脉络丛上皮单层细胞——一种来自猪脑的细胞培养模型。
Choroid plexus epithelial monolayers--a cell culture model from porcine brain.
作者信息
Baehr Carsten, Reichel Valeska, Fricker Gert
机构信息
Ruprecht-Karls-University, Institute of Pharmacy and Molecular Biotechnology, 69120 Heidelberg, Germany.
出版信息
Cerebrospinal Fluid Res. 2006 Dec 21;3:13. doi: 10.1186/1743-8454-3-13.
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 ohms cm2. 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 microl/cm2/h over 2-3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing) 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 blood-cerebrospinal fluid barrier (BCSFB).
背景
本研究的目的是建立一种体外脉络丛（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）以及从脑脊液到血液的分泌转运。
方法
通过对新鲜分离的猪脉络丛组织进行机械和酶处理来分离细胞。培养上皮细胞单层，并测定脑脊液分泌和跨上皮电阻。评估f-肌动蛋白以及脉络丛标记蛋白转甲状腺素蛋白（TTR）的表达。通过逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（Western-blot）和免疫荧光技术研究外排蛋白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
以高纯度分离出脉络丛上皮细胞，并培养形成汇合的单层。滤膜培养的单层显示跨内皮电阻（TEER）值在100至150欧姆·平方厘米范围内。在形态学上，细胞显示出典型的f-肌动蛋白网络，并高表达TTR。培养的细胞能够在2至3小时内以48.2±4.6微升/平方厘米/小时的速率分泌脑脊液。ABC外排蛋白Mrp1表达于细胞单层和完整组织的基底外侧（面向血液）膜中。P-糖蛋白在顶端（面向脑脊液）膜中仅显示低表达，但更多位于顶端下细胞区室。这一发现与P-糖蛋白底物维拉帕米和罗丹明123缺乏定向排泄相平行。
结论
已证明可以分离和培养脉络丛上皮细胞，细胞生长形成完整的单层，充分分化且具有类似于体内组织的特性。因此，所建立的原代猪脉络丛模型能够研究复杂的转运过程，可作为分析外源性物质跨血脑脊液屏障（BCSFB）转运的可靠工具。
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