STIMULUS/SECRETION COUPLING IN DISEASED LACRIMAL GLAND

干燥综合征是导致泪水缺乏的干眼症的主要原因。干燥综合征是一种自身免疫性疾病，几乎只发生在女性(&gt；90%)，与泪腺中广泛的淋巴细胞聚集和上皮细胞破坏有关，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。本建议的长期目标是，利用干燥综合征的海洋模型，确定泪腺中淋巴细胞聚集对神经功能、细胞内信号通路以及蛋白质和液体分泌的影响。第二个目标是测试细胞因子，特别是白细胞介素1β(IL-1β)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)治疗是否能恢复泪腺功能。为实现这些目标，已提出以下具体目标：(1)确定淋巴球聚集开始和进展过程中，泪腺神经和神经递质的功能；(2)确定是否所有信号通路都随着淋巴球聚集的开始和进展而在泪腺中上调，以及阻断这种积累是否阻止上调；(3)确定神经刺激疾病小鼠泪腺诱导的蛋白质和液体分泌是否发生改变。泪腺切片或腺泡将从患病和对照雌性小鼠身上制备。用高效液相色谱法测定泪腺神经末梢神经递质的释放。用Western blotting和ELISA法测定IL-1β的含量。淋巴细胞聚集对泪腺信号通路的影响将通过以下测量来确定：(1)使用钙离子敏感探针测量[Ca~(2+)]的变化；(2)使用酶免疫分析(EIA)试剂盒测量[cAMP]的变化；(3)使用免疫荧光显微镜测量PKC位置的变化。泪腺分泌将通过体外测定，或通过测量腺泡或小叶分泌的过氧化物酶或新合成的蛋白质，或在体内通过插管泪道或使用Schirmer试验来确定。IL-1-ra将被给予S.C.(21天释放微丸)在患病动物的包膜区域。采用双抗体夹心法测定血清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.