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.     

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。     

Increased Expression of Intercellular Adhesion Molecule-1,Vascular Cellular Adhesion Molecule-1 and Leukocyte Common Antigen in Diabetic Rat Retina

Purpose: To understand the expression and distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1(VCAM-1)and CD45 (Leukocyte Common Antigen) in the control nondiabetic and various courses of diabetic rats retina. To explore the role of adhesion molecules (AMs) and the adhesion of leukocytes to vascular endothelial cells via AMs in diabetic retinopathy(DR).Methods: Sixty healthy adult male Wistar rats were randomly divided into diabetic groups (induced by Streptozotocin, STZ) and normal control groups. Rats in these two groups were further randomly divided into 3, 7, 14, 30, 90 and 180 days-group, including 5 rats respectively. The immunohistochemical studies of ICAM-1, VCAM-1 and CD45 were carried out in the retinal digest preparations or retinal paraffin sections, and the results were analyzed qualitatively, semi-quantitatively.Results: No positive reaction of VCAM-1 was found, and weak reactions of ICAM-1, CD45 were found in nondiabetic rats retina. The differ     

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.     

Co-localization of junction-associated proteins of the human blood--aqueous barrier: occludin, ZO-1 and F-actin

Recent studies in mouse and rabbit eyes have begun to identify the molecular constituents of tight and adherens junctions that represent the structural equivalent of the blood--aqueous barrier (BAB). These species are commonly used as experimental models to examine the pathobiology of anterior uveitis, an inflammatory condition in which the junctions of the BAB are compromised. Because it was unclear whether major molecular elements of the junctions in these species were the same as those in humans, the goal of this study was to determine if the junction related proteins ZO-1 and occludin are present in normal human ciliary epithelium and iridial vascular endothelium. To determine their presence, sections of human anterior uvea were probed in 14 normal, human, eyebank eyes immunolabelled with antibodies to ZO-1, and occludin, and confocal microscopy was used to examine them. Phalloidin staining for F-actin was also assessed. ZO-1 and occludin were both localized along the apico-lateral surfaces of the non-pigmented ciliary epithelium and the interendothelial clefts of iris blood vessels. In both locations, the distribution of occludin was more focal than seen for ZO-1. ZO-1 was also found along the apical surfaces between the pigmented and non-pigmented ciliary epithelial cell layers. The distribution of these proteins supports the notion that occludin is more specifically associated with tight junctions than is ZO-1 in the normal human BAB. No change in this distribution was found with increasing age. These data are consistent with findings reported previously in rabbit ciliary epithelium and iridial vascular endothelium, indicating the relevance of experimental induced uveitis studies in rabbit, as a model of BAB breakdown in human uveitis.     

可溶性细胞间黏附分子-1在Graves眼病患者外周血中的表达

目的：研究可溶性细胞间黏附分子-1（soluble intercellular adhesion molecule-1，sICAM-1）在活动期Graves眼病（Graves opthalmopathy，GO）患者治疗前后处周血中的表达，探讨sICAM-1在Graves眼病中的作用。方法：42例活动期Graves眼病患者根据伴有或不伴有甲亢分为两组。同时设20正常人对照组。抽取所有患者激素治疗前后的血液，用Elisa方法检测血清中sICAM-1的浓度同正常健康人做对比。结果：GO患者血清中的sICAM-1浓度显著高于对照组，伴甲亢者比不伴甲亢者血清中的sICAM-1浓度高，二者同对照组比较差异有显著意义。激素治疗后比治疗前显著降低，差异有显著意义；但与对照组相比差异无显著性。结论：sICAM-1参与了Graves眼病的发生，其血清浓度同Graves眼病的活动性有关。检测患者血清中的sICAM-1浓度对评估Graves眼病的活动性有一定的临床意义。     

翼状胬肉组织中Bcl-2和ICAM-1的表达

目的:研究翼状胬肉组织中Bcl-2和细胞间粘附分子–1(ICAM-1)的表达,以及Bcl-2和ICAM-1表达间的关系。方法:应用免疫组织化学法检测30例翼状胬肉组织中Bcl-2和ICAM-1的表达,并与正常结膜组织进行比较。结果:翼状胬肉组织中Bcl-2和ICAM-1的表达均明显高于正常结膜组织(χ2=5.92和χ2=6.65,P<0.01);翼状胬肉的临床分型与Bcl-2和ICAM-1的阳性表达有关﹙P<0.05﹚;翼状胬肉组织中Bcl-2和ICAM-1阳性表达间存在非常显著正相关﹙r=0.628,P<0.01﹚。结论:翼状胬肉组织中Bcl-2和ICAM-1的表达增加,在翼状胬肉的发生和发展中可能通过调控细胞增殖和细胞凋亡起着重要的作用。     

ICAM-1和VCAM-1在糖尿病视网膜病变中的作用

糖尿病会造成视网膜很多异常改变,其中包括细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)和血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)的上调,这显示了炎症反应在糖尿病视网膜病变(diabetic retinopathy,DR)发展中发挥了一些作用。本文针对粘附分子ICAM-1和VCAM-1在DR的血管内皮细胞的损伤及其在炎症反应中发挥的重要作用进行了简要分析。     

