CAJ | 학술논문

目的检测甲氨蝶呤(MTX)诱导叶酸代谢障碍神经管畸形(NTDs)小鼠胚胎神经组织的基因组拷贝数变异(CNVs),探讨叶酸代谢障碍与NTDs发生的分子遗传学机制.方法采用本实验室已建立的MTX诱导的叶酸代谢障碍NTDs C57BL/6J小鼠模型,于孕第7.5天采用MTX干预,收集孕鼠血清及NTDs小鼠胚胎神经组织;用NimbleGen高分辨率微阵列比较基因组杂交(array-CGH)芯片对NTDs小鼠胚胎神经组织进行全基因组CNVs分析,反转录(RT)-PCR验证新发现的CNVs;用液相色谱串联质谱法(LC-MS/MS)与酶学方法检测孕鼠血清中叶酸与其相关代谢产物水平及二氢叶酸还原酶(DHFR)活性.结果 array-CGH分析发现,NTD小鼠胚胎神经组织全基因组存在3个高可信度CNVs,分别位于XqE3、XqA1.1-qA2和XqA1.1染色体,RT-PCR验证了这3个CNVs的存在.与对照组相比,MTX干预后,NTDs孕鼠血清中5-甲基四氢叶酸、5-甲酰四氢叶酸、S-腺苷甲硫氨酸水平及DHFR活性明显降低,差异均有统计学意义(P均＜0.05).结论 MTX诱导的NTDs小鼠胚胎神经组织中存在明显的CNVs,叶酸代谢紊乱可通过CNVs导致小鼠胚胎神经管发育障碍.
목적 검측갑안접령(MTX)유도협산대사장애신경관기형(NTDs)소서배태신경조직적기인조고패수변이(CNVs),탐토협산대사장애여NTDs발생적분자유전학궤제.방법 채용본실험실이건립적MTX유도적협산대사장애NTDs C57BL/6J소서모형,우잉제7.5천채용MTX간예,수집잉서혈청급NTDs소서배태신경조직;용NimbleGen고분변솔미진렬비교기인조잡교(array-CGH)심편대NTDs소서배태신경조직진행전기인조CNVs분석,반전록(RT)-PCR험증신발현적CNVs;용액상색보천련질보법(LC-MS/MS)여매학방법검측잉서혈청중협산여기상관대사산물수평급이경협산환원매(DHFR)활성.결과 array-CGH분석발현,NTD소서배태신경조직전기인조존재3개고가신도CNVs,분별위우XqE3、XqA1.1-qA2화XqA1.1염색체,RT-PCR험증료저3개CNVs적존재.여대조조상비,MTX간예후,NTDs잉서혈청중5-갑기사경협산、5-갑선사경협산、S-선감갑류안산수평급DHFR활성명현강저,차이균유통계학의의(P균＜0.05).결론 MTX유도적NTDs소서배태신경조직중존재명현적CNVs,협산대사문란가통과CNVs도치소서배태신경관발육장애.
Objective To detect the genomic copy number variations(CNVs) of mice embryonic neural tissue with neural tube defects (NTDs) induced by methotrexate (MTX),and investigate the molecular genetic mechanisms between folic acid metabolism disorders and NTDs pathogenesis.Methods C57BL/6J NTD mice model was induced by MTX on gestational day 7.5,and the maternal serum and NTD embryonic neural tissues were collected;the array-comparative genomic hybridization(array-CGH) assay was utilized to analyze the whole genomic CNVs in NTD embryonic neural tissues;reverse transcription polymerase chain reaction(RT-PCR) was used to confirm the positive results;the maternal serum concentrations of folic acid and related metabolites and dihydrofolate reductase (DHFR) activity were detected by liquid chromatography-tandem mass spectrometry and enzymatic methods,respectively.Results ArrayCGH and RT-PCR results showed the 3 high confidence CNVs on XqE3,XqA1.1-qA2 and XqA1.1 in the NTD embryonic neural tissues.The NTD maternal serum concentrations of 5-methyltetrahydrofolate,5-formyltetrahydrofolic acid,S-adenosyl methionine and DHFR activity were reduced significantly compared with the control group,and there were statistical differences(all P ＜0.05).Conclusions There are obvious CNVs in embryonic neural tissue of NTD mice induced by MTX and folic acid dysmetabolism might cause mice embryonic neural tube developmental disorders through CNVs.