CORNEAL ENDOTHELIAL CELL REGENERATION

这项建议的目的是绕过穿透性角膜移植(Pkp)。 通过刺激原位角膜内皮细胞(CEC)的生长。这个 这一策略的理论基础是基于观察到的病毒 导入SV40大T抗原基因可挽救体外培养的人CEC 来自衰老的核不扩散。这些永垂不朽(更恰当地说， 寿命延长的细胞保留了观察到的形态和免疫学特征 在正常成人CEC的原代培养中。 由于这种永生化基因可以诱导角膜的增殖 我们建议在体外培养内皮细胞。来检验一个假设 永生化蛋白被引入细胞后可以诱导有限的增殖。 细胞。因此，与基因治疗方法相关的许多负面特征 通过专注于特定的蛋白质可以避免使用转化基因 引言。蛋白质的半衰期决定了有限的存在 启动一种短暂的增殖状态，然后随着蛋白质 正常降解后，细胞恢复到“正常”状态。 因此，我们建议确定是否具体引入 强大的细胞内丝裂原(SV40)大T抗原可刺激有限的 正常不分裂的角膜内皮细胞在体内的增殖 细胞从而维持角膜功能，并消除了许多 角膜移植。为了让这个系统在角膜内皮细胞中发挥作用， 我们需要解决两个重要的问题，为此，我们提出 遵循特定的目标； 1.验证纯化的SV40大T抗原可以 在培养细胞中引入，并将这一策略扩展到人角膜 内皮细胞(HCEC)。将确定是否引入了 SV40T抗原定位于细胞核并能刺激DNA合成 不能复制的细胞。 2.验证单纯疱疹病毒L颗粒三角洲假说 可作为一种有效的“进入工具”，用于特定的交付 将幼虫T抗原蛋白导入细胞并验证其在两种细胞中的有效性 培养的细胞和动物模型。

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.