Generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model.
Generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model.
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DOI：
10.3791/4022
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发表时间：
2012-08-29
期刊：
影响因子：
--
通讯作者：
Förster CY
中科院分区：
文献类型：
--
作者：
Burek M;Salvador E;Förster CY
上皮细胞和内皮细胞（EC）正在构建保护组织免受外部和内部环境影响的细胞旁屏障。由EC、星形胶质细胞终足、周细胞和基底膜组成的血脑屏障（BBB）负责脑实质的保护和稳态。体外血脑屏障模型是在细胞水平研究血脑屏障结构和功能的常用工具。到目前为止，已经在不同的实验室建立了相当数量的不同的体外血脑屏障模型用于研究。通常，从牛、猪、大鼠或小鼠脑组织中获得细胞（在Wilhelm等人的综述中详细讨论1）。人体组织样本仅在有限数量的实验室或公司中提供2，3。虽然原代细胞制备耗时，EC培养物可能因批次而异，但永生化EC系的建立是科学兴趣的焦点。在这里，我们提出了一种建立永生化的新生小鼠脑微血管EC系的方法。我们描述的程序一步一步列出所使用的试剂和解决方案。我们实验室建立的方法允许在4到5周内分离同质永生化内皮细胞系。脑微血管内皮细胞系cEND 4（来自大脑皮层）和cerebEND 5（来自小脑皮层）是根据Förster实验室的这一程序分离的，并已有效地用于解释BBB的不同生理和病理过程。使用cEND和cerebEND，我们已经证明这些细胞对糖皮质激素4，6 -9和雌激素治疗10以及促炎介质如TNF α 5，8有反应。此外，我们还在EC水平上研究了多发性硬化症11和缺氧12，13的病理学。cEND和cerebEND细胞系可被认为是研究血脑屏障结构和功能、EC对不同刺激的细胞反应以及EC与淋巴细胞或癌细胞相互作用的良好工具。
Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest. Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.
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