角膜上皮树突状细胞免疫学特性研究的现状

角膜上皮内存在的朗格罕细胞型树突状细胞具有吞噬、加工、递呈抗原及激发T细胞的能力.这些细胞是"职业的"抗原递呈细胞,在眼表免疫系统中起关键的免疫监视作用.近期,有研究证明在正常的角膜缘上皮基底层存在具有慢周期特性的、表达角膜缘干细胞标识物-ATP粘连转运蛋白家族G2成员(ABCG2)的树突状细胞.进一步研究发现,中央角膜炎症反应早期可以诱导角膜缘上皮基底层表达主要组织相容性复合体Ⅱ型抗原的树突状细胞密度增加并向角膜中央迁移.这些研究提示角膜缘上皮基底层内存在的树突状细胞是角膜炎症反应发生过程中的重要参与者.     

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.     

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures

BACKGROUND/AIM: Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. METHODS: The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. RESULTS: Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. CONCLUSIONS: These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.     

Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness

The concept that corneal epithelium stem cells reside in limbus has been recognized for more than a decade, but isolation of these stem cells has not been accomplished. This study was an initial attempt to isolate a population of human limbal epithelial cells enriched for certain putative stem cell properties based on their phenotype. Epithelial cells harvested from fresh human limbal rings and their primary cultures were allowed to adhere to collagen IV-coated dishes for 20 min and 2 hr, sequentially. The rapidly adherent cells (RAC), slowly adherent cells and non-adherent cells were evaluated for certain stem cell properties: (a) BrdU-label retention, (b) expression of basal cell (integrin beta1, p63, ABCG2) and differentiation (involucrin, keratin 12) markers, and (c) colony forming efficiency (CFE) and growth capacity on a 3T3 fibroblast feeder layer. Among unfractionated cells and the three selected populations, the RAC, accounting for about 10% of whole population, were enriched 5-fold in BrdU label-retaining cells, displayed the highest number of integrin beta1 and p63 positive and involucrin negative cells, expressed high levels of DeltaNp63 and ABCG2 mRNA, and lacked involucrin and K12 expression, and possessed the greatest CFE and growth capacity. These findings demonstrated for the first time that human limbal epithelial cells with stem cell properties can be partially enriched by their adhesiveness to collagen IV. The RAC population enriched for certain putative stem cell properties may prove useful in the future for transplantation to diseased and damaged corneas with limbal stem cell deficiency.     

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。     

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。     

Improved transduction of human corneal epithelial progenitor cells with cell-targeting adenoviral vectors

The development of vectors and techniques to transfer therapeutic genes to corneal epithelium has broad clinical applications. To determine if adenoviral (Ad5) vectors could be tailored to increase transduction of corneal epithelial progenitor cells expressing epidermal growth factor receptor (EGFR), the feasibility of targeting gene therapy vectors to genetically modify primary cultured human corneal epithelial cells (PHCEC) was evaluated. PHCECs were cultured from human limbal explants and transduced with Ad5 vectors containing the enhanced green fluorescent protein (GFP) reporter cassette to mediate gene transfer. The efficiencies of transduction with different Ad5 dosages and different time periods of exposure were compared. Metabolically biotinylated Ad5 vectors were retargeted to PHCECs using biotinylated epidermal growth factor (EGF) as a cell-targeting ligand. Phenotypes and function assays of transduced cells were determined by real-time PCR and BrdU incorporation. Ad5 vectors transduced approximately 50-93% of PHCEC at 10-100 PFU/cell in a dose-dependent manner and the transgene persisted for more than 2 weeks in vitro. Retargeting of biotinylated Ad5 with EGF increased transduction of EGFR and ABCG2-expressing corneal epithelial progenitor cells up to nine-fold and reduced transduction of K12 and involucrin-expressing differentiated corneal epithelial cells and had higher BrdU incorporation indexes. These data provide proof of principle that ligand-bearing modified Ad5 vectors can target a population of corneal epithelial progenitor cells for corneal gene therapy.     

羊膜为载体的人角膜缘上皮细胞移植治疗大鼠角膜碱烧伤的研究

目的研究人角膜缘上皮细胞的生物学特性,探讨体外培养的人角膜缘上皮细胞移植进行眼表重建的临床应用价值及前景。方法体外培养人角膜缘上皮细胞,通过免疫荧光染色法和逆转录聚合酶链反应（RT．PCR）法分析表型,Brdu掺入法检测慢周期（slow—cycling）细胞。建立大鼠角膜碱烧伤模型,随机分为单纯对照组、羊膜移植（AMT）组及羊膜为载体的角膜缘上皮细胞移植（LSAT）组,术后通过裂隙灯显微镜观察、角膜切片HE染色及免疫荧光染色等方法对各组大鼠眼表恢复情况进行评价。结果培养早期大多数角膜缘上皮细胞p63和K19染色阳性,仅少数细胞K3染色阳性;随培养时间的延长,K3阳性细胞增多,部分细胞同时表达p63和K3。RT—PCR示培养的角膜缘上皮细胞中p63和K12均有阳性表达,而角膜上皮细胞仅表达K12。Brdu掺入法检测到慢周期细胞。动物实验表明,LSAT可以显著减轻角膜病变,使角膜恢复完整性和透明性,眼表评分各项指标与AMT组和对照组比较差异均有统计学意义。LSAT组大鼠角膜切片染色示大部分上皮细胞抗人核和K3染色阳性。结论体外培养的人角膜缘上皮细胞中p63不仅表达于角膜缘干细胞,在短暂扩充细胞中也有一定量的表达;慢周期细胞的存在证实了培养的角膜缘上皮细胞中角膜缘干细胞的存在,p63和K19阳性、K3阴性的细胞为角膜缘干细胞;羊膜为载体的体外培养的人角膜缘上皮细胞移植可有效重建眼表,具有较高的临床应用价值与发展前景。     

