论文部分内容阅读
目的探讨长期使用核苷类似物是否产生皮层神经元线粒体DNA D-loop区损伤。方法 7周龄Balb/C小鼠50只,分为5组(对照组和4个实验组),每组10只。对照组腹腔内注射双蒸水,实验组腹腔内分别注射司他夫定(D4T)50 mg/kg、齐多夫定(AZT)100 mg/kg、拉米夫定(3TC)50 mg/kg和去羟肌苷(DDI)50 mg/kg,每周5次,连续6周。应用激光捕获显微切割技术捕捉小鼠大脑皮层神经元,对其线粒体D-loop区PCR扩增子克隆和测序。结果与对照组相比,实验组小鼠神经元线粒体D-loop区序列突变增加,与参考序列的平均距离,AZT组为0.0021,D4T组为0.0047,3TC组为0.0024和DDI组为0.0053,对照组为0.0012。D4T组和DDI组的核苷酸距离明显高于对照组(P<0.001)。D-loop序列单碱基的转换率AZT组为0.0018,D4T组为0.0033,3TC组为0.0021,DDI组为0.0037,显著高于对照组(P<0.001)。4种核苷类似物诱导的D-loop区序列点突变主要类型是转换,特别是“A→G”和“T→C”的转换。结论长期暴露于核苷类似物可导致小鼠皮层神经元线粒体DNA D-loop区实变。
Objective To investigate whether long-term use of nucleoside analogues can induce D-loop injury in mitochondrial DNA of cortical neurons. Methods Fifty Balb / C mice of 7 weeks old were divided into 5 groups (control group and 4 experimental groups), 10 in each group. The control group received intraperitoneal injection of double distilled water. The experimental group received intraperitoneal injection of 50 mg / kg of Stavudine (D4T), 100 mg / kg of AZT, 50 mg / kg of lamivudine (3TC) And didanosine (DDI) 50 mg / kg, 5 times a week for 6 weeks. Laser capture microdissection technique was used to capture mouse cerebral cortical neurons, cloned and sequenced the PCR amplicons of mitochondrial D-loop region. Results Compared with the control group, the mitochondrial D-loop region of the neurons in the experimental group was increased. The average distance from the reference sequence was 0.0021 in the AZT group, 0.0047 in the D4T group, 0.0024 in the TC group and 0.0053 in the DDI group, Group is 0.0012. The nucleotide distance between D4T group and DDI group was significantly higher than that of control group (P <0.001). The single-base conversion rate of the D-loop sequence was 0.0018 in the AZT group, 0.0033 in the D4T group, 0.0021 in the TC group, and 0.0037 in the DDI group, which was significantly higher than that in the control group (P <0.001). The main types of point mutations in the D-loop region sequence induced by the four nucleoside analogues are transitions, especially the transforms of “A → G” and “T → C”. Conclusion Long-term exposure to nucleoside analogs leads to consolidation of mitochondrial DNA D-loop region in mouse cortical neurons.