RNA干扰沉默神经轴突导向因子受体4基因增加血肿瘤屏障通透性

RNA interference-induced Robo4 gene silencing increases the permeability of blood-tumor barrier

目的研究神经轴突导向因子受体(Robo4)对血肿瘤屏障(BTB)通透性的影响。方法建立了体外BTB模型,应用Real-time PCR和Western blot检测Robo4在正常人脑微血管内皮细胞和胶质瘤微血管内皮细胞中的表达变化。设计合成针对Robo4基因的小干扰RNA,转染至人脑微血管内皮h CMEC/D3细胞,下调体外血肿瘤屏障模型内皮细胞中Robo4的表达,跨内皮电阻测量系统和辣根过氧化物酶渗透试验分析血肿瘤屏障通透性变化;Western blot和免疫荧光法检测h CMEC/D3细胞中紧密连接相关蛋白occludin和ZO-1的表达和分布变化。结果和正常人脑微血管内皮细胞相比,Robo4在胶质瘤微血管内皮细胞中的表达显著上调。下调体外血肿瘤屏障模型内皮细胞Robo4的表达后,TEER值显著降低,辣根过氧化物酶透过率显著增高;同时胶质瘤微血管内皮细胞中紧密连接相关蛋白occludin和ZO-1的表达显著降低,在细胞膜上呈不连续分布。结论 RNA干扰沉默Robo4表达能够显著降低紧密连接相关蛋白occludin和ZO-1的表达,增加BTB通透性。

Objective To investigate the effect of Robo4 on the blood-tumor barrier（BTB） permeability. Methods After establishing the BTB model in vitro, the expressions of Robo4 in h CMEC/D3 cells were detected by Realtime PCR and Western blot. Small interfering RNA targeting Robo4 was transfected into h CMEC/D3 cells and the transendothelial electrical resistance of BTB and HRP flux were analyzed. Meanwhile, the expression and distribution of tight junction associated proteins occludin and ZO-1 were detected by Western blot and immunofluorescence staining. Results The expression level of Robo4 was significantly increased in the glioma co-cultured endothelial cells of BTB model compared with normal endothelial cells. The downregulation of Robo4 in endothelial cells of BTB model led to decreased transendothelial electric resistance（TEER） values and increased HRP flux, down-regulated the expression of tight junction proteins occludin and ZO-1,as well as the discontinuous distribution on the membrane. Conclusion RNA interference-induced Robo4 gene silencing increases the permeability of blood-tumor barrier by reducing the expression of occludin and ZO-1.