钟基因在不同胎龄小鼠胚胎和孕鼠的表达规律  

Expression of the clock gene in embryonic and maternal tissues of mice

作　　者：闫银弟 罗旭光[2] 杨艳萍[1] 李海荣[1] 崔慧林[1] 曹锡梅[1] Yan Yindi;Luo Xuguang;Yang Yanping;Li Hairong;Cui Huilin;Cao Ximei(Department of Histology and Embryology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China;Department of Microbiology and Immunology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China)

机构地区：[1]山西医科大学组织学与胚胎学教研室,山西省太原市030001 [2]山西医科大学微生物免疫学教研室,山西省太原市030001

出　　处：《中国组织工程研究》2019年第1期96-102,共7页Chinese Journal of Tissue Engineering Research

基　　金：山西省自然科学基金青年基金(2014021028-1);项目负责人:曹锡梅;山西医科大学科技创新基金(01201401);项目负责人:曹锡梅;山西医科大学基础医学院331基础医学科技培植基金计划(201413);项目负责人:曹锡梅~~

摘　　要：背景:胚胎发育过程中,周期性节律出现的时间和作用至今尚未完全研究清楚。目的:观察小鼠胚胎发育过程中钟基因在不同胎龄胚胎、孕鼠肝脏和骨骼肌的时空表达特点。方法:收集ICR小鼠胎龄10-17 d全胚胎,用抗CLOCK和抗Per1多克隆抗体对胎龄10-17 d小鼠胚胎心脏、肺、肝和体壁骨骼肌进行免疫组织化学染色;胎龄10-17 d小鼠胚胎、孕鼠肝脏和股四头肌的c DNA作模板,定量RT-PCR分析钟基因BMAL1、CLOCK、Per1和肌调节因子myogenin、Tcap、MAZ的表达规律。结果与结论:(1)小鼠胚胎发育早期,全胚的钟基因相对表达较低;(2)免疫组织化学显示胎龄10至11d,心脏未见CLOCK和Per1阳性细胞,孕鼠子宫蜕膜组织中可见略高于背景的CLOCK弱阳性细胞和Per1强阳性细胞;胎龄13 d,左心房底壁和左右支气管气道平滑肌清晰可见CLOCK阳性细胞;胎龄14 d,体壁骨骼肌出现CLOCK和Per1阳性细胞;胎龄15d始,全胚的钟基因表达明显升高,但未呈现周期性节律波动,Z线相关蛋白Tcap表达显著增高,提示肌节发育,肺叶内细支气管壁可见CLOCK阳性细胞,右心房出现少量Per1阳性细胞;胎龄16至17 d,肺叶内终末细支气管气道平滑肌呈CLOCK和Per1阳性,右心房出现Per1强阳性细胞;(3)胎龄10-17 d肝脏内始终未见CLOCK和Per1阳性细胞;(4)胚胎发育过程中MAZ表达水平始终高于myogenin和Tcap,可能参与骨骼肌细胞的分化,钟基因在孕鼠骨骼肌呈稳定周期性节律表达;(5)结果表明,小鼠胚胎发育过程中钟基因的表达和细胞发育成熟相关;心脏、肺和骨骼肌发育可能受钟基因的调节,心房和心室发育不同步,右心室发育晚;小鼠胚胎昼夜节律和功能活动变化可能受孕鼠骨骼肌影响。BACKGROUND:Expression and mechanisms of periodic rhythm emergence during mammalian development are not fully understood.OBJECTIVE:To study the spatio-temporal expression characteristics of clock gene in mouse embryos at the gestational age,maternal livers,and maternal skeletal muscles during the development of the mouse.METHODS:The whole embryos from pregnant 10-17-day ICR mice were collected.The mouse heart,lung,liver and skeletal muscle of body underwent immunohistochemical staining by anti-CLOCK and-Per1 antibodies.Total RNA was extracted using TRIZol from embryos,quadriceps and liver.BMAL1,CLOCK,Per1,myogenin,Tcap and MAZ mRNA were examined by RT-qPCR.RESULTS AND CONCLUSION:The levels of BMAL1,CLOCK and Per1 expression in mouse embryo in the early developmental stages were lower.Immunohistochemical analysis showed that the heart of the early embryos did not express CLOCK at gestational days 10 and 11.However,apparent signals against Per1 and weak expression of CLOCK were observed in the maternal uterus tissue(decidua)of embryos at gestational days 10 and 11.At gestational day 13,CLOCK positive cells showed C-shaped patterns in the wall of the left and right bronchi.At the same time,CLOCK positive cells were detected in the bottom of left atrium and Per1 positive cells were detected in the left ventricular myocardium.At gestational day 14,CLOCK and Per1 positive cells were easily observed in skeletal muscle of body wall.At gestational day 15,immunohistochemical analysis showed a small number of Per1 positive cells were observed in the bottom of right atrium.The cross section of bronchial branches in the lungs increased obviously.At the same time,the expression levels of BMAL1,CLOCK and Per1 mRNA in mouse embryo were increased,especially Per1.However,circadian molecular rhythms could not be found.We found the level of Tcap expression at gestational day 15 significantly increased.The data suggest that sarcomeres in muscle develop rapidly.During development from gestational day 16 to gestational day 17,CLOCK and

关 键 词：昼夜节律 BMAL1 CLOCK 免疫组织化学 RT-PCR 心脏 肝脏 骨骼肌 胚胎 组织建构 

分 类 号：R466[医药卫生—临床医学] R394.1