应用激光扫描共聚焦显微镜观察晶体上皮细胞中的游离钙

目的用钙的荧光指示剂ｆｌｕｏ－３和激光扫描共聚焦显微镜（ｌａｓｅｒｓｃａｎｎｉｎｇｃｏｎｆｏｃａｌｍｉｃｒｏｓｃｏｐｙ，ＬＳＣＭ）观察大白鼠的晶体上皮细胞（ｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌ，ＬＥＣ）中的游离钙。方法ｆｌｕｏ－３着染细胞后，应用ＬＳＣＭ观察细胞内ｆｌｕｏ－３与钙螯合后的荧光分布，最后用钙离子载体Ａ２３１８７和重金属离子Ｍｎ＋＋作校正，将ｆｌｕｏ－３与钙结合显示的荧光强度转换为［Ｃａ＋＋］ｉ值。结果ＬＥＣ中的游离钙主要分布在细胞核中。大白鼠晶体上皮细胞中的［Ｃａ＋＋］ｉ为２５９．７９±４９．２４ｎｍｏｌ／Ｌ。结论ｆｌｕｏ－３与ＬＥＣ孵育后能着染细胞。用ＬＳＣＭ能直观地观察细胞内钙的分布，并能通过校正得到细胞内游离钙的绝对值。这种成熟的方法，为进一步对ＬＥＣ中游离Ｃａ＋＋的研究提供技术准备     

生长因子与晶体上皮细胞

白内障在致盲眼病的发生率中占首位。随着白内障囊外摘除术及人工晶体植入术的广泛开展 ,术后患者短期内视力可提高至较可观的水平。但术后 4 0 %的成年人和几乎 10 0 %的儿童发生晶体后囊混浊 (后发性白内障 ) ,导致视力再度下降。残留的晶体上皮细胞增殖是其形成原因。近年来的研究表明生长因子在促进晶体上皮细胞增殖、迁移、分化方面发挥着重要作用〔1～ 3〕。本文就生长因子与晶体上皮细胞的关系作一综述。一、生长因子概述生长因子是机体在有丝分裂原作用下 ,由体细胞产生的 ,在体内和体外对细胞的生长具有促进或抑制作用的物质。除促…     

晶体上皮细胞单克隆抗体的动物实验研究：第一部分晶体上皮细胞…

试图以免疫学方法制备特异性晶体上皮细胞膜单克隆抗体来抑制晶体上皮细胞的生长，以探索防止后发障的发生。共分四部分：１。晶体上皮细胞的培养；２，晶体上皮细胞膜单克隆抗体的制备；３，抗体的免疫学检测，４，所制备的单克隆抗体对动物和人的晶体上皮细胞的抑制作用。报告家兔晶体上细胞的原代和继代培养的方法并进行讨论。     

人工晶体植入术后晶体上皮细胞的增殖和反应

人工晶体植入术后晶体上皮细胞的增殖和反应天津医学院世界人工晶体中国天津培训中心李筱荣，张惠敏，袁佳琴日本藤田保健卫生大学病院眼科张永品白内障是目前致盲的首要病因，囊外摘除和人工晶体植入无疑是现代白内障治疗的主要方法。尽管手术技巧、医疗器械和医学材料科...     

IL-1和TNF-α对晶体上皮细胞增殖及胞浆内游离Ca^2＋浓度的影响

目的研究白细胞介素１（ｉｎｔｅｒｌｅｕｋｉｎ１,ＩＬ－１）、肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα,ＴＮＦ－α）对体外培养的小牛晶体上皮细胞（ｂｏｖｉｎｅｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌ,ＢＬＥＣ）增殖和胞浆内游离Ｃａ２＋浓度的影响,探讨它们在后囊混浊形成中的作用及其影响细胞增殖的机理。方法采用ＭＴＴ比色法测定ＩＬ－１及ＴＮＦ－α对细胞增殖的影响,用荧光指示剂Ｆｕｒａ－２测定它们对胞浆内游离Ｃａ２＋的影响。结果ＩＬ－１１０２～１０５ｎｇ／ｍｌ、ＴＮＦ－α１０２～１０４Ｕ／ｍｌ可显著促进细胞增殖,并增加胞浆内游离Ｃａ２＋浓度。结论推测ＩＬ－１、ＴＮＦ－α参与了白内障术后的后囊混浊形成;它们影响细胞增殖的作用机理之一是通过胞浆内游离Ｃａ２＋。     

