Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA.
Am J Physiol Cell Physiol. 2012 May 15;302(10):C1492-503. doi: 10.1152/ajpcell.00392.2011. Epub 2012 Mar 7.
Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.
完整隐窝上皮的生理学研究受到体内可及性问题和标准原代培养中去分化的限制。最近，对小鼠肠道干细胞的研究产生了一种在三维凝胶悬浮中的原代肠道培养物，该培养物再现了隐窝结构和上皮分化（Sato T、Vries RG、Snippert HJ、van de Wetering M、Barker N、Stange DE、Van Es JH、Abo A、Kujala P、Peters PJ、Clevers H. Nature 459:262-265, 2009）。我们通过关注囊性纤维化跨膜电导调节剂 Cftr 的转运活性，研究了小鼠肠道隐窝培养物（称为“肠类器官”）在隐窝上皮生理学研究中的应用。肠类器官具有多个隐窝，其中杯状细胞和潘氏细胞分化良好，在暴露于毒蕈碱激动剂卡巴胆碱时会脱颗粒。改良的生长培养基提供了一种隐窝增殖率，如通过 5-乙炔基-2'-脱氧尿苷标记测量，与体内增殖相似。免疫印迹显示，与新鲜分离的隐窝相比，新鲜分离的隐窝与原代和第 1 代肠类器官的 Cftr 表达相当。其他转运体的 mRNA 表达中明显的肠类器官差异主要与新鲜分离的隐窝中绒毛上皮的污染有关。微电极分析显示，cAMP 刺激 WT 而不是 Cftr 敲除（KO）小鼠的肠类器官上皮的膜去极化。形态学和微荧光研究分别显示，WT 肠类器官上皮中 Cftr 依赖性细胞收缩和较低的细胞内 pH 与 Cftr KO 上皮或用 Cftr 抑制剂 172 处理的 WT 上皮形成对比。我们得出结论，肠类器官的隐窝上皮表现出 Cftr 的表达和活性，再现了体内隐窝上皮。肠类器官提供了一种适合于再生隐窝上皮的生理学研究的原代培养模型。
