高胆红素血症新生大鼠脑组织凋亡诱导因子、细胞色素C表达的实验研究

Experimental Study on Expression of Apoptosis-inducing Factor and Cytochrome C in Brain Tissue of Neonatal Rats with Hyperbilirubinemia

目的:探讨高胆红素血症新生大鼠脑组织凋亡诱导因子(AIF)、细胞色素C(Cty-C)表达的实验研究。方法:将120只清洁级7 d龄SD大鼠随机分为对照组、模型组、Z-VAD-FMK干预组,每组40只,3组分别于造模后6、24、48、72、96 h断头取脑组织,每时间点取8只。模型组制备采用胆红素腹腔注射法(100μg/g)。Z-VAD-FMK干预组在模型组基础上即刻腹腔注射Z-VAD-FMK 3μg/g(处死前每日1次)。分别应用原位缺口末端标记法(TUNEL)、免疫组化法测定脑组织海马(CA1)区不同时间点细胞凋亡和Cty-C和AIF表达情况。结果:模型组、Z-VAD-FMK干预组海马区脑组织6 h开始出现细胞凋亡和Cty-C、AIF表达,模型组、Z-VAD-FMK干预组6 h细胞凋亡指数分别为17.7±2.4、12.9±1.1,Cty-C阳性细胞平均灰度值分别为140.5±11.3、142.9±13.1,AIF阳性细胞平均灰度值分别为169.5±11.9、170.7±10.0;随时间延长,细胞凋亡及Cty-C、AIF表达逐渐增多,96 h达高峰,模型组、Z-VAD-FMK干预组96 h细胞凋亡指数分别为38.0±6.0、33.6±3.8,Cty-C阳性细胞平均灰度值分别为109.6±8.0、127.3±9.8;AIF阳性细胞平均灰度值分别为139.7±9.4、141.0±7.1;对照组各时间点海马区脑组织未见明显细胞凋亡和Cty-C、AIF表达。结论:高胆红素血症所致脑损伤机制与线粒体损伤Cty-C、AIF表达增多有关,且非Caspase依赖性细胞凋亡也在一定程度上参与了此进程。

Objective:To investigate experimental study on expression of apoptosis-inducing factor(AIF)and cytochrome C(Cty-C)in brain tissue of neonatal rats with hyperbilirubinemia.Methods:A total of 120 clean SD rats aged 7 days were randomly divided into control group,model group,and Z-VAD-FMK intervention group.Brain tissue was extracted from the three groups at 6,24,48,72,and 96 h after modeling.The model group was prepared by intraperitoneal injection of bilirubin(100μg/g).The Z-VAD-FMK intervention group was immediately intraperitoneally injected with Z-VAD-FMK 3μg/g on the basis of the model group(once a day before death).Apoptosis and the expression of Cty-C and AIF in hippocampus(CA1)were measured by in situ notch end labeling(TUNEL)and immunohistochemistry at different time points.Results:Apoptosis and Cty-C and AIF expression began to appear in hippocampus brain tissue of model group and Z-VAD-FMK intervention group at 6 h.The apoptotic indices of the model group and Z-VAD-FMK intervention group at 6 h were(17.7±2.4)and(12.9±1.1),respectively.The average gray values of Cty-C positive cells were(140.5±11.3)and(142.9±13.1),and the average gray values of AIF positive cells were(169.5±11.9)and(170.7±10.0),respectively.Apoptosis and expressions of Cty-C and AIF gradually increased with time,and reached the peak at 96 h.The apoptotic indices of the model group and Z-VAD-FMK intervention group at 96 h were(38.0±6.0)and(33.6±3.8),respectively.The average gray values of Cty-C positive cells were(109.6±8.0)and(127.3±9.8),respectively.The average gray values of AIF positive cells were(139.7±9.4)and(141.0±7.1),respectively.There were no obvious apoptosis and Cty-C and AIF expression in hippocampal brain tissue of control group at all time points.Conclusion:The mechanism of brain injury induced by hyperbilirubinemia is related to the increased expression of Cty-C and AIF in mitochondrial injury.Non-Caspase-dependent apoptosis also participated in this process with certain exten.