【摘要】 背景:以往的研究发现,感染性脑损伤时脑组织中白细胞介素1和肿瘤坏死因子α含量明显增加,并与脑损害呈正相关关系,其能否导致血脑屏障通透性增高?目的:观察肿瘤坏死因子α对体外血脑屏障模型通透性的影响及其可能的调控机制。设计:体外细胞模型对照实验。单位:中南大学湘雅医院儿科,中南大学湘雅医学院生化系。材料:选用20只生后7d健康SD大鼠,雌雄不拘,清洁级,由中南大学湘雅医学院动物部提供;肿瘤坏死因子α购自sigma公司;DMEM液体培养基、胎牛血清购自Hyclone;Rhok的特异性拮抗剂Y-27632购自Alexis公司,兔抗人Ⅷ因子相关抗原购自Zymed公司;小鼠抗大鼠神经胶质纤维酸性蛋白单抗(GFAP)购自Neomarkers公司。方法:实验于2004-03/2005-04在中南大学湘雅医院完成。利用脑微血管内皮细胞与星型胶质细胞共培养建立体外大鼠血脑屏障模型,共培养至10d开始干预,实验分为模型组、Y-27632对照组、肿瘤坏死因子α干预组和Y-27632预处理组。模型组仅给予血脑屏障模型制备,肿瘤坏死因子α干预组向血脑屏障模型内加入肿瘤坏死因子α0.01g/L干预5h;Y-27632预处理组指向血脑屏障模型内加入Y-2763230μmol/L预处理1h后,再予肿瘤坏死因子α0.01g/L干预5h,Y-27632对照组造模后仅加入Y-27632干预,方式同Y-27632预处理组。取分组处理后待用模型,分别于30,60,120,240min采用γ计数仪检测125Ⅰ-牛血清白蛋白的通透量观察肿瘤坏死因子α对血脑屏障通透性的影响。主要观察指标:各组干预后不同时间点体外大鼠血脑屏障模型对125Ⅰ-牛血清白蛋白的通透量。结果:处理后30,60,120,240min,肿瘤坏死因子α干预组体外血脑屏障模型125Ⅰ-BSA通透量均高于其他组别(P<0.01),240min时达高峰;处理后30,60min,Y-27632预处理组125Ⅰ-BSA通透量低于肿瘤坏死因子α干预组(P<0.01),表现出拮抗肿瘤坏死因子α的作用,自120min后,Y-27632预处理组与Y-27632对照组比较,差异也有显著性意义(P<0.05)。结论:肿瘤坏死因子α可导致血脑屏障通透性增高,Y-27632预处理能早期逆转肿瘤坏死因子α对血脑屏障通透性的影响。更多还原

【Abstract】 BACKGROUND:The levels of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are increased during infectious brain edema,and are positively relevant to the degree of brain damage. However,whether TNF-α can enhance blood brain barrier (BBB) permeability remains unclear,especially in vitro. OBJECTIVE: To understand the changes and possible mechanism of the BBB permeability induced by TNF-α in vitro. DESIGN: Randomized controlled cell model study in vitro. SETTING:Department of Pediatrics,Xiangya Hospital,Central South University; Department of Biochemistry,Xiangya Medical College,Central South University. MATERIALS: Twenty 7-day-old healthy Sprague-Dawley rats,of clean grade and either gender,were provided by the Animal Center,Xiangya Hospital,Central South University. TNF-α was purchased from sigma Company; DMEM fluid medium and fetal bovine serum were purchased from Hyclone Company; Y-27632 was purchased from Alexis Company,and rabbit anti-human factor Ⅷ -related antigen was purchased from Zymed Company; Mouse anti-rat glial fibrillary acidic protein (GFAP) was purchased from Neomarkers. Other biochemical reagents were imported (Sigma Company). METHODS: This experiment was carried out in the Xiangya Hospital,Central South University between March 2004 and April 2005. Brain microvascular endothelial cells and astrocytes were co-cultured 10 days to set up rat models of BBB in vitro. Then,the cells were divided into 4 groups: model group(BBB models were prepared),TNF-α group ( BBB model incubated with 0.01 g/L TNF-α for 5 hours),Y-27632 pretreated group ( BBB model incubated with 30 μmol/L Y-27632 for 1 hour before 0.01g/L TNF-α challenge ) and Y-27632 control group (BBB models only incubated with Y-27632 as those in the Y-27632 pretreated group). The effect of TNF-α on BBB permeability was observed by detecting the 125Ⅰ-BSA,which passed through the inserts at each time point (30,60,120 and 240 minutes) using γ radioimmunoassay counter. MAIN OUTCOME MEASURES: The 125Ⅰ-BSA,which passed through the inserts in rat models of BBB at different time points after intervention. RESULTS: The 125Ⅰ-BSA,which passed through the inserts in rat models of BBB,was all significantly higher in the TNF-α group than in the other groups at 30,60,120 and 240 minutes after intervention,respectively (P < 0.01),and reached the peak at 240 minutes; The 125Ⅰ-BSA,which passed through the inserts,was lower in the Y-27632 pre-treated group than in the TNF-α group at 30 and 60 minutes after intervention (P < 0.01). There was also significant difference in 125Ⅰ-BSA permeation between Y-27632 pretreated group and Y-27632 control group after 120 minutes (P < 0.05). CONCLUSION: TNF-α can increase BBB permeability,and Y-27632 pretreatment can early reverse the effect of TNF-α on BBB permeability.更多还原