A new system for storing donor corneas

A new system was designed for the fixation of corneal donor discs in culture media, which can be applied with commercially available 50 ml culture flasks. The disposable cornea-"spoon" fixates the cornea in a position near the bottom of the culture dish, thus allowing for an evaluation by slitlamp, specular microscope, and inverted phase microscope without opening the bottle. Using disposable materials and omitting manipulations like suture-fixating the cornea, corneal preservation has been significantly simplified.     

不同浓度聚维酮碘致GFP转基因大鼠角膜毒性损伤的研究

目的　评估不同浓度的聚维酮碘（polyvinylpyrrolidone-iodine，PVP-I）对角膜的毒性损害。方法　30只GFP转基因大鼠随机分为对照组、1 g·L^-1 PVP-I组和5 g·L^-1 PVP-I组，后2组局部滴PVP-I眼液建立损伤模型，对照组采用生理盐水滴眼，以体视荧光显微镜观察各组不同时间点角膜荧光强度改变，并取各组角膜组织进行固定、包埋和切片，以HE染色或荧光显微镜观察角膜组织病理改变，采用TUNEL染色分析有无细胞凋亡。结果　体视显微镜观察发现，与对照组相比，PVP-I组角膜片随时间推移其荧光强度明显减弱。HE染色及荧光显微镜检测进一步证实PVP-I组出现角膜上皮细胞剥脱、基质细胞数量减少等病理变化，TUNEL染色提示，5 g·L^-1 PVP-I组角膜出现上皮细胞凋亡。结论　PVP-I局部滴眼可造成明确的角膜损伤，PVP-I临床应用的安全性尚有待进一步探讨。     

组织工程角膜片移植治疗无菌性角膜溃疡的实验研究

目的探讨组织工程角膜片治疗角膜溃疡的可行性及疗效。方法将16只新西兰大白兔制作成角膜溃疡模型,随机分为2组,每组8只,分别作单纯羊膜移植和以羊膜为载体的组织工程角膜片移植。术后进行大体观察、裂隙灯观察、组织学观察、扫描电镜观察。结果羊膜为载体组织工程角膜片移植治疗角膜溃疡,平均7.5周溃疡完全愈合,角膜恢复透明性和屈光性,较单纯羊膜移植组(平均9周)角膜溃疡修复快,效果好结论羊膜为载体构建的组织工程角膜片可以有效治疗角膜溃疡。     

体外培养重建角膜组织的技术探讨

目的：探讨体外重建角膜组织的三维培养技术。方法：原代培养角膜上皮,基质和内皮细胞,利用硫酸软骨素,透明质酸钠,I型胶原蛋白和微孔材料,建立三维培养系统,体外重建角膜组织结构,观察非醛交联,醛交联和微孔材料等三类人工角膜的透明度,常规病理组织学检测细胞在三维培养系统中生长状态。结果：非醛交联类重建人工角膜质地较软,透明度较低,光镜可见纤维结构,醛交联类重建人工角膜质地较韧,透明性较高,光镜可见均质结构,非吸收微孔材料类细胞长人较少,较难体外重建人工角膜,结论：利用生物工程技术能够体外重建人工角膜,非醛交联类结构较规则,醛交联类透明度较高。     

兔角膜碱烧伤的共焦显微镜及病理观察

目的采用角膜共焦显微镜活体动态观察兔角膜碱烧伤的病理改变,组织化学方法观察角膜组织病理学变化。方法NaOH造成兔角膜碱烧伤模型,烧伤后4d、20d、36d行角膜共焦显微镜检查及组织病理学检查。结果碱烧伤后4d,角膜即有炎性细胞浸润,出现少许CD4+、CD8+细胞;碱烧伤后20d,角膜基质中炎性细胞浸润明显,CD4+、CD8+细胞增多,瘢痕及新生血管形成;碱烧伤后36d,炎性细胞浸润减少,CD4+、CD8+细胞消失,仍可见瘢痕及新生血管。结论角膜共焦显微镜活体观察碱烧伤后角膜的创伤修复,反映角膜的病理状态,有助于指导临床治疗。     

