C3胞外酶抑制缓激肽诱导的血肿瘤屏障通透性的增加

C_3 Exoenzyme Inhibits Bradykinin-induced Selective Increase of the Permeability of Blood-tumor Barrier

目的研究Ras基因家族成员A(RhoA)的特异性抑制剂C3胞外酶(exoenzyme)对缓激肽诱导的血肿瘤屏障通透性的作用。方法应用C3 exoenzyme预处理大鼠原代脑微血管内皮细胞后,测量跨内皮阻抗值(TEER)、辣根过氧化物酶(HRP)流量,分析血肿瘤屏障的通透性的改变;应用Western blot检测紧密连接相关蛋白(ZO-1)的表达;应用免疫荧光法观察原代大鼠脑微血管内皮细胞紧密连接相关蛋白ZO-1和丝状肌动蛋白(F-actin)结构和分布的改变。结果 C3 exoenzyme显著抑制缓激肽诱导TEER值的降低、HRP流量的升高及ZO-1的表达,抑制ZO-1由内皮细胞的边缘向细胞质转移,抑制丝状肌动蛋白由细胞膜边缘向细胞中央区分布,分布于细胞边缘的F-actin明显增加,应力纤维形成明显减少。结论 RhoA能够介导缓激肽开放血肿瘤屏障。

Objective To investigate the effect of C3 exoenzymea,specific Ras homolog gene family member A（RhoA）inhibitoro,n bradykinin（BK）-induced increase in blood-tumor barrier（BTB）permeability.Methods Rat brain microvascular endothelail cells（RB-MECs）were pretreated with C3 exoenzyme and then treated with BK for 15 minutes.Horseradish peroxidase（HRP）flux and TEER assays were employed to reveal BTB permeability.The protein expression level of ZO-1 was observed by Western blot.The stress fiber formation and distribution of filamentous actin（F-actin）and ZO-1 were assessed by immunofluorescence microscopy.Results C3 exoenzyme could par-tially inhibit endothelial leakage and restored normal TEER values in RBMECs.The expression level of ZO-1 protein decreased significantly and it was prevented by C3 exoenzyme.C3 exoenzyme inhibited BK-induced relocation of ZO-1 from cellular borders into the cytoplasm as well as stress fiber formation in RBMECs.Conclusion RhoA plays a key role in BK-induced openning of blood-tumor barrier.