缓激肽开放血肿瘤屏障的作用及其与TNF-α、MAPK以及NF-κB的关系研究  

Study of relation between bradykinin-induced blood-tumor barrier opening and TNF-α/MAPK/NF-κB

作　　者：秦丽娟[1] 薛一雪[2] 谷艳婷[3] 张田[1] 孙娜[1] 张伟[1] 张志勇[4] 张文丽[5] 郭静[5] 

机构地区：[1]河北联合大学基础医学院生理学教研室,河北唐山063000 [2]中国医科大学基础医学院神经生物学教研室,辽宁沈阳110001 [3]沈阳药科大学生命科学与生物制药学院生理学教研室,辽宁沈阳110016 [4]河北联合大学附属医院神经外科,河北唐山063000 [5]河北联合大学医学实验研究中心,河北唐山063000

出　　处：《中国药理学通报》2012年第9期1276-1280,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金项目(No 81101912);国家教育部新教师基金(No 20102134120007);河北省卫生厅科学研究基金项目(No 20100465;20110165);唐山市科学技术研究与发展计划项目(No 111302048b);河北联合大学大学生创新性实验计划项目(No X2011035);河北联合大学博士启动基金项目(No BS09012);辽宁省博士启动基金(No20101109)

摘　　要：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。Aim To assess the effect and mechanism of tumor necrosis factor-α （TNF-α induced by bradykinin （BK） from glioma cells to an in vitro blood-tumor barrier. Methods Radioimmunoassay was used to dynamically monitor the contents of TNF-α in nutrient fluid for C6 cells after BK treatment. In vitro bloodtumor barrier model was established, then the influence of conditioned medium of C6 that was treated by BK （C6CM） was observed on levels of mitogenactivated protein kinase （MAPK） mRNA by RT-PCR method for brain microvascular endothelial cell （ BMEC ） , contents of nuclear transcription factor kappaB （ NF-κB ） was observed by immunohistochemistry, as well as permeability of blood-tumor barrier was detected by Evans blue. And immunofluorescence technique was used todetect the expression of occluding. Results For C6 cells, contents of TNF-α in the nutrient fluid increased obviously after BK treatment, with the maximal achieved at 120 min. C6CM acted on in vitro bloodtumor barrier, levels of MAPK mRNA and contents of NF-κB were both decreased, and reached the maximum at 120 min. The expression of occluding showed the same trend. Conclusions BK promotes C6 cell to release TNF-α, which decreases expression of NF-KB and levels of MAPK mRNA in BMEC,then reduces the expression of occluding. All these mechanisms may explain the effect of TNF-α on opening the blood-tumor barrier by BK.

关 键 词：缓激肽 肿瘤坏死因子-α. 紧密连接蛋白 血瘤屏障 MAPK NF-ΚB 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R730.264[医药卫生—基础医学]