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

1)鼻息肉内容物SDS-聚丙烯酰胺凝胶电泳分析鼻息肉内容物从慢性鼻窦炎患者手术切除鼻息肉，用盐水冲洗，并解剖收集内容物渗出液。用磷酸盐缓冲盐水稀释渗出液，并离心以除去细胞成分。将上清液进行12.5%SDS-聚丙烯酰胺凝胶电泳以分析蛋白质组成。蛋白质组分没有从堆积凝胶进入分离凝胶，表明高含量的大分子如糖胺聚糖。然后用透明质酸酶、软骨素酶或角蛋白酶处理上清液，并再次进行SDS-聚丙烯酰胺凝胶电泳。2）鼻息肉上皮细胞分泌囊泡的胞吐作用手术切除的鼻息肉用细胞膜标记物FM 4 - 6进行双重染色 关于我们 4和分泌囊泡标记物lysotracker绿色，并在共聚焦激光扫描显微镜下观察。激发波长为488 nm，发射波长为650 nm（FM 4 - 64）和540 nm（lysotracker绿色）。绿色荧光逐渐被红色荧光所取代，提示细胞内吞-胞吐循环的发生。垂直面观察，上皮细胞的基底面无明显的胞吐作用。3）Ussing小室法测定鼻息肉上皮层的短路电流将鼻息肉置于Ussing小室中，测定短路电流以估计主动离子转运。一些息肉显示出约20 μ A/cm ^2的短路电流，电阻约为100 Ω cm ^2，但大多数息肉没有显示出可检测到的短路电流，电阻为15 - 40 Ω cm ^2。说明鼻息肉上皮细胞间的紧密连接受损，可能存在离子的主动转运，但短路电流可能无法检测到。4）在超微结构水平上观察到紧密连接和示踪剂的泄漏。将生物素化磷脂负载在鼻息肉的顶端表面上，然后加入抗生物素蛋白-金。然后在透射电子显微镜下检查样品，然而，在上皮细胞之间的细胞间隙中未观察到明显的渗漏。少

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