从开矿和超微结构对干燥保存角膜内皮细胞活性的评价

为从形态和超微结构上观察干燥保存角膜内皮细胞是具有活性，应用０．２％茜素红和０．２５％台寺对干燥保存不同时间的兔、猴和人角膜内皮细胞分别进行活性染色，并与正常新鲜才作对照。同时，在电镜下对干燥保存角膜及其新鲜标本进行超微结构观察。结果，新鲜角膜六角形细胞边界清楚，核呈淡蓝色；干燥保存角膜，六角形细胞边界不清，但核的染色及分布密度一新鲜者几无区别。电镜下，干燥保存角肛六角形镶嵌状细胞边界看不清，但     

用茜素红—锥蓝联合染色法对兔角膜内皮细胞活性的评价

利用茜素红—锥蓝联合染色法,对正常新鲜角膜和干燥保存角膜内皮细胞进行染色。正常新鲜角膜内皮细胞为六角形,边界呈桔红染色,核淡蓝色、蚕豆状;而保存角膜内皮细胞则仅可看到淡蓝色、蚕豆状细胞核。提示保存角膜与新鲜角膜内皮细胞染色所见有明显区别。     

保存兔角膜内皮细胞形态学的研究

保存不同时间的兔角膜内皮细胞,SEM:六角形边界不清,仅可见细胞轮廓,细胞有水肿,细胞间连接处有空泡。TEM:核膜尚完整,胞浆内有空泡,还有细胞器存在。术后48天及11个月植片,可见六角形边界的内皮细胞,呈镶嵌型排列,细胞核膜完整,细胞器丰富。提示:保存角膜内皮细胞形态学上的改变在活体房水中的再水合作用或培育下是可以恢复的。     

不同染色剂对兔眼角膜内皮细胞影响的研究

目的 观察并比较荧光素钠、吲哚靛青绿，台盼蓝，亚甲蓝，龙胆紫5种不同的前囊膜染色剂对兔眼角膜内皮细胞的影响。方法 新鲜离体兔眼角膜内皮滴入各实验试剂，0.25%台盼蓝，0.2%茜素红双重染色，光镜下观察角膜内皮细胞的活性，计数内皮细胞的死亡数，透射电镜检查，观察角膜内皮细胞超微结构的变化。结果 1%亚甲蓝组角膜内皮细胞失去正常的六边形结构，可见较多着色死亡细胞，细胞的死亡率与对照组相比，有统计学差异（P〈0.01）。电镜下，1%亚甲蓝组可观察到内皮细胞的胞膜不完整，胞浆内有空泡形成等变化。结论 1%亚甲蓝溶液对兔眼角膜内皮细胞的损伤作用。     

真空冷冻干燥保存兔角膜内皮细胞酶的实验研究

目的　通过光/电镜酶组织细胞化学方法测定兔角膜内皮细胞酶活性,探讨真空冷冻干燥保存法(冻干法)对于兔角膜内皮细胞活性的影响。方法　按随机及配对原则将兔眼分为新鲜对照组、冷冻对照组、冻干实验组。对冷冻组进行角膜深低温冷冻保存。对冻干组进行真空冷冻干燥保存。将冷冻及冻干保存的兔角膜片分别复水及复温后,与新鲜的角膜片同时进行三磷酸腺苷酶(AT Pase)、琥珀酸脱氢酶(SDH)的光镜及电镜酶组织细胞化学染色,观察三组酶的活性是否有差异。结果　在新鲜组、冷冻组、冻干组中,ATPase及DSH的酶组织化学染色在光镜观察下均呈阳性反应。三组样本内皮细胞ATPase和SDH酶的平均积分光密度值(IOD)差异具有统计学意义(P <0 .0 1)冻干组活性较低。电镜酶组织细胞化学染色见三组样本角膜内皮细胞膜上均有电子致密颗粒沉着。结论　真空冷冻干燥保存法可使离体角膜内皮细胞保持一定的活性。与新鲜及冷冻保存的兔角膜相比,活性较低。但可以推测冻干法有望成为一种新的角膜长期保存方法     

