干扰雄性小鼠睾丸Clock基因对胎鼠发育的影响及机制研究  

Effects and Mechanisms of Interference of the Male Mouse Testicular Clock Gene on the Development of Fetal Mice

作　　者：梁鑫 宋林江[1] 刘驰 何煊 江舟[2] 陈浩然[1] 成姝婷 王正荣[2] LIANG Xin;SONG Linjiang;LIU Chi;HE Xuan;JIANG Zhou;CHEN Haoran;CHENG Shuting;WANG Zhengrong(School of Medical and Life Sciences/Reproductive&Women-Children Hospital,Chengdu University of Traditional Chinese Medicine,Chengdu 610041,China;West China School of Basic Medical Sciences&Forensic Medicine,Sichuan University,Chengdu 610041,China)

机构地区：[1]成都中医药大学医学与生命科学学院/附属生殖妇幼医院,成都610041 [2]四川大学华西基础医学与法医学院,成都610041

出　　处：《四川农业大学学报》2021年第3期378-384,共7页Journal of Sichuan Agricultural University

基　　金：四川省科技计划项目(2021JDZH0040);成都市科技局项目(2019-YF05-00218-SN);四川省卫生健康委课题(19PJ033);成都中医药大学自然科学基金预研项目(ZKYY2021);成都中医药大学杏林学者学科人才科研提升计划医院专项(2020yky02)。

摘　　要：【目的】研究干扰雄性小鼠睾丸Clock基因后,对小鼠胚胎着床及后期胎鼠发育的影响,并从甲基化的角度探讨引起胎鼠发育异常的原因。【方法】将成年雄性小鼠分为干扰组和对照组,分别于睾丸中注射干扰质粒和阴性质粒,与经过促排处理的雌鼠合笼,研究比较两组胚胎着床情况,观察两组胎鼠的外形及体重等指标的差异;观察胎鼠组织结构,比较两组是否存在差异;分析胎鼠总甲基化水平,同时测定甲基转移酶(DNMTs)水平,比较两组是否存在差异。【结果】:①与对照组比较,干扰组胚胎着床数下降(14.5 vs.20.33),胎儿体重减轻,死胎和异常胎数增多(P<0.05)。②干扰组胎鼠的心脏和脊柱发育分节出现异常。③11.5~16.5 dpc阶段胎鼠总甲基化水平,干扰组较对照组显著降低(8.59 vs.18.25);干扰组甲基转移酶DNMT3B-4的相对IOD值低于对照组(0.124 vs.0.141),有统计学意义。【结论】干扰雄性小鼠睾丸Clock基因,可导致胚胎着床数下降,胎鼠发育异常增多,体重减轻。甲基转移酶DNMT3B-4水平下调及总甲基化水平下降,可能是影响胎鼠发育异常的原因。本研究表明Clock基因与胎鼠发育密切相关,初步探讨引起不育的机制,为揭示该类疾病发病机制提供依据。【Objective】To study the effects of interference with testicular Clock gene on mouse embryo implantation and late fetal development,and to explore the causes of abnormal fetal development from the perspective of methylation.【Method】The adult male mice were divided into interference group and control group.The interference plasmid and negative plasmid were injected into the testis,respectively.These mice were in the same cage with female mice treated with ovulation stimulation.Then,the embryo implantation situation of the two groups was compared,and the differences of the shape,weight and the histological structure of the two groups were observed.The total methylation level of fetal rats was analyzed,and the levels of methyltransferases(DNMTs)were measured.【Result】①Compared with the control group,the number of embryo implantation was decreased(14.5 vs.20.33),fetal weight was decreased,and the number of dead and abnormal was fetuses increased in the interference group(P<0.05).②In the interference group,the development of heart and spine was abnormal.③The total methylation level of fetal rats in 11.5-16.5 DPC stage in the experimental group was significantly lower than that in the control group(8.59 vs.18.25);the relative IOD value of methyltransferase dnmt3 b-4 in the experimental group was lower than that in the control group(0.124 vs.0.141),with statistical significance.【Conclusion】The interference with testicular Clock gene in male mice can lead to the decrease of embryo implantation number,the increase of fetal development abnormality and weight loss.The down-regulation of methyltransferase DNMT3 B-4 level and the decrease of total methylation level may be the reasons for the abnormal development of fetal rats.This study showed that Clock gene was closely related to the development of fetal mice,and preliminarily explored the mechanism of infertility,so as to provide the basis for revealing the pathogenesis of this kind of disease.

关 键 词：CLOCK基因 小鼠 胚胎着床 DNA甲基转移酶(DNMTs) 近日节律 DNA甲基化 

分 类 号：Q593+4[生物学—生物化学] S865.1[农业科学—野生动物驯养]