Pin1抑制剂对小鼠植入前胚体外发育的影响

摘要/Abstract

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

关键词: Pin1, 胡桃醌, 2-细胞阻滞, 实时定量PCR, 免疫荧光染色

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.

Key words: Pin1, Juglone, 2-cell block, Real-time PCR, Immunofluorescence staining

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]. , 2016, 47(1): 87-94.

参考文献

[1] Lu KP, Hanes SD, Hunter T. A human peptidyl-prolyl isomerase essential for regulation of mitosis [J]. Nature, 1996,380(6574):544-547.
[2] Campbell HD, Webb GC, Fountain S, et al. The human pin1 peptidyl-prolyl cis/trans isomerase gene maps to human chromosome 19p13 and the closely related pin1l gene to 1p31 [J]. Genomics, 1997,44(2):157-162.
[3] Macias MJ, Hyvonen M, Baraldi E, et al. Structure of the ww domain of a kinase-associated protein complexed with a proline-rich peptide [J]. Nature, 1996,382(6592):646-649.
[4] Ryo A, Nakamura M, Wulf G, et al. Pin1 regulates turnover and subcellular localization of beta-catenin by inhibiting its interaction with apc [J]. Nat Cell Biol, 2001,3(9):793-801.
[5] Saegusa M, Hashimura M, Kuwata T. Pin1 acts as a modulator of cell proliferation through alteration in nf-kappab but not beta-catenin/tcf4 signalling in a subset of endometrial carcinoma cells [J]. J Pathol, 2010,222(4):410-420.
[6] Ryo A, Suizu F, Yoshida Y, et al. Regulation of nf-kappab signaling by pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/rela [J]. Mol Cell, 2003,12(6):1413-1426.
[7] Toko H, Hariharan N, Konstandin MH, et al. Differential regulation of cellular senescence and differentiation by prolyl isomerase pin1 in cardiac progenitor cells [J]. J Biol Chem, 2014,289(9):5348-5356.
[8] Cheng CW, Chow AK, Pang R, et al. Pinibits apoptosis in hepatocellular carcinoma through modulation of the antia1 inhpoptotic function of survivin [J]. Am J Pathol, 2013,182(3):765-775.
[9] Liou YC, Ryo A, Huang HK, et al. Loss of pin1 function in the mouse causes phenotypes resembling cyclin d1-null phenotypes [J]. Proc Natl Acad Sci U S A, 2002,99(3):1335-1340.
[10] Wang JZ, Zhang Y. Inflammationconfiguration-specific immunotherapy targeting cis pthr231-pro232 tau for alzheimer disease [J]. J Neurol Sci, 2015,348(1-2):253-255.
[11] Sultana R, Boyd-Kimball D, Poon HF, et al. Oxidative modification and down-regulation of pin1 in alzheimer's disease hippocampus: A redox proteomics analysis [J]. Neurobiol Aging, 2006,27(7):918-925.
[12] Kanatsu-Shinohara M, Onoyama I, Nakayama KI, et al. Skp1-cullin-f-box (scf)-type ubiquitin ligase fbxw7 negatively regulates spermatogonial stem cell self-renewal [J]. Proc Natl Acad Sci U S A, 2014,111(24):8826-8831.
[13] Cohen M, Wuillemin C, Bischof P. Trophoblastic p53 is stabilised by a cis-trans isomerisation necessary for the formation of high molecular weight complexes involving the n-terminus of p53 [J]. Biochimie, 2008,90(6):855-862.
[14] Hamatani T, Carter MG, Sharov AA, et al. Dynamics of global gene expression changes during mouse preimplantation development [J]. Developmental cell, 2004,6(1):117-131.
[15] Uchida T, Takamiya M, Takahashi M, et al. Pin1 and par14 peptidyl prolyl isomerase inhibitors block cell proliferation [J]. Chem Biol, 2003,10(1):15-24.
[16] Fila C, Metz C, van der Sluijs P. Juglone inactivates cysteine-rich proteins required for progression through mitosis [J]. J Biol Chem, 2008,283(31):21714-21724.
[17] Paulsen MT, Ljungman M. The natural toxin juglone causes degradation of p53 and induces rapid h2ax phosphorylation and cell death in human fibroblasts [J]. Toxicol Appl Pharmacol, 2005,209(1):1-9.
[18] Hennig L, Christner C, Kipping M, et al. Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone [J]. Biochemistry, 1998,37(17):5953-5960.
[19] Bogolyubova IO. Transcriptional activity of nuclei in 2-cell blocked mouse embryos [J]. Tissue Cell, 2011,43(4):262-265.
[20] Bellier S, Chastant S, Adenot P, et al. Nuclear translocation and carboxyl-terminal domain phosphorylation of rna polymerase ii delineate the two phases of zygotic gene activation in mammalian embryos [J]. EMBO J, 1997,16(20):6250-6262.
[21] Schultz RM. The molecular foundations of the maternal to zygotic transition in the preimplantation embryo [J]. Hum Reprod Update, 2002,8(4):323-331.
[22] Chu DP, Tian S, Qi L, et al. Abnormality of maternal-to-embryonic transition contributes to mehp-induced mouse 2-cell block [J]. J Cell Physiol, 2013,228(4):753-763.
[23] Shin SW, Tokoro M, Nishikawa S, et al. Inhibition of the ubiquitin-proteasome system leads to delay of the onset of zga gene expression [J]. J Reprod Dev, 2010,56(6):655-663.
[24] Qiu JJ, Zhang WW, Wu ZL, et al. Delay of zga initiation occurred in 2-cell blocked mouse embryos [J]. Cell Res, 2003,13(3):179-185.