Creation of an In Vitro Brain Barrier Transport System

描述(申请人提供)：通过两个主要屏障，即血脑屏障(BBB)和血脑脊液屏障(BCB)，大脑可以很好地保护大脑免受血源性异物(药物、营养物质和毒素)的伤害。血脑屏障位于脑室，其表面积约为血脑屏障的一半，其功能是产生脑脊液，并在血液和脑脊液之间运输异物。我们最近建立了一种新型的永生化脉络膜上皮细胞系，命名为Z310细胞系。该细胞系具有亲代原代细胞的基本形态，当生长在半透膜上时，形成限制细胞旁渗漏标记[14C]蔗糖自由运动的单层。虽然通过跨上皮电阻(TEER)或细胞旁[14C]蔗糖渗漏来衡量细胞单层的紧密性仍有待改善，但我们相信该细胞系在体外显示出巨大的前景，作为一种独特的血脑屏障运输系统，因为在当前的神经毒理学和神经药理学研究领域还没有这样的脑细胞来源的运输模型。为了建立这个新的系统，我们假设Z310上皮细胞单层的紧密性可以通过改变培养液的化学成分，通过诱导和促进紧密连接组装，和/或通过遗传调控紧密连接蛋白的表达来改善。因此，我们的具体目标是通过修改培养基组分，如使用无血清或星形胶质细胞条件培养基来降低Z310屏障模型的细胞旁通透性，通过在细胞培养液中应用紧密连接诱导剂来改善屏障结构的紧密性，并在现有的Z310细胞中敲入编码与紧密连接相关的蛋白质或调节蛋白的特定基因片段。我们进一步设计了一系列实验来验证该模型系统。本申请中提出的研究如果成功，尽管存在显著的风险，但将导致建立一种新的体外血-脑/脑-脑-脑脊液模型，用于材料向脑内的转运研究，并将对药物和毒物的中枢神经系统转运的药理学和毒理学研究、中枢神经系统药物开发和脑部疾病的病因学研究产生重大影响。

DESCRIPTION (provided by applicant): Brain is well protected against blood-borne xenobiotics (drugs, nutrients, and toxins) by two major barriers, i.e., blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier (BCB). The BCB, whose surface area is about one-half of the BBB, is located in brain ventricles and functions to produce CSF and transport xenobiotics between blood and CSF. We have recently established a novel immortalized choroidal epithelial cell line, named Z310 cell line. This ceil line possesses the essential morphology of the parent primary cells and, upon growing on a semipermeable membrane, forms a monolayer that restricts the free movement of paracellular leakage marker, [14C]sucrose. While the tightness of the cell monolayer, as measured by trans-epithelial electrical resistance (TEER) or paracellular leakage of [14C]sucrose, remains to be improved, we are convinced that this cell line shows a great promise as a unique in vitro blood-brain barrier transport system, as there has been no such brain cell-derived transport model in the current neurotoxicology and neuropharmacology research field. To create this novel system, we hypothesize that the tightness of the monolayer of Z310 epithelial cells can be improved by alteration of the chemical components of the culture media, by induction and promotion of tight junction assembly, and/or by genetic modulation of the expression of tight junction proteins. Thus, our specific aims are to reduce the paracellular permeability of the Z310 barrier model by modifying the culture medium components, such as using serum-free or astrocyte-conditioned culture media, to improve the tightness of the barrier structure by application of tight-junction inducing agents in cell culture medium, and to knock-in the specific gene fragments that encode the proteins or regulatory proteins associated with tight junctions in existing Z310 cells. We further design a series of experiments to validate this model system. The studies proposed in this application, if successful despite the notable risk, will lead to the creation of a novel in vitro blood-brain/CSF model for transport study of materials into brain and should have significant impact on pharmacological and toxicological investigation of CNS transport of drugs and toxicants, CNS drug development, and etiological research of brain diseases.