High molecular weight secretory products of the human pancreatic ductal epithelium and the effect of secretin on their discharge.
High molecular weight secretory products of the human pancreatic ductal epithelium and the effect of secretin on their discharge.
复制标题
人胰腺导管上皮的高分子量分泌产物以及促胰液素对其分泌的影响。
DOI：
10.1097/00006676-199203000-00002
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Palmiter-Thomas,P
中科院分区：
文献类型：
--
作者：
SarrasJr,MP;Huff,JK;Palmiter-Thomas,P
据报道，胰腺导管上皮主细胞分泌高分子量(HMW)糖结合物，如粘蛋白等进入导管管腔。我们使用一株人胰腺癌细胞系(PANC-1)作为分离HMW分泌产物的来源，该细胞系保留了正常导管主细胞的一些形态和生化特征。本研究旨在分离这些HMW分泌产物，通过生化和免疫学方法对其进行部分鉴定，并确定促胰液素对其合成和分泌的影响。我们的结果表明，当PANC-1细胞生长在胶原蛋白包裹的珠子上时，在特定的无血清培养条件下，至少可以从培养液中分离出三种HMW分泌产物。根据十二烷基硫酸钠聚丙烯酰胺凝胶电泳法分析，这些分泌物的质量约为200、280和370 kDa。在凝胶染色模式中，200 kDa的物种所占比例较小。针对370 kDa分泌产物的多克隆抗体与280 kDa物种发生交叉反应。370 kDa的分泌产物被硫酸盐化，与小麦胚芽凝集素(WGA)的结合表明370 kDa是一种糖偶联物。280 kDa和200 kDa物种的硫酸盐化程度比370 kDa物种小得多，280 kDa和200 kDa物种不能清楚地证明WGA结合。然而，糖苷酶和选择性降解研究表明，这三个物种都含有糖胺多糖部分。针对370 kDa分泌产物的抗体定位于人胰腺癌的上皮细胞，而不是肿瘤组织中的其他细胞类型。该抗体还可与正常人胰腺导管上皮发生交叉反应，并可定位于中央腺泡细胞、小叶内导管上皮和小叶间导管上皮。该抗体与人小肠或大肠的杯状细胞不发生交叉反应，表明对胃肠道粘蛋白没有广泛的反应性。ELISA法和脉冲追逐免疫沉淀法研究表明，10−8~10−11M促胰液素刺激PANC-1细胞后，这些高分子量分泌产物的含量和合成均增加。我们在超微结构水平上观察到，分泌素的刺激与细胞质中大量膜结合的小泡的出现有关。促胰液素的最佳浓度范围为10 kDa~10 M。超微结构免疫细胞化学技术测定，这些囊泡的含量与分泌产物的抗体呈免疫反应。这些结果表明，胰腺导管上皮主细胞产生HMW分泌产物，这种分泌产物(A)可能是胰腺所特有的，(B)在其合成和释放方面对促胰液素刺激有反应。
Principal cells of the pancreatic ductal epithelium have been reported to secrete high molecular weight (HMW) glycoconjugates such as mucins into the ductal lumen. We used a human pancreatic carcinoma cell line of ductal origin (PANC-1) which has retained some of the morphological and biochemical characteristics of normal ductal principal cells as a source for isolation of HMW secretory products. The present study was designed to isolate these HMW secretory products, partially characterize them through biochemical and immunological approaches, and determine the effects of secretin on their synthesis and discharge from PANC-1 cells. Our results indicated that when PANC-1 cells are grown on collagen-coated beads in defined serum-free medium, at least three HMW secretory products could be isolated from the medium. These secretory products had a mass of approximately 200, 280, and 370 kDa based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The 200–kDa species made up proportionally less of the three in the gel-staining pattern. Polyclonal antibodies raised to the 370–kDa secretory product cross-reacted with the 280–kDa species. The 370–kDa secretory product was sulfated and wheat germ agglutinin (WGA) binding indicated that the 370–kDa species was a glycoconjugate. The 280–kDa and 200–kDa species were sulfated to a much lesser degree than the 370–kDa species and WGA binding could not be clearly demonstrated with the 280 kDa or 200 kDa species. Glycosidase and selective degradation studies, however, indicated that all three species contained glycosaminoglycan moieties. Antibodies raised to the 370–kDa secretory product localized to the epithelium of human pancreatic carcinomas but not to other cell types in this neoplastic tissue. The antibody also cross-reacted with the ductal epithelium of normal human pancreas and could be localized to centroacinar cells, the epithelium of intralobular ducts, and the epithelium of interlobular ducts. The antibody did not cross-react with goblet cells of the human small or large intestine, indicating no generalized reactivity to gastrointestinal mucins. ELISA and pulse-chase immunoprecipitation studies indicated an increase in the cellular content and synthesis of these HMW secretory products after stimulation of PANC-1 cells with 10− 8-to 10− 11 M secretin. We correlated secretin stimulation with the appearance of numerous membrane bound vesicles throughout the cytoplasm as monitored at the ultrastructural level. Optimal secretin concentrations were in the range of 10− 10 M. The content of these vesicles was immunoreactive to the antibody raised against the 370–kDa secretory product as determined by ultrastructural immunocytochemical techniques. These results indicate that principal cells of the pancreatic ductal epithelium produce HMW secretory products that (a) may be unique to the pancreas and (b) are responsive to secretin stimulation in terms of their synthesis and discharge.