Dissertation > Medicine, health > Pharmacy > Pharmacology

Construction of Stably Transfected HEK-293 Cell Line Expressing Human OATP1B1-wild/Variant Alleles

Author QuBoXuan
Tutor ZengSu
School Zhejiang University
Course Pharmaceutical Analysis
Keywords OATP1B1 expressing stably variant alleles Human Embryonic Kidney 293(HEK293)
Type Master's thesis
Year 2011
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During recent years, plasma membrane influx and efflux transporters are increasingly recognized as the key determinants in determining the rate and extent of intestinal absorption of drugs and the hepatic drug clearance during both first-pass and elimination phases.Members of the organic anion-transporting polypeptide(OATP) family are drug uptake transporters that have been found capable of mediating the active cellular influx of a variety of amphipathic compounds. OATP1B1 (previously known as OATP2, OATP-C; encoded by the SLCO1B1 gene) is specially expresed at the basolateral membrane of human hepatocyte where it mediates the uptake of its substrates from blood into the liver. Considering its liver-specific tissue distribution pattern and the capacity for transporting a large number of structurally divergent compounds, it is likely that OATP-C plays an important role in the hepatocellμLar uptake of endogenous compounds and xenobiotics such as bilirubin, bilirubin-glucuronide, bile acids, estrone sulfate and estradiol-17β-D-glucuronide. Furthermore, several statins, methotrexate, the antibiotics benzylpenicillin, and rifampicin are the substrates of OATP1B1.Several sequence variations or SNPs have been discovered in the SLCO1B1 gene encoding OATP1B1, some associated with altered transport activity in vitro and in vivo. The 521T>C(Val174Ala) SNP has been associated with markedly reduced uptake activity in vitro of the OATP1B1 substrates.Taking into account the important role of OATP1B1(wide and variant alleles) in the hepatocellular uptake of so many drugs, our study focused on the OATP1B1*1a and OATP1B1*lb/*15 (the alleles’frequency separately are 73.4%and 14.0%in Chinese people), and constructed the HEK-293 cell line expressing stably human OATP1B1-wild/variant alleles as a basement for further study on the uptake mechanisms and properties of OATP1B1 in vitro cell models.Objective:To establish Human Embryonic Kidney 293 (HEK293) cell lines with stable expression of human organic anion transporting polypeptide (OATP1B1) wild and variant alleles and to screen an effective model to study drug transportation.Methods:OATP1B1*1a sequence was amplified through RT-PCR with the extracted total RNA as templates from human liver, then subcloned into the plasmid pMD19-T and verified by sequencing. OATP1B1*1b/15 mutants sequences were obtained by site-directed mutation PCR with pMD19-T/OATP1B1*1a as templates. Choose the right ones, connected to the plasmid pcDNA3.1(+), then obtained the plasmids pcDNA3.1 (+)/OATP1B1*1a/*1b/* 15 finally.The plasmids pcDNA3.1 (+)/OATP 1B1* 1a/* 1b/* 15 were transfected into HEK293 cell line using LipofectamineTM2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Rosuvastatin, the OATP1B1 substrate, was used to study the profiles of uptake to test OATP1B1 activities, which was performed with HEK293 and HEK-OATP1B1*1a/*1b/*15 monoclone cells. The expression of OATP1B1 messenger ribonucleic acid (mRNA) and protein were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results:The RT-PCR, the amount of intracellular Rosuvastatin and Western blot result indicated high human OATP1B1 expression in transfected cells comparing with controls. The HEK-293 cell line expressing stably human OATP1B1-wild/mutants (HEK-OATP1B1*1a/*1b/*15) cell lines are effective models to study drug transportation in vitro.

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