High expression of p63 combined with a large N/C ratio defines a subset of human limbal epithelial cells: implications on epithelial stem cells

PURPOSE: To characterize human limbal epithelial cells based on the expression levels of nuclear protein p63 and the nucleus-to-cytoplasm (N/C) ratio. METHODS: Limbal, peripheral, and central corneal epithelia were separated from the stroma by Dispase II and subsequently were treated with trypsin to obtain single-cell suspensions. Cytospin smears of the cell suspensions were double immunostained for p63 and then stained for any one of the markers (acidic cytokeratins [AE1], K5, K3, or connexin 43 [Cx43]). They were counterstained with propidium iodide. More than 100 cells from each zone were analyzed for p63 expression levels and nuclear/cellular area using quantitative confocal microscopy. RESULTS: A gradient of p63-positive cells was observed in corneal and limbal epithelial cells. The percentage of p63-positive cells and the level of p63 expression were significantly higher in the limbal than in the peripheral or central corneal epithelium. Two-parameter (p63 levels and N/C ratio) analysis revealed the presence of a distinct population of small cells with higher levels of p63 and a large N/C ratio in the limbal epithelium. Such limbal epithelial cells were positive for AE1 and K5 but negative for K3 and Cx43. CONCLUSIONS: These results suggest that this distinct group of small cells in the limbal epithelium with greater N/C ratio, expressing high levels of nuclear protein p63, probably represent corneal epithelial stem cells.     

Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims

Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes. Successful cultures in each medium developed predominantly small (10 μm) tightly packed cells. Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19. Expression of these keratins in situ was confirmed by immunohistochemistry. Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3. However, intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims. These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea.     

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.     

Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche

BACKGROUND/AIMS: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. METHODS: Systematic serial 5-7 microm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the "stem cell" marker ABCG2 transporter protein. RESULTS: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. CONCLUSIONS: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.     

Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.     

Identification and characterization of limbal stem cells

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SC) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SC biology remains the ability to identify stem cells in situ and in vitro. Until recently, the identification of limbal stem cells mainly has been based on general properties of stem cells, e.g. lack of differentiation, prolonged label-retaining, indefinite capacity of proliferation exemplified by the clonogenic assay as well as their special role in corneal wound healing. During the last years, a number of molecular markers for the limbal SC compartment has been proposed, however, their role in distinguishing limbal SC from their early progeny is still under debate. Data reported from the literature combined with our own recent observations suggest, that the basal epithelial cells of the human limbus contain ABCG2, K19, vimentin, KGF-R, metallothionein, and integrin alpha9, but do not stain for K3/K12, Cx43, involucrin, P-cadherin, integrins alpha2, alpha6, and beta4, and nestin, when compared to the basal cells of the corneal epithelium. A relatively higher expression level in basal limbal cells was observed for p63, alpha-enolase, K5/14, and HGF-R, whereas there were no significant differences in staining intensity for beta-catenin, integrins alphav, beta1, beta2, and beta5, CD71, EGF-R, TGF-beta-RI, TGF-beta-RII, and TrkA between limbal and corneal basal epithelial cells. Therefore, a combination of differentiation-associated markers (e.g. K3/K12, Cx43, or involucrin) and putative SC-associated markers (e.g. ABCG2, K19, vimentin, or integrin alpha9) may provide a suitable tool for identification of human limbal SC. While most putative SC markers label the majority of limbal basal cells and, therefore, may not distinguish SC from progenitor cells, only ABCG2 was strictly confined to small clusters of basal cells in the limbal epithelium. At present, ABCG2 therefore appears to be the most useful cell surface marker for the identification and isolation of corneal epithelial SC. Moreover, the characteristics of the specific microenvironment of corneal SC, as provided by growth factor activity and basement membrane heterogeneity in the limbal area, could serve as additional tools for their selective enrichment and in vitro expansion for the purpose of ocular surface reconstruction.     

Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane.

