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 D4T 0 μmol/L组、D4T25 μmol/L组和D4T 50 μmol/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).D4T0 μmol/L组、D4T 25μmol/L组和D4T 50 μmol/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,D4T 25 μmol/L组与D4T 0μmol/L组、D4T 50 μmol/L组与D4T25 μmol/L组之间差异有统计学意义(t=4.06、4.35,均P＜0.01)；D4T 0 μmol/L组、D4T 25 μmol/L组与D4T 50 μmol/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,D4T 25 μmol/L组与D4T 0μmol/L组、D4T 50μmol/L组与D4T 25 μmol/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,D4T 25 μmol/L组和D4T50μmol/L组TK2 mRNA拷贝数分别是D4T 0 μmol/L组的0.34和0.08倍,D4T 25mol/L组与D4T 0 μmol/L组、D4T 50 mol/L组与D4T 25 μmol/L组之间差异有统计学意义(Z=-3.28、-4.25,均P＜0.01)；D4T 25μmol/L组和D4T 50 μmol/L组COX-2拷贝数分别是D4T 0 μmol/L组的1.01和1.12倍,D4T 25 μmol/L组与D4T 0 μmol/L组、D4T 50 μmol/L组与D4T 25 μmol/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,但对线粒体含量无影响.

abstractsObjective 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 Wilcoxon 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 0μmol/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 group and 0.08 in 50 μmol/L D4T treatment group. The difference was statistically significant between 25 μmol/L D4T treatment group and 0μmol/L D4T treatment group (Z=- 3.28,P＜0.01) and between 50 μmol/L D4T treatment group and 25 μmol/L D4T treatment group (Z=-4.25,P＜0.01).Compared with 0μmol/L D4T treatment group,the relative fold changes of COX-2 copies were 1.01 in 25 μmol/L D4T treatment group and 1.12 in 50 μmol/L D4T treatment group.The differences were not significant among the three groups (Z=0.98 and 1.24,respectively; both P＞0.05).Conclsion It suggests that short-term exposure to D4T may result in neuron apoptosis,neurite shrink and down-regulated expression of TK2,but the level of mitochondrial DNA copies keeps stable.