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。     

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.     

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.     

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.     

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。     

Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation.

The authors studied the distribution of specific keratins within the superior, inferior, medial, and lateral regions of human limbus and cornea to determine whether the limbal epithelium exhibits regional heterogeneity in its microstructure. A corneal epithelial basic keratin (K3), recognized by monoclonal antibody AE5, was immunohistochemically undetectable in the basal layers of the limbus in these four regions, but was seen in all layers in the central cornea. The pattern of immunostaining with another monoclonal antibody, AE1, which recognizes several acidic keratins, was complementary to AE5 staining in that AE1 recognized a similar heterogeneity in the limbal epithelial cells. AE1 immunoreacted with the basal cells of the limbus, but not those of the central corneal epithelium. Limbal characteristics, as defined by AE1-positive and AE5-negative staining, extended deeply into peripheral cornea in the superior and inferior regions, but to a lesser extent in the lateral and medial regions. The broader regions of epithelium with limbal characteristics in the superior and inferior regions raises the possibility that these regions play an important role in corneal epithelial maintenance and wound healing.     

角膜缘干细胞的研究

角膜缘干细胞是位于角膜缘基底上皮层底的一类特殊类型的上皮细胞,随着细胞培养技术的发展,角膜缘干细胞体外培养后移植用于治疗由于角膜缘干细胞缺乏或者功能不全引起的眼表疾病成为研究的热点。本文就其解剖学定位、生物学特性、组织工程化角膜的基础性研究及其临床应用做一综述。     

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.     

Comparison of limbal and peripheral human corneal epithelium in tissue culture

Peripheral human corneal epithelium grows better in tissue culture than central epithelium, but it is not known whether ocular limbal epithelium grows even better than does the peripheral corneal epithelium. In this work we compared the growth kinetics of limbal and peripheral human corneal epithelial cells in tissue culture. Four 1-2 mm2 explants, removed from the limbus or from peripheral cornea (1-2 mm inside the limbus) of eye bank eyes, were grown to confluence in primary culture. Cells were then passaged at 2 X 10(5) cells per dish. At intervals thereafter, the cells were counted in a hemocytometer to determine plating efficiency and growth curves. Mitotic activity was determined 4 days after passaging by labeling cultures with 3H-thymidine and counting aliquots using the hemocytometer and scintillation counter. In the primary cultures, limbal epithelium grew as small, uniformly polygonal cells. Peripheral corneal cells grew to a variety sizes. The 24 hr plating efficiency and doubling time of limbal epithelial cells were 47 +/- 8% and 80 +/- 14 hr, respectively, while those of peripheral corneal cells were 41 +/- 10% (P less than 0.1) and 131 +/- 25 hr (P less than 0.001). The mitotic activity of limbal cells was significantly higher than that of peripheral (2.9 +/- 1.2 vs. 0.8 +/- 0.6) (P less than 0.01). These results indicate that human ocular limbal epithelium grows better in culture than does peripheral human corneal epithelium.     

Cytokeratin patterns in corneal, limbal, and conjunctival epithelium. An immunofluorescence study with PKK-1, 8.12, 8.60, and 4.62 anticytokeratin antibodies.

The authors examined immunofluorescently the specific cytokeratin staining patterns of corneal, limbal, and conjunctival epithelium with PKK-1, 8.12, 4.62, and 8.60 monoclonal anticytokeratin antibodies. Observations were made on unfixed frozen postmortem human tissue. The PKK-1 antibody stain was observed in all layers of corneal epithelium but only in suprabasal layers of limbal and conjunctival epithelium. By contrast, the 8.12 antibody stain was observed only in the superficial layer of corneal epithelium but through all layers of limbal and conjunctival epithelium. The 4.60 antibody stain was seen in focal areas of corneal and limbal epithelium and through all layers of conjunctival epithelium. The 8.60 antibody stain was not present in the three epithelia. These immunofluorescence studies showed unique cytokeratin patterns among layers in corneal, limbal, and conjunctival epithelium.     

Limbal stem cells of the corneal epithelium

Stem cells have certain unique characteristics, which include longevity, high capacity of self-renewal with a long cell cycle time and a short S-phase duration, increased potential for error-free proliferation, and poor differentiation. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. Stem cells for the cornea reside at the corneoscleral limbus. The limbal palisades of Vogt and the interpalisade rete ridges are believed to be repositories of stem cells. The microenvironment of the limbus is considered to be important in maintaining the stemness of stem cells. Limbal stem cells also act as a "barrier" to conjunctival epithelial cells and normally prevent them from migrating on to the corneal surface. Under certain conditions, however, the limbal stem cells may be partially or totally depleted, resulting in varying degrees of stem cell deficiency with resulting abnormalities in the corneal surface. Such deficiency of limbal stem cells leads to "conjunctivalization" of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. This results in ocular discomfort and reduced vision. Partial stem cell deficiency can be managed by removing the abnormal epithelium and allowing the denuded cornea, especially the visual axis, to resurface with cells derived from the remaining intact limbal epithelium. In total stem cell deficiency, autologous limbus from the opposite normal eye or homologous limbus from living related or cadaveric donors can be transplanted on to the affected eye. With the latter option, systemic immunosuppression is required. Amniotic membrane transplantation is a useful adjunct to the above procedures in some instances.     