PURPOSE: Stem cell (SC)-containing limbal basal epithelium and transient amplifying cell (TAC)-containing corneal basal epithelium lie on different mesenchymal matrices. The gap junction protein connexin 43 (Cx43) is absent in the limbal basal epithelium but is present in the corneal basal epithelium, suggesting that the expression of Cx43 denotes SC differentiation into TACs. Amniotic membrane (AM) can expand limbal epithelial progenitor cells in vivo and in culture for subsequent corneal surface reconstruction. In this study, the modulation of Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of ex vivo expanded human limbal epithelial (HLE) cells on intact and epithelially denuded AM was investigated. METHODS: HLE cells were expanded on intact (i.e., remaining devitalized amniotic epithelium) or epithelially denuded AM (EDTA-treated). Cx43 expression and 24-hour 5-bromo-2'-deoxyuridine-5'monophosphate (BrdU) labeling index (percentage) were determined by double immunostaining. GJIC was investigated by a scrape-loading dye transfer assay. In a subset of cultures Cx43 and K3 keratin as well as BrdU-retaining nuclei were analyzed in the stratified epithelium obtained 5 days after subcutaneous transplantation in NIH bg-nu-xidBR mice of AM cultures continuously labeled with BrdU for 7 days. RESULTS: The outgrowth rate, overall, was significantly higher on EDTA-treated AM than on intact AM (P < 0.05). Cx43 was expressed in 12.4% +/- 14.5% (n = 5) on intact and 57.5% +/- 18.2% (n = 5) on EDTA-treated AM (P < 0.05). The BrdU labeling index was 2.4% +/- 0.9% (n = 5) for the intact AM group, which was significantly less than 22.5% +/- 8.2% (n = 5) for EDTA-treated AM (P < 0.05). BrdU-labeled cells did not express Cx43. The dye transfer assay revealed reduced GJIC on both AM-cultured groups compared with the control culture on plastic (P < 0.002). GJIC on intact AM (17%) was reduced compared with that on EDTA-treated AM (27%; P = 0.42). After xenotransplantation, the basal layer of the stratified epithelium was Cx43 and K3 keratin negative and retained BrdU on intact AM, resembling characteristics of the limbal basal epithelium in vivo. In contrast, that of EDTA-treated AM was Cx43 and K3 keratin positive without BrdU retention, resembling characteristics of the corneal epithelium in vivo. CONCLUSION: These data indicate that denudation of the devitalized amniotic epithelium to expose its basement membrane might be a microenvironmental cue to promote TAC differentiation. The model system described herein is ideal for future exploration of the exact mechanistic operation in the microenvironmental niche that maintains the "stemness" of limbal SCs as well as in the signal that promotes corneal TAC differentiation.     

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。     

Expansion of conjunctival epithelial progenitor cells on amniotic membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells. Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture. After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase. Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.     

Characterisation of rat corneal cells that take up soluble antigen: an in vivo and in vitro study

The aim of the present study was to determine the capacity of resident corneal and limbal dendritic cells (DC) and macrophages to capture antigen (Ag) in vivo and compare this to their capacity in vitro to take up Ag during organ culture conditions. To investigate Ag uptake in vivo 3 microl (30 microg) of fluorescently labelled Dextran, bovine serum albumin (BSA) or ovalbumin (OVA) were either placed on the intact ocular surface or injected into the anterior chamber (AC) or subconjunctival space of the Lewis rat eye. The presence of Ag+ cells in the cornea was assessed using intravital fluorescence video microscopy. Animals were sacrificed 24h after Ag injections or topical application and the distribution and phenotype of Ag+ cells were analysed ex vivo by fluorescence and confocal microscopic analysis of immunostained and unstained corneal tissue wholemounts or frozen sections. Corneal buttons and corneoscleral rims from naive Lewis rats were placed in organ culture conditions in the presence of LPS with or without FITC-Dextran for 48 h and 72 h. The explants were examined by epi-fluorescence microscopy and the phenotype of Ag+ cells in the supernatant from the organ cultures was analyzed by flow cytometry using a range of macrophage and DC markers. In vivo observations and microscopic examination of corneas 24h following Ag topical application failed to reveal evidence of Ag retention by ocular cells. Those in which Ag had been placed in the AC or subconjunctival space revealed Ag+ cells within the corneal stroma. The distribution of Ag+ cells displayed a centripetal gradient, the most marked uptake of Ag being by cells in the circumferential limbal zone. Immunophenotypic studies revealed that Ag uptake was predominantly performed by cells that were CD68+, CD172+ but rarely MHC class II+, a profile characteristic of macrophages. Occasional Ag+ keratocytes were noted. In vitro studies of corneal buttons placed in culture conditions revealed that cells from the limbal zone, but not the central cornea, were able to take up Ag from the supernatant. Significant numbers of the cells that had migrated from the corneal buttons and captured fluorescent labelled Ag in the presence of LPS were revealed by flow cytometry to consist of CD163+ and CD11b+ macrophages, but none expressed the DC markers CD11c or OX62 and they were also generally MHC class II(-). In conclusion the present study revealed that macrophages and keratocytes in the corneal stroma and limbal episcleral tissue have the capacity to internalise fluorescent mock Ag injected into the AC or subconjunctival space or in culture conditions. The failure to demonstrate significant Ag trapping ability by corneal or limbal stromal or epithelial DC may either be due to the rarity of such cells or their lack of Ag trapping ability.     

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.