荧光素染色和角膜上皮细胞的生理状态。

Fluorescein staining and physiological state of corneal epithelial cells.

机构信息

Brien Holden Vision Institute, Sydney, Australia; School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia.

Brien Holden Vision Institute, Sydney, Australia; School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia; Vision Cooperative Research Centre, Sydney, Australia.

出版信息

Cont Lens Anterior Eye. 2014 Jun;37(3):213-23. doi: 10.1016/j.clae.2013.11.003. Epub 2013 Dec 12.

DOI:10.1016/j.clae.2013.11.003

PMID:24332360

Abstract

PURPOSE

To evaluate the physiological status of corneal epithelial cells exhibiting fluorescein staining.

METHODS

Fluorescein staining properties of corneal epithelial cells under normal and stressed conditions were studied using cell-culture (human corneal limbal epithelial cells - HCLE) and organ-culture (rabbit) models. Stress stimuli comprised exposure to hypotonicity, hypertonicity, preservatives, scratch, and alkaline wounding. In addition to fluorescein, cells were stained with Hoechst-33342 (HO), Propidium-iodide (PI), and Annexin-V (AN-V) to identify live, dead and apoptotic cells. Clinical-slit-lamp and fluorescence confocal-microscopic (FCM) observations were performed. FCM images were quantified for fluorescence intensity using Image-J software.

RESULTS

Healthy HCLE cells uniformly took up fluorescein to a moderate degree with a mean grey value of 62 ± 24 (mean ± SD) on a scale of 0-256 (no unit). Fluorescence levels similar to those observed prior to stress were associated with healthy cells. Apoptotic cells showed the highest fluorescence (138 ± 38). Dead cells showed minimal fluorescence (23 ± 7) that was similar to the background (20 ± 11, p>0.05). Observations in whole rabbit eyes were in general agreement with these cell culture findings.

CONCLUSIONS

The clinical observation of corneal staining with fluorescein suggests the presence of epithelial cells that are undergoing apoptosis but does not indicate dead cells. Under in vitro or ex vivo conditions, healthy cells took up fluorescein at levels that were lower than those of apoptotic cells and thus, are not likely to be perceived as exhibiting staining during clinical observation. Sodium fluorescein may be considered as a probe for apoptotic epithelial cells.

摘要

目的

评估表现为荧光素染色的角膜上皮细胞的生理状态。

方法

使用细胞培养（人角膜缘上皮细胞-HCLE）和器官培养（兔）模型研究正常和应激条件下角膜上皮细胞的荧光素染色特性。应激刺激包括暴露于低渗、高渗、防腐剂、划痕和碱性创伤。除了荧光素，还使用 Hoechst-33342（HO）、碘化丙啶（PI）和 Annexin-V（AN-V）对细胞进行染色，以识别活细胞、死细胞和凋亡细胞。进行临床裂隙灯和荧光共聚焦显微镜（FCM）观察。使用 Image-J 软件对 FCM 图像的荧光强度进行量化。

结果

健康的 HCLE 细胞均匀摄取荧光素，程度适中，在 0-256 灰度级上的平均灰度值为 62±24（平均值±标准差）。与应激前观察到的相似的荧光水平与健康细胞相关。凋亡细胞显示出最高的荧光（138±38）。死细胞显示出最小的荧光（23±7），与背景相似（20±11，p>0.05）。整个兔眼的观察结果与这些细胞培养发现基本一致。