摘要
目的探讨高龄妊娠对子代大鼠海马甲基化水平的影响。方法将12月龄及3月龄SD雌性大鼠分别与3月龄SD雄性大鼠合笼,将其子代分为高龄子代组(AMA组)和适龄子代组(对照组),留取子代生后第7、14、28天的大脑及海马标本。实时荧光定量PCR检测两组子代海马DNA甲基转移酶(Dnmt)1、Dnmt3A、Dnmt3B mRNA表达水平,Western blotting检测两组子代海马Dnmt1、Dnmt3A、Dnmt3B蛋白表达水平,免疫荧光及ELISA法检测两组子代海马5-甲基胞嘧啶(5-mC)的定位及表达水平。结果实时荧光定量PCR检测结果显示,AMA组生后第28天海马Dnmt1 mRNA表达水平明显低于对照组(P<0.05)。Western blotting检测结果显示,AMA组生后第7天海马Dnmt3A蛋白表达水平明显高于对照组(P<0.05),第28天海马Dnmt1、Dnmt3A蛋白表达水平明显低于对照组(P<0.05)。免疫荧光检测结果显示,5-mC表达于海马DG、CA1、CA3区的细胞核内,AMA组生后第7、14天海马5-mC表达水平明显高于对照组(P<0.05)。ELISA检测结果显示,AMA组生后第7、14、28天海马5-mC表达水平较对照组升高,但差异无统计学意义(P>0.05)。结论高龄妊娠可导致子代海马Dnmts表达紊乱,海马整体甲基化水平升高,可能是高龄妊娠子代脑功能障碍的潜在机制之一。
Objective To investigate the effects of advanced pregnancy on DNA methylation level of hippocampus in the offspring rats.Methods 12-and 3-month-old female SD rats were mated with 3-month-old male SD rats.The offspring(s)from older mother were marked as AMA group,and those from younger mother were marked as control group.The brain and hippocampus samples of the offsprings were taken at the 7th,14th and 28th day after birth.The mRNA levels of DNA methyltransferase(Dnmt)1,Dnmt3A and Dnmt3B were detected by real-time fluorescent quantitative PCR(RT-PCR).The protein expression levels of Dnmt1,Dnmt3A and Dnmt3B were measured by Western blotting.Immunofluorescence and ELISA were used to detect the localization and expression level of 5-methylcytosine(5-mC)in hippocampus.Results RT-PCR showed the Dnmt1 mRNA level was markedly lower in hippocampus of AMA group than that in control group on day 28 after birth(P<0.05);Western blotting analysis showed the expression level of Dnmt3A protein was obviously higher in hippocampus of the AMA group than that in control group on day 7 after birth(P<0.05),while the expression levels of Dnmt1 and Dnmt3A were lower in AMA group than those in control group on day 28 after birth(P<0.05).Immunofluorescence test showed that the 5-mC level in hippocampus was significantly higher in AMA group than that in control group on day 7 and 14 after birth(P<0.05).ELISA showed that the 5-mC level in hippocampus increased slightly but without statistical significance in AMA group than that in control group on day 7,14 and 28 after birth(P>0.05).Conclusion Advanced pregnancy may lead to the expression disorder of Dnmts and the increased DNA methylation level in offspring's hippocampus,which may be one of the potential mechanisms of offspring's brain dysfunction.
作者
潘亚男
韩慰
杨静
程莉
蒋莉
Pan Ya-Nan;Han Wei;Yang Jing;Cheng Li;Jiang Li(Department of Neurology,Children’s Hospital of Chongqing Medical University/National Clinical Research Center for Children's Health and Disease/Ministry of Education Key Laboratory of Developmental Diseases in Children,hongqing 400014,China;Chongqing Key Laboratory of Cognitive Developmental and Transformation of Learning and Memory Disorders,Chongqing 400014,China;Institute of Pediatrics,Children’s Hospital of Chongqing Medical University,Chongqing 400014,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2021年第5期456-461,共6页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(81873792)。
关键词
高龄妊娠
子代大鼠
海马
DNA甲基化
advanced pregnancy
offspring rat
hippocampus
DNA methylation
