Isolated canine ileal mucosal cells in short-term culture: a model for study of neurotensin release.
Isolated canine ileal mucosal cells in short-term culture: a model for study of neurotensin release.
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短期培养中分离的犬回肠粘膜细胞：神经降压素释放研究的模型。
DOI：
10.1152/ajpgi.1986.250.3.g374
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Soll,AH
中科院分区：
文献类型：
--
作者：
Barber,DL;Buchan,AM;Walsh,JH;Soll,AH
我们开发了一种新的技术，用于在短期培养中保持分离的含有神经降压素样免疫反应(NTLI)的肠粘膜细胞，以确定NTLI释放的调节。免疫组织化学和放射免疫分析显示，经离心洗脱分离的胶原酶分散的回肠粘膜细胞富含含NTLI的细胞。蛙皮素(BBS)能迅速刺激新鲜分离细胞释放NTLI。然而，对新鲜分离细胞的研究受到高、不稳定的基础释放测量和添加的[~3H]神经降压素-(1-13)快速降解的限制。因此，建立了富含淋洗剂的NTLI细胞的培养体系。48h后，NTLI细胞选择性地黏附在胶原基质上，占培养中活细胞的40%。培养的NTLI细胞形态类似于肠组织切片中的神经降压素细胞，其分泌颗粒直径为292+/-14 nm。蛙皮素刺激NTLI分泌呈剂量依赖性增加，持续时间超过120min。在这些细胞培养中，添加的[~3H]-神经降压素-(1-13)在120min内降解最小。对培养上清液的高压液相色谱分析表明，神经降压素-(1-13)是对BBS刺激作出反应时释放的神经降压素的主要分子形式。综上所述，我们建立了肠神经降压素细胞的原代培养方法，为研究多肽释放的调控提供了模型。蛙皮素对NTLI释放的刺激验证了该分离细胞系统的功能反应性。
We have developed a new technique for maintaining isolated enteric mucosal cells containing neurotensinlike immunoreactivity (NTLI) in short-term culture to determine the regulation of NTLI release. Collagenase-dispersed ileal mucosal cells, separated by centrifugal elutriation, were enriched for NTLI-containing cells as determined by immunohistochemistry and radioimmunoassay. Bombesin (BBS) rapidly stimulated NTLI release from freshly isolated cells. However, studies with freshly isolated cells were limited by high, unstable basal release measurements and rapid degradation of added [3H]neurotensin-(1-13). A culture system of elutriator-enriched NTLI cells was therefore developed. After 48 h NTLI cells selectively adhered to the collagen substrate and constituted 40% of the viable cells in culture. The morphology of NTLI cells in culture closely resembled neurotensin cells in intestinal tissue sections, with a secretory granule diameter of 292 +/- 14 nm. Bombesin stimulated a dose-dependent increase in NTLI secretion over 120 min. In these cell cultures, degradation of added [3H]-neurotensin-(1-13) was minimal over 120 min. High-pressure liquid chromatographic analysis of culture supernatants characterized neurotensin-(1-13) as the primary molecular form of neurotensin released in response to BBS stimulation. In conclusion, we have established a primary culture of enteric neurotensin cells that provides a model for studying the regulation of peptide release. Bombesin stimulation of NTLI release verified the functional responsiveness of this isolated cell system.