Control of Conjuctival Goblet Cell Mucin Production

性状（由申请方提供）：由结膜杯状细胞合成和分泌的粘蛋白通过保护眼表免受外部环境影响，为眼表提供了关键的防线。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，即杯状细胞对给定刺激的反应（快速反应）和结膜中存在的杯状细胞总数（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并补充泪膜粘液层来治疗眼表粘蛋白缺乏症。本建议的重点是确定：（1）角膜中的感觉神经是否刺激表皮生长因子（EGF）从结膜释放，可能是从杯状细胞释放，以刺激杯状细胞增殖和（2）EGF刺激这种增殖所使用的信号通路。杯状细胞增殖将通过角膜感觉神经刺激物在体内刺激，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EGF受体的活化。结膜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。将使用传代的大鼠和人结膜杯状细胞来确定EGF是否通过激活1）p44/p42促分裂原活化蛋白激酶、（2）磷脂酰肌醇-3激酶或（3）PKC同种型来刺激增殖。将通过生物化学和免疫组织化学测定来测量增殖。药理学抑制剂和中和抗体以及显性阴性和组成型活性腺病毒将用于抑制或刺激特定信号传导途径。将通过免疫沉淀、蛋白质印迹和免疫荧光显微镜技术测量单个信号传导组分。在粘蛋白缺乏的疾病如麻醉性角膜、疱疹性角膜炎和神经营养性角膜炎中，以及在衰老和LASIK手术中，杯状细胞增殖的神经和生长因子调节的激活受损。刺激结膜杯状细胞增殖的信号通路的研究将导致治疗的发展，以补充这些疾病中的粘蛋白层。

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.