内皮—单核细胞激活多肽酶Ⅱ增强血肿瘤屏障通透性的作用位点

摘要

目的 研究内皮—单核细胞激活多肽Ⅱ(EMAP-Ⅱ)增强血肿瘤屏障(BTB)的作用机制.方法 采集出生3～5 d的Wistar胎鼠大脑皮质,应用酶消化法及葡聚糖离心法获得脑微血管段后,接种于培养皿中进行脑微血管内皮细胞(BMEC)的原代培养；将BMEC与C6脑胶质瘤细胞共培养,构建体外BTB模型；实验随机分为4组(每组6例):对照组、EMAP-Ⅱ组、寡霉素组和寡霉素+EMAP-Ⅱ组.测定跨内皮阻抗值和辣根过氧化物酶流量,评估各组BTB通透性变化；Western blot检测BMEC上紧密连接(TJ)相关蛋白ZO-1的表达水平；双重免疫荧光法检测EMAP-Ⅱ和α-ATP合成酶在BMEC上的分布.结果 EMAP-Ⅱ组BTB通透性较对照组和寡霉素组显著增高(P＜0.01)；与对照组和寡霉素组比较,EMAP-Ⅱ组TJ相关蛋白ZO-1的表达水平显著降低(P＜ 0.01)；EMAP-Ⅱ组与寡霉素组比较,EMAP-Ⅱ的作用受到ATP合成酶抑制剂寡霉素的显著抑制(P＜0.01);EMAP-Ⅱ与α-ATP合成酶在BMEC膜上共定位.结论 EMAP-Ⅱ通过开放TJ增强BTB的通透性,BMEC膜上的α-ATP合成酶是其作用的位点.%Objective To gain an insight into the mechanisms for endothelial monocyte-activating polypeptide- Ⅱ (EMAP- Ⅱ )-induced en-hancement in the blood-tumor barrier ( BTB) permeability. Methods Relatively pure cerebral microvcsscl fragments were obtained from the cortex of 3-5 days old wistar rats through carefully dissection, enzyme digestion, and dextran centrifugation. The fragments were then seeded on dishes and cultured primarily. In vitro BTB models were constructed by co-culture rat brain microvascular endothelial cells (BMEC) with C6 glioma cells. Confluent monolayers of co-cultured BMEC were randomly divided into 4 groups (each n = 6): control, EMAP-Ⅱ, oligomycin and oligomycin + EMAP- Ⅱ groups. Transendothelial electric resistance values and horseradish peroxidase flux were measured to evaluate changes in the RTB permeability. The expression level of tight junction (TJ)-related protein ZO-1 in BMEC was measured by western blot assay. Double imrnunofluorescence was used to identify the expression and distribution of EMAP- Ⅱ and α-ATP synthase in BMEC. Re-sults Compared with control and oligomycin groups,the BTB permeability of EMAP-Ⅱ group was increased significantly (P < 0.01) .while the expression level of TJ-related protein ZO-1 was significantly decreased (P < 0.01). These effects of EMAP- Ⅱ were significantly inhibited by pretreatment with oligomycin,the inhibitor of ATP synthase (P< 0.01). Moreover,our data showed that both EMAP-Ⅱ and α-ATP syn-thase were expressed on the surface of BMEC. In addition, EMAP-Ⅱ was found co-localized with α-ATP synthase. Conclusion EMAP-Ⅱ could increase the BTB permeability by opening TJ, and α-ATP synthase on the surface of BMEC might serve as the functional target.