应用跨角膜内皮细胞膜电位评价角膜保存液的保存效果

目的角膜透明性的维持决定于角膜内皮细胞的功能，内皮细胞膜上的钠泵不断地进行着离子转运，从而维持角膜的正常水合状态，这种由离子转运所产生的电位差（跨角膜内皮细胞膜电位）直接反映着角膜内皮细胞的功能状态。本研究通过测定跨角膜内皮细胞膜电位评价角膜保存液的保存效果。方法使用Ｕｓｓｉｎｇ灌流小室分别测定了新鲜兔去上皮角膜片和保存于ＭＫ液、Ｋ液和高钾保存液不同时间的兔去上皮角膜片的跨角膜内皮细胞膜电位。结果新鲜兔角膜跨角膜内皮细胞膜电位值是０．４～０．５±０．１ｍＶ；随着保存时间的延长，３种保存液所保存的角膜片电位值均出现下降，二者呈线性关系。当保存至ｄ１２时，高钾保存液所保存的角膜其电位值最接近新鲜角膜。结论高钾保存液维持角膜内皮细胞活性的作用优于ＭＫ液和Ｋ液。     

100例儿童角膜内皮细胞分析

目的：观察４～１０岁儿童中央区角膜内皮细胞密度及形态学特征。方法：应用非接触型角膜内皮显微镜进行观察。结果：儿童中央区角膜内皮细胞平均密度及六角形细胞百分率明显高于成年人（Ｐ＜０．０１），平均细胞面积明显小于成年人（Ｐ＜０．０１）；远视性屈光不正儿童内皮细胞平均密度高于非远视者（０．０１＜Ｐ＜０．０５）。结论：儿童角膜内皮细胞在正常人群中平均密度最高、形态最佳，并具有最大的自身扩潜能力；随着年龄增长，角膜内皮细胞将发生一系列规律性变化。     

超声乳化白内障摘除术的角膜内皮细胞观察

用角膜内皮细胞分析仪对５８例（６９眼）超声乳化白内障摘除术行内皮细胞观察。手术前和术后３－６月的内皮细胞密度分别为２５８１．９±３４３．２个／ｍｍ２和２２７４．６±４０６．７个／ｍｍ２，细胞丧失率为１１．９％。平均细胞面积分别为３４２．１８±１３９．２１μｍ２和３９３．０３±２０３．４５μｍ２，变异系数分别为４０．６８％和５１．７％，六角形细胞比率分别为５５．３±１３．６％和４６．４±１７．１％，均有显著性差异。角膜厚度分别为５６１．８±３６．７μｍ和５７０．１±３４．４μｍ，无显著性差异。术后矫正视力０．５以上者占８９．７％，１．０以上者占６１．８％。     

角膜中期保存法

应用角膜中期保存法（Ｋ液，ＣＳＭ，Ｄｅｘｓｏｌ及Ｏｐｔｉｓｏｌ）对８５只动物角膜及５只人角膜进行保存试验，保存时间为３、８、１０、１４及２０天。应用０．２５％锥蓝及０．２％茜素红双染色，保存１０天以内的动物及人角膜内皮完整性良好，１４天的供体角膜部分出现水肿，内皮细胞丢失率为５－１０％，保存２０天的供体角膜在部分出现水肿，内皮细胞丢失率在３０－４０％。对保存８及１０天之动物角膜进行电镜检查，其超微     

波动的压力对兔角膜内皮细胞的影响

目的　观察波动的压力对角膜内皮细胞的影响。方法　将体外培养的第一代兔角膜内皮细胞分为两组:Ａ组为压力波动组（压力设置为:１５ｍｍＨｇ～２５ｍｍＨｇ～２０ｍｍＨｇ～１０ｍｍＨｇ,每个压力持续６ｈ;１ｋＰａ＝７．５ｍｍＨｇ）;Ｂ组为３０ｍｍＨｇ压力组;Ｃ组为无压力组。三组细胞分别培养２４ｈ。免疫细胞化学染色法鉴定原代角膜内皮细胞形态;台盼蓝-茜素红联合染色检测细胞活性,ＨＥ染色观察细胞形态;Ｗｅｓｔｅｒｎ-ｂｌｏｔｔｉｎｇ检测细胞中Ｂｃｌ-２和Ｐ５３蛋白的表达水平。结果　获取的所有细胞证实为角膜内皮细胞表型,无角膜上皮细胞及基质细胞污染。三组细胞分别培养２４ｈ后,经台盼蓝-茜素红染色和ＨＥ染色证实:两个压力培养组的细胞活性较无压力培养组明显下降,其中压力波动组的细胞活性低于３０ｍｍＨｇ压力组。同时无压力组、３０ｍｍＨｇ压力组和压力波动组细胞中Ｐ５３蛋白的相对表达量分别为０．１５０±０．００５、０．２５３±０．０１４、０．６７０±０．０１９,差异有统计学意义（Ｐ＜０．０５）。结论　证实了高压力及非生理性波动的压力对角膜内皮细胞均具有损伤作用。     

Endothelial cell damage in human and rabbit corneas stored in K-Sol without antioxidants.

