缓激肽诱发胶质瘤细胞释放的肿瘤坏死因子-α对体外血瘤屏障的影响

In Vitro Effect of TNF-α Expression Induced by Bradykinin in Glioma Cells on the Blood-Tumor Barrier

目的:探讨缓激肽(bradykinin,BK)诱发胶质瘤细胞释放的肿瘤坏死因子-α(TNF-α)对体外血瘤屏障的影响及其作用机制。方法:分别用缓激肽和生理盐水作用于C6胶质瘤细胞及神经胶质细胞后,放射免疫法检测0、5、10、15、30和60 min时培养液内TNF-α的浓度。用原代培养的脑微血管内皮细胞(BMEC)和C6细胞共培养构建血瘤屏障模型。将缓激肽处理C6细胞不同时间点的条件培养液(C6CM)作用于屏障模型后,动态观察屏障的通透性及跨内皮阻抗(TER)的改变,并用免疫荧光技术检测脑微血管内皮细胞的紧密连接蛋白occludin的表达,RT-PCR法检测屏障模型脑微血管内皮细胞中核因子-κB(NF-κB)的变化。结果:缓激肽作用于C6细胞后,培养液内TNF-α浓度较对照组明显增加(P<0.05),而神经胶质细胞无明显变化。C6CM作用于血瘤屏障模型后,血瘤屏障通透性增加,跨内皮阻抗减小,而内皮细胞间的紧密连接蛋白occludin表达和核因子-κB的转录水平降低,直至30 min后NF-κB转录水平逐渐增强。结论:缓激肽诱发了胶质瘤细胞释放TNF-α,释放的TNF-α可通过NF-KB影响紧密连接蛋白occludin表达而影响血瘤屏障通透性。

Objective： To assess the in vitro effect and mechanism of tumor necrosis factor-or （ TNF - α ） expression induced by bradykinin （ BK ） in glioma cells on the blood-tumor barrier （ BTB ）. Methods： A radioimmunoassay was used to dynamically monitor the TNF-α content in a nutrient fluid at 0, 5, 10, 15, 30, and 60 min time points after BK treatment of C6 cells and glial cells. An in vitro BTB model was established using a coculture of brain microvascular endothelial cells （ BMEC ） and C6 cells. The effect of a BK- treated C6 conditioned medium （ C6CM ） on the levels of nuclear transcription factor kappa B （ NF -κB ） mRNA was investigated by PCR. The occludin expression, as well as the transendothelial resistance （ TER ） and permeability of BTB, was also investigated via the immunofluorescence technique. Results： The TNF-α content in the nutrient fluid of the C6 cells clearly increased after BK treatment compared with that of the control group; however, the glial cells showed no such result. C6CM acted on BTB in vitro, resulting in the decrease in the NF-κB mRNAand occludin expression levels until the minimum was reached at 15 min. Meanwhile, the permeability of BTB increased, whereas TER decreased. Conclusion： BK induced the C6 cells to release TNF-α TNF-α in turn inhibited NF-κB transcription and reduced the occlu- din expression. These mechanisms may explain the effects of TNF-α n the opening of BTB using BK.