晶体上皮细胞单克隆抗体的动物实验研究：第二部分 晶体上皮细胞膜单克…

通过将免疫小鼠的脾细胞与ＨＧＰＲＴ缺陷的ＮＳ－１骨髓瘤细胞进行细胞融合，并经ＨＡＴ培养液对融合的杂交瘤细胞进行选择培养，再经二次限界稀释法对此杂交瘤细胞进行克隆化。     

人工晶体植入术后晶体上皮细胞增殖的抑制

人工晶体植入术后晶体上皮细胞增殖的抑制天津医科大学世界人工晶体中国天津培训中心李筱荣，孙慧敏，袁佳琴日本藤田保健卫生大学病院眼科张永品囊外摘除和人工晶体植入术是当今白内障治疗的主要方法，但术后后囊混浊、人工晶体偏位及纤维蛋白反应等仍是未解决的并发症，...     

添加晶体皮质提高晶体上皮细胞原代培养成功率

目的介绍一种提高晶体上皮细胞原代培养成功率的方法。方法基本步骤为将一晶体前囊膜剪碎后加纯胎牛血清０．５ｍｌ和冰冻过的新生牛晶体皮质０．５～１ｍｌ，密封培养２４～４８ｈ后添加约３ｍｌ含１０％胎牛血清的ＤＭＥＭ培养液继续培养。结果用该方法原代培养新生牛、幼年兔和人胚胎及角膜移植后的中青年供体标本，成功率为１００％，老年白内障手术取出标本成功率为８７％。结论添加晶体皮质可提高晶体上皮细胞原代培养成功率。     

晶体上皮细胞单克隆抗体的动物实验研究第一部分晶体上皮细胞的培养

试图以免疫学方法制备特异性晶体上皮细胞膜单克隆抗体来抑制晶体上皮细胞的生长，以探索防止后发障的发生。共分四部分：１．晶体上皮细胞的培养；２．晶体上皮细胞膜单克隆抗体的制备；３．抗体的免疫学检测；４．所制备的单克隆抗体对动物和人的晶体上皮细胞的抑制作用。报告家兔晶体上皮细胞的原代和继代培养的方法并进行讨论。     

猕猴白内障晶体上皮细胞超微结构研究

目的 探讨猕猴白内障晶体上皮细胞的超微结构,为将猕猴白内障作为人类白内障理想的动物模型提供病理学研究资料。方法 采用透射电镜对猕猴正常眼和猕猴白内障眼的晶体上皮细胞超微结构进行观察。结果 猕猴白内障眼晶体上皮细胞发生明显的变化：线粒体肿胀、空化、嵴消失,甚至形成凹空细胞;细胞水肿,细胞质溶解,甚至细胞崩解破坏;细胞核固缩、畸形,核膜间隙与核孙消失,核内异染色质浓集、周边化。结论 猕猴白内障上皮细胞     

In vivo three-dimensional confocal laser scanning microscopy of the epithelial nerve structure in the human cornea

