中国法医学杂志
中國法醫學雜誌
중국법의학잡지
CHINESE JOURNAL OF FORENSIC MEDICINE
2009年
6期
379-382
,共4页
汪岩%卢延旭%耿广军%喻洪江
汪巖%盧延旭%耿廣軍%喻洪江
왕암%로연욱%경엄군%유홍강
法医毒理学%咖啡因%乳鼠%脑皮质神经元%损伤作用
法醫毒理學%咖啡因%乳鼠%腦皮質神經元%損傷作用
법의독이학%가배인%유서%뇌피질신경원%손상작용
forensic toxicology%caffeine%neonatal rats%cortical neuron%damage effect
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%CO_2、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 咖啡因对乳鼠腩皮质神经元凋亡有促进作用.
目的 攷察咖啡因對乳鼠腦皮質神經元凋亡的作用.方法 取齣生後2～3d的乳鼠腦皮質神經元,在37℃、5%CO_2、100%相對濕度的培養箱中培養7d後,分彆加入終濃度為300μmol/L和1 000μmol/L的鹽痠咖啡因培養液,繼續培養6～36h後,流式細胞儀測定細胞內鈣離子濃度、線粒體膜電位和細胞凋亡率,酶標儀測定Caspase-9的活性,電鏡和Hoechst 33258熒光染色觀察細胞的形態學改變.結果 與正常組相比較,300μmol/L和1 000μmol/L鹽痠咖啡因組在給藥後6h的鈣離子平均熒光彊度明顯增彊(P<0.05),由正常值43.13±2.02分彆增加到45.28±1.16和46.92±1.99;在給藥後8h的線粒體膜電位下降最明顯(P<0.05),由正常值443.58±11.77分彆下降到289.53±16.47和165.14±14.72;在給藥後10h的Caspase-9活性最高(P<0.05),由正常值1.00±0.000分彆增加到5.33±1.02和8.33±0.92;在給藥後36h的細胞凋亡率明顯升高(P<0.05),由正常值4.94±1.74分彆增加到15.98±2.03和18.70±2.09;在給藥後24h熒光顯微鏡下見典型凋亡小體.結論 咖啡因對乳鼠腩皮質神經元凋亡有促進作用.
목적 고찰가배인대유서뇌피질신경원조망적작용.방법 취출생후2～3d적유서뇌피질신경원,재37℃、5%CO_2、100%상대습도적배양상중배양7d후,분별가입종농도위300μmol/L화1 000μmol/L적염산가배인배양액,계속배양6～36h후,류식세포의측정세포내개리자농도、선립체막전위화세포조망솔,매표의측정Caspase-9적활성,전경화Hoechst 33258형광염색관찰세포적형태학개변.결과 여정상조상비교,300μmol/L화1 000μmol/L염산가배인조재급약후6h적개리자평균형광강도명현증강(P<0.05),유정상치43.13±2.02분별증가도45.28±1.16화46.92±1.99;재급약후8h적선립체막전위하강최명현(P<0.05),유정상치443.58±11.77분별하강도289.53±16.47화165.14±14.72;재급약후10h적Caspase-9활성최고(P<0.05),유정상치1.00±0.000분별증가도5.33±1.02화8.33±0.92;재급약후36h적세포조망솔명현승고(P<0.05),유정상치4.94±1.74분별증가도15.98±2.03화18.70±2.09;재급약후24h형광현미경하견전형조망소체.결론 가배인대유서남피질신경원조망유촉진작용.
Objective To examine the effect of Caffeine on the cultured cortical neuron apoptosis in neonatal rats.Methods The primary cerebral cortex neurons for cultures were obtained from neonatal mice 2-3 days after birth,Caffeine reconstituted at final concentrations 300μmol/L and 1 000μmol/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% CO_2 and 100% relative humidity,the intracellular 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-1inked 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;mitochondrial membrane potentials were reduced most significandy(P<0.05).from the base value 443.58 ±11.77 down to 289.53±16.47 and 165.14±14.72,respectively at 8h.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 36h.The apoptotic bodies were observed at 24 h after administration of 300 μmol/L and 1000 μmol/L Caffeine.Conclusion Caffeine may promote neuronal apoptosis in neonate mice.
