Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived.
Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived.
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DOI：
10.1038/labinvest.2013.145
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发表时间：
2014-02
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影响因子：
--
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为了建立人结肠肿瘤的培养模型，我们建立了从正常人结肠组织和腺瘤中分离和体外维持完整的结肠隐窝的方法。隐窝用简单、无血清、低钙(0.15 mm)的培养液进行三维Matrigel培养。取自正常人粘膜的完整结肠隐窝保存了3-5天，保留了原位的隐窝样结构，呈现出明显的底部和顶端。腺瘤组织的异常结构可以通过多个通道(长达几个月)保持不变，并伴有扩张的芽/小管。肠干细胞(Lgr5)、生长(Ki67)、分化(E-cadherin、细胞角蛋白20(CK20)和粘蛋白2(MUC2))和上皮周转(Bax、裂解Caspase-3)的免疫组织化学标记与功能变化相一致。正常隐窝中的上皮细胞遵循从增殖到分化再到溶解的生理顺序，在空间和时间上都是合适的。新分离的隐窝中有少量基底细胞表达LGr5，培养1~3d后未检测到LGr5的表达。培养24小时后，正常结肠组织中的隐窝在隐窝底部仍有较强的Ki67和MUC2的表达，随着时间的推移，Ki67的表达逐渐减弱，直到3-4天时Ki67才不表达。分化标记CK20在同一时期增加，最终在整个隐窝变得强烈。在腺瘤来源的结构中，所有进展阶段的标志物在培养的整个时间都持续存在。培养数月后，LGR5在少数细胞中有表达。Ki67和MUC2主要与增殖萌发区相关，而CK20定位于亲本结构。这种正常和腺瘤隐窝的体外培养模型为了解人结肠上皮细胞的生长和分化过程提供了一个容易获得的工具。
In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca2+ (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3–5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1–3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3–4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.
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影响因子：
6.6
作者：
Grossmann, J;Walther, K;Rogler, G
通讯作者：
Rogler, G
影响因子：
4.8
作者：
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Brugge, JS
影响因子：
2.2
作者：
Li, AH;Goto, M;Yonezawa, S
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Yonezawa, S
影响因子：
158.5
作者：
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通讯作者：
Greenberg, ER
DOI：
10.1007/s11626-009-9247-9
发表时间：
2010-02-01
影响因子：
2.1
作者：
Dame, Michael K.;Bhagavathula, Narasimharao;Varani, James
通讯作者：
Varani, James