CAJ | 학술논문

目的：探讨齐多夫定（AZT）的中枢神经毒性及其相关机制。方法原代培养小鼠大脑皮层神经元，分别用0 mmol/L、50 mmol/L、100 mmol/L的AZT作用于细胞，TUNEL法检测细胞凋亡，免疫荧光观察细胞形态，实时定量PCR（qPCR）检测依赖p53的p53R2、抑癌基因p21、胸苷激酶（TK2）mRNA表达及线粒体DNA含量，蛋白印迹检测p53R2与p21蛋白的表达。结果 AZT 0 mmol/L组（对照组）、50 mmol/L组和100 mmol/L组细胞凋亡率分别为（11.9±3.37）%、（24.3±8.94）%和（54.7±17.9）%，差异具有统计学意义（χ2=5.19、19.33，P均＜0.01）；突起长度分别为（869.21±177.75）mm、（495.76±175.20）mm和（120.38±47.12）mm，且差异均具有统计学意义（F=19.558，P=0.002）；real-time qPCR检测结果显示AZT 50 mmol/L和100 mmol/L组p53R2、TK2 mRNA拷贝数分别是对照组的0.42和0.04倍、0.52和0.29倍，组间差异均具有统计学意义（Z=-4.54、-6.65、-4.33和-5.24，P均＜0.01）；AZT 50 mmol/L和100 mmol/L组p21 mRNA拷贝数分别是对照组的0.98和0.86倍，差异无统计学意义（P＞0.05）。线粒体含量用环氧化酶2（COX-2）拷贝数评估，AZT 50 mmol/L组和100 mmol/L组分别是对照组的0.92和0.87倍，差异无统计学意义（P＞0.05）。蛋白印迹显示对照组、AZT 50 mmol/L组和100 mmol/L组p53R2蛋白含量依次降低，而p21蛋白含量无差异。结论 AZT可诱导神经元凋亡，抑制突起形成，推测其机制与p53R2表达降低有关。短期接触AZT可抑制TK2的表达，但对线粒体DNA含量无影响。
목적：탐토제다부정（AZT）적중추신경독성급기상관궤제。방법원대배양소서대뇌피층신경원，분별용0 mmol/L、50 mmol/L、100 mmol/L적AZT작용우세포，TUNEL법검측세포조망，면역형광관찰세포형태，실시정량PCR（qPCR）검측의뢰p53적p53R2、억암기인p21、흉감격매（TK2）mRNA표체급선립체DNA함량，단백인적검측p53R2여p21단백적표체。결과 AZT 0 mmol/L조（대조조）、50 mmol/L조화100 mmol/L조세포조망솔분별위（11.9±3.37）%、（24.3±8.94）%화（54.7±17.9）%，차이구유통계학의의（χ2=5.19、19.33，P균＜0.01）；돌기장도분별위（869.21±177.75）mm、（495.76±175.20）mm화（120.38±47.12）mm，차차이균구유통계학의의（F=19.558，P=0.002）；real-time qPCR검측결과현시AZT 50 mmol/L화100 mmol/L조p53R2、TK2 mRNA고패수분별시대조조적0.42화0.04배、0.52화0.29배，조간차이균구유통계학의의（Z=-4.54、-6.65、-4.33화-5.24，P균＜0.01）；AZT 50 mmol/L화100 mmol/L조p21 mRNA고패수분별시대조조적0.98화0.86배，차이무통계학의의（P＞0.05）。선립체함량용배양화매2（COX-2）고패수평고，AZT 50 mmol/L조화100 mmol/L조분별시대조조적0.92화0.87배，차이무통계학의의（P＞0.05）。단백인적현시대조조、AZT 50 mmol/L조화100 mmol/L조p53R2단백함량의차강저，이p21단백함량무차이。결론 AZT가유도신경원조망，억제돌기형성，추측기궤제여p53R2표체강저유관。단기접촉AZT가억제TK2적표체，단대선립체DNA함량무영향。
Objective To investigate the central neurotoxicity and the related mechanism of nucleoside analog reverse transcriptase inhibitors-zidovudine (AZT). Methods Mouse primary cortical neurons were cultured and treated with different concentrations of AZT. Neuron apoptosis was analyzed by TUNEL assay and neurite lengths change was conifrmed by anti-MAP2 immunolfuorescence. Mitochondrial DNA copies which were usually evaluated through COX-2 and the mRNA expression of thymidine kinase 2 (TK2), p53R2 and p21 were tested by real-time polymerase chain reaction (qPCR). The protein expression of p53R2 and p21 was tested with Western blot. Results The rate of neuronal apoptosis increased as AZT concentration, with (11.9 ± 3.37)%(control) vs (24.3 ± 8.94)%(50 mmol/L) vs (54.7 ± 17.9)%(100 mmol/L) (χ2=5.19, 19.33;P<0.01). The average neurite lengths were (869.21 ± 177.75) mm in control and (495.76 ± 175.20) mm in 50 mmol/L AZT treatment group and (120.38 ± 47.12) mm in 100 mmol/L AZT treatment group. There were signiifcant differences between the two groups (F=19.558, P=0.002). Compared with control group, the relative fold change of p53R2 mRNA copies were 0.42 in 50 mmol/L AZT treatment group and 0.04 in 100 mmol/L AZT treatment group via real-time qPCR. The difference were significant between each two groups (Z=-4.54 and-6.65, P<0.01), but the difference of p21 mRNA change was not signiifcant (0.98 vs 0.86;Z=1.11 vs 1.21, P>0.05). Simultaneously, p53R2 protein expression rather than p21 signiifcantly decreased according to AZT concentration by Western blot. The relative fold changes of COX-2 were 0.92 in 50 mmol/L AZT treatment group and 0.87 in 100 mmol/L AZT treatment group. The differences were not signiifcant (Z=0.63 and 0.71, P>0.05). But TK2 mRNA expression signiifcantly decreased according to AZT concentration and the fold changes of it were 0.52 in 50 mmol/L AZT treatment group and 0.29 in 100 mmol/L AZT treatment group (Z= - 4.33 and - 5.24, P<0.01). Conclusions Short-term exposure to AZT may result in neuron apoptosis and neurite shrink by low expression of p53R2. Simultaneously, the expression of TK2 mRNA is low, but mitochondrial DNA maintain stable.