CONTROL OF CONJUNCTIVAL GOBLET CELL MUCIN PRODUCTION

粘液层主要由结膜杯状细胞分泌，是眼睛表面的关键保护层，保护眼睛免受病原体和环境的挑战。为了保护眼表，杯状细胞必须能够应对外界的挑战。这些外部挑战激活了角膜中的感觉神经，以刺激围绕杯状细胞的副交感神经和交感神经。这些神经释放神经递质，与杯状细胞膜上的受体相互作用，激活细胞内信号通路，诱导杯状细胞粘蛋白分泌。本研究的长期目标是确定、表征副交感神经递质和交感神经递质激活的信号通路中特定的细胞生化过程的功能和活性，并刺激杯状细胞粘蛋白分泌和杯状细胞增殖。本提案的重点将是胆碱能激动剂(副交感神经通路)，这是结膜的主要刺激途径之一。将确定胆碱能激动剂是否：(1)激活钙/钙调蛋白依赖的途径来刺激杯状细胞粘蛋白的分泌；(2)激活PKC依赖的途径来刺激杯状细胞的粘蛋白分泌；(3)激活MAP激酶途径和应激反应转录因子NF-kappaB和AP-1来诱导杯状细胞的增殖，以及(4)介导神经诱导的杯状细胞的增殖。将大鼠结膜体外培养，用酶联凝集素测定法测定杯状细胞粘蛋白的分泌。该方法将与免疫沉淀、Western blotting、共聚焦免疫荧光显微镜、ELISA法和凝胶迁移率改变分析相结合，以确定信号通路各个组成部分的识别、定位和活性，并通过溴-2脱氧尿苷标记来测量细胞增殖。杯状细胞产生的粘蛋白受到严格的调控，因为粘蛋白分泌的增加或减少与眼表疾病有关。神经刺激对杯状细胞的分泌和增殖尤为重要。在粘蛋白分泌过多的疾病中，如巨乳头性结膜炎，持续的刺激性刺激会激活角膜的感觉神经，刺激结膜的传出神经，从而增加杯状细胞的分泌和增殖。在粘蛋白缺乏的疾病中，如麻醉性角膜和疱疹病毒性角膜炎，角膜中的感觉神经功能障碍，阻止传出通路的激活，阻止杯状细胞的分泌和增殖。对刺激杯状细胞分泌和增殖的信号通路的细胞成分的研究将有助于设计针对黏液过度分泌和黏液缺乏的疾病的杯状细胞特异性治疗方法。

The mucus layer, secreted primarily by the conjunctival goblet cells, is a critical protective layer for the ocular surface, shielding it from pathogenic and environmental challenges. To defend the ocular surface, goblet cells must be able to respond to the external challenges. These external challenges activate sensory nerves in the cornea to stimulate parasympathetic and sympathetic nerves that surround the goblet cells. These nerves release neurotransmitters that interact with receptors in the goblet cell membranes and activate intracellular signaling pathways to induce goblet cell mucin secretion. The long term objective of this research is to identify, characterize the function of, and determine the activity of the specific cellular biochemical processes in the signaling pathways activated by parasympathetic and sympathetic neurotransmitters and to stimulate goblet cell mucin secretion and goblet cell proliferation. The focus of the present proposal will be cholinergic agonists (parasympathetic pathway), one of the major stimulatory pathways in the conjunctiva. It will be determined if cholinergic agonists: (1) activate a Ca2+/calmodulin-dependent pathway to stimulate goblet cell mucin secretion; (2) activate a PKC-dependent pathway to stimulate goblet cell mucin secretion; (3) activate the MAP kinase pathway and the stress responsive transcription factors NF-kappaB and AP-1 to induce goblet cell proliferation, and (4) mediate neurally-induced goblet cell proliferation. Rat conjunctiva will be incubated in vitro and an enzyme-linked lectin assay used to measure secretion of goblet cell mucin. This method will be combined with immunoprecipitation, Western blotting, confocal immunofluorescence microscopy, ELISA, and electrophoretic mobility shift assay to determine identify, location, and activity of the individual components of the signaling pathways and bromo-2 deoxyuridine labeling to measure cell proliferation. Mucin production by the goblet cells is tightly regulated as either an increase or decrease in mucin secretion is associated with ocular surface disease. Neural stimulation of goblet cell secretion and proliferation is especially important. In diseases of mucin overproduction, such as giant papillary conjunctivitis, a constant irritative stimulus would activate sensory nerves in the cornea to stimulate efferent nerves in the conjunctiva to increase goblet cell secretion and proliferation. In diseases of mucin deficiency, such as anesthetic cornea and herpetic keratitis, sensory nerves in the cornea are rendered dysfunctional preventing activation of the efferent pathway and blocking goblet cell secretion and proliferation. Study of the cellular components of the signaling pathways that stimulate goblet cell secretion and proliferation will lead to the design of goblet cell-specific therapies for diseases of both mucus overproduction and deficiency.