Quantitative Targeted Absolute Proteomic Analysis of Transporters, Receptors and Junction Proteins for Validation of Human Cerebral Microvascular Endothelial Cell Line hCMEC/D3 as a Human Blood鈥揃rain
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- 作者：Sumio Ohtsuki ; Chiemi Ikeda ; Yasuo Uchida ; Yumi Sakamoto ; Florence Miller ; Fabienne Glacial ; Xavier Decleves ; Jean-Michel Scherrmann ; Pierre-Olivier Couraud ; Yoshiyuki Kubo ; Masanori Tachikawa ; Tetsuya Terasaki
- 刊名：Molecular Pharmaceutics
- 出版年：2013
- 出版时间：January 7, 2013
- 年：2013
- 卷：10
- 期：1
- 页码：289-296
- 全文大小：283K
- 年卷期：v.10,no.1(January 7, 2013)
- ISSN：1543-8392
文摘
Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood鈥揵rain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na+/K+ ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and 纬-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.