Transient immortalisation of human cells using human corneal endothelium (posterior corneal epithelium) as a model

终末分化的细胞的特征在于离开细胞周期，这是一个积极维持的过程，在操作时可能是可逆的。人角膜内皮细胞（human corneal endothelial cells，HCEC）是一种处于有丝分裂后的细胞，在体内几乎没有增殖活性，在体外增殖能力有限。年龄、变性、疾病或手术干预可导致角膜内皮细胞的损失，并因此需要角膜移植，但质量足够的供体角膜的数量有限。开发一种方法，在体外和原位增殖的HCEC，而不损失其典型的形态特征，以提高质量不足的供体角膜，并分析这些细胞在更详细的将大大加快角膜的研究。在本项目中，应鉴定细胞生长因子和病毒/细胞癌基因，其允许使用逆转录病毒基因转移稳定表达后的原代HCEC的简单和可重现的永生化。随后，将分析这些鉴定的因子通过瞬时转基因表达扩增HCEC原代培养物的能力，所述瞬时转基因表达是以下两种策略暂时促进增殖的结果：1）非整合逆转录病毒基因转移后的瞬时转基因表达，和2）在生长培养基中补充重组嵌合蛋白，所述重组嵌合蛋白通过蛋白转导结构域被HCEC摄取并产生它们的细胞内增殖促进活性。

Terminally differentiated cells are characterised by leaving the cell cycle, an actively maintained process that may be reversible upon manipulation. Human corneal endothelial cells (HCEC) are such post-mitotic cells, which show almost no proliferative activity in vivo and only limited proliferation in vitro. Age, degeneration, diseases or surgical interventions can cause a loss of corneal endothelial cells and by this the need for a corneal transplant, but the number of qualitatively sufficient donor corneas is limited. To develop a method for in vitro and in situ multiplication of HCEC without loss of their typical morphological features in order to improve the quality of insufficient donor corneas and also to analyse these cells in more detail would greatly expedite corneal research. In this project, cellular growth factors and viral/cellular oncogenes shall be identified, which allow simple and reproducible immortalisation of primary HCEC after stable expression using retroviral gene transfer. Subsequently, such identified factors will be analysed regarding their ability to expand primary cultures of HCEC by transient transgene expression as a result of temporary promotion of proliferation following two strategies: 1) transient transgene expression after non-integrating retroviral gene transfer, and 2) supplementation of the growth medium with recombinant chimeric proteins that are taken up by HCEC through protein transduction domains and develop their proliferation promoting activity intracellularly.