人血脑屏障体外实验模型的建立及缺氧-复氧对其通透性的影响

目的建立人血脑屏障体外实验模型，并探讨缺氧-复氧对血脑屏障模型通透性的影响。 方法将分离、纯化的人脑微血管内皮细胞（HB-MVEC）在细胞插入器上培养至汇合状态，以液面试漏试验确定血脑屏障模型的形成，并通过形态学检查、跨内皮细胞电阻（TEER）测定和辣根过氧化物酶（HRP）通透率对血脑屏障模型进行鉴定；以未达汇合状态的HB-MVEC及培养至汇合状态的人脐静脉内皮细胞（HUVEC）作为对照。观察缺氧-复氧处理（缺氧2h和复氧1、2、4、8、24h）和缺氧-复氧时存在白细胞激活产物（缺氧2h后在有白细胞激活产物存在情况下复氧1h）对血脑屏障模型通透性的影响，以及前列腺素E、α1抗胰蛋白酶和丹参单体764-3对血脑屏障模型的保护作用。各项实验均观察3孔细胞。 结果HB-MVEC在细胞插入器培养至汇合状态后，液面试漏试验呈阳性；扫描电镜观察显示细胞间无间隙，透射电镜检查证实细胞间存在紧密连接。血脑屏障模型、未达汇合状态HB-MVEC和HUVEC的TEER分别为（46.0±1.3）、（30.8±1.4）、（7.5±2.1）Ω/cm^2；向细胞插入器内加入含HRP的培养基培养1h后，HRP通透率分别为0.17%±0.03%、0.26%±0.04%和0.94%±0.07%；缺氧2h和复氧1、2、4、8、24h HRP通透率分别为3.97%±0.94%、6.06%±0.75%、7.17%±0.18%、7.96%±0.47%、8.57%±0.62%、10.37%±0.78%。血脑屏障模型缺氧2h后在有白细胞激活产物的情况下复氧1h，其HRP通透率为8.87%±0.76%，明显高于无白细胞激活产物组（7.20%±0.87%）；而前列腺素E、α1抗胰蛋白酶和丹参单体764-3能够减弱这种情况下的血脑屏障模型通透性增加，3组的HRP通透率分别为7.08%±0.89%，6.01%±0.57%和5.53%±0.62%。 结论应用HB-MVEC可以构建血脑屏障体外模型，缺氧-复氧明显增加血脑屏障模型的通透性，前列腺素E、α1抗胰蛋白酶和丹参单体764-3具有保护血脑屏障

Establishment of in vitro human blood-brain barrier model and effect of hypoxia-reoxygenation on its permeability

Objective To establish an in vitro model of brain blood barrier (BBB) and investigate the effect of hypoxia-reoxygenetion on the permeability of BBB. Methods A BBB model was established by culturing isolated and purified human brain microvascular endothelial cells (HB-MVECs) to confluence on a cell culture insert system, and then tested by 4-hour water-leaking test, and identified by morphological examination, transendothelial electrical resistant (TEER) determination, assessment of permeability of BBB for horseradish peroxidase (HRP). Nonconfluent HB-MVECs and confluent human umbilical vein endothelial cells (HUVECs) were set as controls. The permeability of the BBB was tested at 2 hours of hypoxia, and 1, 2, 4, 8, and 24 hours after reoxygenation. The effect of leukocytes activation products on the permeability of BBB was detected by adding supernatants of activated leukocytes, prostaglandin E, alpha-1 antitrypsin, or Salvia miltiorrhiza monomer 764-3 into the BBB culture media 1 hour after reoxygenation. Three-well plates were used for each experiment. Results When the HB-MVECs were confluent in the culture insert system, 4-hour water-leaking test was positive; scanning electron microscopy showed no gap between the cells, and transmission electron microscopy confirmed that the cells tightly connected. The TEER over confluent HB-MVECs, nonconfulent HB-MVECs, and HUVECs was (46.0 ± 1.3), (30.8 ± 1.4), and (7.5 ± 2.1) Ω/cm2, respectively. After being cultured with HRP for 1 hour, the permeability of the three groups was 0.17% ± 0.03%, 0.26% ± 0.04%, and 0.94% ± 0.07%. The permeability of BBB for HRP was 3.97% ± 0.94%, 6.06% ± 0.75%, 7.17% ± 0.18%, 7.96% ± 0.47%, 8.57% ± 0.62%, and 10.37% ± 0.78% respectively at 2 hours of hypoxia, and 1, 2, 4, 8, and 24 hours after reoxygenation. One hour after reoxygenation, the permeability for HRP of the BBB that was co-cultured with leukocytes activation products (added at 2 hours of hypoxia), was 8.87% ± 0.76%, which was significantly higher than that of the BBB cultured without leukocytes activation products (7.20% ± 0.87%). In addition, the increased permeability could be reduced by prostaglandin E (7.08% ± 0.89%), alpha-1 antitrypsin (6.01% ± 0.57%), and Salvia miltiorrhiza monomer 764-3 (5.53% ± 0.62%). Conclusions In vitro model of BBB can be established by using HB-MVECs. The permeability of BBB model can be increased by hypoxia-reoxygenation. Prostaglandin E, alpha-1 antitrypsin, and 764-3 have protective effect on the BBB model.