The development of basic technologies for the cellular therapy of corneal epithelial cells by the regulation of cellular senescence and epigenetic changes

在本研究中，为了建立从有限数量的自体角膜上皮细胞体外扩增的角膜上皮细胞移植的方法，我们研究了培养的角膜上皮细胞的细胞衰老和表观遗传学变化，以及针对这些培养相关缺陷的可能对策。我们发现，角膜上皮细胞分离后，角蛋白12基因的表达显著下降，这可能是由于MAPK等信号通路的激活伴随着角蛋白12启动子的表观遗传变化。研究发现，引入SV40大T抗原可以显着促进角膜上皮细胞的增殖，SV40大T抗原可以抑制P53和Rb的肿瘤抑制通路。相当有趣的是，当角膜上皮干细胞被转染SV40大T抗原时，发现内源性hTERT基因被激活，这被认为是导致这些细胞致瘤转化的风险。

In this study, in order to establish the method to transplant corneal epithelial cells which are amplified ex vivo from the limited number of autologous corneal epithelial cells, we investigated the cellular senescence and epigenetic changes of the cultured corneal epithelial cells as well as the possible countermeasures against these culture-associated drawbacks. We have found that the expression of the keratin 12 gene significantly decreased rapidly after the cell dissociation of the corneal epithelial cells, which was found to be due to the activation of several signaling pathways such as MAPK accompanied with the epigenetic changes of the keratin 12 promoter. The proliferation of the corneal epithelial cell was found to be significantly enhanced by the introduction of SV40 large T antigen which is known to inhibit p53 and Rb tumor- suppressive pathways. Quite interestingly, when corneal epithelial stem cells were transfected with the SV40 large T antigen, the endogenous hTERT gene was found to be activated, which is thought to be the risk to lead the tumorigenic transformation of these cells.