组织工程角膜三维构建的实验研究

目的以人胚胎干细胞（hESC）诱导细胞为种子细胞,以脱细胞猪角膜基质（APCM）为支架三维构建生物工程角膜,以期用于穿透性角膜移植,解决角膜供体极度匮乏的难题。方法实验研究。无菌条件下将新鲜猪角膜组织置于0.5% SDS溶液中4 ℃脱细胞 24 h,获取APCM。将hESCs与人角膜基质细胞通过Transwell共培养5 d,获取眼周间充质干细胞（POMPs）,再于人晶状体上皮细胞源性条件培养基继续培养14 d获取角膜内皮样细胞并进行鉴定和筛选纯化。将纯化后扩增的角膜内皮样细胞接种于APCM构建角膜内皮植片,并移植入角膜内皮功能失代偿动物模型进行泵功能评估;采用人角膜缘干细胞（LSCs）来源的条件培养基培养hESCs 12 d,诱导其分化人角膜上皮样细胞并筛选鉴定,将其与APCM构建的角膜上皮植片移植于LSC失代偿动物模型的角膜缘,观察其眼表修复能力。结果诱导的人角膜内皮样细胞表达内皮细胞相关标记物vimentin、N-cadherin、Na+/K+ATP酶和ZO-1。构建的角膜内皮植片能够促使角膜内皮功能失代偿动物的角膜逐渐恢复透明。构建的角膜上皮细胞植片具有4～5层细胞复层结构,类似于正常角膜上皮,且能够一定程度上修复LSC失代偿动物模型眼表。结论采用hESCs诱导分化来源的细胞与APCM构建的人角膜内皮植片和人角膜上皮植片具有类似于正常角膜的功能,为全层生物角膜的构建提供了良好的实验和理论基础,具有良好的临床应用前景。     

Specular microscopic observation of human corneal epithelial abnormalities

The diameter of the cone lens of the specular microscope was enlarged from its original size of 3.5 mm to 6.0 mm. This modification enabled the authors to take clear pictures of the corneal epithelium with a specular microscope (SM) contact lens, because tears could be excluded from the photographic field by exerting even pressure on the cornea. The normal corneal epithelium showed three kinds of cells with no abnormally shaped or reflex cells. As spindle-shaped cells were mainly observed in keratoconus patients, post-penetrating keratoplasty patients, and postepikeratophakia patients, this suggests that these cells may be an indicator of wound healing. Large cells were observed in diabetic aphakic patients, aphakic extended-wear soft contact lens wearers, and aphakic patients using 0.5% indomethacin eye drops; thus, the presence of large cells may indicate suppressed metabolism and mitosis. Colonies of small cells were observed in keratoconjunctivitis patients. Cells with nuclei and irregular cell patterns were observed in all diseased conditions. There are several common abnormal patterns in the corneal epithelium that are observable under the specular microscope. Modified specular microscopic observation can detect these subtle alterations at the cellular level.     

Optical principles for estimation of endothelial cell density with the non-contact specular microscope

In non-contact specular microscopy of corneal endothelium, endothelial cell density is overestimated due to the angle of observation and the curvature of cornea. On the basis of theoretical considerations, it is concluded that if the angle of observation is kept at a small value the curvature of cornea contributes only insignificantly to this overestimation, the major determinant being the angle of observation. When an angle of 46 degrees is chosen between slit illumination and optical axis of the microscope it is calculated that estimates of endothelial cells should be multiplied with a factor of 0.959 to correct for the angle effect. Six eye were photographed with both a contact and non-contact specular microscope and endothelial cell density estimated. Mean observed difference in cell counts was +5.5% without correction and +1.2% with correction for angle effect. 95% confidence limits for the difference with correction were -3.7 and +6.0% respectively showing that estimates of endothelial cell density obtained with the non-contact specular microscope agree closely with those obtained by contact specular microscopy when corrected for angle of observation.     

