Choroid plexus epithelial monolayers - a cell culture model from porcine brain
Choroid plexus epithelial monolayers - a cell culture model from porcine brain
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DOI：
10.1186/1743-8454-3-13
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发表时间：
2006-12-21
影响因子：
7.3
通讯作者：
Fricker, Gert
中科院分区：
文献类型：
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作者：
Baehr, Carsten;Reichel, Valeska;Fricker, Gert
背景：本研究的目的是建立体外脉络膜丛（CP）上皮细胞培养模型，用于研究蛋白质介导的药物从血液到脑脊液（CSF）的转运。方法：对新鲜分离的猪神经丛组织进行机械和酶处理，分离细胞。培养上皮细胞单层，测定脑脊液分泌和经上皮细胞耐药性。观察f-actin和脉络膜丛标志蛋白转甲状腺素（TTR）的表达。采用RT-PCR、Western-blot和免疫荧光技术研究输出蛋白p-糖蛋白（Pgp、Abcb1）和多药耐药蛋白1 （Mrp1、Abcc1）的表达，并通过转运和摄取实验评估其功能活性。结果：获得高纯度脉络膜丛上皮细胞，培养成融合单层。滤过物培养的单层显示出跨内皮阻力（TEER）值在100至150 Omega cm(2)之间。形态学上，细胞呈典型的f-actin网状结构，TTR的表达率较高。培养的细胞能够在2-3小时内以48.2 +/- 4.6 μ l/cm(2)/h的速率分泌CSF。abc输出蛋白Mrp1在细胞单层和完整组织的基底外侧（面向血液）膜中表达。p -糖蛋白仅在根尖（脑脊液导向）膜内低表达，而更多地位于根尖下细胞区室。这一发现与p糖蛋白底物维拉帕米和罗丹明缺乏定向排泄相一致。结论：CP上皮是可以分离培养的，细胞发育成完整的单层，完全分化，具有与活体组织相似的特性。因此，建立的猪初级CP模型可以研究复杂的转运过程，可以作为分析异种生物通过血脑脊液屏障（BCSFB）转运的可靠工具。
Background: The goal of the present study was to develop an in vitro choroid plexus (CP) epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF) and vice versa.Methods: Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR), were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1) and multidrug resistance protein 1 (Mrp1, Abcc1) was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments.Results: Choroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER) values in the range of 100 to 150 Omega cm(2). Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 +/- 4.6 mu l/cm(2)/h over 2-3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (bloodfacing) membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed) membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123.Conclusion: It was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic transport across the bloodcerebrospinal fluid barrier (BCSFB).