司他夫定中枢神经毒性研究  

Short-term exposure to stavudine results in neuron apoptosis, neurite shrink and down-regulated expression of thymidine kinase 2

作　　者：张玉林[1] 石英[1] 乔录新[1] 张宏海[1] 孙玉[1] 丁渭[1] 张彤[1] 吴昊[1] 陈德喜[1] 

机构地区：[1]首都医科大学附属北京佑安医院感染科,100069

出　　处：《中华传染病杂志》2012年第1期4-9,共6页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（30770742、30870853、30910103915）;国家“十一五”重大科技专项（2008ZX10001-007）

摘　　要：目的探讨核苷类抗HIV药物司他夫定（D4T）的中枢神经毒性。方法原代培养小鼠大脑皮层神经细胞，应用不同浓度D4T作用于细胞，采用乙酰甲氧基钙黄绿素-（AM）和碘化丙啶（PI）染色法检测神经细胞凋亡，免疫荧光法检测神经元细胞形态学变化，实时定量PCR检测环氧合酶-2（Cox-2）拷贝数来评估细胞线粒体DNA含量，同时检测胸苷激酶2（TK2）mRNA表达的变化。采用卡方检验、t检验和Wilcoxon非参数检验进行统计分析。结果D4T0μmol／L组、D4T25μmol／L组和D4T50μmol／L组凋亡细胞所占比例分别为24．9％±8．2％、26．5％±10．6％和51．3％±12．4％，后者与前两者比较，差异有统计学意义（x2=7．25、6．93，均P〈0．01）。D4T0μmol／L组、D4T25μmol／L组和D4T50μmol／L组平均每个神经元神经突起的数量分别为11．2±3．6、8．6±2．8和4．3±2．4，D4T25μmol／L组与D4T0μmol／L组、D4T50μmol／L组与D4T25μmol／I。组之间差异有统计学意义（t=4．06、4．35，均P〈0．01）；D4T0μmol／L组、D4T25μmol／L组与D4T50μmol／L组神经突起长度分别为（319．9±100．2）、（298．3±83．9）和（258．4±82．2）μm，D4T25μmol／L组与D4T0μmol／L组、D4T50μmol／L组与D4T25μmol／L组之间差异有统计学意义（t=4．58、4．65，均P〈0．01）。PCR相对定量显示，D4T25μmol／L组和D4T50μmol／L组TK2mRNA拷贝数分别是D4T0μmol／L组的0．34和0．08倍，D4T25μmol／L组与D4T0μmol／L组、D4T50μmol／L组与D4T25μmol／L组之间差异有统计学意义（Z=-3．28、-4．25，均P（0．01）；D4T25μmol／L组和D4T50μmol／L组COx-2拷贝数分别是D4T0μmol／L组的1．01和1．12倍，D4T25μmol／L组与D4T0μmol／l。组、D4T50μmol／L组与D4T25μmol／L组之间差异均无统计学意义（Z=0．98、1．24，均P〉0．05）。结论短期接触D4T可诱导神经元凋亡，抑制其突起形成，并可抑制TK2表达，但对线Objective To investigate the central neurotoxicity induced by nucleoside analog reverse transcriptase inhibitors （NRTIs）-stavudine （D4T）. Methods Mouse primary cortical neurons were cultured and treated with different concentrations of stavudine. Neuron apoptosis was analyzed by calcein/acetomethoxy/propidium iodide （AM/PI） staining. Morphological change of neuron was confirmed by immunofluorescence. Mitochondrial DNA copies which were usually evaluated through Cycloxygenase 2 （COX-2） and thymidine kinase2 （TK2） mRNA were determined by real-time quantitative polymerase chain reaction. Chi-square test, student t test and Wileoxon nonparameter test were used to analyze the data. Results Neuronal apoptosis observed in 50 μmol/L D4T treatment group was more significant than that in 0μmol/L D4T treatment group and 25 μmol/L D4T treatment group （51.3%±12.4% vs 24.9%±8.2% and 26.5%± 10. 6%, respectively; X2 =7.25 and 6.93, respectively, both P〈0.01）. The average neurite numbers of each neuron were 11.2±3.6 in 0 μmol/L D4T treatment group, 8.6±2.8 in 25 μmol/L D4T treatment group and 4.3±2.4 in 50 μmol/L D4T treatment group. The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group （t = 4. 06, P 〈0. 01） and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group （t = 4. 35, P〈 0. 01）. Furthermore, the average lengths of neuritis were （319.9 ± 100.2） μm in 0 μmol/L D4T treatment group, （298. 3 83.9） μm in 25 μmol/L D4T treatment group and （258.4±82.2） μm in 50 μmol/L D4T treatment group. The difference was statistically significant between 25 μmol/L D4T treatment group and 0 μmol/L D4T treatment group （t=4. 58, P〈0.01） and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group （t = 4. 65, P〈 0. 01）. TK2 mRNA expression dramatically decreased along with the increasing D4T concentration. The fold changes were 0.34 in 25 μmol/L D4T treatment grou

关 键 词：司他夫定 核苷类反转录酶抑制剂 神经毒性 线粒体 获得性免疫缺陷综合征 

分 类 号：R96[医药卫生—药理学]