CORNEAL ENDOTHELIAL CELL REGENERATION

角膜内皮细胞再生

• 批准号: 2430395
• 负责人: TIMOTHY Peter FLEMING
• 金额: $ 17.72万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2430395
• 关键词: DNA replication; animal tissue; cats; cell cycle; cell growth regulation; cell line; cell nucleus; chimeric proteins; corneal endothelium; density gradient ultracentrifugation; electron microscopy; eye regeneration; herpes simplex virus 1; human tissue; laboratory rabbit; liposomes; molecular cloning; organ culture; polymerase chain reaction; protein structure function; simian virus 40; tissue /cell culture; transfection /expression vector; virus antigen; virus protein

The goal of this proposal is to circumvent penetrating keratoplasty (PKP) by stimulating the growth of in situ corneal endothelial cells (CEC). The rationale for this strategy is based on the observation that virally introduced SV40 large T-antigen gene can rescue in vitro cultured human CEC from senescent non-proliferation. These immortalized (more appropriately, life-(extended) cells retain morphologic and immunologic features observed in primary cultures of normal adult CEC. Since this immortalizing gene can induce proliferation of corneal endothelial cells in vitro, we propose. to test the hypothesis that an immortalizing protein can induce limited proliferation when introduced into cells. Thus many negative feature associated with gene therapy approaches using transforming genes can be avoided by focusing on specific protein introduction. The half-life of the protein dictates a limited presence to initiate a transient state of proliferation and then as the protein is normally degraded, the cells return to "normal". Therefore, we propose to determine whether the specific introduction of a potent intracellular mitogen (SV40) large T-antigen) can stimulate limited in vivo proliferation of the normally non-dividing corneal endothelial cells thus maintaining cornea function and abrogate the need for many cornea transplants. For this system to work in corneal endothelial cells, we need to address two important questions and to do this, we propose the following specific aims; 1. Test the hypothesis that purified SV40 large T-antigen can be introduced in cultured cells and extend this strategy to human corneal endothelials cells (HCEC). It will be determined whether the introduced SV40 T-antigen localizes to the nucleus and can stimulate DNA synthesis in non-replicating cells. 2. Test the hypothesis that Herpes Simplex Virus (HSV) delta L-particles can be used as an efficient "entry vehicle" for the specific delivery of protein larvae T-antigen into cells and validate its effectiveness in both cultured cells and in an animal model.

该建议的目的是规避穿透性角膜移植术（PKP） 通过刺激原位角膜内皮细胞（CEC）的生长。 的 这种策略的基本原理是基于观察到， 导入SV 40大T抗原基因对体外培养人CEC的拯救作用 来自衰老的防扩散。 这些不朽的（更恰当地说， 寿命-（延长）细胞保留观察到的形态学和免疫学特征 在正常成人CEC的原代培养物中。 由于该永生化基因可以诱导角膜上皮细胞增殖， 内皮细胞在体外，我们建议。来验证一个假设， 永生化蛋白在引入到细胞中时可以诱导有限的增殖， 细胞 因此，许多与基因治疗方法相关的负面特征 通过聚焦于特定蛋白质，可以避免使用转化基因 导论. 蛋白质的半衰期决定了蛋白质的有限存在， 启动短暂的增殖状态，然后当蛋白质 正常降解后，细胞恢复“正常”。 因此，我们建议确定是否具体引入一个 有效的细胞内有丝分裂原（SV 40）大T抗原）可以刺激有限的 正常不分裂的角膜内皮细胞的体内增殖 细胞，从而维持角膜功能，并消除了许多需要 角膜移植 为了使这个系统在角膜内皮细胞中起作用， 我们需要解决两个重要问题，为此，我们建议 具体目标; 1. 验证纯化的SV 40大T抗原可以 并将该策略扩展到人类角膜 内皮细胞（HCEC）。 将决定是否引进 SV 40 T抗原定位于细胞核，可刺激细胞内DNA合成。 非复制细胞 2. 检验单纯疱疹病毒（HSV）δ L颗粒 可作为一种有效的“入境车辆”， 蛋白幼虫T抗原进入细胞，并验证其有效性， 培养的细胞和动物模型。