Expression of the 55-kD/64-kD corneal keratins in ocular surface epithelium.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells. Immunofluorescence microscopic analysis of frozen sections of guinea pig and bovine tissue revealed that the 55-kD antiserum labeled corneal and limbal epithelium. In addition, the antiserum stained a subpopulation of peripheral limbal cells that were distributed in both basal and suprabasal layers of the epithelium. The monoclonal antibody AE5 was used to investigate the distribution of the 64-kD polypeptide in guinea pig and bovine tissue. Immunoblotting analysis revealed that AE5 antibodies recognized a 64-kD polypeptide in guinea pig cornea, but recognized a 66-kD polypeptide in bovine cornea. Immunofluorescence microscopic analysis of guinea pig tissue revealed that AE5 antibodies labeled suprabasal layers of corneal epithelium, in suprabasal layers of limbal epithelium, and in groups of cells in the peripheral limbal epithelium. We discuss the possibility that the ocular epithelial cells recognized by either the 55-kD or the 64-kD antibodies in the peripheral limbus may play a role in the reepithelialization of the cornea after wounding.     

角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外的所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

Conjunctival transdifferentiation is due to the incomplete removal of limbal basal epithelium

Previous studies have shown that using n-heptanol to create a total corneal epithelial defect beyond the limbus results in two different healing patterns with an unpredictable incidence. Between 14-68% of these wounded rabbit corneas (n = 287, combining various reports) showed extensive vascularization and conjunctivalization, whereas the remaining were not vascularized and had conjunctival transdifferentiation with a cornea-like epithelium. To investigate the role of the limbal epithelium in these two healing patterns, the authors treated rabbit eyes for various durations with n-heptanol and additional scraping. Histology showed that treatment for up to 120 seconds removed both the corneal and conjunctival epithelia but left the limbal basal cells intact. To prove viability, they cultured the treated limbal explants on collagen gel. After 14 days of culture, increased stratification of the limbal epithelium and an epithelial outgrowth onto the corneal stroma was observed. The latter was proven to be of corneal origin (positive to AE-5 but negative to AM-3 monoclonal antibody staining). The authors then surgically removed the entire limbal zone including 2 mm of peripheral cornea and 3 mm of adjacent conjunctiva in addition to n-heptanol debridement of the entire corneal epithelium in 54 rabbit eyes and observed a high incidence (96%) of corneal vascularization and conjunctivalization of the resultant epithelial phenotype (positive to AM-3, but negative to AE-5 monoclonal antibody staining). These results support the hypothesis that corneal epithelial stem cells are located in the limbus and indicate that an incomplete removal of the basal limbal epithelium by n-heptanol leads to unvascularized corneas with conjunctival transdifferentiation. Conversely, complete removal of such cells results in corneal vascularization and conjunctivalization.     

Differentiation in cultured limbal epithelium as defined by keratin expression.

The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AE1, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AE1. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AE1 binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AE1. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AE1. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells.     

The effect of rigid gas permeable contact lens wear on proliferation of rabbit corneal and conjunctival epithelial cells.

