Equilibrative nucleoside transporter 1 in the blood-brain barrier: an in vitro study using the hCMEC/D3 cell line
Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
Introduction: Equilibrative nucleoside transporter 1 (ENT1) in the blood-brain barrier (BBB) plays a crucial role in the transportation of nucleosides and nucleoside analogs, which modulate various physiological functions in the central nervous system (CNS), making it a potential therapeutical target in neurological disorders. The human immortalized brain endothelial cell line hCMEC/D3 is a well-characterized in vitro BBB model.
Aim: The objective of this study was to validate the hCMEC/D3 as a model for the study of ENT1-targeting drugs in BBB by performing expression and functional analyses.
Materials and Methods: Gene expression of ENT1 and a number of other major BBB transporters in hCMEC/D3 were analyzed by quantitative real-time PCR. ENT1 protein was detected by immunocytochemistry and western blotting. Lastly, ENT1 was functionally characterized by a radioisotope uptake assay using gemcitabine and NBTI as substrate and inhibitor respectively.
Results: The real-time PCR showed a relatively high RNA expression level of ENT1 (7%) in hCMEC/D3. ENT1 protein expression was unable to be proved in the cell line by immunofluorescence staining and western blotting, due to failing in antibody validation. In the uptake assay, the total gemcitabine radioactivity was significantly lower in cells with NBTI with a 3.7-fold decrease, proving the function of ENT1.
Conclusions: This study showed potential usefulness of the hCMEC/D3 cell line as a model in discovering and optimizing ENT1-targeting drugs. Further confirmatory studies regarding the protein expression are needed.
Place, publisher, year, edition, pages
2014. , 32 p.
Equilibrative nucleoside transporter 1, ENT1, blood-brain barrier, BBB, hCMEC/D3
Medical and Health Sciences Pharmaceutical Sciences
IdentifiersURN: urn:nbn:se:uu:diva-231526OAI: oai:DiVA.org:uu-231526DiVA: diva2:744852
University of California, San Francisco
Master of Science Programme in Pharmacy