Wilmer Eye Institute, The Johns Hopkins Medical Institutions, Smith 5011 400 N. Broadway, Baltimore, MD 21231, USA.
Graefes Arch Clin Exp Ophthalmol. 2012 Jan;250(1):103-10. doi: 10.1007/s00417-011-1805-7. Epub 2011 Sep 1.
To characterize the morphology and gene expression of transformed murine corneal endothelial cells.
Primary immortomouse corneal endothelial cells were continuously cultured before and after cryopreservation. Morphologic assessment, real time-reverse transcriptase polymerase chain reaction ((RT)-PCR) and immunofluorescence studies were performed using newly cultured cells, cells that had been continuously in culture for 1 year, and cryopreserved cells, to assess for structural and functional integrity. The expression of corneal endothelial markers zonula occludens-1 (ZO1), NaK-ATPase and collagen VIII (α2) (COL8A2), and myofibroblast markers Desmin, alpha smooth muscle actin (αSMA), and Vimentin was assessed and compared by both RT-PCR and immunofluorescence.
Cells in culture formed a monolayer, and exhibited a polygonal shape after reaching confluence. Cells retained this morphology during the full observation time of 12 months and when reused after cryopreservation. Immunofluorescence experiments exhibited positive staining for NaK-ATPase and COL8A2 with low variability between the three groups. In RT-PCR experiments, ZO1, COL8A2 and Desmin were increased in fresh and thawed cells, αSMA was decreased, and NaK-ATPase and Vimentin remained unchanged, compared to 12-month-old cells. Comparing fresh and thawed cells, COL8A2 was increased in thawed cells, while Desmin was increased in fresh cells.
Using the immortomouse strain, murine corneal endothelial cells can be propagated over a long time period and be used after cryopreservation. Cells retain the expression of NaK-ATPase, but show some decline in ZO1 and COL8A2 over time and after cryopreservation. The expression of myofibroblast markers suggests an endothelial-to-mesenchymal transformation process in culture.
描述转化的鼠角膜内皮细胞的形态和基因表达。
在冷冻保存前后，连续培养原代永生化鼠角膜内皮细胞。使用新培养的细胞、连续培养 1 年的细胞和冷冻保存的细胞进行形态评估、实时逆转录聚合酶链反应（RT-PCR）和免疫荧光研究，以评估结构和功能的完整性。通过 RT-PCR 和免疫荧光评估角膜内皮标志物紧密连接蛋白-1（ZO1）、NaK-ATP 酶和胶原 VIII（α2）（COL8A2）以及肌成纤维细胞标志物结蛋白、α 平滑肌肌动蛋白（αSMA）和波形蛋白的表达，并进行比较。
培养的细胞形成单层，在达到汇合后呈多边形。当在 12 个月的整个观察时间内以及在冷冻保存后重新使用时，细胞保持这种形态。免疫荧光实验显示 NaK-ATP 酶和 COL8A2 的阳性染色，三组之间的变异性较小。在 RT-PCR 实验中，与 12 月龄细胞相比，新鲜和解冻细胞中 ZO1、COL8A2 和结蛋白增加，αSMA 减少，NaK-ATP 酶和波形蛋白不变。与新鲜细胞相比，解冻细胞中 COL8A2 增加，而新鲜细胞中结蛋白增加。
使用永生化鼠株，可长时间繁殖鼠角膜内皮细胞，并可在冷冻保存后使用。细胞保留 NaK-ATP 酶的表达，但随着时间的推移和冷冻保存后，ZO1 和 COL8A2 的表达略有下降。肌成纤维细胞标志物的表达提示在培养过程中存在内皮到间充质转化过程。
