组织工程上皮移植对碱烧伤角膜新生血管的影响

目的：评价组织工程上皮移植在碱烧伤角膜缘干细胞缺乏症中对角膜新生血管的抑制作用。

方法：回顾性非随机的病例研究。2006/2011年我院收治的19例(23眼)完全性角膜缘干细胞缺乏症的碱烧伤患者, 10例13眼行组织工程上皮移植,9例10眼行羊膜移植。所有患者在手术前后均用裂隙灯观察角膜新生血管情况,在术后第21,60d对角膜新生血管进行评分比较。

结果：术后第21d和术后第60d组织工程上皮移植组和羊膜移植组角膜新生血管均较术前明显减少( P<0.05),在术后两个评价时间点,组织工程上皮移植组平均角膜新生血管分数明显低于羊膜移植组。

结论：对碱烧伤所致角膜缘干细胞缺乏的患者,组织工程上皮移植抑制角膜新生血管的作用明显好于羊膜移植。     

Quantification of corneal neovascularization after ex vivo limbal epithelial stem cell therapy

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animal-free culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization, vessel caliber (VC), and invasive area (IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity (BCVA), epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction (improvement) of all three parameters of corneal neovascularization [neovascular area (NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31% (P=0.035), invasion area 29.37% (P=0.018) and VC 14.29% (P=0.072). BCVA improved in all eyes (mean follow-up, 76±21mo). Epithelial transparency improved significantly from 2.00±0.93 to 0.88±1.25 (P=0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing and monitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method, successful cases could be differentiated from failed cases.     

角膜新生血管中细胞与分子的研究进展

多种眼部损伤可诱导角膜新生血管形成,促进疾病发展,造成角膜水肿、视力受损,甚至失明,因此抑制角膜新生血管有助于延缓疾病进程并降低角膜损伤,具有十分重要的临床意义。本文将对参与角膜新生血管形成的细胞及分子作最新的系统论述,并分析可能的抑制靶点,以期为科研及临床提供参考。     

In situ immunohistochemical analysis of cell adhesion molecules on human corneal endothelial cells.

Interaction of leucocytes with human corneal endothelial cells (HCECs) can be observed in several clinicopathological conditions, such as uveitis, keratitis, and corneal graft rejection. Since leucocyte-endothelial cell interactions involve various adhesion receptors we have analysed the expression and distribution pattern of the neural cell adhesion molecule (NCAM), the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1), the endothelial leucocyte adhesion molecule-1 (ELAM-1), and the cluster of differentiation antigen-44 (CD44) on flat preparations of normal and organ-cultured HCECs. NCAM and ICAM were constitutively expressed on HCECs whereas VCAM-1, ELAM-1, and CD44 were absent from normal HCECs. However flat mounts of HCECs from organ-culture preserved corneas showed a mosaic-like distribution pattern of VCAM-1 and ELAM-1 positive cells and garland-like clusters of CD44 positive cells. We suggest that modulation of ELAM-1, VCAM-1, and CD44 expression on HCECs may contribute to the regulation of leucocytes-HCECs interaction in the case of anterior segment inflammation.     

细胞间粘附分子在角膜疾病中的表达

目的：探讨细胞间粘附分子（Intercellular Adhesion Molecule-1,ICAM-1）在感染性角膜疾病和角膜移植免疫排斥中的表达及作用。方法：应用抗细胞间粘附分子ICAM-1的单克隆抗体,对42例角膜移植所取下的病变角膜进行免疫组织化学研究。角膜组织连续冰冻切片,进行SABC免疫组化及HE染色,研究细胞间粘附分子的表达与炎性细胞分布以及炎症程度的关系。结果：各种感染性角膜疾病、角膜变性以及角膜移植排斥过程中,ICAM-1在角膜上皮,尤其是基底细胞、角膜细胞、内皮细胞和血管化的基质层新生血管内皮细胞中均有表达,在炎性细胞包括单核细胞、巨噬细胞及淋巴细胞浸润部位表达增强;ICAM-1表达强度与炎症程度相平行。结论：ICAM-1在感染性、变性角膜病及角膜移植排斥中均有表达,在炎性细胞浸润部位表达增强;I-CAM-1对白细胞募集于炎症部位、炎症的发生、发展具有重要的中介作用。     

