Cryopreservation and long-term culture of transformed murine corneal endothelial cells.
Cryopreservation and long-term culture of transformed murine corneal endothelial cells.
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转化小鼠角膜内皮细胞的冷冻保存和长期培养。
DOI：
10.1007/s00417-011-1805-7
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Jun,AlbertS
中科院分区：
文献类型：
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作者：
Engler,Christoph;Kelliher,Clare;Chang,Sungdong;Meng,Huan;Jun,AlbertS
目的研究转基因小鼠角膜内皮细胞的形态学和基因表达特征。使用新培养的细胞、已连续培养1年的细胞和冻存细胞进行形态学评估、真实的时间逆转录酶聚合酶链反应（（RT）-PCR）和免疫荧光研究，以评估结构和功能完整性。RT-PCR和免疫荧光法检测角膜内皮细胞标志物封闭小带1（ZO 1）、Na K ATP酶和VIII型胶原（α2）（COL 8A 2），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（Vimentin）的表达。在12个月的完整观察时间内以及在冷冻保存后重复使用时，细胞保持这种形态。免疫荧光实验显示NaK-ATP酶和COL 8A 2阳性染色，三组之间的变异性较低。在RT-PCR实验中，与12月龄的细胞相比，新鲜和解冻的细胞中ZO 1、COL 8A 2和Desmin增加，αSMA减少，NaK-ATPase和Vimentin保持不变。比较新鲜和解冻的细胞，COL 8A 2在解冻的细胞中增加，而结蛋白在新鲜cells.ConclusionsUsing immartomouse strain，小鼠角膜内皮细胞可以在一个较长的时间内增殖，并在冻存后使用。细胞保留NaK-ATPase的表达，但随着时间的推移和冷冻保存后，ZO 1和COL 8A 2显示出一定程度的下降。肌成纤维细胞标志物的表达表明培养物中存在内皮细胞向间充质细胞转化的过程。
PurposeTo characterize the morphology and gene expression of transformed murine corneal endothelial cells.MethodsPrimary 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.ResultsCells 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.ConclusionsUsing 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.