Pin1抑制剂对小鼠植入前胚体外发育的影响

连秀丽; 宋婵婵; 贺琳; 程霄翔; 陈俊明; 杜娟; 刘玥; 王世鄂

解剖学报 ›› 2016, Vol. 47 ›› Issue (1) : 87-94.

组织学胚胎学发育生物学

Pin1抑制剂对小鼠植入前胚体外发育的影响

连秀丽,宋婵婵,贺琳,程霄翔,陈俊明,杜娟,刘玥,王世鄂

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Effects of Pin1 antagonist on mouse preimplantation embryo development in vitro

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摘要

目的观察Pin1在小鼠植入前胚中的定位分布及其抑制剂胡桃醌（Juglone）对小鼠植入前胚体外发育的影响，为进一步阐明Pin1在植入前胚中的作用机制提供理论依据。方法免疫荧光染色观察不同发育阶段植入前胚中Pin1的分布；收集小鼠1-细胞胚，以KSOM培养液为对照组，以培养液中添加有不同浓度的Juglone为实验组，观察各组1-细胞胚体外发育情况；实时定量PCR检测体外发育2-细胞胚内干细胞转录因子（Sox2、Oct4、c-Myc和Klf4）mRNA表达水平。结果小鼠不同发育阶段植入前胚中都存在Pin1的表达，胞质和胞核内均有分布，且核内荧光着色明显比胞质强；10μmol/L和25μmol/L的Juglone持续作用93h（hCG后27h-120h）使1-细胞胚阻滞于2-细胞阶段，极少能过渡至4-细胞胚 (P<0.001)。25μmol/L Juglone短时间作用18h（hCG后27h至45h）同样使1-细胞胚发育阻滞于2-细胞胚 (P<0.001)；25μmol/L Juglone组2-细胞胚内Sox2 mRNA表达水平低于KSOM组（P

Abstract

Objective To elucidate the further function of Pin1 in mouse preimplantation embryos, We observed the distribution of Pin1 in mouse preimplantation embryos and the effect of Pin1 antagonist (Juglone) on the development of mouse preimplantation embryo in vitro. Methods The immunofluorescence technique was used in the present study in order to localize the distribution of pin1 protein in the preimplanration mouse embryo. The 1-cell embryos of KM mice were cultured in KSOM culture medium supplemented various concentrations pin1 inhibitor (Juglone), respectively. The different development potential was observed under an inverted microscope. The mRNA expression levels of Sox2, Oct4, Klf4 and c-Myc were detected by Real-time PCR. Results Pin1 protein was distributed in both cytoplasm and nucleus of mouse preimplantation embryos. The immunofluorescent intensities in nuclei were higher than those in the complasma. The 1-cell embryos treated with 10μmol/L and 25μmol/L Juglone for 93h (from post hCG 27h to 120h) in vitro could hardly develop to 4-cell embryos (P<0.001). The 1-cell embryos treated with 25μmol/L Juglone for 18h (from post hCG 27h to 45h) in vitro were arrested at 2-cell stage (P<0.001). In 25μmol/L Juglone group, the Sox2 mRNA levels were significantly low compared with KSOM group (P<0.05), while the mRNA levels of Oct4, c-Myc and klf4 were no significant differences compared with KSOM group (P>0.05). Conclusion Pin1 is mainly distributed in the nucleus of mouse preimplantation embryos, the results suggest Pin1 can participate in gene transcription. The development rate of mouse preimplantatiom embryos from 2-cell to 4-cell is markedly decreased and the mRNA expression levels of stem cell factor (Sox2) are down-regulated by Juglone treatment, which indicates Pin1 may play a important role in the early development of mouse preimplantation embryos by regulating zygotic genome activation.

导出引用

连秀丽宋婵婵贺琳程霄翔陈俊明杜娟刘玥王世鄂. Pin1抑制剂对小鼠植入前胚体外发育的影响[J]. 解剖学报. 2016, 47(1): 87-94

Effects of Pin1 antagonist on mouse preimplantation embryo development in vitro[J]. Acta Anatomica Sinica. 2016, 47(1): 87-94

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