Isolation, functional characterization, and transcriptome of Mastomys ileal enterochromaffin cells
Isolation, functional characterization, and transcriptome of Mastomys ileal enterochromaffin cells
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DOI：
10.1152/ajpgi.00552.2005
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发表时间：
2006-11-01
影响因子：
4.5
通讯作者：
Champaneria, M. C.
中科院分区：
文献类型：
--
作者：
Kidd, M.;Modlin, I. M.;Champaneria, M. C.
虽然肠嗜铬（EC）细胞是小肠的主要神经内分泌调节细胞之一，缺乏纯化的细胞系统，排除了表征的细胞和有限的精确的生理评价。我们开发的方法，以获得一个纯粹的群体Mastomys回肠EC细胞，评估其功能调节，并定义了转录组。将回肠乳鼠翻转，末端结扎，链霉蛋白酶-胶原酶消化，Nycodenz梯度离心，并通过吖啶橙标记的细胞的荧光激活细胞分选（FACS）收集EC细胞。通过色氨酸羟化酶和嗜铬粒蛋白A的免疫染色、特异性EC细胞标志物、5-羟色胺含量、EC细胞标志物基因表达和电子显微镜检查证实富集。用实时荧光定量RT-PCR法检测垂体腺苷酸环化酶激活多肽（PACAP）、生长抑素和胃泌素受体的表达。培养FACS分选后的活细胞，并通过ELISA测量毛喉素、异丙肾上腺素、乙酰胆碱、GABA（A）、PACAP-38和胃泌素对5-羟色胺分泌的影响。进行FACS分选的细胞的基因芯片亲和分析以获得EC细胞转录组。流式细胞术产生了EC细胞的> 70倍富集，5-羟色胺含量为240 +/-22 ng/mg蛋白质。通过色氨酸羟化酶免疫染色，制备物纯度为99 +/- 0.7%。血管活性肠肽/PACAP受体1（VPAC（1））和生长抑素受体2存在，而PACAP受体1（PAC（1））和CCK 2受体未检测到。毛喉素、异丙肾上腺素和PACAP-38刺激血清素分泌的EC 50值分别为5 × 10（-10）、4.5 × 10（-10）和1.2 × 10（-9）M。异丙肾上腺素刺激的cAMP水平与未刺激的细胞相比接近3.5 +/- 0.62倍（EC 50接近10（-9）M）。奥曲肽、乙酰胆碱和GABA（A）抑制5-羟色胺分泌，IC 50值分别为3 × 10（-11）、3 × 10（-10）和2.9 × 10（-10）M。胃泌素对5-羟色胺的分泌无影响。幼稚EC细胞转录组显示高表达的EC细胞标志物基因，其他小肠细胞类型的标志物基因的缺失，以及包括胆碱能、肾上腺素能、多巴胺能、胆碱能、GABA能和前列腺素受体的受体谱。我们能够分离活的回肠EC细胞的均质制备物（> 99%），并证明了5-羟色胺分泌的调节以及建立了正常的EC细胞转录组。将这种方法应用于正常和患病的人回肠将有助于阐明EC细胞的病理生理学。
Although the enterochromaffin (EC) cell is one of the primary neuroendocrine regulatory cells of the small intestine, the lack of a purified cell system has precluded characterization of the cell and limited precise physiological evaluation. We developed methodology to obtain a pure population of Mastomys ileal EC cells, evaluated their functional regulation, and defined the transcriptome. Mastomys ilea were everted, end ligated, pronase-collagenase digested, and Nycodenz gradient centrifuged, and EC cells were collected by fluorescence-activated cell sorting (FACS) of acridine orange-labeled cells. Enrichment was confirmed by immunostaining of tryptophan hydroxylase and chromogranin A, specific EC cell markers, serotonin content, EC cell marker gene expression, and electron microscopy. Pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin, and gastrin receptor expression was determined by real-time RT-PCR. Live post-FACS-sorted cells were cultured, and the effects of forskolin, isoproterenol, acetylcholine, GABA(A), PACAP-38, and gastrin on serotonin secretion were measured by ELISA. GeneChip Affymetrix profiling of FACS-sorted cells was undertaken to obtain the EC cell transcriptome. FACS produced a > 70-fold enrichment of EC cells with a serotonin content of 240 +/- 22 ng/mg protein. Preparations were 99 +/- 0.7% pure by immunostaining for tryptophan hydroxylase. Vasoactive intestinal peptide/PACAP receptor 1 (VPAC(1)) and somatostatin receptor 2 were present, whereas PACAP receptor 1 (PAC(1)) and CCK2 receptors were undetectable. Forskolin, isoproterenol, and PACAP-38 stimulated serotonin secretion at EC50 values of 5 X 10(-10), 4.5 X 10(-10), and 1.2 X 10(-9) M, respectively. Isoproterenol stimulated cAMP levels by similar to 3.5 +/- 0.62-fold vs. unstimulated cells (EC50 of similar to 10(-9) M). Octreotide, acetylcholine, and GABA(A) inhibited serotonin secretion with IC50 values of 3 X 10(-11), 3 X 10(-10), and 2.9 X 10(-10) M, respectively. Gastrin had no effect on serotonin secretion. The naive EC cell transcriptome revealed highly expressed EC cell marker genes, the absence of marker genes for other small intestinal cell types, and a receptor profile that included cholinergic, adrenergic, dopaminergic, serotoninergic, GABAergic, and prostaglandin receptors. We were able to isolate homogeneous preparations (> 99%) of live ileal EC cells and demonstrated regulation of serotonin secretion as well as established the normal EC cell transcriptome. Application of this methodology to normal and diseased human ileum will facilitate the elucidation of the pathophysiology of EC cells.