Establishing a method to isolate rat brain capillary endothelial cells by magnetic cell sorting and dominant mRNA expression of multidrug resistance-associated protein 1 and 4 in highly purified rat brain capillary endothelial cells.
OBJECTIVE
To establish a method for isolating highly purified brain capillary endothelial cells (BCECs) from rat brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs.


METHODS
The cells were prepared from the capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis.


RESULTS
From five rats, 2.3 x 10(6) cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat brain. The contamination by neurons and astrocytes was markedly less than in the brain capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively.


CONCLUSIONS
This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-brain barrier.
Ohtsuki
Sumio
S
Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Sendai, Japan.
Yamaguchi
Hirofumi
H
Asashima
Tomoko
T
Terasaki
Tetsuya
T
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000032:analysis / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000235:genetics; D000818:Animals; D001253:Astrocytes / Q000737:chemistry; D001921:Brain / Q000098:blood supply*; D002196:Capillaries / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D062507:Claudin-5; D042783:Endothelial Cells / Q000737:chemistry* / Q000276:immunology; D005434:Flow Cytometry; D015870:Gene Expression; D018189:Immunomagnetic Separation / Q000379:methods*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis* / Q000235:genetics; D009474:Neurons / Q000737:chemistry; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D042787:Receptor, TIE-2 / Q000032:analysis / Q000235:genetics
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D062507:Claudin-5; D008565:Membrane Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D012333:RNA, Messenger; D042787:Receptor, TIE-2; C489542:Abcc4 protein, rat; C492375:Abcc5 protein, rat; C491510:Cldn5 protein, rat; C095122:multidrug resistance protein 3; C421109:multidrug resistance-associated protein 1