【摘要】 目的:利用特异性发夹结构Wee1BshRNA,探讨蛋白激酶Wee1B对小鼠受精卵早期发育的影响。方法:超排卵方法获得小鼠1-细胞期受精卵并显微注射质粒,实验分为未注射组、TE缓冲液注射组、Control shRNA注射组和Wee1BshRNA注射组,收集各组卵细胞后用RT-PCR和Western blotting方法检测Wee1B shRNA的干涉效能;荧光显微镜观察受精卵的形态变化、计数卵裂率;放射自显影测定Wee1B活性。结果:与TE缓冲液和Control shRNA注射组比较,Wee1BshRNA注射组小鼠1-细胞期受精卵Wee1BmRNA水平和蛋白表达显著降低;与其他3组比较,Wee1BshRNA注射组Wee1B的激酶活性明显降低;未注射组、TE缓冲液注射组和Control shRNA注射组在hCG注射33h后1-细胞期受精卵的卵裂率分别为71.91%±1.74%、72.90%±2.25%和72.28%±2.06%,3组间比较差异无统计学意义(P>0.05);而Wee1BshRNA注射组的卵裂率提高18.00%,为90.00%±0.77%,与其他3组比较差异均有统计学意义(P<0.01)。结论:Wee1B表达沉默可提高小鼠1-细胞期受精卵的卵裂率,说明Wee1B负调控小鼠受精卵有丝分裂的进程。更多还原

【Abstract】 Objective To study the influence of protein kinase Wee1Bin the early development of mouse embryos by the means of specific Wee1B shRNA.Methods The mouse one-cell-stage embryos were collected after superovulation,and microinjected with TE buffer,control shRNA or Wee1B shRNA respectively,then the specificity of Wee1B shRNA was examined by the methods of RT-PCR and Western blotting.After RNA interference,the morphological changes and cleavage rate of mouse embryos were observed under fluorescence microscope,and the Wee1Bactivity of mouse embryos was detected by autoradiography.Results Compared with TE buffer and Control shRNA groups,the expression levels of Wee1BmRNA and protein in Wee1BshRNA group were significantly reduced,and the activity of Wee1B was also decreased.The cleavage rates of mouse one-cellstage embryos in non-injection,TE buffer,Control shRNA groups 33hafter hCG injection were 71.91 % ±1.74%,72.90%±2.25%,and 72.28% ±2.06%,and there was no obvious difference among the three groups(P>0.05);the cleavage rate in Wee1BshRNA group was 90.00%±0.77% at 33h,which was markedly higher than those of the controls(P<0.01).Conclusion Wee1BshRNA can promote the onset of mitosis of mouse onecell-stage embryos,suggestting that Wee1Bnegatively regulates mitosis during early embryogenesis in mouse onecell-stage embryos.更多还原