Migration of cells in the rat corneal epithelium

The migration of cells in the rat corneal epithelium was studied using continuous labelling with tritiated thymidine [3H]TdR, during a 24 h period. Most mitoses resulted in 2 new basal cells. Cells leaving the basal cell layer moved vertically to the surface in all areas of the corneal epithelium. The first labelled cells reached the surface 3 days after the first injection of [3H]TdR. No stream of cells from the conjunctiva to the cornea in the limbal areas was observed, and no centripetal migration of epithelial cells in the cornea could be observed. After a cell has lost its attachment to the basement membrane, it is committed to be exfoliated in a few days without undergoing mitosis. Thus the slow centripetal migration of epithelial cells and the exchange of centrally located cells, as indicated by clinical findings and experimental studies, can only be explained by migration of basal cells.     

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。     

人角膜上皮干细胞的识别

目的 探讨人角膜上皮干细胞的分子标记。方法 对人角膜和角膜缘部位行组织学检查以分析角膜缘解剖结构。对人角膜切片和整个角膜组织行免疫组织化学染色以检测中央角膜和角膜缘部位未分化标记,如核蛋白p63、乳腺癌抵抗蛋白（ABCG2,BCRP1）、烯醇化酶α、整合素拍、胡及β1、表皮生长因子受体（EGFR）、细胞角蛋白19（CK19）、14（CK14）及转铁蛋白受体（CDT1）的表达,经荧光显微镜和激光扫描共焦显微镜观察。对角膜中部和角膜缘上皮细胞的mRNA进行半定量逆转录聚合酶链反应（RT—PCR）和原位杂交以检测其相关基因的表达。结果 角膜缘部位横向切片显示角膜缘上皮细胞为乳头放射状排列,对应于Vogt栅栏环境。未分化标记整合素β1、EGFR、烯醇化酶α及CK19在角膜缘基底细胞胞质染色较表层细胞更强;p63、ABCG2、整合素胡蛋白仅见于角膜缘基底部上皮细胞。激光扫描共焦显微镜观察和RT—PCR结果显示角膜缘表达p63、ABCG2、整合素胡蛋白及mRNA。原位杂交显示p63仅表达于角膜缘基底层细胞。结论 角膜缘上皮呈乳头放射状排列,角膜缘干细胞群具有复合标记：p63表达于细胞核、ABCG2表达于胞质、整合素胡表达于胞膜。采用这些标记复合体,可将角膜缘干细胞群与其他上皮细胞区分。     

Wistar rat palpebral conjunctiva contains more slow-cycling stem cells that have larger proliferative capacity: implication for conjunctival epithelial homeostasis

PURPOSE: To determine the location of conjunctival epithelial stem cells. METHODS: Wistar rats received daily injection of 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks followed by a 1-month BrdU-free period before death. After the rats were sacrificed, the orbital contents and eyelids were exenterated en bloc, fixed in buffer formalin, and embedded in paraffin. To compare the proliferative capacity of ocular epithelial cells, 1.0% phorbol myristate (TPA) in petrolatum was topically applied once daily to both eyes of Wistar rats for 12 days. After 6, 12, 18, and 24 hours and 2, 4, 6, 8, 10, and 12 days of TPA treatment, rats were administered BrdU intraperitoneally 7 hours before they were sacrificed. The ocular epithelium was fixed and processed for immunochemistry, and the labeling index (LI) of every epithelial zone was determined. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were found in bulbar, fornical, and palpebral epithelia and mucocutaneous junctions, as well as in limbal epithelia. The greatest numbers of LRCs were identified in palpebral epithelium. Under normal situations, in conjunctiva the LI was lowest in palpebral epithelium (2.1 +/- 0.5) compared with bulbar (2.2 +/- 0.5), fornical (2.3 +/- 0.4) epithelia and mucocutaneous junction (3.4 +/- 0.9), respectively. In cornea, the LI was lowest in limbal epithelium (1.8 +/- 0.7) compared with central corneal epithelium (3.5 +/- 0.6). Twenty-four hours after TPA treatment, an 8.2-fold increase in the palpebral epithelial basal cell labeling index was noted compared with 4.7-fold, 5.7-fold, and 3.8-fold increases in bulbar, fornical, and mucocutaneous junction epithelial basal cell labeling indices, and a sevenfold increase in the limbal basal cell labeling indices compared with a 2.1-fold increase in the corneal basal cell labeling index, respectively. Limbal and palpebral epithelia maintained a significantly greater proliferative response (5.5-to 6.3-fold increase, respectively) during chronic stimulation than corneal, bulbar, fornical epithelia, and mucocutaneous junction (0.6- to 2.3-fold increase, respectively). CONCLUSIONS: In Wistar rat conjunctiva, slow-cycling cells are primarily located in palpebral epithelium, which has greater proliferative capacity than other conjunctival epithelia. This finding means that, in the Wistar rat, the conjunctival epithelial stem cells are mainly located in palpebral epithelium. These data open new perspectives in ocular epithelial development and are relevant in conjunctival wound repair.     

Cell kinetics during healing of corneal epithelial wounds

After removing a circular area of the central corneal epithelium of the rat eye, the labelling indices and the mitotic rates were measured at various times after wounding, both in the cornea and in the adjacent conjunctival epithelium. The proliferative response was most marked in the corneal epithelium adjacent to the wound, but there was also a definite response in the epithelium covering the denuded areas, and in the conjunctival epithelium. The study demonstrated that the conjunctival epithelium. The study demonstrated that the conjunctiva itself plays a role in the healing of a central corneal epithelial wound. The similarities in the cellular response may indicate that both epithelia are under the influence of the same growth-suppressing factors (chalones), and must be looked upon as a unit. However, no support was found for the theory that the limbal area serves as a generative organ for the corneal epithelium.