Thomsen Maj Schneider, Birkelund Svend, Burkhart Annette, Stensballe Allan, Moos Torben
Laboratory of Neurobiology, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
Laboratory of Medical Mass Spectrometry, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
J Neurochem. 2017 Mar;140(5):741-754. doi: 10.1111/jnc.13747. Epub 2016 Sep 8.
The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)] α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789.
脑血管基底膜对于血脑屏障（BBB）的发育、稳定性及屏障完整性都很重要，而脑毛细血管内皮细胞（BCEC）、周细胞和血脑屏障的星形胶质细胞对此的贡献可能很大。本研究的目的是分析四种不同的小鼠血脑屏障体外模型，以检测小鼠BCEC（mBCEC）主要基底膜蛋白的表达及可能的分泌情况。mBCEC、周细胞和神经胶质细胞（主要是星形胶质细胞和小胶质细胞）取自C57BL/6小鼠的脑。mBCEC以单培养、与周细胞或混合神经胶质细胞共培养，或与周细胞和混合神经胶质细胞进行三培养的方式生长。通过跨内皮电阻（TEER）测量和甘露醇被动通透性来验证血脑屏障模型的完整性。使用RT-qPCR、质谱和免疫细胞化学分析基底膜蛋白的表达。与单培养相比，mBCEC与周细胞、混合神经胶质细胞或两者共培养显著提高了TEER，低被动通透性与高TEER相关。mBCEC在体外表达所有主要的基底膜蛋白，如层粘连蛋白-411、层粘连蛋白-511、胶原蛋白[α1(IV)]α2(IV)、集聚蛋白、基底膜聚糖以及巢蛋白1和2。层粘连蛋白α5亚基表达增加与添加血脑屏障诱导因子（氢化可的松、Ro 20-1724和pCPT-cAMP）相关，而胶原蛋白IV α1表达增加主要与cAMP水平升高相关。总之，体外培养的BCEC可连贯地形成血脑屏障并表达基底膜蛋白，作为成熟的一个特征。本期封面图片：doi: 10.1111/jnc.13789。
