The Role of BCL-2 in Corneal Epithelial Cell Shedding

描述(由申请人提供)：这项建议的主要目标是获得必要的培训，以发展成为一名独立的调查员，将我在视觉科学方面的背景与分子生物学的基础相结合。这一目标将通过在导师詹姆斯·V·杰斯特博士、H.Dwight Cavanagh博士和曾傑瑞·W·谢伊博士的指导下，通过教学和实践相结合的实验室培训来实现。这项建议的第二个目标是获得所需的知识和分子技术，以回答以下假设：支持生存的癌基因bcl2的核定位负责调节角膜上皮细胞分化和凋亡的最后阶段。表面细胞凋亡是维持角膜上皮细胞动态平衡的重要组成部分，因此也是视力清晰的先决条件。在正常人中，抗凋亡蛋白BCL-2定位于细胞核，并在表面细胞凋亡之前消失，提示BCL-2在介导表面细胞脱落中起作用。端粒化的人角膜上皮细胞(HTCEpi)在培养过程中也表现出在凋亡之前核bcl2的消失，从而为研究体外调控上皮细胞脱屑的机制(S)提供了一个新的模型。在这个方案中，这个模型将被用来检测四个特定的目标：1)hTCEpi构建概括了正常的人角膜上皮细胞的分化和脱落，关于bcl2的表达，定位，以及在TUNEL标记之前的消失。2)bcl2表达增强导致细胞存活率增加，bcl2核定位持续，表面细胞脱落减少，而bcl2表达降低导致细胞存活率下降，bcl2核定位丧失，表面细胞脱落增加。3)bc1-2调控环和丝氨酸70磷酸化位点是调控表面细胞脱落的关键。4)导致细胞凋亡增加的事件，如紫外光通过bcl2的磷酸化而使表面细胞发生凋亡。

DESCRIPTION (provided by applicant): The primary objective of this proposal is to gain the necessary training to develop into an independent investigator, integrating my background in the vision sciences with the fundamentals of molecular biology. This objective will be met by a combination of didactic and practical laboratory training under the guidance of mentors Dr. James V. Jester, Dr. H.Dwight Cavanagh, and Dr. Jerry W. Shay. The second objective of this proposal focuses on acquiring the knowledge and molecular techniques required to answer the hypothesis that: nuclear localization of the pro-survival oncogene, BCL-2, is responsible for regulating the final stages of corneal epithelial differentiation and apoptosis. Surface cell apoptosis is a critical component to the maintenance of corneal epithelial homeostasis and is thus a prerequisite for clear vision. In the normal human, the anti-apoptotic protein, BCL-2, localizes to the nucleus and disappears prior to surface cell apoptosis, suggesting a role for BCL-2 in mediating surface cell shedding. Telomerized human corneal epithelial (hTCEpi) cells have also been shown in culture to exhibit disappearance of nuclear BCL-2 prior to apoptosis, thus providing a novel model for studying the mechanism(s) responsible for regulating epithelial desquamation in vitro. In this proposal, this model will be used to examine four specific aims: 1) That an hTCEpi construct recapitulates normal human corneal epithelial cell differentiation and shedding regarding expression, localization, and disappearance of BCL-2 prior to TUNEL labeling. 2) That enhanced expression of BCL-2 results in increased cell survival, persistence of nuclear BCL-2 localization, and decreased surface cell shedding, whereas reduced expression of BCL-2 results in decreased cell survival, loss of nuclear BCL-2 localization, and increased surface cell shedding. 3) That the regulatory loop of BCL-2 and the serine 70 phosphorylation site are critical to regulating surface cell shedding. 4) That events leading to increased apoptosis such as UV light signal surface cell apoptosis through the phosphorylation of BCL-2.