STIMULUS/SECRETION COUPLING IN DISEASED LACRIMAL GLAND

患病泪腺中的刺激/分泌耦合

• 批准号: 6138222
• 负责人: DRISS ZOUKHRI
• 金额: $ 24.52万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138222
• 关键词: RNase protection assay; Sjogren's syndrome; cataract; cytokine receptors; electron microscopy; enzyme activity; enzyme linked immunosorbent assay; fiber cell; fluorescence microscopy; high performance liquid chromatography; immunoprecipitation; in situ hybridization; innervation; interleukin 1; laboratory mouse; lacrimal disorder; lens; lipoxygenase; neurotransmitter antagonist; organ culture; polymerase chain reaction; protein kinase C; receptor coupling; secretion; stimulus /response; western blottings

Sjogren's syndrome is the leading cause of aqueous tear deficient dry eye. Sjogren's syndrome is an autoimmune disease, which occurs almost exclusively in females (>90%), is associated with extensive lymphocytic accumulation in the lacrimal gland and destruction of epithelial cells resulting in insufficient fluid and protein secretion leading to dry eye. To date there are no treatments or cures for this disease. The long term objective of the present proposal is to determine, using marine models of Sjogren's syndrome, the effect of the lymphocytic accumulation in the lacrimal gland on nerve functionality, intracellular signaling pathways, and protein and fluid secretion. A second objective is to test the role of cytokines, especially interleukin-1beta (IL-1beta), in the impaired function of the lacrimal gland in Sjogren's syndrome, and if IL-1 receptor antagonist (IL-1-ra) treatment can restore lacrimal gland function. To obtain these goals, the following specific aims have been proposed: (1) determine functionality of nerves and neurotransmitters in the lacrimal gland after the onset and during the progression of the lymphocytic accumulation; (2) determine if all signaling pathways are up-regulated in the lacrimal gland with the onset and progression of lymphocytic accumulation, and if blocking this accumulation prevents the up-regulation; and (3) determine if protein and fluid secretion induced by neural stimulation of the lacrimal gland of diseased mice is altered. Lacrimal gland slices or acini will be prepared from diseased and control female mice. The release of neurotransmitter from lacrimal gland nerve endings will be measured by HPLC. The amount of IL-1beta will be determined by western blotting and ELISA. The effect of lymphocytic accumulation on lacrimal gland signaling pathways will be determined by measuring: (1) changes in [Ca2+], using Ca2+ sensitive probes; (2) changes in [cAMP] using an enzyme immunoassay (EIA) kit, and (3) changes in PKC location using immunofluorescence microscopy. Lacrimal gland secretion will be determined either in vitro, by measuring peroxidase or newly synthesized proteins secretion from acini or lobules, or in vivo by cannulating lacrimal ducts or using the Schirmer test. IL-1-ra will be given s.c. (21-day release pellets) in the capsular region of diseased animals. Serum levels of IL-1-ra will be determined by ELISA.

干燥综合征是水状泪液缺乏性干眼症的主要原因。干燥综合征是一种自身免疫性疾病，其几乎仅发生在女性（>90%）中，与泪腺中广泛的淋巴细胞积聚和上皮细胞的破坏相关，导致液体和蛋白质分泌不足，从而导致干眼。 到目前为止，还没有治疗或治愈这种疾病。 本提案的长期目标是使用干燥综合征的海洋模型来确定泪腺中淋巴细胞积聚对神经功能、细胞内信号传导通路以及蛋白质和液体分泌的影响。 第二个目的是检测细胞因子，特别是白细胞介素-1 β（IL-1 β）在干燥综合征泪腺功能受损中的作用，以及IL-1受体拮抗剂（IL-1-ra）治疗是否可以恢复泪腺功能。 为了实现这些目标，已经提出了以下具体目标：（1）确定在淋巴细胞积累的开始和进展期间泪腺中神经和神经递质的功能;（2）确定是否所有信号传导途径在淋巴细胞积累的开始和进展时在泪腺中上调，以及是否阻断这种积累阻止了上调;和（3）确定由患病小鼠的泪腺的神经刺激诱导的蛋白质和液体分泌是否改变。 将从患病和对照雌性小鼠制备泪腺切片或腺泡。 将通过HPLC测量泪腺神经末梢的神经递质释放。 将通过蛋白质印迹和ELISA测定IL-1 β的量。 将通过测量以下指标来确定淋巴细胞蓄积对泪腺信号传导途径的影响：（1）使用Ca 2+敏感探针测量[Ca 2 +]的变化;（2）使用酶免疫测定（EIA）试剂盒测量[cAMP]的变化;以及（3）使用免疫荧光显微镜测量PKC位置的变化。 通过测量腺泡或小叶的过氧化物酶或新合成的蛋白质分泌在体外测定泪腺分泌，或通过对泪管插管或使用Schirmer试验在体内测定泪腺分泌。 IL-1-ra将皮下给药。（21天释放丸剂）在患病动物的囊区。 将通过ELISA测定IL-1-ra的血清水平。