Variation of peptide transporter (PepT1 expression in Caco-2 cells as a function and HPT1) of cell origin
Variation of peptide transporter (PepT1 expression in Caco-2 cells as a function and HPT1) of cell origin
复制标题
DOI：
10.1002/jps.20062
复制
发表时间：
2004-07-01
影响因子：
3.8
通讯作者：
Kissel, T
中科院分区：
文献类型：
--
作者：
Behrens, I;Kamm, W;Kissel, T
Caco-2细胞培养物广泛用于小肠药物转运的体外模型，尽管不同实验室的主动转运底物存在很大差异。因此，我们比较了三种不同的Caco-2克隆：（1）来自美国培养组织保藏中心（ATCC），（2）来自海德堡的德国癌症研究中心（DKFZ），和（3）来自马尔堡的大学医院，在不同传代次数下的形态、多层和紧密连接形成，以及肽转运蛋白HPT 1和PepT 1的表达。我们通过测量跨上皮电阻确定紧密连接形成，通过共聚焦激光扫描显微镜确定多层形成，通过RT-PCR确定PepT 1和HPT 1的表达，间接免疫荧光和PepT 1底物头孢拉定的渗透性。不同克隆之间的形态和TEER值变化很大。PepT 1和HPT 1的表达按以下顺序增加：HD > ATCC > MR。间接免疫荧光显示在ATCC-细胞中的转运蛋白的不均匀分布，而在HD-细胞中是均匀的。在MR细胞中仅发现非常弱的表达。而在ATCC-细胞中的转运蛋白的表达随着传代次数的增加而降低，在HD-细胞中则增加。表达水平与头孢拉定的转运一致。PepT 1和HPT 1的表达受培养条件的强烈影响。在相同的培养条件下，海德堡（HD）Caco-2细胞似乎是一种适当的体外细胞培养模型，用于主动转运药物的转运，因为传代间变化较低，并且转运蛋白在整个单层中分布均匀。(C)2004 Wiley-Liss，Inc.和美国药剂师协会
Caco-2 cell cultures area widely used in vitro model for the small intestinal drug transport, although large differences have been reported for actively transported substrates from different laboratories. Therefore, we compared three different Caco-2 clones: (1) from the American Culture Tissue Collection (ATCC), (2) from the German Cancer Research Center (DKFZ) in Heidelberg, and (3) from the University Hospital in Marburg in different passage numbers regarding their morphology, multilayers, and tight junction formation, as well as expression of the peptide transporters, HPT1 and PepT1. We determined tight junction formation by measurement of the transepithelial electrical resistance, multilayer formation by confocal laser scanning microscopy, the expression of PepT1 and HPT1 by RT-PCR, indirect immunofluorescence and the permeability of the PepT1 substrate, cephradine. Morphology and TEER-values varied strongly between the different clones. The expression of PepT1 and HPT1 increased in the following order: HD > ATCC > MR. Indirect immunofluorescence revealed a heterogeneous distribution of the transporters in ATCC-cells, whereas it was homogeneous in HD-cells. Only a very weak expression was found in MR-cells. While in ATCC-cells expression of transporters decreased with increasing passage number, it increased in HD-cells. Expression levels were congruent with the transport of cephradine. Expression of PepT1 and HPT1 was strongly affected by the culture conditions. Under identical culture conditions, Heidelberg (HD) Caco-2 cells seemed to be an appropriate in vitro cell culture model for the transport of actively transported drugs, because interpassage changes are low and the transporter distribution was homogeneous throughout the monolayer. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.