Control of Conjuctival Goblet Cell Mucin Production

描述（由申请人提供）：由结膜杯状细胞合成和分泌的粘蛋白为眼表提供了一道关键的防线，保护眼表免受外部环境的侵害。结膜杯状细胞粘蛋白产生的减少对眼表是毁灭性的，并导致一系列眼表疾病。粘蛋白的产生取决于两个过程，即在特定刺激下分泌粘蛋白的杯状细胞的百分比（快速反应）和存在于结膜中的杯状细胞总数（长期反应）。因此，该项目的长期目标是通过刺激杯状细胞分泌和增殖，从而增加粘蛋白的产生和补充泪膜的黏液层来治疗眼表粘蛋白缺缺性疾病。本研究的重点是确定：(1)角膜中的感觉神经是否刺激结膜（可能是杯状细胞）释放表皮生长因子（EGF），从而刺激杯状细胞增殖；(2)表皮生长因子刺激这种增殖的信号通路。在体内，角膜感觉神经刺激会刺激杯状细胞的增殖，并通过免疫荧光显微镜测量增殖的杯状细胞，以确定是否使用了EGF受体的激活。结膜碎片将用于确定神经激活是否释放EGF，培养的杯状细胞用于确定这些细胞是否释放EGF。传代的大鼠和人结膜杯状细胞将被用来确定EGF是否通过激活1)p44/p42丝裂原激活的蛋白激酶，(2)磷脂酰肌醇-3激酶，或(3)PKC异构体来刺激增殖，增殖将通过生化和免疫组织化学分析来测量。药理学抑制剂和中和抗体，以及显性阴性和组成性活性腺病毒将用于抑制或刺激特定的信号通路。个体信号成分将通过免疫沉淀、免疫印迹和免疫荧光显微镜技术进行测量。在诸如麻醉性角膜、疱疹性角膜炎和神经营养性角膜炎等黏液缺乏疾病中，以及在衰老和LASIK手术中，激活神经和生长因子对杯状细胞增殖的调节受到损害。刺激结膜杯状细胞增殖的信号通路的研究将导致治疗这些疾病补充粘蛋白层的发展。

DESCRIPTION (provided by applicant): Mucins synthesized and secreted by conjunctival goblet cells provide a critical line of defense for the ocular surface by protecting it from the external environment. A decrease in conjunctival goblet cell mucin production is devastating to the ocular surface and results in a spectrum of ocular surface diseases. Mucin production depends upon two processes, the percentage of goblet cells secreting mucins in response to a given stimulus (rapid response) and the total number of goblet cells present in the conjunctiva (long-term response). Thus the long-term goal of this project is to treat diseases of ocular surface mucin deficiency by stimulating goblet cell secretion and proliferation thus increasing mucin production and replenishing the mucous layer of the tear film. The focus of the present proposal is to determine: (1) if sensory nerves in the cornea stimulate epidermal growth factor (EGF) release from the conjunctiva, perhaps from the goblet cells, to stimulate goblet cell proliferation and (2) the signaling pathways used by EGF to stimulate this proliferation. Goblet cell proliferation will be stimulated in vivo by a corneal sensory nerve stimulus and proliferating goblet cells measured by immuno-fluorescence microscopy to determine if activation of the EGF receptor is used. Pieces of conjunctiva will be used to determine if activation of nerves releases EGF and cultured goblet cells used to determine if these cells release EGF. Passaged rat and human conjunctival goblet cells will be used to determined if EGF stimulates proliferation by activating 1) p44/p42 mitogen-activated protein kinase, (2) phosphatidylinositol-3 kinase, or (3) PKC isoforms, Proliferation will be measured by a biochemical and an immunohistochemical assay. Pharmacological inhibitors and neutralizing antibodies, as well as dominant negative and constitutively active adenovirus will be used to inhibit or stimulate specific signaling pathways. Individual signaling components will be measured by immunoprecipitation, western blotting, and immunofluorescence microscopy techniques. In diseases of mucin deficiency such as anesthetic cornea, herpetic keratitis, and neurotrophic keratitis, as well as in aging and LASIK surgery, activation of neural and growth factor regulation of goblet cell proliferation is impaired. Study of the signaling pathways that stimulate conjunctival goblet cell proliferation will lead to the development of treatments to replenish the mucin layer in these diseases.