Human and rabbit corneas were stored at 4 degrees C in K-Sol with and without antioxidants (ascorbic acid, reduced glutathione, alpha-tocopherol, and retinol acetate) for two to three weeks. All the corneas were then examined visually and by scanning electron microscopy. They appeared clear and slightly oedematous. Scanning electron micrographs were used to grade corneal endothelial cell morphology in a masked manner in terms of cell shape, cell borders, cell swelling, and apical holes. Corneas stored in K-Sol without antioxidants showed changes in cell shape, cell borders, and apical holes. Human corneas showed more morphological changes than rabbit corneas. The results suggest that antioxidants in K-Sol have an important role in the preservation of endothelial cell morphology.     

HAI EB-3000XYZ型眼库内皮显微镜对供体角膜内皮的评估

目的 评价HAI EB-3000XYZ型眼库内皮显微镜对供体角膜内皮细胞检测的可靠性。方法 选取供体角膜材料35例,年龄11～50岁;按供体死亡时间分为3组:A组≤6h;B组7～12h;C组>12h。术前分别对3组行内皮显微镜检查,PKP术后对剩余角膜环行台盼蓝-茜素红联合染色。对两者所测得的内皮细胞密度进行比较;对内皮显微镜测得的内皮细胞六边形比例和染色测得的活性率行相关分析。结果 在A、B两组中,内皮显微镜检测得的内皮细胞密度与染色结果无明显差别;C组两者之间的差别有显著统计学意义(P=0.000)。3组的内皮细胞六边形比例与活性率之间均没有明显的相关性。结论 HAI EB-3000XYZ型眼库内皮显微镜对死亡时间在12h以内的供体角膜内皮细胞密度检测较为准确,而对死亡12h以上的供体材料的检测则存在一定误差。     

Organ-culture preservation of human corneas

Human corneas were preserved up to 40 days in a modified tissue culture medium at 31 degrees C. The corneal endothelium was examined by light microscopy before and after culture. After staining with trypan-blue the number of dead cells was counted and by swelling of the intercellular borders in a 1.8 per cent sucrose solution the cellular mosaic was observed. A loss of endothelial cells was found varying from 0-30 per cent. During culture the stroma increased considerably in thickness. Prior to transplantation the cornea was thinned during 24 h in culture medium containing 5 per cent Dextran T500. The combination of the organ culture procedure and the evaluation of the endothelium enables preservation of human corneas for at least 30 days. In addition the quality of the endothelium is guaranteed and the transport of corneas can be carried out at room temperature.     

Assessment of endothelial cell density in bovine corneas after osmotically induced dilation of intercellular spaces

Corneal endothelial cells were visualized in intact bovine eyes by specular microscopy of unstained and stained cell borders. The corneas were excised, and flat corneal preparations were studied by reflected and transmitted light. In the excised corneas, the cell borders were visualized by osmotically induced dilation of intercellular spaces, alizarin red staining, and a combination of alizarin red and trypan blue staining. In whole eyes and in excised corneas, the estimates of endothelial cell densities varied by less than 3% from one method of visualization to the next. Estimates of endothelial cell densities obtained in intact eyes at 1 mm Hg were highly correlated to, but 13.6% lower than, estimates in excised corneas. Estimates of endothelial cell density obtained at intraocular pressures of 50 mm Hg were 7.7% lower than estimates obtained at 1 mm Hg.     

锥兰联合茜素红染色证实保存后的兔角膜内皮细胞活性

我们应用锥兰联合茜素红染色证实保存后的兔角膜内皮细胞活性。我们随机的将离体的新西兰白兔眼球分为四组，每组５只。将房水抽空，随即前房内注入Ｃ３Ｆ８（全氟乙烷，惰性气体）气体，于湿房４℃条件下分别保存５，７，１０，１４天，然后取下带巩膜环的角膜片，内皮面向上放在角膜容器内，将０．２５％锥兰溶液滴于内皮面，１分半钟后将染料洗净，再将０．２％的茜素红溶液滴于内皮面，染色１分半钟，将染料洗净，将角膜片放于２     