细胞间黏附分子-1在糖尿病大鼠视网膜中的表达及意义

目的探讨细胞间黏附分子-1(ICAM-1)在糖尿病大鼠视网膜中的表达及ICAM-1多克隆抗体(ICAM-1Pab)的治疗作用。方法Wistar大鼠40只随机分成4组正常对照组(CON)、糖尿病组(DM)、糖尿病(ICAM-1PAb)组(DM(ICAM-1PAb))和糖尿病(生理盐水)组(DM(saline)),每组10只。大鼠腹腔注射链脲佐菌素(STZ)诱发糖尿病模型。DM(ICAM-1PAb)组右眼结膜下注射ICAM-1PAb,连续1周。制作大鼠视网膜冰冻及石蜡切片分别行免疫组化SP染色和常规HE染色,对SP染色结果作计算机图像分析。结果ICAM-1在大鼠视网膜血管内皮细胞胞膜有表达。SP染色结果采用计算机图像分析系统处理。其中DM组分别与CON组、DM(ICAM-1PAb)组比较有显著性差异(P<0.01)。DM组视网膜血管内有较多附壁的白细胞。结论早期糖尿病大鼠视网膜ICAM-1表达上调,ICAM-1PAb通过阻断ICAM-1,抑制白细胞黏附,改善糖尿病大鼠视网膜微循环。     

甲状腺相关眼病患者血清sICAM-1、sVCAM-1及微小RNA-146a的表达

目的：探讨甲状腺相关眼病( thyroid associated ophthalmopathy,TAO)患者血清可溶性细胞间黏附分子-1( serum soluble intercellular adhesion molecules -1, sICAM-1)、可溶性血管细胞黏附分子-1( soluble vascular cell adhesion molecule-1,sVCAM-1)及外周血单个核细胞( peripheral blood mononuclear cells, PBMC )中微小 RNA-146 a的表达及其意义。
　　方法：选取2014-06/2015-12在我院诊治的 TAO患者37例( TAO组)、甲状腺功能亢进不伴有眼病患者40例(非眼病组)、健康人群30例(健康组),检测各组血清sICAM-1、sVCAM-1及PBMC中微小RNA-146 a表达。结果：TAO组的血清 sICAM-1(366.14±67.28μg/L)、sVCAM-1(211.07±27.45μg/L)水平显著高于非眼病组(286.62±51.09μg/L、179.83±25.09μg/L)和健康组(234.51±38.969μg/L、164.51±22.57μg/L),差异有统计学意义(P<0.05),TAO组PBMC中微小RNA-146a (0.071±0.016)低于非眼病组(0.381±0.084)和健康组(1.105±0.216),差异有统计学意义(P<0.05);非眼病组的血清sICAM-1、sVCAM-1水平显著高于健康组(P<0.05),非眼病组PBMC中微小RNA-146a表达低于健康组( P<0.05)。轻度组的血清sICAM-1、sVCAM-1水平显著低于中重度组和极重度组( P<0.05),轻度组 PBMC中微小RNA-146 a表达高于中重度组和极重度组( P<0.05);中重度组的血清sICAM-1、sVCAM-1水平显著低于极重度组( P<0.05),中重度组 PBMC中微小 RNA-146 a表达高于极重度组(P<0.05)。
　　结论：血清 sICAM-1、sVCAM-1在 TAO 患者中高表达, PBMC中微小RNA-146 a在TAO患者低表达,并且与患者的病情程度有关。     

VCAM-1和MCP-1在实验性变态反应性脑脊髓炎大鼠模型视神经内的表达

目的观察实验性变态反应性脑脊髓炎大鼠模型视神经内血管内皮细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)和单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)的表达变化,为研究视神经炎(optic neurikis,ON)的发病机制提供新的实验依据。方法用豚鼠脑脊髓匀浆和完全弗氏佐剂诱导Wistar大鼠,建立实验性变态反应性脑脊髓炎模型,将50只Wistar大鼠随机分为对照组和免疫后7d、12d、18d、25d组,通过行为学观察对各组进行神经功能评分,通过HE染色法观察各组视神经的病理形态学改变,采用免疫组织化学染色,观察各组大鼠VCAM-1和MCP-1的表达情况。结果神经功能评分显示,与对照组相比,免疫组大鼠随着时间的延长,神经功能评分增加,至18d,达到最大的1.9分。HE染色结果显示,对照组大鼠视神经组织结构正常,而免疫组大鼠视神经组织出现结构的异常,免疫后18d,病变程度达最高峰,这和功能评分的结果相符。免疫组织化学结果显示,对照组没有VCAM-1和MCP-1表达;免疫后7d,VCAM-1和MCP-1开始表达,阳性细胞数分别为11.45±8.65和47.86±9.65;免疫后12d,VCAM-1和MCP-1表达均进一步增加,阳性细胞数分别为17.34±5.76和86.45±5.61,与对照组比较差异均有统计学意义(均为P<0.05);免疫后18d,VCAM-1和MCP-1表达达到高峰,阳性细胞数分别为32.53±9.56和153.56±12.47,与对照组比较差异均有显著统计学意义(均为P<0.01);免疫后25d,VCAM-1和MCP-1表达减少,阳性细胞数分别为21.23±7.58和116.49±8.83。结论 VCAM-1和MCP-1参与了ON的发病过程,ON是一种免疫因素介导的疾病。     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