角膜缘上皮细胞分化与增殖及细胞周期素表达

目的:探讨角膜缘上皮细胞分化、增殖能力与细胞周期素D1、E、A表达水平的关系.方法:应用消化培养法分别培养人角膜缘及角膜中央上皮细胞,并将角膜缘上皮细胞传至第3代,将角膜中央上皮细胞传至第1代.分别选用抗细胞周期素D1、E、A的抗体,通过免疫组织化学染色检测各代细胞表达细胞周期素D1、E、A的水平.结果:角膜缘上皮原代细胞表达细胞周期素D1、E、A的水平最高,传代培养时细胞周期素的表达水平逐代下降,至第3代已基本上检测不到细胞周期素的表达.角膜中央上皮原代细胞仅极少数表达细胞周期素D1、E、A,传至第1代时已失去表达细胞周期素的能力.各代细胞表达细胞周期素D1、E、A的水平与其增殖能力一致.结论:细胞周期素D1、E、A表达水平的高低可反映角膜缘上皮细胞增殖能力的强弱.高度表达细胞周期素D1、E、A的角膜缘上皮细胞可能就是角膜缘干细胞.细胞周期素可能成为干细胞的一种新的免疫学或生化标记.     

角膜缘干细胞标记物研究现状

当前视觉科学研究领域及眼科临床存在的主要难题包括:缺乏适当的移植材料治疗眼表疾病以及青光眼、视网膜黄斑疾病等的视功能保护和有效治疗等。随着干细胞和组织工程学研究的推广,越来越多的研究人员已经认识到干细胞标记物的重要性。角膜缘干细胞是角膜上皮的唯一细胞来源,对维持角膜上皮的动态稳定和角膜透明性具有极其重要的作用。而角膜缘干细胞标记物的确立对建立规范的角膜缘干细胞分离纯化和培养方法、进一步研究其生物学特性起着决定性的作用。本文就该研究领域现状及前景作一简要综述。     

角膜缘niche细胞与基质细胞维持角膜缘干细胞功能的对比研究

目的比较角膜缘niche细胞(limbal niche cells,LNCs)与角膜缘基质细胞(limbal stromal cells,LSCs)在维持角膜缘干细胞功能上的不同特性。方法将LNCs和LSCs分别从6个角膜缘组织分离,并在相同的条件下培养、传代。LNCs与LSCs经丝裂霉素C(mitomycin C,MMC)处理后分为LNCs组与LSCs组作为饲养细胞分别与角膜缘干细胞共培养,比较两组角膜缘干细胞克隆形成率(colony-forming efficiency,CFE)、上皮细胞复层化以及细胞标志物和部分基因的表达。结果 LNCs组角膜缘干细胞CFE(6.57±1.54)%高于LSCs组(1.43±0.47)%。LNCs组细胞复层上皮数(4～5层)多于LSCs组(2～3层)。角膜缘干细胞克隆与免疫荧光染色及mRNA半定量分析结果显示,LNCs组比LSCs组表达了更多干细胞标志物ΔNp63,能更有效地维持角膜缘干细胞的细胞特性。逆转录PCR分析结果显示,LNCs组与LSCs组都分泌了一些维持角膜缘干细胞生长的生长因子,但LNCs组比LSCs组高表达上皮型钙黏蛋白(E-cad...     

Leukocyte adhesion molecules in rejected corneal allografts

Leukocyte adhesion molecules are believed to play a key role in the selective recruitment of different leukocyte populations to inflammatory sites. In this study, we investigated the presence and distribution of intercellular ad hesion molecule-1 (ICAM-1), E-selectin (endothelial leukocyte adhesion molecule-1) and vascular cell adhesion molecule-1 (VCAM1) in 12 rejected corneal allografts and compared the presence of these adhesion molecules with the composition of the associated inflammatory infiltrates. ICAM-1 was focally expressed on corneal epithelial cells and its expression was increased on keratocytes, corneal and vascular endothelial cells particulary at the site of dense infiltration with mononuclear leukocytes. E-selectin was present on endothelial cells of vessels in the stroma of rejected corneal allografts which were characterized by dense infiltration with T cells and macrophages. VCAM-1 was predominantly expressed on inflammatory cells of the macrophage/monocyte lineage, but only sporadically on vascular endothelial cells in thet stroma of vascularized rejected corneal allografts. Our results suggest that ICAM-1, E-selectin and VCAM-1 may all be involved in the pathogenesis of corneal allograft rejection, particulary in the generation of the inflammatory infiltrates.     

