In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model
In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model
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DOI：
10.1016/s0928-0987(03)00205-7
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发表时间：
2004-01-01
影响因子：
4.6
通讯作者：
Brodin, B
中科院分区：
文献类型：
--
作者：
Bravo, SA;Nielsen, CU;Brodin, B
本研究旨在探讨培养时间对hPEPT1在Caco-2细胞单层转运的影响。首先比较了在标准培养液和快速培养4天模型中生长的Caco-2细胞的多肽转运活性。用生长因子和激动剂混合培养的快速培养4d的Caco-2细胞模型，比在常规培养4d培养的Caco-2细胞对多肽的摄取能力低，被认为不适合进行多肽转运研究。在标准的21天模型中，肽转运活性以及单层完整性和组织形态作为培养时间的函数被评估。用[C-14]-甘氨酸肌氨酸([C-14]-Gly-Sar)研究了多肽的转运活性。通过测量跨上皮电阻(TEER)和[H-3]-甘露醇通透性来评价单层完整性。用激光共聚焦扫描显微镜(CLSM)和常规染色/免疫组织化学染色观察组织形态和hPEPT1的表达。在常规培养液中生长的Caco-2细胞在3-4天后开始融合。甘露醇通透性从第5天到第21天下降，TEER稳步上升，直到今天第21天。顶端hPEPT1摄取活性在培养21天的细胞中似乎最大，而基侧摄取在培养12天后已经达到最大。在所研究的一些传代中，在培养25天的细胞中观察到hPEPT1转运活性的二次增加。大载体介导的跨皮细胞多肽流量成分从第14天起就很明显。(C)2003年爱思唯尔公司。保留所有权利。
The aim of the present study was to investigate the influence of culture time on hPEPT1-mediated transport in Caco-2 cell monolayers. Peptide transport activity in Caco-2 cells grown in standard media and in a "rapid" 4-day model was first compared. The rapid 4-day Caco-2 cell model, cultured using a cocktail of growth factors and agonists, displayed lower peptide uptake capacity than Caco-2 cells grown for 4 days in conventional media, and was judged to be unsuitable for peptide transport studies. Peptide transport activity as well as monolayer integrity and tissue morphology were evaluated in the standard >21 days model as a function of the culture time. Peptide transport activity was studied using [C-14]-glycylsarcosine ([C-14]-Gly-Sar). Monolayer integrity was evaluated by transepithelial electrical resistance (TEER) measurements and [H-3]-mannitol permeabilities. Tissue morphology and hPEPT1 expression were studied using confocal laser scanning microscopy (CLSM) and conventional staining/immunostaining. Caco-2 cells grown in conventional media became confluent after 3-4 days. Mannitol permeability decreased from day 5 to 21 and TEER increased steadily until similar today 21. Apical hPEPT1 uptake activity appeared to be maximal in cells cultured for >21 days, whereas basolateral uptake reached a maximum already after 12 days in culture. In some of the passages studied, a secondary increase in hPEPT1 transport activity was observed in cells grown for >25 days. A large carrier-mediated transepithelial peptide flux component was evident from day 14. (C) 2003 Elsevier B.V. All rights reserved.