短期使用齐多夫定抑制p53R2表达及诱导小鼠神经元凋亡的研究

Short-term exposure to zidovudine results in low expression of p53R2 and neuronal apoptosis in mouse cortical neuronal cultures

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 0mmol/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。

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 ＆#177; 3.37）%（control） vs （24.3 ＆#177; 8.94）%（50 mmol/L） vs （54.7 ＆#177; 17.9）%（100 mmol/L） （χ2=5.19, 19.33;P＆lt;0.01）. The average neurite lengths were （869.21 ＆#177; 177.75） mm in control and （495.76 ＆#177; 175.20） mm in 50 mmol/L AZT treatment group and （120.38 ＆#177; 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＆lt;0.01）, but the difference of p21 mRNA change was not signiifcant （0.98 vs 0.86;Z=1.11 vs 1.21, P＆gt;0.05）. Simultaneously, p53R2 protein expression rather＆amp;nbsp;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＆gt;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＆lt;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.