Creation of an In Vitro Brain Barrier Transport System

体外脑屏障运输系统的创建

• 批准号: 7035380
• 负责人: WEI ZHENG
• 金额: $ 20.65万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035380
• 关键词: biological transport; blood brain barrier; brain metabolism; cell line; cerebrospinal fluid; gene expression; genetic manipulation; glucocorticoids; growth media; interleukin 15; laboratory rat; membrane model; membrane permeability; model design /development; protein localization; protein protein interaction; tight junctions; tissue /cell culture; transfection

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.

描述（由申请人提供）：大脑通过两个主要屏障得到良好保护，免受血液传播的外源性物质（药物、营养素和毒素）的影响，即，血脑屏障（BBB）和血脑脊液（CSF）屏障（BCB）。BCB的表面积约为BBB的一半，位于脑室中，其功能是产生CSF并在血液和CSF之间运输异生物质。我们最近建立了一个新的永生化脉络膜上皮细胞系，命名为Z310细胞系。该细胞系具有亲本原代细胞的基本形态，并且在半透膜上生长时，形成限制细胞旁渗漏标记物[14 C]蔗糖自由移动的单层。虽然通过跨上皮电阻（TEER）或[14 C]蔗糖的细胞旁渗漏测量的细胞单层的紧密性仍有待改善，但我们相信该细胞系作为独特的体外血脑屏障转运系统显示出巨大的前景，因为在当前的神经毒理学和神经药理学研究领域中还没有这样的脑细胞源性转运模型。为了创建这种新的系统，我们假设可以通过改变培养基的化学成分，通过诱导和促进紧密连接组装，和/或通过遗传调节紧密连接蛋白的表达来改善Z310上皮细胞单层的紧密性。因此，我们的具体目的是通过改变培养基组分，例如使用无血清或星形胶质细胞条件培养基，来降低Z310屏障模型的细胞旁渗透性，通过在细胞培养基中应用紧密连接诱导剂来改善屏障结构的紧密性，并敲入编码与现有Z310细胞中紧密连接相关的蛋白质或调节蛋白质的特异性基因片段。我们进一步设计了一系列的实验来验证这个模型系统。本申请中提出的研究如果成功，尽管存在显著的风险，将导致创建一种新的体外血脑/CSF模型，用于材料向脑中的转运研究，并将对药物和毒物的CNS转运的药理学和毒理学研究、CNS药物开发和脑疾病的病因学研究产生重大影响。

项目成果

TWIST-1 is overexpressed in neoplastic choroid plexus epithelial cells and promotes proliferation and invasion.

• DOI: 10.1158/0008-5472.can-08-3176
• 发表时间: 2009-03-15
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Hasselblatt M;Mertsch S;Koos B;Riesmeier B;Stegemann H;Jeibmann A;Tomm M;Schmitz N;Wrede B;Wolff JE;Zheng W;Paulus W
• 通讯作者: Paulus W