ICAM-1含量变化与兔角膜移植排斥反应的实验研究

目的：观察细胞间粘附分子-1（intercellular adhesionmolecule-1,ICAM-1）含量变化与角膜移植排斥反应的关系。 方法：利用缝线诱发新西兰白兔角膜新生血管（corneal neovascularization,CNV）后,制作穿透性角膜移植（penetrating keratoplasty,PKP）模型4组,术后记录移植排斥指数（rejection index,RI）;记录植片存活时间;应用酶联免疫吸附（ELISA）双抗体夹心法检测房水及外周血中sICAM-1的含量,免疫组织化学染色法观察ICAM-1的表达。 结果：排斥组植片平均存活12.4±1.3d;正常房水和血清中均可检测到少量的sICAM-1,它们的浓度分别为（16.6±3.6）ng/L（,95.2±6.3）ng/L。术后排斥组房水及外周血中sICAM-1含量即升高,至排斥反应前达最高水平分别为（53.9±19.2）ng/L,（378.8±30.6）ng/L,在观察期内维持高水平,排斥反应发生时,免疫组化染色ICAM-1呈强阳性表达。 结论：ICAM-1在角膜移植免疫排斥反应中发挥重要作用,术后外周血中sICAM-1浓度变化对排斥反应的发生有一定预测和早期诊断作用。     

Knockdown of IQGAP-1 Enhances Tight Junctions and Prevents P. aeruginosa Invasion of Human Corneal Epithelial Cells

ABSTRACT

Purpose

To determine the role of IQ-domain GTPase-activating protein1 (IQGAP-1) in tight junctions of human corneal epithelial cells (HCECs) and its effect against P. aeruginosa (PAK) invasion.     

Distribution of intercellular adhesion molecule-1 on leukocytes and corneal endothelium after endotoxin stimulation in rats

After stimulation with Salmonella typhimurium endotoxin, the intercellular adhesion molecule-1 (ICAM-1) was studied on the corneal endothelium and associated leukocytes in rats using immunoscanning electron microscopy. Two hundred g of the endotoxin was injected in Lewis rats. The corneae were excised at 0-h and 16-h-postinjection time (n = 5, respectively). The corneae were prepared in hypothermic University of Wisconsin (UW) solution for immunoscanning electron microscopy. Histotopographical examination visualized ICAM-1 antigen on cytoplasmic processes of the corneal endothelium, arranged along microfolds, especially at the peaks. In the leukocytes, ICAM-1 was located primarily in morphologically non-specialized domains of the cell body surface, and only rarely scattered on the surface of microvillar projections. We concluded that the endotoxin stimulation can increase ICAM-1 in both corneal endothelium and associated leukocytes. Increased ICAM-1 may be an important factor for the leukocytes to form clustering and adhering to the corneal endothelium.     

细胞间黏附分子与角膜移植免疫

角膜移植免疫是多种免疫细胞和免疫分子参与的复杂的免疫反应过程。近来研究表明ICAM1在角膜移植免疫排斥反应的发生机制中起重要作用。ICAM1协同抗原递呈,促使免疫细胞在植片聚集,触发和促进移植排斥反应的发生和发展,引发粒细胞聚集于角膜基质层,导致组织损伤、破坏、角膜新生血管化使移植失败。目前研究抗ICAM1药物抑制角膜移植排斥反应已进入实验阶段。就细胞间黏附分子与角膜移植免疫的研究现状进行综述。     

角膜移植排斥反应中ICAM-1含量的变化

目的:探讨细胞间粘附分子-1(intercellular adhesion mole-cule-1,ICAM-1)在血液和房水中的含量变化与角膜移植排斥反应的关系。方法:缝线法诱发兔角膜新生血管化(corneal neovascular-ization,CNV)后,制作穿透性角膜移植(penetrating kerato-plasty,PKP)模型4组,A组:正常对照组;B组:自体穿透性角膜移植组;C组:同种异体穿透性角膜移植组;D组:新生血管化穿透性角膜移植组。术后记录植片存活时间和移植排斥指数(rejection index,RI);ELISA法检测房水及外周血中可溶性ICAM-1(sICAM-1)的含量,免疫组化法观察ICAM-1的表达。结果:B、C组在观察期内未见排斥反应发生。D组发生排斥反应,角膜植片平均存活12.4±1.3d,术后房水及外周血中sICAM-1含量即升高,至排斥反应前达最高水平分别为53.9±19.3ng/L,378.8±30.6ng/L,在排斥反应发生时,免疫组化染色发现角膜组织中ICAM-1呈强阳性表达。结论:血液和房水中的ICAM-1含量可以是角膜移植免疫排斥反应发生的指标,术后监测ICAM-1浓度变化对排斥反应的发生有一定预测和早期诊断作用。