to identify and determine the spatial organization of several major AJC-associated proteins in normal human and feline corneal endothelium. METHODS: Fresh corneal tissue was obtained from 4 recipient buttons removed during penetrating keratoplasty (two from keratoconus patients, and two from patients with post-traumatic stromal scarring) as well as from 16 cat eyes. En bloc double- and triple-labeling of corneas was performed using phalloidin, and mouse, rat or rabbit antibodies to ZO-1, occludin, pan-cadherin, alpha-catenin, beta-catenin and plakoglobin (gamma-catenin). The 3-D localization of the proteins was then determined in situ using laser confocal microscopy. RESULTS: Similar staining patterns were obtained for the corneal endothelium of normal cat corneas and fresh human buttons. Apically, f-actin was arranged into dense peripheral bands (DPB) in individual cells that were separated from those in adjacent cells. Diffuse phalloidin staining also extended from the DPB into the cytoplasm apically. Although weaker, phalloidin staining also appeared to be associated with the basolateral cell borders. The adherens junction protein, cadherin, formed a thin pericellular band at the apical cell junctions between the DPB. In addition, cadherin staining also appeared to extend along the basolateral cell borders in a convoluted pattern. Staining for alpha-catenin, beta-catenin and plakoglobin each showed a nearly identical organization as cadherin. ZO-1 formed a single apical band that was localized between the DPB; however, no basolateral ZO-1 staining was detected. Interestingly, the distribution of ZO-1 was discontinuous around the cell, with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. Positive staining for occludin was not detected in either human or feline corneal endothelium. CONCLUSIONS: The composition and organization of the AJC of corneal endothelium appears to be different from that of classical transport epithelia; these findings may be related to functional differences between these two cell types.     

Cytokeratin patterns in corneal, limbal, and conjunctival epithelium. An immunofluorescence study with PKK-1, 8.12, 8.60, and 4.62 anticytokeratin antibodies.

The authors examined immunofluorescently the specific cytokeratin staining patterns of corneal, limbal, and conjunctival epithelium with PKK-1, 8.12, 4.62, and 8.60 monoclonal anticytokeratin antibodies. Observations were made on unfixed frozen postmortem human tissue. The PKK-1 antibody stain was observed in all layers of corneal epithelium but only in suprabasal layers of limbal and conjunctival epithelium. By contrast, the 8.12 antibody stain was observed only in the superficial layer of corneal epithelium but through all layers of limbal and conjunctival epithelium. The 4.60 antibody stain was seen in focal areas of corneal and limbal epithelium and through all layers of conjunctival epithelium. The 8.60 antibody stain was not present in the three epithelia. These immunofluorescence studies showed unique cytokeratin patterns among layers in corneal, limbal, and conjunctival epithelium.     

In situ localization of cytoskeletal elements in the human trabecular meshwork and cornea

The authors compared cytoskeletal elements of the in situ human trabecular-meshwork cell with in situ human corneal cells using indirect immunofluorescence staining for tubulin and intermediate filaments (vimentin, cytokeratin, and desmin) and NBD-phallacidin staining for f-actin using both fixed frozen and unfixed frozen sections from postmortem eyes. Both f-actin and tubulin were found throughout the cell body of trabecular-meshwork cells, keratocytes, corneal endothelium, and corneal epithelium. The f-actin staining pattern was concentrated at the cell periphery of these four cell types. Vimentin stain was intensely localized in focal areas of the trabecular-meshwork cell, keratocytes, and throughout the corneal endothelium. A general anticytokeratin antibody was intensely localized in corneal epithelium and endothelium. However, PKK-1 anticytokeratin antibody was seen only in superficial layers of corneal epithelium and not in corneal endothelium. The 4.62 anticytokeratin antibody was not observed in either corneal epithelium or endothelium. None of these three cytokeratin antibodies were seen in trabecular-meshwork cells or keratocytes. Desmin stain was not noted in any of these cell types. In general, cytoskeletal staining of unfixed frozen sections showed a similar staining pattern for f-actin and tubulin but a more uniform and intense staining pattern for vimentin and cytokeratin compared with fixed frozen material. The authors conclude that these cytoskeletal stains can differentiate human trabecular-meshwork cells from cells of the cornea in situ.     

Expression of interleukin-1 receptor antagonist in human cornea

The purpose of this study was to confirm the expression of interleukin-1 receptor antagonist (IL-1 Ra) in the human cornea. Four samples of human ex vivo corneal epithelium were obtained from patients undergoing photorefractive keratectomy. RT-PCR was performed using mRNA isolated from the corneal epithelium and oligo-dT primers. PCR was performed on the cDNA products using primers specific for human IL-1 Ra. The PCR products were subcloned and sequenced. Human cornea sections were prepared from eyes enucleated for choroidal melanoma. Immunocytochemistry was performed using goat anti-mouse polyclonal IL-1 Ra IgG and NL-577 conjugated donkey anti-goat IgG. IL-1 Ra mRNA was expressed in all ex vivo corneal epithelium samples as confirmed by sequencing of the PCR products. Immunofluorescence studies revealed strongest expression of IL-1 Ra in the superficial apical layer of corneal epithelium. Expression of IL-1 Ra may represent an endogenous mechanism of down-regulating the effects of epithelial- and tear-derived IL-1α and IL-1β on the intact epithelium in the unwounded cornea and stromal cells after injury.     

