咖啡因对乳鼠脑皮质神经元凋亡的作用  

The effect of caffeine on the primarily cultured cortical neuron apoptosis in neonate mice

作　　者：汪岩[1] 卢延旭[1] 耿广军[2] 喻洪江[3] 

机构地区：[1]中国刑事警察学院法医学系,辽宁沈阳110035 [2]沈阳市公安局交警支队,辽宁沈阳110031 [3]中国人民公安大学,北京100038

出　　处：《中国法医学杂志》2009年第6期379-382,共4页Chinese Journal of Forensic Medicine

摘　　要：目的考察咖啡因对乳鼠脑皮质神经元凋亡的作用。方法取出生后2-3d的乳鼠脑皮质神经元,在37℃、5%CO2、100%相对湿度的培养箱中培养7d后,分别加入终浓度为300μmol/L和1 000μmol/L的盐酸咖啡因培养液,继续培养6-36h后,流式细胞仪测定细胞内钙离子浓度、线粒体膜电位和细胞凋亡率,酶标仪测定Caspase-9的活性,电镜和Hoechst 33258荧光染色观察细胞的形态学改变。结果与正常组相比较,300μmol/L和1 000μmol/L盐酸咖啡因组在给药后6h的钙离子平均荧光强度明显增强（P〈0.05）,由正常值43.13±2.02分别增加到45.28±1.16和46.92±1.99;在给药后8h的线粒体膜电位下降最明显（P〈0.05）,由正常值443.58±11.77分别下降到289.53±16.47和165.14±14.72;在给药后10h的Caspase-9活性最高（P〈0.05）,由正常值1.00±0.000分别增加到5.33±1.02和8.33±0.92;在给药后36h的细胞凋亡率明显升高（P〈0.05）,由正常值4.94±1.74分别增加到15.98±2.03和18.70±2.09;在给药后24h荧光显微镜下见典型凋亡小体。结论咖啡因对乳鼠脑皮质神经元凋亡有促进作用。Objective To examine the effect of Caffeine on the cultured conical neuron apoptosis in neonatal rats. Methods The primary cerebral cortex neurons for cultures were obtained from neonatat mice 2 - 3 days after birth, Caffeine reconstituted at final concentrations 300μmol/L and 1000μmol/L was added to the cell cultures and continuously co-incubated for 6 - 36 h, respectively after the cortical neurons were continuously cultivated 7 days after incubation under temperature of 37℃ incubator with 5% CO2 and 100% relative humidity, the intraeellular calcium concentration, mitochondrial membrane potential and apoptosis rate were determined by the flow cytometry. The activity of Caspase-9 was assayed by enzyme-labeled instrument, and Caspase-9 activity by the enzyme-linked analyzer. Cell morphological changes were observed under electron microscope and fluorescent microscope after being stained with Hoechst 33258 fluorescent dye. Results Compared with the control group, the average increase in intracellular calcium fluorescence intensity was most significant（P 〈 0.05 ） , which elevated from the normal value 43.13 ± 2.02 to 45.28 ± 1.16 and 46.92 ± 1.99,respectively at 6 h ; mitoehondrial membrane potentials were reduced most significantly（ P 〈0.05） , from the base value 443.58 ± 11.77 down to 289.53 ± 16.47 and 165.14 ± 14.72, respectively at 8 h. Caspase-9 activity was peaked（P 〈0.05） , from the normal value 1.00 ± 0. 000 to 5.33 ± 1. 02 and 8.33 ± 0.92, respectively at 10 h. The neuronal apoptosis ratio was increased significantly （ P 〈0.05 ）, from the normal value 4.94 ± 1.74 to 15.98 ± 2.03 and 18.70 ± 2.09, at 36 h. The apoptotic bodies were observed at 24 h after administration of 300μmol/L and 1 000μmol/L caffeine. Conclusion Caffeine may promote neuronal apoptosis in neonate mice.

关 键 词：法医毒理学 咖啡因 乳鼠 脑皮质神经元 损伤作用 

分 类 号：D919.1[医药卫生—法医学]