碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III， Lig3）基因是碱基切除修复通路中的关键基因，在胚胎发育过程中发挥重要作用，通过研究Lig3基因在叶酸代谢障碍状态下的表达情况，探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周，18-20 g），雌雄1:1合笼，孕鼠随机分为实验组和对照组，孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX，二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型，对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上，应用实时荧光定量聚合酶链反应(Real time quantitative PCR，RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61），而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合，而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现，与对照组相比，叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时，在MTX处理的神经干细胞中，Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加，表明细胞凋亡增加。结论：在叶酸代谢障碍前提下，Lig3表达降低，DNA修复功能减弱，细胞凋亡增加，导致NTDs的发生，为NTDs及出生缺陷的防控提供新思路。

Effect of Base Excision Repair-DNA ligase III on Mice Embryonic Neural Development

ABSTRACT Objective: DNA ligase III (Lig3) gene as a key gene of the base excision repair pathway plays an important role in the process of embryonic development. Here, the study was to evaluate the effect of Lig3 gene on the mice embryonic neural tube development in the state of folate metabolism disorder. Methods: C57BL / 6J adult mice (8-9 weeks, 18-20g) with specific pathogen free (SPF) were used. The pregnant mice were randomly divided into experimental group and control group, At 7.5 days of gestation, the experimental group was intraperitoneally injected with 4.5 mg/kg methotrexate(MTX, dihydrofolate reductase inhibitor) to induce the mice model of neural tube defects (NTDs) with folate metabolism disorder, and the control group was intraperitoneally injected with equal volume of normal saline. The development of fetal mice was observed under stereomicroscope on 10.5 days of gestation. At the same time, a mice neural stem cell model with folate metabolism disorder was established by using 200 nM MTX. Based on the successful establishment of the model, the expression level of base excision repair pathway related gene Lig3 and cleaved- caspase3 were studied by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot. Results: The incidence of NTDs in embryonic mice was 31.1% (19/61) after 4.5 mg/kg body weight MTX treatment, while there was no occurrence of NTDs in normal control group. Under the stereomicroscope, it can be seen that the neural tube of NTDs embryonic mice were not closed, while the normal embryonic mice were well developed. RT-qPCR showed that the expression level of Lig3 mRNA in NTDs embryonic neural tissue of mice with folate metabolism disorder was significantly lower than that of the control group(P<0.05). Western blot showed that compared with the control group, the level of Lig3 protein in the neural tissue of NTDs embryonic mice with folate metabolism disorder decreased significantly(P<0.05). Meanwhile, the expression of Lig3 in neural stem cells treated with MTX was significantly lower than that in the control group(P<0.05). The expression of apoptosis related protein cleaved caspase-3 in NTDs embryonic mice neural tissue and cell model after MTX treatment were increased significantly, which suggest that the apoptosis was increased. Conclusion: The low levels of Lig3 causes the decreased DNA repair ability and the increased apoptosis that results in NTDs induced by folate metabolism disorder, which could offer new insight for preventing and controlling NTDs and birth defects.