Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology.
Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology.
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DOI：
10.1053/j.gastro.2015.11.047
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发表时间：
2016-03
期刊：
影响因子：
29.4
通讯作者：
Donowitz M
中科院分区：
文献类型：
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作者：
Foulke-Abel J;In J;Yin J;Zachos NC;Kovbasnjuk O;Estes MK;de Jonge H;Donowitz M
人肠隐窝来源的肠样体是一种肠道离子转运模型，需要通过与细胞培养和动物模型的比较来验证。我们利用人的小肠小肠样，在未分化和分化培养的基础和调节条件下，研究中性Na+吸收和刺激液体和阴离子的分泌，以证明它们与离子转运生理学和病理生理学的功能相关性。人体肠道组织标本取自约翰·霍普金斯医院的内窥镜活检或手术切除。分离隐窝，在培养中繁殖肠样，诱导分化，并用慢病毒载体转导。用qRT-PCR、免疫印迹或免疫荧光分析表征隐窝标志物、表面细胞酶和膜离子转运蛋白。我们使用多光子和延时共聚焦显微镜在基础和调节条件下监测细胞内pH和肠腔扩张。随着WNT3a的退出，肠样组织分化，产生的隐窝标志减少，绒毛样特征增加。NHE3活性在未分化和分化的肠样体中相似，并受已知的抑制物、第二信使和细菌肠毒素的影响。在未分化和分化的肠样体中，Forsklin诱导的肿胀(FIS)完全依赖于CFTR，部分依赖于NHE3和NKCC1的抑制。在无HCO3-−缓冲液中，用Forsklin增加cAMP可引起肠样细胞内酸化。CAMP诱导的肠样PHi酸化是十二指肠HCO3−分泌的一部分，似乎需要CFTR1和NBCE1。未分化的或隐窝样的，以及分化的或绒毛样的，代表隐窝-绒毛轴上不同的点；它们可以用来表征沿着小肠垂直轴的电解质运输过程。十二指肠肠样病变模型显示，NBCE1可能是肠道粘膜治疗分泌性腹泻的靶点。
Human intestinal crypt-derived enteroids are a model of intestinal ion transport that require validation by comparison with cell culture and animal models. We used human small intestinal enteroids to study neutral Na+ absorption and stimulated fluid and anion secretion under basal and regulated conditions in undifferentiated and differentiated cultures to demonstrate their functional relevance to ion transport physiology and pathophysiology. Human intestinal tissue specimens were obtained from endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypts were isolated, enteroids were propagated in culture, induced to undergo differentiation, and transduced with lentiviral vectors. Crypt markers, surface cell enzymes, and membrane ion transporters were characterized using qRT-PCR, immunoblot, or immunofluorescence analyses. We used multiphoton and time-lapse confocal microscopy to monitor intracellular pH and luminal dilatation in enteroids under basal and regulated conditions. Enteroids differentiated upon withdrawl of WNT3A, yielding decreased crypt markers and increased villus-like characteristics. NHE3 activity was similar in undifferentiated and differentiated enteroids, and was affected by known inhibitors, second messengers, and bacterial enterotoxins. Forskolin-induced swelling (FIS) was completely dependent on CFTR and partially dependent upon NHE3 and NKCC1 inhibition in undifferentiated and differentiated enteroids. Increases in cAMP with forskolin caused enteroid intracellular acidification in HCO3−- free buffer. cAMP-induced enteroid pHi acidification as part of duodenal HCO3− secretion appears to require CFTR and NBCE1. Undifferentiated or crypt-like, and differentiated or villus-like, human enteroids represent distinct points along the crypt–villus axis; they can be used to characterize electrolyte transport processes along the vertical axis of the small intestine. The duodenal enteroid model revealed that NBCE1 might be a target in the intestinal mucosa for treatment of secretory diarrheas.