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息肉对鼻息息含量的分析超过了慢性鼻窦炎的患者，用盐水洗涤并进行了解剖以收集含量泡沫。用磷酸盐缓冲盐水稀释积液，并离心以去除细胞成分。将上清液进行12.5％的SDS-聚丙烯酰胺凝胶电泳，以分析蛋白质组成。蛋白质成分未进入与堆叠凝胶分离的凝胶，表明大量的大分子（例如糖胺聚糖）。然后将上清液用透明质酸酶，软骨蛋白酶或角质酶处理，并再次接受SDS-聚丙烯酰胺凝胶电泳。 However, the same result was obtained, implying low contents of hyaluronic acid, chondroitin sulfates and keratan sulfate.2) Exocytosis of secretary vegetables of nasal polyp epithelial cellsSurgically Excellent nasal polyps were double-stained with a cell membrane marker, FM4-6 … More 4, and a secret venue marker, lysotracker green, and was observed under a共聚焦激光扫描显微镜。兴奋波长为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取代，表明发生内吞作用 - 细胞增多症周期。观察垂直平面的观察表明，在上皮细胞的基底外侧表面没有证据胞吐作用。3）通过将室方法Nasasal息肉放置在USSISING室中，测量鼻息盘的短回路电流，并测量短路电流，以估算活跃的离子运输。一些息肉显示出约20μA/cm^2的短路电流，电阻约为100ΩCM^2，但大多数没有可检测的短路电流，电阻为15-40ΩCM^2。这表明，活性离子运输可能在整个鼻息息层上存在，但是由于上皮细胞之间的紧密连接的损害，短路电流可能是无法检测的。4）观察紧密连接和截然词在超微结构水平上泄漏。传输电子微观显微镜揭示了紧密的EPITHICTIAL ePITICTICTIAL的EPITICTICTICTICTICTICTICTICTICTICTICTICTIAL。将生物素基化的磷脂加载在鼻息肉的顶部表面，然后加入抗生蛋白蛋白脂。然后在透射电子显微镜下检查样品，但是，在上皮细胞之间的细胞间空间中未看到明显的泄漏。较少的