Expression of leukocyte adhesion molecules in human subfoveal choroidal neovascular membranes treated with and without photodynamic therapy

PURPOSE: The purposes of this study were to investigate the immunostaining of the leukocyte adhesion molecules intercellular adhesion molecule (ICAM)-1 and E-selectin in subfoveal choroidal neovascular membranes (CNVMs) surgically excised from patients with age-related macular degeneration (AMD) and to determine whether prior photodynamic therapy (PDT) alters their immunostaining. METHODS: The localization of ICAM-1 and E-selectin in 10 subfoveal CNVMs was determined by immunohistochemistry. Membranes were also immunostained for CD31 to assess vascularity. RESULTS: Significantly higher numbers of CD31-staining vessels per unit membrane area were found in the peripheral regions of the membranes compared with the central regions (P = 0.05). ICAM-1 immunoreactivity in the CNVMs was found predominantly on RPE cells, but also on small vessels in the periphery. ICAM-1 staining was significantly more intense in the peripheral, more cellular areas of the membranes than in the central, more fibrotic regions (P = 0.04). ICAM-1 staining in the periphery of the CNVMs was greater than that in choroidal vessels and the RPE of the normal control eye. ICAM-1 immunostaining grade in peripheral regions of the CNVMs decreased with the increasing number of PDT treatments (P = 0.05). Some of the CNVMs also stained for E-selectin in RPE cells and small vessels in the periphery. CONCLUSIONS: In subfoveal CNVMs from patients with AMD, there is increased immunostaining for leukocyte adhesion molecules, particularly in the peripheral, more cellular regions where angiogenesis may be ongoing. Increasing numbers of PDT treatments may be associated with decreased ICAM-1 immunostaining in the proliferating edges of the CNVMs.     

人羊膜匀浆提取液对角膜碱烧伤后新生血管PEDF和VEGF表达及角膜新生血管的影响

目的:探讨人羊膜匀浆提取液对角膜碱烧伤后新生血管形成过程中色素上皮衍生因子(PEDF)和血管内皮生长因子(VEGF)表达及角膜新生血管的影响.方法:选取2015-06/2016-06在佛山爱尔眼科医院治疗的角膜碱烧伤患者32例37眼,随机分为A、B两组.其中A组17例19眼,采用40mg/L人羊膜匀浆提取液治疗,B组15例18眼,采用3g/L泼尼松龙滴眼液治疗.在治疗不同的时间点(1、4、7、14、21、28d)观察角膜新生血管的生长,同时检测新生血管形成过程中PEDF及VEGF的表达水平.结果:A组患者在使用人羊膜匀浆提取液治疗后,PEDF表达水平显著高于B组,差异有统计学意义(P=0.001),在治疗28d后,PEDF表达水平达到了0.721依0.314,而B组患者PEDF表达水平仅有0.538依0.253,两组患者PEDF表达水平间的差异具有统计学意义( P<0.05 );A组VEGF表达水平在不同的时间点检测时均低于B组,在治疗28 d后,A组患者VEGF 表达水平为0.152依0.020,B 组患者VEGF表达水平为0.302依0.031,两组患者VEGF表达水平间的差异具有统计学意义(P<0.05);A组患者角膜新生血管数量显著低于B组患者,差异有统计学意义( P<0郾05 ).结论:人羊膜匀浆提取液可以促进患者角膜碱烧伤后新生血管形成过程中PEDF表达,抑制VEGF的表达和角膜新生血管的增殖.     

诱导型一氧化氮合成酶在大鼠角膜新生血管模型的表达及意义

目的:研究诱导型一氧化氮合成酶(iNOS)在大鼠角膜新生血管模型的表达和意义。方法:将SD大鼠随机分为正常组,角膜新生血管对照组,地塞米松球结膜下注射组,采用缝线诱导角膜新生血管模型。用裂隙灯数字图像处理系统观察第8d模型组新生血管生长情况,RT-PCR检测3组iNOSmRNA的表达,免疫组织化学方法检测iNOS蛋白质水平的表达。结果:在正常大鼠角膜上皮基底膜和基质层即表达iNOS,新生血管对照组其表达明显增强,球结膜下注射地塞米松组iNOS在新生血管角膜组织中则受到显著抑制,iNOSmRNA水平和蛋白质水平表达有一致性。结论:iNOS表达水平与炎症性角膜新生血管明显相关,地塞米松球结膜下注射抑制iNOS表达与其抑制炎症性角膜新生血管可能有关。     

Microcarrier cell culture of neonatal human corneal endothelium.

