Wilson S E, Li Q, Weng J, Barry-Lane P A, Jester J V, Liang Q, Wordinger R J
Eye Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Invest Ophthalmol Vis Sci. 1996 Jul;37(8):1582-92.
Previous studies have suggested that the disappearance of anterior keratocytes after injury to the overlying epithelium is mediated by apoptosis. The authors examined the expression of the apoptosis-related modulators, Fas (receptor), Fas ligand, Bax, Bcl-2, Bcl-XL, and interleukin-1 beta converting enzyme (ICE) in corneal cells as candidate mediators of this response and tested the effect of Fas receptor-stimulating antibody on corneal stromal fibroblast cells in vitro.
Reverse-transcription-polymerase chain reaction was used to detect FAS, FAS ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNA expression in primary cultures of human corneal epithelial, stromal fibroblast, and endothelial cells. Immunohistochemistry was applied to detect Fas and Fas ligand proteins in fresh-frozen sections of normal human cornea. The effect of FAS-stimulating monoclonal antibody on first-passage stromal fibroblasts was studied using a DNA fragmentation assay, the live-dead assay with fluorescent microscopy, toluidene blue staining with light microscopy, and electron microscopy.
FAS, Fas ligand, Bax, Bcl-2, Bcl-XL, and ICE mRNAs are expressed in all three major cell types of the cornea. Fas protein is expressed in corneal epithelial, keratocyte, and endothelial cells in fresh-frozen human cornea. Fas ligand protein, however, was detected in corneal epithelial and endothelial, but not keratocyte, cells. Fas-stimulating antibody induced first-passage stromal fibroblast cell death with morphologic changes and DNA fragmentation consistent with apoptosis.
The Fas system (Fas and Fas ligand) modulators and final common pathway mediators of apoptosis are expressed in corneal cells. The distribution of Fas (epithelial, keratocyte, and endothelial cells) and Fas ligand (epithelial and endothelial cells) protein expression in fresh-frozen corneal tissue suggests that Fas ligand expressed in corneal epithelial and endothelial cells modulates functions in keratocyte cells and, possibly, autocrine-juxtacrine functions in epithelium and endothelium. The Fas-Fas ligand system is expressed in the cornea and could have important functions in normal corneal physiology and in the pathophysiology of corneal disease, including modulation of keratocyte apoptosis after epithelial injury.
以往研究表明，角膜上皮损伤后前基质细胞的消失是由凋亡介导的。作者检测了凋亡相关调节因子Fas（受体）、Fas配体、Bax、Bcl-2、Bcl-XL和白细胞介素-1β转换酶（ICE）在角膜细胞中的表达，作为这种反应的候选介质，并在体外测试了Fas受体刺激抗体对角膜基质成纤维细胞的作用。
采用逆转录聚合酶链反应检测人角膜上皮细胞、基质成纤维细胞和内皮细胞原代培养物中FAS、FAS配体、Bax、Bcl-2、Bcl-XL和ICE mRNA的表达。应用免疫组织化学检测正常人角膜新鲜冰冻切片中Fas和Fas配体蛋白。采用DNA片段化分析、荧光显微镜活死细胞检测、光学显微镜甲苯胺蓝染色和电子显微镜研究FAS刺激单克隆抗体对第一代基质成纤维细胞的作用。
FAS、Fas配体、Bax、Bcl-2、Bcl-XL和ICE mRNA在角膜的所有三种主要细胞类型中均有表达。Fas蛋白在人角膜新鲜冰冻切片的角膜上皮细胞、基质细胞和内皮细胞中表达。然而，Fas配体蛋白在角膜上皮细胞和内皮细胞中检测到，但在基质细胞中未检测到。Fas刺激抗体诱导第一代基质成纤维细胞死亡，其形态学变化和DNA片段化与凋亡一致。
Fas系统（Fas和Fas配体）及凋亡的最终共同途径介质在角膜细胞中表达。新鲜冰冻角膜组织中Fas（上皮细胞、基质细胞和内皮细胞）和Fas配体（上皮细胞和内皮细胞）蛋白表达的分布表明，角膜上皮细胞和内皮细胞中表达的Fas配体调节基质细胞的功能，并可能调节上皮细胞和内皮细胞的自分泌-旁分泌功能。Fas-Fas配体系统在角膜中表达，可能在正常角膜生理及角膜疾病的病理生理中发挥重要作用，包括调节上皮损伤后基质细胞的凋亡。
