Zhao Wanzhu, Han Linjiang, Bae Younsoo, Manickam Devika S
Department of Pharmaceutical, Administrative and Social Sciences, Duquesne University.
Department of Pharmaceutical Sciences, University of Kentucky.
J Vis Exp. 2019 Aug 19(150). doi: 10.3791/58900.
The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.
血脑屏障（BBB）由内皮细胞组成，这些内皮细胞在体循环和大脑之间形成一道屏障，以阻止非必需离子和有毒物质的交换。紧密连接（TJ）有效地封闭了单层细胞中的细胞旁间隙，从而形成完整的屏障。本研究描述了一种基于LY的荧光测定法，该方法可用于测定其表观渗透系数（Papp），进而可用于确定hCMEC/D3单层细胞中汇合单层的形成动力学以及由此产生的紧密连接屏障完整性。我们进一步证明了该测定法在确定转染细胞中TJ功能完整性方面的额外用途。我们基于LY Papp测定法的数据表明，接种在Transwell装置中的hCMEC/D3细胞在培养7天后有效地限制了LY的细胞旁转运。作为所提出测定法的额外用途，我们还证明DNA纳米颗粒转染不会改变hCMEC/D3单层细胞中LY的细胞旁转运。
