STIMULUS/SECRETION COUPLING IN DISEASED LACRIMAL GLAND

干燥综合征是导致水性泪液缺乏性干眼的主要原因。干燥综合征是一种自身免疫性疾病，几乎只发生在女性中（>90%），与泪腺中广泛的淋巴细胞积聚和上皮细胞的破坏有关，导致液体和蛋白质分泌不足，导致干眼。到目前为止，还没有治疗或治愈这种疾病的方法。本研究的长期目标是利用干燥综合征的海洋模型，确定泪腺淋巴细胞积聚对神经功能、细胞内信号通路以及蛋白质和液体分泌的影响。第二个目的是测试细胞因子，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，提出了以下具体目标：(1)确定泪腺中神经和神经递质在淋巴细胞积聚发生后和发展过程中的功能；(2)确定泪腺中所有信号通路是否随着淋巴细胞积聚的发生和进展而上调，以及阻断这种积聚是否会阻止上调；(3)测定神经刺激患病小鼠泪腺诱导的蛋白和分泌液分泌是否发生改变。将从患病和对照组的雌性小鼠身上制备泪腺切片或腺泡。用高效液相色谱法测定泪腺神经末梢神经递质的释放。western blotting和ELISA检测il -1 β的含量。淋巴细胞积聚对泪腺信号通路的影响将通过测量来确定：(1)[Ca2+]的变化，使用Ca2+敏感探针；(2)使用酶免疫测定（EIA）试剂盒检测[cAMP]的变化，(3)使用免疫荧光显微镜检测PKC位置的变化。泪腺分泌可以在体外通过测量过氧化物酶或腺泡或小叶新合成的蛋白质分泌来测定，也可以在体内通过管状泪管或使用Schirmer试验来测定。IL-1-ra将在患病动物的包膜区域给予s.c（21天释放微丸）。ELISA检测血清IL-1-ra水平。

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.