Merjava Stanislava, Neuwirth Ales, Mandys Vaclav, Jirsova Katerina
Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, General Teaching Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic.
Exp Eye Res. 2009 Sep;89(3):426-31. doi: 10.1016/j.exer.2009.04.009. Epub 2009 May 4.
The aim of this study was to determine if cytokeratins (CKs) 8 and 18--typical epithelial cell markers--are constitutively expressed in adult human corneal endothelium. Cryosections, paraffin-embedded sections and corneal endothelial imprints obtained from eleven adult human corneal discs not suitable for transplantation were used. Different fixative solutions were applied before indirect immunofluorescent or enzymatic staining was performed with antibodies against CK8 (Chemicon), CK18 (Dako and Sigma) and CK8/18 (Novocastra). Semi-quantitative RT-PCR and Western blotting (mRNA or proteins were isolated from Millicell membranes) were used to determine cytokeratin mRNA and protein levels. Approximately 50% of the corneal endothelial cells were positive for CK8 (Chemicon), CK18 (Sigma) and the CK pair 8/18 (Novocastra) in the endothelium when acetone was used for fixation. Four and 52% CK18-positive cells were observed using immunofluorescent and enzymatic immunohistochemistry, respectively, when the CK18 antibody from Dako was used. No signal was detected when 4% formalin or 10% paraformaldehyde was used as a fixative, irrespective of the antibody used. CK8 and CK18 proteins and mRNAs were detected in the endothelium of all tested corneas by Western blotting or semi-quantitative RT-PCR, respectively. We detected both CK8 and CK18 in the endothelium of all specimens at both the protein and mRNA levels. These results clearly demonstrate that cells of the corneal endothelium express CKs 8 and 18 and share some features with simple epithelia.
本研究的目的是确定细胞角蛋白（CKs）8和18（典型的上皮细胞标志物）是否在成人角膜内皮中组成性表达。使用了从11个不适用于移植的成人角膜片中获得的冰冻切片、石蜡包埋切片和角膜内皮印片。在使用抗CK8（Chemicon）、CK18（Dako和Sigma）和CK8/18（Novocastra）抗体进行间接免疫荧光或酶染色之前，应用了不同的固定剂溶液。使用半定量RT-PCR和蛋白质印迹法（从Millicell膜中分离mRNA或蛋白质）来确定细胞角蛋白的mRNA和蛋白质水平。当使用丙酮固定时，约50%的角膜内皮细胞在CK8（Chemicon）、CK18（Sigma）以及内皮中的CK8/18对（Novocastra）方面呈阳性。当使用Dako公司的CK18抗体时，分别通过免疫荧光和酶免疫组织化学观察到4%和52%的CK18阳性细胞。当使用4%甲醛或10%多聚甲醛作为固定剂时，无论使用何种抗体均未检测到信号。分别通过蛋白质印迹法或半定量RT-PCR在所有测试角膜的内皮中检测到CK8和CK18蛋白及mRNA。我们在所有标本的内皮中均在蛋白质和mRNA水平检测到了CK8和CK18。这些结果清楚地表明，角膜内皮细胞表达CK8和CK18，并与单层上皮细胞具有一些共同特征。