PURPOSE: To study the effect of rigid contact lens oxygen transmissibility on cell proliferation of the corneal, limbal, and conjunctival epithelium in vivo following 2 days of extended wear in the rabbit model. METHODS: Fourteen adult New Zealand White rabbits were divided equally into two groups. Each group was assigned to one of two test rigid gas permeable (RGP) contact lenses (Dk/Ltotal = 10 and 97) with uniform thickness (0.15 mm) and diameter (14.0 mm). One eye of each rabbit randomly received a contact lens for two days (48 hrs) extended wear, and the fellow eye was used as a control. Rabbits were injected intravenously with 5-bromo-2-deoxyuridine (200 mg/kg) in sterile phosphate buffered saline (pH 7.4) 24 hours before being killed. Corneas with a limbal rim of episclera and overlying conjunctiva were fixed in situ and excised. Nuclei labeled with BrdU were detected with a monoclonal anti-BrdU antibody and an FITC-conjugated secondary antibody. Digital images were collected and BrdU-labeled nuclei of whole-mount corneas were counted from superior limbus to inferior limbus using epifluorescence microscopy. RESULTS: Twenty-four hours after intravenous injection of BrdU, labeled nuclei were confined to and appeared as pairs in the basal epithelial layer. The density of BrdU-labeled nuclei were found to be 258 +/- 42, 167 +/- 43, 372 +/- 64, and 310 +/- 46 (pairs/mm2, mean +/- SD, n = 14) in normal controls for adjacent conjunctiva, limbus, peripheral cornea, and central cornea, respectively. By contrast,there was significant 81.35% (low Dk)and 22.46% (ultra-high Dk) suppression of cell proliferation in the central cornea after two days lens wear (n = 7). In addition, significant increases in the labeling of limbal and conjunctival epithelium were also noted. CONCLUSIONS: Significantly less BrdU labeling of epithelial cells at the normal rabbit limbus was noted as compared to the peripheral and central cornea (P < 0.05) and is consistent with the presence of slow-cycling limbal basal cells and the limbal stem cell theory; however, this is the first report of up-regulation of limbal cell proliferation induced by contact lens wear. This study also revealed, for the first time, that short-term extended wear of RGP lenses inhibits central corneal epithelial cell proliferation. This effect was significantly more pronounced for a low-oxygen vs. a hyper-oxygen transmissible test lens. This data also suggests that corneal epithelial layer thinning seen following extended contact lens wear may be explained, in part, by suppression of basal epithelial cell proliferation. Further study is clearly necessary to validate and extend these preliminary findings.     

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells.     

In vivo corneal confocal microscopic findings of palisades of Vogt and its underlying limbal stroma

PURPOSE: To demonstrate the corneal confocal microscopic findings of limbal palisades of Vogt and its underlying limbal stroma. METHODS: Two unrelated healthy subjects (a 56-year-old man and a 40-year-old man) with prominent palisades of Vogt were enrolled in this study. A detailed examination with confocal microscopy was performed in addition to a routine slit-lamp biomicroscopy. Cell sizes of epithelial basal layers of both the central and limbal (beneath the palisades of Vogt) were measured and statistically analyzed. RESULTS: In both subjects, confocal microscopy in the region of the palisades of Vogt revealed normal appearance of corneal superficial layers. However, in some images, undulant basal epithelial layer was observed. In the superficial stromal layer adjacent to the corneal epithelium, we noted corneal nerves that seemed to terminate at the epithelial basal layers. The mid-stromal layers showed a highly reflective spatter-like pattern as well as numerous dark striae in a branching pattern. The corneal endothelial layer seemed normal. The average cell size of epithelial basal cells beneath the palisades of Vogt was significantly smaller than those of the central cornea (P = 0.015 and 0.005 in cases 1 and 2, respectively). CONCLUSIONS: In vivo corneal confocal microscopy is useful in observing limbal stromal microstructures. Further investigations of pathologic corneal limbus may be useful in elucidating the mechanisms contributing to corneal limbal epithelial stem cell deficiency.     

Localization of corneal epithelial stem cells in the developing rat.

A monoclonal antibody, 4G10.3, was developed that preferentially binds limbal basal cells in adult rat, rabbit, and human corneas. These cells were hypothesized to be the stem cells for the corneal epithelium. The antibody 4G10.3 was localized by immunofluorescence microscopy in rats 1 d and 1, 1.5, 2, 3, 4, and 6 wk of age. Until 1.5 wk, 4G10.3 bound intensely to all basal cells in the cornea and the limbus. At 2 wks, the basal cells at the central cornea abruptly changed their shape from flattened or ovoid to large and cuboidal and bound 4G10.3 with greatly reduced intensity. Increased stratification of epithelium also was seen. Cells binding 4G10.3 gradually became sequestered to the limbal area after 2 wk, concomitant with increased stratification. At 4 and 6 wk, 4G10.3 binding was identical to that in adult corneas with only limbal basal cells showing positive binding. Basal cells in the limbal epithelium did not decrease their intense binding of 4G10.3 or change their ovoid cellular shape from 1 d through adult life. These results suggest that, during development, stem or stem-like cells are localized throughout the basal layer of the corneal and limbal epithelium. As the cornea matures, these cells are sequestered in the limbus at the same time that stratification of the epithelium and shape changes occur in the basal cells.     

