肿瘤坏死因子-α在常压高体积分数氧致新生大鼠脑细胞凋亡中的作用  

Role of Tumor Necrosis Factor-α in Pathogenesis of Normobaric Hyperoxia-Induced Brain Cell Apoptosis in Newborn Rats

作　　者：李易娟[1] 岳智慧[1] 陈玮琪[1] 庄思齐[1] 陈述枚[1] 

机构地区：[1]中山大学附属第一医院儿科,广州510080

出　　处：《实用儿科临床杂志》2009年第18期1392-1395,共4页Journal of Applied Clinical Pediatrics

基　　金：广东省医学科研基金项目资助(A2008162)

摘　　要：目的制备常压高体积分数氧(高氧)性脑损伤7日龄新生大鼠动物模型,探讨TNF-α在高氧性脑细胞凋亡发生中的作用。方法取112只体质量12～18 g的7日龄SD大鼠,随机分为2组:高氧组(依高氧暴露时间分为高氧暴露2、6、12、24 h组,每组14只)和空气对照组(各时间点新生大鼠数目与高氧组相同)。高氧组新生大鼠与母鼠一起置于氧箱中,调节氧流量(3 L/min)使箱内氧体积分数维持在(800±50)mL/L,用数字式测氧仪进行监测。空气对照组置于同一室内空气中,氧体积分数为210 mL/L。高氧/空气开始暴露24 h后各处死10只大鼠,取其脑组织常规脱水、包埋、切片,脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记(TUNEL)法观察其脑组织细胞凋亡指数,免疫组织化学染色法检测其脑组织半胱氨酸蛋白酶-3(Caspase-3)、TNF-α的表达水平。高氧暴露后2、6、12、24 h各处死4只大鼠,取左脑组织提取RNA,反转录聚合酶链反应(RT-PCR)检测TNF-αmRNA,以β-actin为内参,计算相对单位,并与空气对照组比较。结果高氧2 h组脑细胞凋亡指数较空气对照组增加(F=88.39 P<0.01),随着吸氧时间的延长,细胞凋亡渐增多,以高氧12、24 h组细胞凋亡最明显(F=119.57,153.48 Pa<0.01)。随着吸氧时间的延长,新生大鼠脑组织TNF-α、Caspase-3的表达水平明显升高,而TNF-αmRNA的表达在吸氧后2 h开始上升(P<0.05),12 h达高峰(P<0.01),24 h开始下降。TNF-α表达水平与脑细胞凋亡程度、Caspase-3表达水平均呈正相关(r=0.986,0.982 Pa<0.01)。结论常压高氧可引起新生大鼠脑细胞凋亡和TNF-αmRNA及其蛋白的过度表达。TNF-α与高氧性脑细胞凋亡的发生密切相关,TNF-α可能通过Caspase-3途径诱导脑细胞凋亡。Objective To establish normobaric hyperoxia - induced brain injury animal models, and explore the effects of TNF -α on the pathogenesis of hyperoxia - induced neural cell apoptosis in the newborn rats. Methods One hundred and twelve 7 - day - old SD rats weighting 12 - 18 g were randomly assigned into 2 groups ：hyperoxia group（ further divided into 2, 6, 12, 24 hours subgroups according to the exposure time, n = 14 ） and air control group. For hyperoxic group, the pups were kept in a chamber containing [ （800 ± 50） mL/L of oxygen （3L/min） ] with their dams for 2 -24 h. The oxygen concentration was monitored with digital oxygen monitor. The rats in air control group were placed in air environment with 210 mL/ML oxygen exposure,and then 10 rats were killed in each group at 24 h after the beginning of oxygen/air exposure. The brains were dehydrated, embedded and sliced for terminal deoxynucleotide transferase - mediated dUTP nick end labeling （TUNEL） staining to calculate the apoptotic index （AI）. Immunohistochemistry staining was performed to detect expression of TNF -α, Caspase - 3 of the brains. Four rats were decapitated in each group at 2, 6, 12, 24 h after the beginning of oxygen/air exposure. The total cel- lular RNA of the left hemispheres was isolated and the expression of TNF - α mRNA was measured by reverse transcription polymerase chain reaction（ RT - PCR）. The relative ratio of optical density in hyperoxia group was calculated with β - actin as internal control and compared with that of normoxic group. All the data were analyzed with SPSS 10.0 software. Results The AI in 2 h hyperoxia group was significantly higher than that in air control group （ F = 88.39 P 〈 0.01 ）. The AI in hyperoxia group was increased gradually with the prolongation of exposure to hyperoxia and reached the highest level after exposure to oxygen for 12,24 h（F = 119. 57,153.48 P 〈 0.01 ）. TNF - α mRNA expression began increase at 2 h （ P 〈 0.05 ） and maintained at high levels until

关 键 词：肿瘤坏死因子-Α 高体积分数氧 脑细胞凋亡 大鼠 新生 

分 类 号：R722.1[医药卫生—儿科]