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目的探讨缓激肽(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机制。方法缓激肽作用于C6细胞后,应用放射免疫法动态监测培养液内TNF-α的含量;构建体外血瘤屏障模型,观察BK作用于C6细胞后的条件培养液(C6CM)对屏障上脑微血管内皮细胞(BMEC)内丝裂素活化蛋白激酶(MAPK)mRNA的转录水平(RT-PCR技术)、NF-κB含量(免疫组织化学技术)及血瘤屏障通透性(伊文思蓝法)的影响;利用免疫荧光技术检测C6CM对体外血瘤屏障模型上occluding表达的影响。结果缓激肽作用于C6细胞后,培养液内TNF-α的含量增加,于120 min时达高峰后减少。C6CM作用于体外血瘤屏障后,脑微血管内皮细胞的MAPK mRNA转录水平及NF-κB含量减少,且均于第120 min时达最低水平后开始增加。与此同时,体外血瘤屏障紧密连接蛋白occluding的表达水平也呈相同的变化趋势。结论缓激肽可诱发C6细胞释放TNF-α,释放的TNF-α可能是通过减少BMEC的MAPK mR-NA转录水平和NF-κB的含量而导致血瘤屏障上occluding的表达减少,进而引起血瘤屏障开放的。
Objective To investigate the mechanism of tumor necrosis factor-α (TNF-α) released from glioma cells induced by bradykinin (BK) in vitro. Methods After the bradykinin was treated in C6 cells, radioimmunoassay was used to dynamically monitor the content of TNF-α in culture broth. The in vitro model of the blood-borne barrier was established to observe the effects of BK on C6 cells in conditioned medium (C6CM) Transcriptional level of mitogen-activated protein kinase (MAPK) mRNA in microvascular endothelial cells (BMECs), NF-κB content (immunohistochemistry) and permeability of the blood vessel barrier (Evans blue method) The effect of C6CM on the occluding expression in the in vitro model of hematological barrier was examined by immunofluorescence technique. Results After bradykinin treatment on C6 cells, the content of TNF-α in the culture fluid increased and reached the peak at 120 min, then decreased. C6CM induced a decrease of MAPK mRNA and NF-κB in brain microvascular endothelial cells, and both began to increase after reaching the lowest level at 120 min. At the same time, the expression level of tight junction protein occluding in the extracorporeal blood-vessel barrier also showed the same trend. Conclusion Bradykinin can induce the release of TNF-α by C6 cells. The release of TNF-α may result in the decrease of occluding expression on the blood-vessel barrier by decreasing the MAPK mR-NA transcriptional level and NF-κB in BMEC, Tumor barrier is open.