Hayashi Kentaro, Nakao Shinobu, Nakaoke Ryota, Nakagawa Shinsuke, Kitagawa Naoki, Niwa Masami
Department of Neurosurgery, Nagasaki University School of Medicine, Nagasaki, Japan.
Regul Pept. 2004 Dec 15;123(1-3):77-83. doi: 10.1016/j.regpep.2004.05.023.
The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytic foot processes. Most research for the in vitro BBB is performed endothelial cells with or without astrocytes. Hypoxia damage to the BBB induces vasogenic brain edema. We have generated a new model of the BBB with brain endothelial cells and pericytes and have examined the effects of hypoxia using this model. Brain microvascular endothelial cells and pericytes were isolated from three-week-old male Wister rats using enzyme and mechanical homogenization. Three models (A: only endothelial monolayer, B: endothelial monolayer with pericytes non-contact condition, and C: contact condition) were made by culturing these cells using Transwell co-culture system and were exposed to hypoxic condition. We evaluated barrier function with transendothelial electrical resistance (TEER) and permeability of Evans blue-albumin and sodium fluorescein. The tightest barrier was observed in the endothelial/pericytic contact model. Despite hypoxia-induced disruption of the barrier in endothelial monolayer and non-contact co-culture models, a minimum of dysfunction was seen in the contact co-culture model. Therefore, it is considered that pericytes effect on the endothelia by secreting factors or through a gap junction. In short, pericytes induce endothelial maturation and a tighter barrier function, which supports the function against the hypoxic injury. Intercellular communication might be important to keep the BBB functional and stabilize in hypoxia.
血脑屏障（BBB）由内皮细胞、周细胞和星形胶质细胞足突组成。大多数体外血脑屏障研究是在有或没有星形胶质细胞的情况下对内皮细胞进行的。血脑屏障的缺氧损伤会诱发血管源性脑水肿。我们利用脑内皮细胞和周细胞构建了一种新的血脑屏障模型，并使用该模型研究了缺氧的影响。采用酶解和机械匀浆法从3周龄雄性Wistar大鼠中分离出脑微血管内皮细胞和周细胞。使用Transwell共培养系统培养这些细胞，构建了三种模型（A：仅内皮单层，B：内皮单层与周细胞非接触条件，C：接触条件），并使其暴露于缺氧环境。我们通过跨内皮电阻（TEER）以及伊文思蓝 - 白蛋白和荧光素钠的通透性来评估屏障功能。在内皮/周细胞接触模型中观察到最紧密的屏障。尽管缺氧导致内皮单层和非接触共培养模型中的屏障破坏，但在接触共培养模型中仅观察到最小程度的功能障碍。因此，认为周细胞通过分泌因子或通过缝隙连接作用于内皮细胞。简而言之，周细胞诱导内皮细胞成熟并形成更紧密的屏障功能，这有助于抵抗缺氧损伤。细胞间通讯对于在缺氧状态下保持血脑屏障功能并使其稳定可能很重要。