Purpose Evaluation of a new method for in vivo visualization of the distribution and morphology of human anterior corneal nerves. Method The anterior cornea was examined to a depth of 100 μm in four human volunteers with a confocal laser scanning microscope (CLSM) using a Rostock Cornea Module (developed in house) attached to a Heidelberg Retina Tomograph II (Heidelberg Engineering, Germany). Optical sections were digitally reconstructed in 3D using AMIRA (TGS Inc., USA). The scanned volumes had a greatest size of 300×300×40 μm and voxel size of 0.78×0.78×0.95 μm. Results The spatial arrangement of the epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. The 3D-reconstruction of the volunteers’ corneas in combination with the oblique sections gave a picture of the nerves in the central human cornea. Thin nerves run in the subepithelial plexus aligned parallel to Bowman’s layer and are partially interconnected. The diameter of these fibres varied between 1.0 and 5 μm. Thick fibres rose out of the deeper stroma. The diameter of the main nerve trunks was 12±2 μm. Branches penetrating the anterior epithelial cell layer could not be visualized. Conclusions 3D-CLSM allows analysis of the spatial arrangement of the anterior corneal nerves and visualization of the epithelium and keratocytes in the living human cornea. The developed method provides a basis for further studies of alterations of the cellular arrangement and epithelial innervation in corneal disease. This may help to clarify alterations of nerve fibre patterns under various clinical and experimental conditions.     

Ocular mucin visualization by confocal laser scanning microscopy

PURPOSE: To describe a new method of visualizing human conjunctiva goblet cell mucin secretion by using a combination of impression cytology and laser scanning microscopy. METHODS: By assembling a Z-stack of confocal microscopy images taken from human impression cytology samples, we obtained 3-dimensional information about the release and spread of goblet cell secretions above the conjunctival surface. After reconstruction and rendering of these images, analysis of the shape and spreading characteristics of the mucins permitted definition of the following parameters related to goblet cell secretion: mucin cloud height as the height of the top of the cloudlike mucin structure visible above the goblet cell opening and spread mucin thickness, which is the thickness of the mucin layer distributed over the surface of the conjunctiva. RESULTS: Several impression cytology samples of control and muco-deficient patients have been analyzed through the confocal laser scanning technique, and significant differences between these groups were found. Mucin cloud height and spread mucin thickness values for controls were 8.81 +/- 4.00 and 2.77 +/- 1.00 microm, respectively (n = 25). These values decreased by approximately 70% and 40%, respectively, for moderately mucodeficient subjects and by 84% and 48% for those with severe mucodeficiency. Classifying those individuals having mucin-related pathology may thus be possible on the basis of application of these techniques. CONCLUSIONS: In summary, we present a method of objectively identifying those individuals with problems associated with either a lack of mucins or a reduction in the distribution of these proteins over the ocular surface.     

Three-dimensional confocal laser scanning microscopy of the corneal nerve structure

PURPOSE: The aim of this study was the evaluation of a technology for in vivo visualization of distribution and morphology of corneal nerves by means of 3D confocal laser scanning microscopy (3D-CLSM). METHOD: The anterior corneas of four human volunteers were examined by an in-house developed confocal laser scanning microscope based on a commercially available instrument (Heidelberg Retina Tomograph II, Heidelberg Engineering GmbH, Germany). Raw stacks were converted using ImageJ (NIH, USA) for 3D-reconstruction using AMIRA 3.1 (TGS Inc, USA). RESULTS: The spatial arrangement of epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. After 3D-reconstruction of volunteers' corneas, volume rendering and selective oblique sections have been done to demonstrate the nerves in the central human cornea. 3D-imaging shows thick nerve bundles rising out of the deeper stroma. The nerves further divide, resulting in fibers that are arranged parallel to Bowman's layer and are partly interconnected. Branches rising up to the superficial cell layer cannot be visualized. Wound healing following refractive surgery can be evaluated. CONCLUSIONS: 3D-CLSM allows in vivo visualization and analysis of the spatial arrangement of the epithelium, nerves and keratocytes of the human cornea. The developed method provides a basis for further studies on the alterations of the cellular arrangement and epithelial innervation in corneal diseases. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.     

