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

终末分化细胞的特征是离开细胞周期，这是一个积极维持的过程，可能在操纵后是可逆的。人角膜内皮细胞是一种有丝分裂后细胞，在体内几乎不具有增殖活性，在体外仅表现出有限的增殖活性。年龄、退化、疾病或手术干预可导致角膜内皮细胞的丢失，从而需要角膜移植，但质量充足的供体角膜的数量有限。为了提高供体角膜的质量，并对供体细胞进行更详细的分析，建立一种在不丢失其典型形态特征的情况下进行体外和原位增殖的方法，将大大加快角膜研究的进程。在这个项目中，将确定细胞生长因子和病毒/细胞癌基因，使原代HCEC在使用逆转录病毒基因转移稳定表达后简单且可重复地永生化。随后，将分析这些已确定的因素通过瞬时转基因表达来扩大HCEC原代培养的能力，这些因素遵循以下两种策略：1)非整合逆转录病毒基因转移后的瞬时转基因表达，以及2)在生长介质中添加HCEC通过蛋白转导结构域摄取的重组嵌合蛋白，并在细胞内开发其促增殖活性。

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.