Control of Conjuctival Goblet Cell Mucin Production

结膜杯状细胞粘蛋白产生的控制

• 批准号: 7433849
• 负责人: Darlene A Dartt
• 金额: $ 41.97万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7433849
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acetylcholine; Adenoviruses; Aging; Agonist; Anesthetics; Binding; Biochemical; Biological Assay; Bromodeoxyuridine; Cell Count; Cell Proliferation; Cell physiology; Cell secretion; Cells; Cholinergic Agonists; Condition; Cornea; Disease; Docking; Dominant-Negative Mutation; EGF gene; Ensure; Environment; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Family; Family member; Film; Fluorescence Microscopy; Goals; Goblet Cells; Grant; Growth Factor; Herpetic Keratitis; Human; Immunofluorescence Microscopy; Immunoprecipitation; In Vitro; Incubated; Individual; Keratitis; Lacrimal gland structure; Laser In Situ Keratomileusis; Lead; Lectin; Measures; Mitogen Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Inhibitor; Mitogen-Activated Protein Kinases; Mucins; Mucous body substance; Nerve; Numbers; Operative Surgical Procedures; Pathway interactions; Phorbol Esters; Play; Process; Production; Proliferating; Protein Isoforms; Protein Kinase C; Protein Kinase C Inhibitor; Proteins; Rattus; Receptor Activation; Recruitment Activity; Regulation; Research Personnel; Role; Sensory Receptors; Signal Pathway; Signal Transduction; Stimulus; Techniques; Western Blotting; afferent nerve; conjunctiva; in vivo; inhibitor/antagonist; member; neural growth; neural stimulation; neutralizing antibody; ocular surface; programs; prototype; response; sensory stimulus; therapy development

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.

描述（由申请人提供）：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的影响，为眼表提供了重要的防线。结膜杯状细胞粘蛋白产生的减少对眼表具有破坏性，并导致一系列眼表疾病。粘蛋白的产生取决于两个过程，即响应给定刺激而分泌粘蛋白的杯状细胞的百分比（快速反应）和结膜中存在的杯状细胞的总数（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产生并补充泪膜粘膜层来治疗眼表粘蛋白缺乏症。本提案的重点是确定：（1）角膜中的感觉神经是否刺激结膜（也许是杯状细胞）释放表皮生长因子（EGF），以刺激杯状细胞增殖；（2）所使用的信号通路EGF 刺激这种增殖。通过角膜感觉神经刺激在体内刺激杯状细胞增殖，并通过免疫荧光显微镜测量杯状细胞的增殖以确定是否使用EGF受体的激活。结膜片将用于确定神经激活是否释放 EGF，培养的杯状细胞将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用于确定 EGF 是否通过激活 1) p44/p42 丝裂原激活蛋白激酶、(2) 磷脂酰肌醇-3 激酶或 (3) PKC 同种型来刺激增殖。生化和免疫组织化学测定。药理学抑制剂和中和抗体以及显性失活和组成型活性腺病毒将用于抑制或刺激特定的信号传导途径。各个信号成分将通过免疫沉淀、蛋白质印迹和免疫荧光显微镜技术进行测量。在粘蛋白缺乏疾病中，如麻醉性角膜、疱疹性角膜炎和神经营养性角膜炎，以及衰老和 LASIK 手术中，杯状细胞增殖的神经和生长因子调节的激活受到损害。对刺激结膜杯状细胞增殖的信号通路的研究将导致开发补充这些疾病中粘蛋白层的治疗方法。