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

以人角膜内皮（后角膜上皮）为模型的人体细胞瞬时永生化

• 批准号: 232772305
• 负责人: Dr. Monika Valtink
• 金额: --
• 依托单位: Institut für Anatomie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232772305?language=en
• 关键词: Transient; immortalisation; human; cells; using

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.

终末分化细胞的特征是离开细胞周期，这是一个主动维持的过程，在操纵后可能是可逆的。人角膜内皮细胞（HCEC）就是这样一种有丝分裂后的细胞，在体内几乎没有增殖活性，在体外也只有有限的增殖。年龄、退行性变、疾病或手术干预可导致角膜内皮细胞的丧失，因此需要角膜移植，但质量足够的供体角膜数量有限。为了提高供体角膜的质量和更详细地分析这些细胞，开发一种不丧失其典型形态学特征的HCEC体外和原位增殖方法将大大加快角膜研究。在这个项目中，需要鉴定细胞生长因子和病毒/细胞癌基因，通过逆转录病毒基因转移稳定表达后，使原发性HCEC能够简单、可重复地永生化。随后，通过以下两种策略暂时促进增殖，将分析这些已确定的因素通过瞬时转基因表达扩大HCEC原代培养的能力：1)非整合逆转录病毒基因转移后的瞬时转基因表达，2)在生长培养基中添加重组嵌合蛋白，这些重组嵌合蛋白通过蛋白质转导域被HCEC吸收，并在细胞内发挥促进增殖的活性。