The phenotype of limbal epithelial stem cells

PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.     

Cytoskeletal antigen expression in ocular mucosa-associated lymphoid tissue.

The human lacrimal gland (LG) and ocular surface contain discrete regions of epithelial cells with specific functions and at different stages of cellular differentiation. Epithelial cells contain cytoskeletal antigens that show a differentiation-dependent pattern of expression. The objective of this study was to characterize the various epithelial cell populations in normal human ocular mucosa-associated lymphoid tissue (MALT; LG, conjunctiva, and cornea) based on their immunohistochemical staining patterns with anticytoskeletal monoclonal antibodies (MoAbs) reactive with cytokeratins (AE-1, AE-2, AE-3, AE-5, AE-14, PKK1, and 34 beta E12), muscle-specific actin (HHF35), and vimentin. AE-1 stained LG (acini, ducts, and myoepithelia) and the full thickness of corneal and conjunctival epithelia. It stained only the superficial and basal limbal epithelium. AE-2 weakly stained all epithelia, except LG acini and proximal intralobular ducts. AE-3 and 34 beta E12 MoAbs had strong immunoreactivity with all MALT epithelia. AE-5 strongly stained the inner cells (suprabasal) of LG central intra- and interlobular ducts and the suprabasal epithelial layers of the cornea. It weakly stained LG myoepithelia and the superficial conjunctival epithelium. AE-14 stained the outer (basal) cells of LG central intra- and interlobular ducts, LG myoepithelia, basal epithelial layers of the limbus and conjunctiva, and all corneal epithelia. PKK1 stained all epithelia, except the basal limbus. HHF35 and the antivimentin MoAbs stained only the LG myoepithelia. The results of these studies indicate that the different epithelia in human ocular MALT may be differentiated by specific patterns of immunoreactivity with anticytoskeletal MoAbs. These MoAbs may be useful molecular markers for identifying ocular MALT epithelia.     

Epithelial dendritic cell distribution in normal and inflamed human cornea: in vivo confocal microscopy study

PURPOSE: To evaluate dendritic cell (DCs) density, distribution, and morphology in central corneal and limbal epithelium in normal subjects and patients with immune-mediated corneal inflammation using in vivo confocal microscopy (IVCM). DESIGN: Comparative case-controlled, observational confocal microscopy study. METHODS: A total of 135 eyes of 135 patients were investigated. Group 1 (normal eyes) included 45 eyes of 45 healthy volunteers, group 2 photorefractive keratectomy (PRK-treated eyes) included 45 myopic eyes of 45 patients treated with PRK, and group 3 (inflamed eyes) comprised 45 eyes of 45 patients affected by immune-mediated corneal inflammation. The central cornea and limbus were examined for epithelial dendritic-shaped cells using laser scanning IVCM. DCs density was calculated using image analysis software. RESULTS: Cells with a branching dendritic morphology were visualized in the basal epithelial layer, basal lamina, and subbasal nerve plexus, in the central cornea, and in the basal layer and basal membrane of the limbal epithelium. The limbal epithelium demonstrated DCs in 93.3%, 89%, and 97.7% of eyes in group 1, 2, and 3, respectively (P = ns). Central epithelial DCs were observed in 20.0% and 13.3% of eyes in group 1 and 2 (P = ns), while in 93.3% of eyes in group 3 (P < .001). DCs were found to be significantly higher at the limbus compared with central cornea in each group (P < .001). Cell densities observed in group 3 were significantly greater than groups 1 and 2, at both locations (P < .05). CONCLUSIONS: Laser scanning IVCM is a useful method for evaluating epithelial DCs distribution at the limbus and central cornea in both healthy and diseased eyes.     

角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

Corneal epithelial wound healing in partial limbal deficiency.

