Development of stably transfected monolayer overexpressing the human apical sodium-dependent bile acid transporter (hASBT)
Development of stably transfected monolayer overexpressing the human apical sodium-dependent bile acid transporter (hASBT)
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DOI：
10.1007/s11095-005-5274-8
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发表时间：
2005-08-01
影响因子：
3.7
通讯作者：
Polli, JE
中科院分区：
文献类型：
--
作者：
Balakrishnan, A;Sussman, DJ;Polli, JE
目的。人顶端钠依赖性胆汁酸转运蛋白（hASBT）代表了前药设计以增加口服药物吸收的潜在目标。不幸的是，现有的单层细胞培养模型不能可靠地表达 hASBT，并且非极化细胞只能进行摄取评估，这限制了前药开发工作。本研究的目的是开发并表征稳定转染的 hASBT-MDCK 细胞系。方法。将编码 hASBT 的 cDNA 克隆到 pcDNA3.1-V5-polyHis-B 中以生成表达质粒，然后将其转染到 MDCK-II 细胞中。根据高 hASBT 活性和单层完整性选择克隆群体。 Western blot证实重组hASBT的表达；使用牛磺胆酸对功能进行表征。结果。在选定的克隆中，hASBT 介导的跨 hASBT-MDCK 单层的牛磺胆酸盐渗透性在有钠的情况下几乎比无钠时高 25 倍（其中 hASBT 不起作用）。在钠存在的情况下，牛磺胆酸盐和甘露醇的渗透性分别为23.0 x 10(-6) cm/s和2.60 x 10(-6) cm/s，表明hASBT具有高功能性和单层完整性。 hASBT-MDCK 单层特性在 6 个月内保持稳定，并且表现出较低的日内变异性。 hASBT-MDCK 的牛磺胆酸摄取和抑制动力学参数与 hASBT-COS7 模型获得的相似，证实了 hASBT-MDCK 中的 hASBT 功能。结论。结果表明，开发的 hASBT-MDCK 系统是 hASBT 转运和抑制研究的有效、高表达、稳定的检测方法。
Purpose. The human apical sodium-dependent bile acid transporter (hASBT) represents a potential target for prodrug design to increase oral drug absorption. Unfortunately, available monolayer cell culture models do not reliably express hASBT, and nonpolarized cells only allow for uptake assessment, which limits prodrug development efforts. The objective of this study was to develop and characterize a stably transfected hASBT-MDCK cell line.Methods. cDNA encoding hASBT was cloned into pcDNA3.1-V5-polyHis-B to generate an expression plasmid that was then transfected into MDCK-II cells. Clonal populations were chosen based on high hASBT activity and monolayer integrity. Western blot confirmed the expression of the recombinant hASBT; functionality was characterized using taurocholic acid.Results. In the selected clone, hASBT-mediated taurocholate permeability across hASBT-MDCK monolayers was almost 25-fold higher with sodium, than without sodium where hASBT is not functional. In the presence of sodium, taurocholate and mannitol permeabilities were 23.0 x 10(-6) cm/sec and 2.60 x 10(-6) cm/s, respectively, indicating high hASBT functionality and monolayer integrity. hASBT-MDCK monolayer properties were stable over 6 months and demonstrated low within-day variability. Taurocholate uptake and inhibition kinetic parameters from hASBT-MDCK were similar to those obtained from hASBT-COS7 model, confirming hASBT functionality in hASBT-MDCK.Conclusions. Results indicate that the developed hASBT-MDCK system is a competent, high-expression, stable assay for hASBT transport and inhibition studies.