丝状角膜炎的共聚焦显微镜观察

目的：应用活体激光共聚焦显微镜（ IVCM）观察丝状角膜炎患者丝状物的组成结构及位置特点,分析角膜丝状物的形成机制。方法收集深圳市眼科医院2014年6月至2016年1月收治的丝状角膜炎患者48例（60只眼）,详细记录患者的病史和相关检查。采用共焦显微镜观察角膜丝状物的组成结构和角膜丝状物附着处的角膜结构。结果活体激光共聚焦显微镜检查显示,丝状物起自前弹力层,由上皮细胞、炎性细胞、黏液、螺旋状条形核心构成,核心结构中亦含有上皮细胞、炎性细胞及纤维状组织。结论丝状角膜炎丝状物的共聚焦表现特点可以为临床治疗方法的选择提供影像的参考依据。     

配戴夜戴型角膜塑形镜两年后角膜激光共聚焦显微镜观察

目的利用激光共聚焦显微镜观察配戴夜戴型角膜塑形镜2年以上患者的角膜组织有无变化。方法回顾性病例研究。选择2009年1月至2012年12月于南京军区总院眼科就诊且配戴夜戴型角膜塑形镜2年的患者30例（60眼）作为戴镜组,未配戴任何形式角膜接触镜的角膜正常患者30例（60眼）作为对照组。对2组对象进行角膜中央部激光共聚焦显微镜观察（观察组摘镜2～3 h后）。数据采用独立样本t检验进行分析。结果配戴角膜塑形镜后角膜形态可发生明显变化,上皮至前基质层可见皱褶,上皮下神经丛疏密不均、扭曲,基质神经及内皮细胞形态正常。上皮下神经丛的分布比对照组稀疏,戴镜组的上皮细胞计数为（4818±148）cells/mm2,前基质细胞计数为（1345±154）cells/mm2,后基质细胞计数为（799±76）cells/mm2,内皮细胞计数为（3025±193）cells/mm2,与对照组相比2组差异无统计学意义。结论长期配戴夜戴型角膜塑形镜对上皮下神经丛数量及眼表形态有影响,对内皮细胞及基质细胞无明显影响。     

Three dimensional analysis of the retinal vasculature using immunofluorescent staining and confocal laser scanning microscopy.

AIM: To undertake a qualitative and quantitative analysis in three dimensions of the human retinal vasculature. METHOD: Fixed and excised whole retinas were permeabilised and subjected to immunofluorescent staining for blood vessel components followed by confocal laser scanning microscopy. Single projection and stereoimages were constructed using computer software. XZ sections through the retina were constructed and the vasculature analysed using appropriate software. RESULTS: Immunofluorescent staining with no discontinuities was present in vessels of all sizes, the confocal images of the capillary network being free of out of focus blur at all depths. Quantitative analysis of XZ sections confirmed the qualitative impression of sharp delineation of the deep retinal capillary plexus, an absence of laminar arrangement of capillaries within the inner retina, and a truncated cone of capillaries around the foveal avascular zone (FAZ) wherein the superficial capillaries approached the FAZ more closely than those in the deeper retina. CONCLUSION: Immunofluorescent staining of the retina and confocal laser scanning microscopy were shown to be useful in analysing accurate three dimensional reconstructions of the normal retinal vasculature without affecting overall tissue architecture.     

佩戴角膜接触镜后角膜变化的激光共焦显微镜观察

目的 应用激光共焦显微镜对长期佩戴角膜软性接触镜的患者活体角膜的组织结构变化进行观察.方法 用激光共焦显微镜对长期佩戴角膜软性接触镜的15例患者进行检查,并选择未戴角膜接触镜者11例进行对照,对两组结果进行比较.结果 1.佩戴角膜软性接触镜组与对照组相比,基底上皮细胞密度减少,为3705.00±447.62个/mm2(P＜0.05),角膜上皮层厚度变薄,为54.3±8.44μm(P＜0.05),并有部分剥脱.2.佩戴角膜软性接触镜组角膜内出现白色点状物,朗汉氏细胞数目多,成树枝状改变.3.角膜软性接触镜组与对照组相比神经纤维数量及密度无明显变化(P＞0.05),但曲折度增加,有分支出现(P＜0.05).结论 佩戴角膜软性接触镜,角膜组织可发生一系列改变,激光共焦显微镜与传统光学共焦显微镜相比,图象清晰,深度定位准确,在疾病的早期诊断、治疗和研究中将起重要作用.