ROLE OF KERATOCAN IN CORNEAL BIOLOGY

描述(申请人提供)：这项资助计划的目标是使用新开发的转基因小鼠模型来1)阐明表达角质形成蛋白的细胞系在成人角膜发育过程中的形态发生和维持角膜基质动态平衡中的作用以及2)研究转化生长因子？受体II型(T？RII)在发育过程中和之后的角化细胞系中介导的信号转导。 为了在体内以角质细胞特异性的方式操纵小鼠基因组，建立了一种新的转基因小鼠系Kera-Cre，用于在体内特定时间对角化阳性细胞系进行Cre介导的遗传功能丧失和/或功能获得。在这个模型系统中，我们将使用Kera-Cre/Stop-DTRed双转基因小鼠在发育过程中和之后特异性地去除角质形成蛋白阳性细胞系，以检验角膜基质细胞在角膜形态发生和维持角膜动态平衡中发挥核心作用的假设。我们还将使用Kera-Cre/TbRII花/花双转基因小鼠来特异性地消除角质形成蛋白表达细胞系中的T？RII信号，以检验一种假设，即转化生长因子？在角膜形态发生过程中，信号在角膜基质细胞中起着至关重要的作用。此外，我们将利用强力霉素诱导的小鼠模型，Kera-RTTA/Teto-Cre/TbRII牙线/牙线三转基因小鼠，特异性地消除角质细胞内的TbRII信号，以验证TGF2信号在成人角膜基质动态平衡中起关键作用的假设。 前述的新型转基因小鼠模型将首次使我们能够大大简化关于角膜细胞系在角膜发育过程中和之后所起作用的研究。这些拟议的研究将为角膜基质细胞在支持角膜基质功能中的作用提供重要的见解。另一个好处是描述了在角膜损伤和角膜细胞丢失后角膜再生过程中TGF2信号通路在角膜细胞内的作用。此外，还将获得关于防止损伤后角膜瘢痕形成的见解，这将有助于设计新的角膜疾病治疗方案。首次在转基因小鼠中开发新的细胞系特异性基因操作，将使我们能够极大地简化关于角膜细胞系在角膜发育过程中和之后所起作用的研究。这些拟议的研究将对角质形成细胞和转化生长因子？的作用提供重要的见解。角膜基质细胞内信号通路对角膜基质功能的支持作用。此外，还将获得关于防止损伤后角膜瘢痕形成的见解，这将有助于设计新的角膜疾病治疗方案。

DESCRIPTION (provided by applicant): The goal of this grant proposal is to use newly developed transgenic mouse models to 1) elucidate the role of keratocan-expressing cell lineage in corneal morphogenesis during development and in the maintenance of corneal stromal homeostasis in adult and 2) to investigate TGF? receptor type II (T?RII)-mediated signaling within keratocan- expressing cell lineage during and following development. To manipulate in vivo the mouse genome in a keratocyte-specific manner, a novel transgenic mouse line, Kera-Cre, has been established to carry out Cre-mediated genetic loss-of-function and/or gain-of-function in keratocan-positive cell lineage at given time in vivo. With this model system, we will use Kera-Cre/STOP-DTRed double- transgenic mice to specifically ablate keratocan-positive cell lineage during and following development to test a hypothesis that keratocytes play a central role in corneal morphogenesis and maintenance of corneal homeostasis. We will also use Kera- Cre/TbRIIfloxed/floxed double-transgenic mice to specifically eliminate T?RII signaling within keratocan-expressing cell lineage to test a hypothesis that TGF? signaling plays critical role within keratocytes during corneal morphogenesis. Additionally, we will utilize doxycyclin-inducible mouse model, Kera-rtTA/tetO-Cre/TbRII floxed/floxed triple- transgenic mice to specifically eliminate TbRII signaling within keratocyte to test a hypothesis that TGF2 signaling plays critical role on corneal stromal homeostasis in adult. The aforementioned novel transgenic mouse models, for the first time, will allow us to considerably streamline research regarding the role of keratocan-positive cell lineages during and following corneal development. These proposed studies will provide important insight into the role of keratocytes in supporting corneal stromal function. Another benefit will be the delineation of the functions mediated by the TGF2 signaling pathway within keratocytes during corneal regeneration following injury and keratocyte loss. Furthermore, insight will be obtained regarding the prevention of corneal scarring following injury, which will aid in the design of novel corneal disease treatment regimens. The development of novel cell lineage-specific gene manipulation in transgenic mice, for the first time, will allow us to considerably streamline research regarding the role of keratocan-positive cell lineages during and following corneal development. These proposed studies will provide important insight into the role of keratocytes and TGF? signaling pathway within keratocytes in supporting corneal stromal function. Furthermore, insight will be obtained regarding the prevention of corneal scarring following injury, which will aid in the design of novel corneal disease treatment regimens.