Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.
Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.
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DOI：
10.1371/journal.pone.0197379
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Pan D
中科院分区：
文献类型：
--
作者：
Dai M;Lin Y;El-Amouri SS;Kohls M;Pan D
原代脑微血管(Brmv)保持了体内显示的细胞特性和分子特征，是血脑屏障(BBB)生物医学研究和脑内药物释放开发/优化的重要工具。不同BrMV样本之间的相对纯度或细胞组成的差异可能会对数据解释和研究结果产生重大影响，特别是对于高通量基因组学和蛋白质组学技术的实验。在本研究中，我们的目的是寻找合适的参考基因(RG)来准确标准化实时定量RT-QPCR分析，并确定合适的标记基因(MG)来评估BrMV样品的相对纯度。在5个看家基因中，β-肌动蛋白被选为最适合的RG，通过对具有一个或两个表达等位基因的小鼠分离的BrMV中α-L艾杜糖醛酸酶的基因水平进行定量验证。4个标记基因在BBB形成的毛细血管内皮细胞中高/选择性地表达，通过RT-qPCR进行纯度评估，结果表明Cldn5和PECAM1是最适合的MGs，细胞成分的免疫荧光分析进一步证实了这两个基因。Plvap被证明是BrMV样本中存在有开窗血管的指示基因。这项研究可能有助于为血脑屏障的总体研究需求奠定基础。
Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, and serve as a vital tool for biomedical research of the blood-brain barrier (BBB) and the development/optimization of brain drug delivery. The variations of relative purities or cellular composition among different BrMV samples may have significant consequences in data interpretation and research outcome, especially for experiments with high-throughput genomics and proteomics technologies. In this study, we aimed to identify suitable reference gene (RG) for accurate normalization of real-time RT-qPCR analysis, and determine the proper marker genes (MG) for relative purity assessment in BrMV samples. Out of five housekeeping genes, β-actin was selected as the most suitable RG that was validated by quantifying mRNA levels of alpha-L-iduronidase in BrMV isolated from mice with one or two expressing alleles. Four marker genes highly/selectively expressed in BBB-forming capillary endothelial cells were evaluated by RT-qPCR for purity assessment, resulting in Cldn5 and Pecam1 as most suitable MGs that were further confirmed by immunofluorescent analysis of cellular components. Plvap proved to be an indicator gene for the presence of fenestrated vessels in BrMV samples. This study may contribute to the building blocks toward overarching research needs on the blood-brain barrier.
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影响因子：
3.7
作者：
Chechi K;Gelinas Y;Mathieu P;Deshaies Y;Richard D
通讯作者：
Richard D
CNS药物发现的血脑屏障的新实验模型。
影响因子：
6.3
作者：
Kaisar MA;Sajja RK;Prasad S;Abhyankar VV;Liles T;Cucullo L
通讯作者：
Cucullo L
影响因子：
4.7
作者：
Gomez-Zepeda, David;Chaves, Catarina;Menet, Marie-Claude
通讯作者：
Menet, Marie-Claude
影响因子：
3.5
作者：
Clarke, LA;Russell, CS;Jirik, FR
通讯作者：
Jirik, FR
DOI：
10.1159/000264398
发表时间：
2010-01-01
期刊：
FRONTIERS IN EATING AND WEIGHT REGULATION
影响因子：
--
作者：
Banks, William A.
通讯作者：
Banks, William A.