The functional characterization of normal and neoplastic human enterochromaffin cells
The functional characterization of normal and neoplastic human enterochromaffin cells
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DOI：
10.1210/jc.2006-0110
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发表时间：
2006-06-01
影响因子：
5.8
通讯作者：
Champaneria, Manish C.
中科院分区：
文献类型：
--
作者：
Modlin, Irvin M.;Kidd, Mark;Champaneria, Manish C.
背景：小肠（SI）功能的神经内分泌调节了解甚少，因为无法获取纯的神经内分泌细胞，而小肠类癌肿瘤的生物学基础未知，因为无法获取肿瘤性人类肠嗜铬（EC）细胞。目的：本研究的目的是确定正常和肿瘤性小肠神经内分泌细胞的分泌调节和转录组。设计：分离和纯化人回肠的EC细胞，并对一种恶性EC细胞类癌细胞系（KRJ - I）进行特性分析。方法：对右半结肠切除术获取的人回肠进行链霉蛋白酶/胶原酶消化和Nycodenz梯度离心，吖啶橙标记后通过荧光激活细胞分选（FACS）对EC细胞进行分选。通过免疫染色、基因表达、血清素（5 - HT）含量和实时逆转录聚合酶链反应（RT - PCR）确定富集情况。培养经FACS分选的正常EC细胞和KRJ - I细胞，并用毛喉素、异丙肾上腺素、乙酰胆碱、γ - 氨基丁酸A（GABAA）、垂体腺苷酸环化酶激活多肽（PACAP）- 38和胃泌素刺激后测量5 - HT分泌。通过Affymetrix基因芯片分析（U133A）获取正常和肿瘤性EC细胞的转录组。结果：通过对色氨酸羟化酶进行免疫染色，FACS产生了100 ± 0.3%（嗜铬粒蛋白A染色）和99 ± 0.7%纯度的EC细胞，富集倍数大于67倍，5 - HT含量为180 ± 18纳克/毫克蛋白质（黏膜为3.5 ± 0.9）。毛喉素和异丙肾上腺素刺激的5 - HT分泌，正常细胞（半数有效浓度EC₅₀，1.8×10⁻⁹ M；5.1×10⁻⁹ M）比肿瘤细胞（EC₅₀，2.1×10⁻⁷ M；8.1×10⁻⁸ M）强10 - 100倍，但PACAP - 38的作用相似（EC₅₀，1×10⁻⁷ M）。异丙肾上腺素刺激的环磷腺苷（cAMP）水平相对于基础水平升高1.6 ± 0.1倍（EC₅₀，2.7×10⁻⁹ M）。乙酰胆碱对正常EC细胞5 - HT分泌的抑制作用比肿瘤细胞更强（半数抑制浓度IC₅₀，3.2×10⁻⁹对1.6×10⁻⁷ M），而GABAA在肿瘤细胞中作用更强（IC₅₀，3.9×10⁻¹⁰对4.4×10⁻⁹ M）。奥曲肽抑制正常细胞但不抑制肿瘤细胞的基础5 - HT分泌。胃泌素对5 - HT分泌无影响。正常和肿瘤转录组的比较显示出共有的神经内分泌和EC细胞特异性标记基因。实时PCR证实肾上腺素能（β1）、生长抑素能（SSTR2）和神经（VPAC1和GABAA）受体在两种细胞类型上均有表达，但PACAP 1型（PAC1）和胆囊收缩素2型（CCK2）未检测到。假定的类癌恶性基因（MTA1和MAGE - D2）是肿瘤性EC细胞转录组所特有的。结论：这些数据支持用于纯化活的人类EC细胞以进行功能特性分析和转录组评估的新方法，这将有助于确定控制小肠类癌分泌和增殖的新靶点。
Context: Neuroendocrine regulation of small intestinal (SI) function is poorly understood because pure neuroendocrine cells are unavailable, whereas the biological basis of SI carcinoid tumors is unknown because neoplastic human enterochromaffin (EC) cells are unavailable.Objective: The objective of this study was to define the secretory regulation and transcriptome of naive and neoplastic SI neuroendocrine cells.Design: EC cells from human ilea were isolated and purified, and a malignant EC cell carcinoid cell line (KRJ-I) was characterized.Methods: Human ilea from right hemicolectomies were pronase/collagenase digested and Nycodenz gradient centrifuged, and EC cells were fluorescence-activated cell sorting (FACS) sorted after acridine orange labeling. Enrichment was defined by immunostaining, gene expression, serotonin (5-HT) content, and real-time RT-PCR. Naive FACS-sorted EC and KRJ-I cells were cultured, and 5-HT secretion was measured after stimulation with forskolin, isoproterenol, acetylcholine, gamma-aminobutyric acid A (GABAA), pituitary adenylate cyclase-activating polypeptide (PACAP)-38, and gastrin. Normal and neoplastic EC cell transcriptomes were acquired by Affymetrix profiling (U133A).Results: FACS produced 100 +/- 0.3% (chromogranin A staining) and 99 +/- 0.7% pure EC cells by immunostaining for tryptophan hydroxylase with greater than 67-fold enrichment and a 5-HT content of 180 +/- 18 ng/mg protein (mucosa, 3.5 +/- 0.9). Forskolin-and isoproterenol-stimulated 5-HT secretion was 10 - 100 times more potent for naive cells (EC(50), 1.8 x 10(-9) M; 5.1 x 10(-9) M) than neoplastic cells (EC(50), 2.1 x 10(-7) M; 8.1 x 10(-8) M), but the effect of PACAP-38 was similar (EC(50), 1 x 10(-7) M). Isoproterenol stimulated cAMP levels 1.6 +/- 0.1- fold vs. basal (EC(50), 2.7 x 10(-9) M). Acetylcholine inhibited naive EC cell 5-HT secretion more potently than neoplastic (IC(50), 3.2 x 10(-9) vs. 1.6 x 10(-7) M), whereas GABAA was more potent in neoplastic cells (IC(50), 3.9 x 10(-10) vs. 4.4 x 10(-9) M). Octreotide inhibited naive, but not neoplastic, basal 5-HT secretion. Gastrin had no effect on 5-HT secretion. Comparison of naive and neoplastic transcriptomes revealed shared neuroendocrine and EC cell-specific marker genes. Real-time PCR confirmed that expression of adrenergic (beta 1), somatostatinergic (SSTR2), and neural (VPAC1 and GABAA) receptors occurred on both cell types, but PACAP type 1 (PAC1) and cholecystokinin type 2 (CCK2) were undetectable. The putative carcinoid malignancy genes (MTA1 and MAGE-D2) were unique to the neoplastic EC cell transcriptome.Conclusion: These data support novel methodology to purify live human EC cells for functional characterization and transcriptome assessment, which will allow identification of new targets to control the secretion and proliferation of SI carcinoids.