大鼠创伤性脑损伤后神经细胞凋亡的动态变化及其与caspase-3基因表达的关系  

Correlation between the changes of neural cell apoptosis and caspase-3 gene expression after the brain traumatic injury in rats

作　　者：吴思荣[1] 惠国桢[2] 李向东[2] 王之敏[3] 胡锦[4] 祁震宇[1] 俞文华[5] 王清[6] 

机构地区：[1]苏州大学附属第一医院急救中心,江苏省苏州215006 [2]苏州大学附属第一医院神经外科,江苏省苏州215006 [3]上海交通大学医学院苏州九龙医院神经外科 [4]上海市第六人民医院神经外科 [5]杭州市第一人民医院神经外科 [6]无锡市第二人民医院神经外科

出　　处：《中华急诊医学杂志》2009年第4期361-366,共6页Chinese Journal of Emergency Medicine

基　　金：基金项目：国家自然科学基金资助项目（30200335）

摘　　要：目的探讨创伤性脑损伤（TBI）后神经细胞凋亡的变化规律及其与caspase-3基因表达的关系。方法成年健康封闭群sD大鼠120只，随机分为对照组8只、损伤组和抑制物组各56只。Feeney法致伤，抑制物组伤后脑内注射5／agcaspase-3抑制剂z—DEVD—fmk。分别在伤后1，6，24，48h和3，7，14d处死取材（每个分析时相点8只大鼠），采集伤灶中心皮质、皮层下白质、海马、齿状回，以及对侧相应部位脑组织，应用原位末端脱氧核糖核酸转移酶介导的脱氧尿苷三磷酸（dUrP）标记法（TUNEL法）和流式细胞术检测神经细胞凋亡的变化；免疫组化法、蛋白印迹（westernblot）和半定量逆转录-聚合酶链式反应（RT-PCR），检测caspase-3蛋白和mRNA的表达；并借助荧光分析试剂盒检测caspase-3活性的变化。所得数据采用SPSS10．1统计软件包进行Spearman等级相关分析和方差分析（SNK-q检验）。结果伤后伤侧各脑区神经细胞凋亡指数（AI）和细胞凋亡百分率（AP）迅速增高，24～48h达峰值，随后逐渐下降，但伤后14d仍高于正常（P〈0．01）。伤后caspase-3蛋白和mRNA的表达明显增加，caspase-3活性迅速上升，峰值在24～48h。其中伤后24h伤侧海马区caspase-3蛋白谱密度与对照组相比增加1484％，caspase-3mRNA的表达量增加1043％，caspase-3活性增加148％；伤后48h伤灶皮层下白质caspase．3蛋白谱密度增加1690％，caspase-3mRNA的表达量增加1181％，caspase-3活性增加183％。Westernblot显示，伤后caspase-3原酶及p17活性亚单位的表达均增强。抑制物组caspase-3蛋白和mRNA的表达均明显下降，caspase-3活性明显降低；同时，AI值和AP值也明显降低。统计学相关分析发现，伤后神经细胞凋亡与caspase-3mRNA和蛋白的表达间呈正相关（r=0．821和r=0．638，P〈0．01），伤后caspase-3在mRNA和蛋白水平的表达间呈正相关（r=0．945，P〈0．01）。结论�Objective To observe the correlation between the changes of neural cell apoptosis and caspase- 3 gene expression in brain tissues following acute severe traumatic injury to brain （TIB）. Method A total of 120 adult Spraque-Dawley rats were divided into a control group （ n = 8）, TIB group （ n = 56） and TIB with administration of caspase-3 inhibitor group （ n = 56）. TIB models of rats were made with Feeney＇ s method. The z-DEVD- fmk （5 gg）, caspase-3 inhibitor, was administered by intracerebral infusion, and the rats were sacrificed 1,6, 24,48 hours and 3, 7, 14 days postinjmy （ n =8 for each interval）. The specimens of the injured cerebral cortex, subeortieal white matter, hippoeampus, dentate gyrus and contralateral corresponding brain tissues were taken for detecting apoptosis of neural cells by the terminal deoxynueleotidyl transferase mediated DUTP nick end labeling （TUNEL） methods and flow eytometry. Caspase-3 mRNA and protein expression were detected by using RT-PCR, irranunohistoehemistry and western blot analysis. The easpase-3 activity was detected by using caspase-3 fluorescent assay kit. Student t-test and Spearman correlation analysis were used to analyze the data with SPSS version 10.1 software package. Results Apoptosis indexes （AI） and the apoptosis percentage （AP） of neural cells in the injured brain regions increased quickly after injury, and reached its peak 24 to 48 hours later, then decreased slowly, but it remained at higher level above that of normal till 14 days later （ P 〈 0.01 ）. The levels of caspase-3 mRNA, caspase-3 protein and caspase-3 activity were increased significantly post injury, and reached its peak at 24 to 48 hours, then it gradually decreased. Compared with control group, the levels of optical density of caspase-3 proreins in the injured hippocampus and subcortical white matter at 24 and 48 hours post injury increased 1484% and 1690%, caspase-3 mRNA expressions increased 1043% and 1180%, and the degreas of caspase-3 activity increas

关 键 词：脑损伤 神经细胞 凋亡 CASPASE-3 z—DEVD—fmk 

分 类 号：R651.15[医药卫生—外科学]