正常中国汉族儿童角膜的共焦显微镜研究

目的采用角膜激光共焦显微镜对正常中国汉族儿童活体角膜各层组织结构进行观察,分析正常中国汉族儿童角膜各层细胞的活体细胞形态学特征及密度。方法 49例6～13岁正常中国汉族儿童中央部角膜进行活体共焦显微镜检查,研究角膜各层结构的图象特点,并分析角膜各层细胞的密度,与成年人进行对比。结果中国汉族儿童正常角膜上皮表层细胞排列疏松,边界清楚,胞体较大,核反光较强,又称翼状细胞;基底层细胞排列紧密,呈规则的蜂巢状,胞质反光弱,正常情况下未见细胞核,细胞平均密度为5947.58±769.3个/mm2。前弹力膜为无细胞结构的膜状物,可见串珠状的神经纤维走行。基质层中可见在相对较暗的背景下明亮的角膜基质细胞核。角膜基质内有神经纤维分布。前基质层角膜细胞平均密度为1621.12±123.26个/mm2,后基质层角膜细胞平均密度为984.71±113.17个/mm2,前后基质层细胞密度有显著性差别（P〈0.05）。后弹力层中无细胞结构,为均一无结构组织。内皮细胞层表现为规则的六边形结构,细胞结构清晰,细胞平均密度为3682.38±251.87个/mm2。左右眼及男女之间角膜各层细胞密度的差异无统计学意义（P=0.341～0.611和P=0.194～0.855）。各层角膜细胞的面积和密度与性别和眼别无差异。中国汉族儿童角膜各层细胞的密度较正常成人高。结论角膜激光共焦显微镜能够在实时、活体和三维空间从细胞水平清晰观察中国汉族儿童活体角膜各层细胞的形态结构,可以对角膜各层结构进行定性和定量分析,在儿童角膜病的研究和临床诊断方面是十分有用的工具。     

Mitosis and cell division in human corneal endothelium

During routine morphologic evaluation of nine human corneas, obtained from cadavers of persons older than 40 years, numerous endothelial cells were observed undergoing changes similar to those seen during mitotic cell division. An inverted phase-contrast microscope was used to examine the corneal endothelium, and trypan blue vital stain was used to assess cellular viability. Changes noted within the cell and the nucleus were documented photomicrographically. These findings support the hypothesis that cell division and mitosis do occur in human corneal endothelium.     

The fate of preserved and transplanted human corneal entothelium

This paper evaluates the numbers of corneal endothelial cells that survive transplantation. The donor endothelium was photographed with a specular microscope both before enucleation and in situ after keratoplasty. The cell density was measured in 16 corneas from donors with choroidal melanoma. Of these donor corneas 12 were cryopreserved, two were preserved in McCarey and Kaufman's (M-K) medium and two were transplanted fresh. The average follow-up period after keratoplasty was 11 months. The mean endothelial cell loss for the whole series was found to be 49%. The mean cell loss for the cryopreserved corneas was 55%. In the four other recipients, with donor corneas that had been stored in M-K medium or transplanted fresh, the mean cell loss was 32%. The corneas preserved in M-K medium had the highest cell density in the transplants, with cell losses of 21 and 22%. Cell losses in the two corneas transplanted fresh were 40 and 44%. Cell losses, in the cryopreserved grafts had a wide variation, 33-77%. No correlation could be found between cell loss and either the age of the donor or the duration of perservation. Freezing and thawing was found to damage a proportion of the cells so that they did not stain with para-nitro-blue tetrazolium (p-NBT) after preservation. Transmission and scanning electron microscopy also revealed changes in the intercellular spaces and some cell disruption in cryopreserved grafts.     

The ultrastructure of contact lens induced changes

The endothelium of 15 human corneas was studied with specular and electron microscopy after exposure to a thick, low water content, soft contact lens (SCL). Five control corneas (no lens wear) were studied using the same methods. SCL wear produced obvious changes in endothelial morphology in 12 of the 15 eyes. With specular microscopy, the changes consisted of an apparent increase in separation of cells and development of areas of loss of membrane reflectivity (blebs). When viewed with electron microscopy, the changes in the same corneas consisted of oedema in the nuclear area of the cells and bulging of the posterior endothelial surface, in some cases over an area of several cells. In 4 cases, the cellular oedema was marked showing both intracellular and intercellular vacuoles. It was concluded that the transient endothelial changes seen with specular microscopy following SCL lens wear were produced by alterations in the contour of the posterior endothelial surface resulting from disturbance to the endothelial environment.