Fresh isolates of neonatal human corneal endothelial cells were maintained in tissue culture using a technique employing collagen-coated, dextran-based microcarrier beads. This method provides large yields of endothelial cells suitable for biochemical and/or transplant studies without exposing the cells to enzymatic passage. Our results suggest that microcarrier culture of human corneal endothelial cells can provide a useful in vitro system for the growth and maintenance of actively mitotic cells.     

Regulation of corneal angiogenesis in limbal stem cell deficiency

Corneal angiogenesis is associated with a variety of corneal diseases, and is sometimes vision threatening. In recent years, with the discovery of major pro- and anti-angiogenic factors in the cornea, details of the angiogenic process are gradually unveiled. Of note, corneal inflammation and neovascularization associated with severe limbal stem cell (LSC) deficiency is a clinically challenging issue in that the condition persists long after the initial insult, and will not improve without transplantation of LSCs. However, to date the molecular mechanism by which LSC transplantation restores corneal avascularity is not fully understood. In addition to discussing major pro-angiogenic factors involved in corneal neovascularization, this review article also focuses on possible molecular mechanisms underlying persistent inflammation and neovascularization following severe LSC deficiency, and anti-angiogenic factors expressed by human limbo-corneal epithelial cells (HLCECs).

Most of the recently discovered corneal anti-angiogenic factors belong to extracellular matrix proteins that aquire angio-inhibitory activity only after proper proteolytic processing. Our recent findings showed that the secretion of endostatin (derived from basement membrane collagen XVIII) and restin (from collagen XV) by HLCECs were enhanced when HLCECs were cultivated on amniotic membrane (AM). This adds to the advantage of transplanting ex vivo expanded HLCECs cultivated on AM in that the anti-angiogenic activity of the epithelial cells is augmented in a physiological way. Furthermore, proteomic profiling of HLCECs and human conjunctival epithelial cells (HCECs) identified a 14-3-3 protein (stratifin) preferentially expressed by HLCECs. In addition to functioning as a cell cycle controller, keratinocyte-derived stratifin induces MMPs which are involved in the generation of restin (by MMP-1) and endostatin (by MMP-3). These findings highlight the significance of delicate epithelial–matrix interactions in the maintenance of corneal avascularity.     

兔角膜缘干细胞的体外培养

目的 探寻兔角膜缘干细胞体外培养方法。方法 分别用20％小牛血清营养液和20％胎牛血清营养液兔角膜缘干细胞行体外培养，观察细胞贴壁生长情况；同时对生长良好的原代细胞进行消化传代，进一步观察传代细胞的形态和生长情况。结果 20％胎牛血清营养液组，角膜缘干细胞在培养48－72h有80％贴壁生长，7－10天形成良好单层，细胞呈圆形、卵圆形或多边形，类似角膜上皮细胞；传代培养细胞形态仍正常，生长良好，但混有成纤维细胞。20％小牛血清营养液培养72h，仅有20％细胞贴壁生长，且细胞形态极不规则，有细胞“拉网”现象，培养14天仍未形成单层。结论 角膜缘干细胞的培养较常规细胞培养难，需要特殊培养基。20％胎牛血清营养液能作为角膜缘干细胞培养的基础液。     

Characterization of corneal endothelium cell cultured on microporous membrane filters.

In these experiments we characterize rabbit and bovine corneal endothelia cell cultured on microporous membrane filters (0.6cm2). Cell cultured bovine or rabbit corneal endothelial cells (subcultures 1-3) were seeded onto Millicell-HA filter inserts. Electrical resistance measured across the cultured monolayers increased steadily through 14 days of culture, reaching 34.2 +/- 0.8 ohm-cm2 (mean +/- SE) for rabbit cells and 33.1 +/- 1.1 ohm-cm2 for bovine cells. Alizarin red staining of the monolayers showed a polygonal morphology comparable to that observed in situ. Transmission electron microscopy showed well developed apical junctional complexes and flaps. Exposure of the monolayers to calcium-free medium resulted in the disruption of intercellular junctions, rounding-up of the cells and a decrease in electrical resistance (to near 0). Transmonolayer fluxes of inulin and dextran correlated well to the resistance measurements. Results of this study demonstrate that corneal endothelium, both bovine and rabbit, grown on filter inserts is comparable in morphology and ultrastructure to corneal endothelium in situ. The cells cultured in this system form functional apical junctional complexes that effect a barrier function comparable to that of the endothelium in situ.     

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.