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)通过SDS-聚丙烯酰胺凝胶电泳分析鼻息肉内容物通过手术切除慢性鼻窦炎患者的鼻息肉，用盐水清洗，解剖以收集内容物渗出液，用磷酸盐缓冲盐水稀释，离心除去细胞成分。上清液进行12.5% SDS-聚丙烯酰胺凝胶电泳分析蛋白质成分，未进入蛋白质成分。将上清液用透明质酸酶、软骨素酶或角蛋白酶处理，并再次进行SDS-聚丙烯酰胺凝胶电泳，这表明得到了相同的结果。透明质酸、硫酸软骨素和硫酸角质素含量低。2) 胞吐作用鼻息肉上皮细胞的分泌囊泡用细胞膜标记物 FM4-6 … More 4 和分泌囊泡标记物 lysotracker green 进行双重染色，并在共焦激光扫描显微镜下观察激发波长。波长为 488 nm，发射波长为 650 nm（FM4-64）和 540 nm（lysotracker 绿色荧光）。逐渐被红色荧光取代，提示发生了胞吞-胞吐循环。垂直平面观察，上皮细胞基底外侧表面没有明显的胞吐作用。3)鼻息肉上皮层短路电流的测量。使用室法将鼻息肉放入使用室中，测量短路电流以估计活性离子传输，一些息肉显示约20的短路电流。 μA/cm^2，电阻约为 100 Ωcm^2，但大多数电阻为 15-40 Ω cm^2 时未检测到短路电流，这表明鼻息肉上皮层可能存在活性离子传输。 ，但由于上皮细胞之间的紧密连接受损，短路电流可能无法检测到。4）超微结构上紧密连接和示踪剂泄漏的观察透射电子显微镜显示上皮细胞的紧密连接不规则，然后将生物素化磷脂加载到鼻息肉的顶端表面，然后添加抗生物素蛋白-金，然后在透射电子显微镜下检查样品。 ，上皮细胞间未见明显渗漏。