连接粘附分子-1在人角膜上皮中的表达

目的：检测连接粘附分子-1（junction adhesion molecule,JAM-1）在正常人角膜上皮各层中的表达及分布特点并与咬合蛋白（occludin）进行比较。 方法：培养人角膜上皮细胞提取细胞总RNA。以逆转录后获得的cDNA为模板PCR扩增目的基因JAM-1及occludin。流式细胞仪检测JAM-1蛋白表达。双重免疫荧光观察JAM-1与occludin在正常人角膜上皮组织的原位表达。 结果：通过RT—PCR在培养人角膜上皮细胞中检测到JAM-1与occludin扩增片段;流式细胞仪检测到JAM-1蛋白表达;双重免疫荧光结果显示occludin染色主要位于表层上皮层细胞之间;而JAM-1荧光染色不仅见于上皮表层,在整个上皮层细胞之间均可见其荧光反应。 结论：Occludin主要位于正常人角膜上皮表层细胞之间,JAM-1在正常人角膜上皮的全层中均有表达。     

Expression of junctional adhesion molecule -1 in human corneal epithelium

AIM: To investigate the expression and distribution of junction adhesion molecule-1(JAM-1) in human corneal epithelium and compare with those of occludin. METHODS: The expression in RNAs of JAM-1 and occludin was revealed by RT-PCR and the presence of protein was analyzed by the FACS method. Double immunofluorescent staining was used to determine the tissue distribution of JAM-1 and occludin in human corneal epithelium. RESULTS: The expression of JAM-1 and occludin was found in cultured human corneal epithelial cells. The double immunofluorescent study showed positive staining for JAM-1 at cell borders in the entire epithelial layer, while relatively extensive staining was seen in the superficial layer, where it coexisted with the expression of occludin. CONCLUSION: JAM-1 is expressed in entire layer of human corneal epithelium encircling the cells.     

Tandem-scanning (confocal) microscopy of the full-thickness cornea

We have utilized a radically new type of optical scanning microscope to study the full-thickness morphology of the intact cornea in an excised human eye bank eye and in freshly sacrificed rabbit eyes in situ. This technology enables one to study corneal morphology layer by layer in extremely thin sections, only disturbing the tissue with an applanating tip. We have demonstrated the cells of the corneal surface, subsurface cells, the topography of Bowman's membrane, corneal lamellae, stromal keratocytes, and the corneal endothelium. The application of this technology lends itself to the in vivo examination of the human cornea. This should aid us greatly in the study of normal morphology, disease states, and the reaction of the cornea in wound healing.     

角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外的所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

Corneal epithelium following intraepikeratophakia

The corneal epithelium in eight patients was surveyed by biomicroscopy and specular microscopy before and after the intraepikeratophakia procedure. Autografting of the epithelium was also examined by electron microscopy. Although the superficial layer of the epithelium was damaged, the wing cell layer remained intact, contributing to the epithelialization of the cornea. Epithelialization of the cornea after surgery was completed in three days in all cases. The epithelium was stained with fluorescein, revealing abnormalities in the superficial layer. This stained superficial epithelium was replaced with new epithelium within seven days, but the new, unstained epithelium exhibited an abnormal pattern when observed under the specular microscope. Abnormally shaped cells, cells with nuclei, and white cells were observed, and normal maturation of the epithelial cells was disrupted. These abnormalities lasted more than eight months in three patients. Patient selection and careful follow-up is advised because of the abnormalities of the epithelium.     

A simple technique for removal of recurring granular dystrophy in corneal grafts.

A 63-year-old man and his 24-year-old son developed recurrences of granular dystrophy in their three corneal grafts within three years after surgery. We regrafted one of the son's corneas. Histologic examination of the corneal button removed during surgery showed an avascular connective tissue layer with the histochemical characteristics of granular dystrophy interposed between the epithelium and Bowman's layer. In the remaining two corneal grafts, we fixed the globe with corneal forceps and removed the opaque dystrophic layer by separating it from the cornea with a spatula. Postoperatively, all three corneal grafts were once more transparent.     

Adaptation of homeostatic ocular surface epithelium to chronic treatment with the opioid antagonist naltrexone

PURPOSE: To determine how ocular surface epithelium adjusts to an increase in cell replication after treatment with the opioid antagonist naltrexone (NTX). METHODS: Adult male rats were given twice daily injections of 30 mg/kg NTX or vehicle for 7 days. Outcomes of NTX administration included DNA synthesis (monitored with BrdU), mitosis (assayed using colchicine), number of cell layers and cell diameter, apoptosis and necrosis, and packing density for the peripheral corneal epithelium, limbus, and conjunctiva. Also, transit time from basal to surface epithelial layers in the peripheral cornea was assessed with [H]thymidine as a marker. RESULTS: DNA synthesis and mitosis in the basal layer of the peripheral corneal epithelium of NTX-treated rats were increased 69% and 85%, respectively, from control levels; no changes in either parameter were recorded in the limbal or conjunctival epithelium (stem cell region). Epithelial thicknesses in the NTX group were increased by 8% to 38% from control subjects, without more cell layers. Packing density in NTX-treated rats was increased from control values by 26% in the basal layer of the limbus and by 12% to 28% in the suprabasal layers of the corneal epithelium, limbus, and conjunctiva. Epithelial cell diameters from corneas of NTX-exposed rats were subnormal in the basal and suprabasal cells of the limbus and conjunctiva. Apoptosis and necrosis were negligible in the epithelium of NTX-treated and control rats. Transit times of peripheral corneal epithelial cells of animals in the NTX group were shortened by 63% from control levels. CONCLUSIONS: These data show that a 1-week treatment with NTX does not induce proliferative pathology or toxicity in ocular surface epithelium, has a minimal effect on stem cell proliferation, and accelerates normal homeostatic processes. Topical application of NTX for stimulation of corneal epithelial wound healing results in no adverse sequelae, thereby supporting the therapeutic role for this drug in the treatment of ocular surface abnormalities.