论文部分内容阅读
目的应用原代分离培养BALB/c小鼠脑微血管内皮细胞(brain microvascular endothelial cells,BMVEC)建立体外血脑屏障模型,探讨跨血脑屏障(blood brainb arrier,BBB)电阻与屏障渗透功能的动态关系以及最佳构建条件。方法用酶消化、机械分离结合密度离心的方法得到原代BALB/c小鼠脑血管内皮细胞,通过培养在具有特殊质材和孔径的Transwell小室上建立BBB体外实验模型,采用倒置显微镜、电镜观察细胞形态结构和生长规律,紧密连接ZO-1蛋白免疫组化检测,比较血脑屏障形成前后膜两侧电阻动态变化与3H葡萄糖通透性的关系等方法,探讨血脑屏障模型的建立及生长特性。结果BMVEC培养至汇合后具有典型的“铺路石”样外观;扫描电镜显示细胞形成致密单层,透射电镜、ZO-1蛋白免疫组化证实细胞间形成光滑、连续、高密度的紧密连接;3H葡萄糖的通透量与实时电阻呈负相关,内皮细胞电阻随着通透性的增加而降低,通透率最低时跨细胞电阻为(346±10)Ω/cm2。结论建立的BBB体外模型在形态学、电阻和通透性方面具备了BBB的基本特性。
Objective To establish an in vitro model of blood-brain barrier (BBB) in primary cultured BALB / c mouse brain microvascular endothelial cells (BMVEC) and explore the dynamic relationship between the resistance of blood brain barrier (BBB) and barrier infiltration As well as the best construction conditions. Methods Cerebral vascular endothelial cells of primary BALB / c mice were obtained by enzymatic digestion, mechanical separation and density centrifugation. The in vitro experimental model of BBB was established by culturing on Transwell chamber with special material and pore size. The inverted microscope and electron microscope Cell morphology and growth, tight junction ZO-1 protein immunohistochemical detection of blood-brain barrier before and after the formation of membrane resistance on both sides of the dynamic changes and 3H glucose permeability of the method to explore the establishment of the blood-brain barrier model and growth characteristic. Results BMVEC cultured to the confluence of the typical “paving stone ” appearance; scanning electron microscopy showed cells forming a dense monolayer, transmission electron microscopy, ZO-1 protein immunohistochemistry confirmed the formation of smooth, continuous, high-density tight junctions The permeation of 3H glucose was negatively correlated with the real-time resistance. The resistance of endothelial cells decreased with the increase of permeability. The trans-cell resistance was (346 ± 10) Ω / cm2 with the lowest permeability. Conclusion The established BBB in vitro model possesses the basic characteristics of BBB in terms of morphology, resistance and permeability.