Previous studies have shown that the corneal epithelial stem cells are located at the limbal basal layer. The limbal stem cells are regarded as the ultimate source for corneal epithelial cell proliferation and differentiation. This paper examines epithelial wound healing in rabbit corneas with partial limbal deficiency (PLD), which was created by the surgical removal of two-thirds of the limbal zone (superior and inferior). Four to eight months after PLD creation, all corneas appeared normal, without vascularization. The residual stem cell capacity then was challenged by two sizes of corneal epithelial debridement created with combined n-heptanol and mechanical scraping. In the first group, two consecutive 6-mm defects were created 1 month apart. After the first wounding, three of eight PLD corneas had delayed wound healing and two of the three had vascularization, as compared to controls (n = 7). After the second wounding, both controls (n = 7) and the remaining PLD (n = 5) corneas showed similar rapid healing. In the second group, a large defect of up to 1 mm within the limbus was created. Healing was completed in 25-40 days in PLD (n = 6) corneas, a more marked delay compared to the 10-12 days for controls (n = 6) (P = 0.001). In addition, all PLD corneas showed increased vascularization and had epithelium of the conjunctival phenotype, verified by the immunofluorescent staining positive to AM-3 monoclonal antibody but negative to AE-5 monoclonal antibody. Thus, a deficiency of limbal stem cells contributes to the triad of conjunctival epithelial ingrowth, corneal vascularization, and delayed healing with recurrent erosion. In PLD, corneal epithelium is still compromised, particularly when a large epithelial cell mass is removed.     

角膜缘干细胞研究新进展

位于角膜最外层的角膜上皮细胞,正常损耗或创伤后,由角膜缘干细胞不断自我更新补充。当损伤等致角膜缘干细胞缺乏时,则会导致角膜溃疡、新生血管、混浊等角膜病变而影响视力。目前治疗这些角膜疾病的重要方式之一为移植体外培养的角膜缘干细胞。本文将从角膜缘干细胞的定位、体外培养、新细胞来源等方面进行综述。     

激光共焦显微镜对正常人眼角膜缘和中央角膜的观察

目的应用激光共焦显微镜对正常人眼角膜缘和中央角膜的组织结构与细胞形态的观察和分析。方法选择15名正常人的28只眼接受常规裂隙灯显微镜和检眼镜检查后,作为正常健康眼入选本研究。使用激光共焦显微镜对其上、下方角膜缘和角膜中央区进行检查,各层角膜图像均被记录,观察组织结构和细胞形态,对细胞密度进行计数并分析。结果所获角膜缘和角膜中央各层平面图像(x,y轴)及纵向断层图像(z轴)均非常清晰,同时获取动态录像。上、下方角膜缘均呈现Vogt栅栏状结构,并动态观察到血细胞在血管内的流动。表层上皮细胞排列非常疏松,边界明亮,胞体发暗,上方和下方角膜缘表层上皮细胞平均密度分别为(812±297)个/mm2和(785±263)个/mm2,二者比较差异无统计学意义(P>0.05)。上皮下可见明亮的Langerhans细胞,形态不规则,呈树枝状,上方和下方角膜缘Langerhans细胞平均密度分别为(288±102)个/mm2和(254±127)/mm2,二者比较差异无统计学意义(P>0.05)。角膜中央表层上皮细胞排列疏松,边界发亮,胞体发暗,细胞平均密度为(1098±315)个/mm2,多于上方和下方角膜缘(P<0.05)。基底上皮细胞排列紧密。上皮下和前基质层可见反光强烈的神经纤维丛,旁边偶见明亮的Langerhans细胞,形态不规则,细胞密度难以计算。浅层的神经纤维细小、弯曲度大、多小分支,深层的神经纤维粗大、弯曲度小、少见分支。基质层暗背景下散在分布细长的基质细胞,边缘欠清,细胞核明亮呈纺锤形。内皮细胞为排列整齐的六边形细胞,胞体发亮,边界发暗。角膜中央全层、基质层、上皮层厚度分别为(543.0±62.9)、(462·0±69.5)、(59.9±11.2)μm。结论激光共焦显微镜不仅可以对角膜进行无创的、实时的、活体的检查,而且与传统的光学共焦显微镜相比,具有高清晰度、确切的深度定位、时间动态观察、纵向断层扫描等优势,更可提供理想的角膜缘图像,对角膜疾病尤其是角膜缘疾病的基础研究与临床诊断将更有价值。     

Expression of K12 keratin in alkali-burned rabbit corneas.

