Mechanism of nasal polyp formation in the light of exocytosis of secretory vesicles of the goblet cells

从杯状细胞分泌囊泡的胞吐作用探讨鼻息肉形成机制

• 批准号: 15591793
• 负责人: SUZUKI Hideaki
• 金额: $ 2.24万
• 依托单位: University of Occupational and Environmental Health
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591793/
• 关键词: Nasal polyp; Goblet cell; Exocytosis; Secretory; Ussing chamber; Tight junction; SDS polyacrylamide electrophoresis; Confocal laser scannine microscope; Ussing chember

1) Analysis of nasal polyp contents by SDS-polyacrylamide gel electrophoresisNasal polyps were surgically excised from patients with chronic sinusitis, washed with saline, and dissected to collect content effusion. The effusion was diluted with phosphate buffered saline, and centrifuged to remove cellular components. The supernatant was subjected to 12.5% SDS-polyacrylamide gel electrophoresis to analyze protein composition. Protein components did not enter to the separating gel from the stacking gel, indicating a high content of macromolecules such as glycosaminoglycans. The supernatant was then treated with hyaluronidase, chondroitinase or keratinase, and again subjected to SDS-polyacrylamide gel electrophoresis. However, the same result was obtained, implying low contents of hyaluronic acid, chondroitin sulfates and keratan sulfate.2) Exocytosis of secretary vesicles of nasal polyp epithelial cellsSurgically excised nasal polyps were double-stained with a cell membrane marker, FM4-6 … More 4, and a secretory vesicle marker, lysotracker green, and was observed under a confocal laser scanning microscope. The excitation wavelength was 488 nm, and the emission wavelengths were 650 nm (FM4-64) and 540 nm (lysotracker green). Green fluorescence was gradually replaced by red fluorescence, suggesting the occurrence of endocytosis-exocytosis cycle. Observation of a vertical plane showed no evident exocytosis on the basolateral surface of the epithelial cells.3) Measurement of short circuit current across the epithelial layer of the nasal polyp by Ussing chamber methodNasal polyps were placed in the Ussing chamber, and short circuit current was measured to estimate an active ion transport. Some polyps showed a short circuit current of about 20 μA/cm^2 with a resistance of about 100 Ωcm^2, but most showed no detectable short circuit current with a resistance of 15-40 Ω cm^2. This suggest that an active ion transport may exist across the nasal polyp epithelial layer, but the short circuit current may be undetectable because of the impairment of the tight junction between the epithelial cells.4) Observation of the tight junction and the leakage of a tracer at the ultrastructural level.Transmission electron microscopy revealed an irregularity of the tight junction of the epithelial cells. Biotinylated phospholipid was loaded on the apical surface of the nasal polyp followed by the addition of avidin-gold. The sample was then examined under a transmission electron microscope, however, no obvious leakage was seen in the intercellular space between the epithelial cells. Less

1) sds -聚丙烯酰胺凝胶电泳分析鼻息肉内容物慢性鼻窦炎患者鼻息肉手术切除，生理盐水冲洗，解剖收集内容物积液。用磷酸盐缓冲盐水稀释积液，离心去除细胞成分。上清液经12.5% sds -聚丙烯酰胺凝胶电泳分析蛋白质组成。蛋白质成分没有进入到分离凝胶和堆积凝胶中，说明糖胺聚糖等大分子的含量很高。然后用透明质酸酶、软骨素酶或角化酶处理上清，并再次进行sds -聚丙烯酰胺凝胶电泳。然而，得到的结果相同，这意味着低含量的透明质酸，硫酸软骨素和硫酸角蛋白。2)鼻息肉上皮细胞分泌性囊泡的分泌情况手术切除的鼻息肉用细胞膜标记物FM4-6…More 4和分泌性囊泡标记物溶踪绿色双染色，在激光共聚焦扫描显微镜下观察。激发波长为488 nm，发射波长为650 nm （FM4-64）和540 nm （lysotracker green）。绿色荧光逐渐被红色荧光所取代，提示发生了内吞-胞吐循环。在垂直平面上观察，上皮细胞基底外侧表面未见明显的胞吐现象。3) Ussing chamber法测量鼻息肉上皮层短路电流将鼻息肉置于Ussing chamber中，测量短路电流以估计活性离子传输。部分息肉的短路电流约为20 μA/cm^2，电阻约为100 Ωcm^2，但大多数息肉的短路电流为15 ~ 40 Ωcm^2。这表明在鼻息肉上皮层可能存在活跃的离子运输，但由于上皮细胞之间紧密连接的损伤，短路电流可能无法检测到。4)在超微结构水平观察示踪剂的紧密连接和渗漏。透射电镜显示上皮细胞紧密连接的不规则性。在鼻息肉的根尖表面加载生物素化磷脂，然后加入亲和金。在透射电镜下观察，上皮细胞间的细胞间隙未见明显渗漏。少