Cell Cycle of Corneal Endothelium

描述（由申请人提供）：角膜内皮细胞负责维持角膜透明度，但这些细胞在整个生命过程中不会分裂。我们的长期目标是开发方法来刺激增殖和增加由于低内皮细胞计数导致视力丧失风险的个体的细胞密度。人角膜内皮细胞可以在器官培养中分裂，这表明多种因素共同在体内施加强大的抗增殖压力。接触依赖性增殖抑制似乎是一个重要的抗增殖因子，这是调节，部分由磷酸酪氨酸磷酸酶（PTP）。受体酪氨酸激酶如EGF受体的激活导致酪氨酸磷酸化（Tyr-PO 4）诱导的细胞周期进入。细胞-细胞接触可以被钙粘蛋白相关蛋白的Tyr-PO 4破坏。PTP抑制Tyr-PO 4事件，表明PTP活性有助于维持接触依赖性增殖抑制。在缺乏促分裂原的情况下，融合内皮细胞中细胞-细胞接触的释放不会刺激细胞进入细胞周期。原钒酸钠（SOV），PTP抑制剂，释放细胞接触，促进细胞周期进入无丝分裂原，这表明PTP活性的抑制可能会增加增殖作用的有丝分裂原，如EGF，在融合内皮细胞。我们假设，通过下调蛋白质表达或抑制内皮细胞中特异性PTP的活性，我们将能够使细胞对促有丝分裂刺激更敏感，从而提供一种方法来增加因内皮细胞计数低而有视力丧失风险的个体的细胞密度。具体的目的提出来测试这一假设包括：I。鉴定角膜内皮细胞中表达的特异性PTP; II.确定每个PTP在调节EGF驱动的促有丝分裂信号传导中的重要性; III.确定钙粘蛋白-连环蛋白复合物是否是角膜内皮中PTP调节的靶标;和IV.检测下调特定PTP的表达或抑制其活性是否会促进原位人角膜内皮细胞的增殖和增加细胞密度。免疫沉淀、Western印迹、RT-PCR、免疫细胞化学、流式细胞术、反义方法和显性阴性突变体的表达将用于实现本提案的目标。

DESCRIPTION (provided by applicant): The corneal endothelium is responsible for maintaining corneal clarity, but these cells do not divide throughout life. Our long-term goal is to develop methods to stimulate proliferation and increase cell density in individuals at risk for vision loss due to low endothelial cell counts. Human corneal endothelial cells CAN divide in organ culture, suggesting that multiple factors together exert a strong anti-proliferative pressure in vivo. Contact-dependent inhibition of proliferation appears to be an important anti-proliferative factor, which is regulated, in part, by phosphotyrosine phosphatases (PTPs). Activation of receptor tyrosine kinases, such as the EGF receptor, leads to tyrosine phosphorylation (Tyr-PO4)-induced cell cycle entry. Cell-cell contacts can be disrupted by Tyr-PO4 of cadherin-associated proteins. PTPs inhibit both Tyr-PO4 events, suggesting that PTP activity helps maintain contact-dependent inhibition of proliferation. Release of cell-cell contacts in confluent endothelium does NOT stimulate cell cycle entry in the absence of mitogens. Sodium orthovanadate (SOV), a PTP inhibitor, releases cell contacts and promotes cell cycle entry without mitogens, suggesting that inhibition of PTP activity might augment the proliferative effect of mitogens, such as EGF, in confluent endothelium. We hypothesize that, by down-regulating the protein expression or inhibiting the activity of specific PTPs in the endothelium, we will be able to make the cells more responsive to mitogenic stimulation, thus providing a method to increase cell density in individuals at risk for vision loss due to low endothelial cell counts. Specific Aims proposed to test this hypothesis include: I. Identify specific PTPs expressed in corneal endothelial cells; II. Determine the importance of each PTP in regulating EGF-driven mitogenic signaling; III. Determine whether the cadherin-catenin complex is a target for PTP regulation in corneal endothelium; and IV. Test if down-regulating the expression or inhibiting the activity of specific PTPs will promote proliferation and increase cell density in human corneal endothelial cells in situ. Immunoprecipitation, Western blotting, RT-PCR, immunocytochemistry, flow cytometry, antisense methods, and expression of dominant-negative mutants will be used to accomplish the objectives of this proposal.