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.     

Adaptation of homeostatic ocular surface epithelium to chronic treatment with the opioid antagonist naltrexone

PURPOSE: To determine how ocular surface epithelium adjusts to an increase in cell replication after treatment with the opioid antagonist naltrexone (NTX). METHODS: Adult male rats were given twice daily injections of 30 mg/kg NTX or vehicle for 7 days. Outcomes of NTX administration included DNA synthesis (monitored with BrdU), mitosis (assayed using colchicine), number of cell layers and cell diameter, apoptosis and necrosis, and packing density for the peripheral corneal epithelium, limbus, and conjunctiva. Also, transit time from basal to surface epithelial layers in the peripheral cornea was assessed with [H]thymidine as a marker. RESULTS: DNA synthesis and mitosis in the basal layer of the peripheral corneal epithelium of NTX-treated rats were increased 69% and 85%, respectively, from control levels; no changes in either parameter were recorded in the limbal or conjunctival epithelium (stem cell region). Epithelial thicknesses in the NTX group were increased by 8% to 38% from control subjects, without more cell layers. Packing density in NTX-treated rats was increased from control values by 26% in the basal layer of the limbus and by 12% to 28% in the suprabasal layers of the corneal epithelium, limbus, and conjunctiva. Epithelial cell diameters from corneas of NTX-exposed rats were subnormal in the basal and suprabasal cells of the limbus and conjunctiva. Apoptosis and necrosis were negligible in the epithelium of NTX-treated and control rats. Transit times of peripheral corneal epithelial cells of animals in the NTX group were shortened by 63% from control levels. CONCLUSIONS: These data show that a 1-week treatment with NTX does not induce proliferative pathology or toxicity in ocular surface epithelium, has a minimal effect on stem cell proliferation, and accelerates normal homeostatic processes. Topical application of NTX for stimulation of corneal epithelial wound healing results in no adverse sequelae, thereby supporting the therapeutic role for this drug in the treatment of ocular surface abnormalities.     

人角膜及角膜缘上皮细胞分化标记的检测

目的检测分化标记在人角膜及角膜缘上皮细胞的表达,以了解角膜及角膜缘细胞分化状态,旨在发现新的角膜上皮干细胞的阴性标记。方法获取人角膜及角膜缘组织,对冰冻切片及整个角膜组织行免疫荧光染色检测分化标记钙粘连素E、角蛋白3(CK3)、角蛋白12(CK12)、缝隙连接蛋白43、巢蛋白(nestin)和包壳蛋白(involucrin)的表达,经荧光显微镜及激光扫描共焦电镜观察,并行半定量RT-PCR以检测其相关分化标记基因的表达。结果分化标记CK3、CK12、缝隙连接蛋白43、巢蛋白和包壳蛋白在角膜和角膜缘上皮的表层细胞表达,角膜缘基底细胞不表达。激光扫描共焦电镜观察及RT-PCR结果显示角膜缘基底上皮细胞不表达细胞CK3、连接蛋白43和巢蛋白,而角膜上皮细胞则明显表达。结论角膜及角膜缘表层上皮较为成熟分化,而角膜缘基底细胞具有未分化细胞的特征,很可能是干细胞的部位。     

Histochemical and morphological study of the regenerating corneal epithelium after limbus-to-limbus denudation

Two typical characteristics of the limbal epithelium, namely, its high mitochondria content and histochemically proven proclivity towards carbonic anhydrase staining, were used to identify regenerating corneal epithelium as originating from the limbus. In addition, the period necessary for metaplasia of limbus epithelial cells into typical corneal epithelia was studied in light of these two criteria. It was proven that, on completion of the morphological transformation, after 7 days the positive carbonic anhydrase reaction was identifiable only in the basal cells of the limbus area, as in those of the control animals. It is concluded that in the early phase of reepithelialization, the forward-proliferating epithelial cells can be directly traced to the basal limbus epithelial cells.