猪脱细胞角膜基质组织结构特点与生物相容性研究

背景构建组织工程化角膜时,载体的选择十分重要。目前有多种载体可供选用,但脱细胞角膜基质是公认的较好载体。目的观察猪脱细胞角膜基质的组织结构特点,评价其与异种角膜基质和上皮细胞的生物相容性。方法取猪角膜组织进行组织块培养,经胰蛋白酶-EDTA酶消化角膜上皮、基质、内皮细胞,支架组织于-20℃冷冻干燥后灭菌保存。猪脱细胞角膜基质石蜡切片行常规苏木精一伊红染色,光学显微镜下观察组织的形态学特征;扫描电镜下观察其组织结构特点;并对其物理特性,如抗拉性、膨胀性、含水量和透明度进行评价。将猪脱细胞角膜基质移植到兔角膜基质层内,同时与体外培养的兔角膜上皮细胞共培养4周,分析其组织和细胞生物相容性。结果经酶消化处理后猪角膜组织中未见上皮、基质和内皮细胞,其胶原纤维直径大小、排列与正常角膜组织相同,抗拉性、膨胀性、含水量和透明度等物理特性与正常猪角膜相似。猪脱细胞角膜基质行异种兔角膜基质层间移植1周时可见轻度水肿,2周后水肿基本消退,4周时透明度较好。猪脱细胞角膜基质与兔角膜基质之间贴附良好,未见炎症反应及新生血管。兔角膜上皮细胞接种于猪脱细胞角膜基质上共培养4周后CK3表达阳性。猪脱细胞角膜基质脱水前与脱水2h、4h后及正常猪角膜基质间的抗拉性、膨胀性、含水量的差异均无统计学意义（P〉0．05）,但脱水2h、4h后及正常猪角膜基质的透明度明显好于脱水前,差异均有统计学意义（P〈0．01）。结论猪脱细胞角膜基质组织结构与正常猪角膜相似,与兔角膜基质和上皮细胞具有良好的生物相容性。     

乳化硅油滴在活体角膜内皮的共焦显微镜表现

目的 采用角膜激光共焦显微镜观察乳化硅油滴在活体角膜内皮面的形态,比较并总结其特点.方法 应用自身对照研究.对近年来在深圳市眼科医院就诊的20例(20只眼)临床硅油乳化的患者采用激光角膜共焦显微镜检查其角膜,分析研究其图像特点,总结乳化硅油滴在活体角膜内皮的共焦显微镜表现.结果 乳化硅油滴在角膜内皮面有着不同的大小和排列方式,镶嵌于角膜内皮面,并伴有角膜内皮细胞的明显水肿增大,内皮细胞的结构不清,仅有模糊轮廓.结论 采用新型的激光角膜共焦显微镜可以观察到乳化硅油滴对角膜内皮细胞的直接损害.该检查是一种对人体无害、可以反复进行,并且连续观察角膜内皮变化的工具,对评估乳化硅油滴对角膜的损害具有重要作用.

Abstract：

Objective To study the emulsified silicone oil droplets in the corneal endothelium by in vivo laser confocal microscopy, compare and summarize its characteristics. Methods The corneas of 20 patients (20 eyes) with silicone oil emulsion were examined by in vivo laser confocal microscopy, analyzed the characteristics of the images, and summarized the performance of emulsified silicone oil droplets in corneal endothelium with confocal microscopy. Results Emulsified silicone oil droplets in the corneal endothelium had a different size and arrangement, embedded in the corneal endothelium and come al endothelial cells associated with increased edema; endothelial cell structure was unclear, with only vague outlines. Conlulusions New type of laser corneal confocal microscope can observe the corneal endothelium damage by emulsified silicone oil drops directly. The inspection is a harmless, can be repeated and continuous observation of corneal endothelial changes in the tools. The laser corneal confocal microscope plays an important role on the assessment of silicon emulsion oil droplets on the cornea in the damage.