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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 investigate the effect of tumor necrosis factor-α (TNF-α) released from glioma cells induced by bradykinin (BK) on the in vitro hematological barrier and its mechanism. Methods: After treated with bradykinin and saline for C6 glioma cells and glial cells respectively, the concentrations of TNF-α in culture medium were detected by radioimmunoassay at 0, 5, 10, 15, 30 and 60 min. The primary culture of brain microvascular endothelial cells (BMEC) and C6 cells were co-cultured to establish a hemoblast barrier model. The C6 cells treated with bradykinin at different time points were applied to the barrier model, and the permeability and trans-endothelial impedance (TER) changes of the barrier were observed dynamically. The expression of endothelial microvascular endothelial cells The expression of tight junction protein occludin was detected by RT-PCR. The changes of nuclear factor-κB (NF-κB) in brain microvascular endothelial cells were detected by RT-PCR. Results: After treated with bradykinin, the concentration of TNF-α in the culture medium was significantly increased (P <0.05) compared with the control group, while the glial cells did not change significantly. After CMCM applied to the blood-borne barrier model, the permeability of the blood-borne barrier was increased and the trans-endothelial impedance was decreased. However, occludin expression and nuclear factor-κB transcription were decreased in endothelial cells until NF-κB Transcriptional levels gradually increased. CONCLUSION: Bradykinin induces the release of TNF-α from glioma cells, and the release of TNF-α can affect occludin expression and affect the barrier permeability of blood-vessel barrier by NF-KB.