不同浓度CB对小鼠纺锤体移植后重构卵细胞骨架及囊胚发育影响的研究

Effects of different concentrations of CB on the reconstruction of egg cytoskeleton and embryo development after spindle transplantation in mice

目的探讨不同浓度细胞松弛素B(cytochalasin B,CB)对小鼠纺锤体移植后重构卵细胞骨架及胚胎发育的影响,筛选出更优的CB浓度。方法8周的C57BL/6雌鼠卵,使用常规剂量、低剂量、中剂量和高剂量的CB做小鼠纺锤体移植,统计小鼠纺锤体移植效率;统计重构小鼠卵体外受精的(in vitro fertilization,IVF)受精率、卵裂率、囊胚率、孵化率;对各组囊胚行免疫荧光染色,荧光显微镜观察计数;重构小鼠卵行免疫荧光染色,共聚焦显微镜观察细胞骨架并分析。结果①低剂量组、中剂量组、高剂量组与常规组相比,小鼠纺锤体移植效率差异无统计学意义(P>0.05)。②高剂量组的受精率低于常规组(P<0.05);高剂量组的囊胚率、孵化率低于常规组、中剂量组(P<0.05)。③高剂量组的囊胚细胞总数少于常规组(P<0.05),中剂量组的囊胚细胞总数多于常规组(P<0.05)。④低剂量组、中剂量组、高剂量组和常规组相比,不同时间重构小鼠卵微丝阳性比率差异无统计学意义(P>0.05);不同时间各组微丝的平均光密度值,组间比较差异有统计学意义(P<0.05),时间点间比较差异有统计学意义(P<0.05),组间的时间变化趋势差异有统计学意义(P<0.05),即不同浓度CB和不同观察时间对小鼠重构卵细胞骨架恢复情况均有影响,且差异有统计学意义。结论本研究选取的CB浓度范围内,中剂量组优于常规组。

Objective To investigate the effects of different concentrations of cytochalasin B(CB)on the reconstruction of egg cytoskeleton and embryo development after oocyte spindle transplantation in mice,and to screen out the better CB concentration.Methods Eight-week-old C57BL/6 female mouse eggs were transplanted using conventional,low,medium,and high doses of CB.The efficiency of spindle transplantation in mice was measured.The fertilization rate of in vitro fertiliazation(IVF),cleavage rate,blastocyst rate and hatching rate of reconstructed mouse eggs were calculated.The blastocysts of each group were stained by immunofluorescence and calculated by fluorescence microscope.Mouse eggs were reconstructed and stained with immunofluorescence.The cytoskeleton was observed and analyzed by confocal microscopy.Results①Compared with the conventional group,there was no difference in the efficiency of spindle transplantation in the low dose,medium dose and high dose groups(P>0.05).②The fertilization rate of high dose group was lower than that of conventional group(P<0.05),and the blastocyst rate and hatching rate of high dose group were lower than those of conventional group and medium dose group(P<0.05).③The total number of blastocyst cells in the high dose group was less than that in the conventional group(P<0.05),and the total number of blastocyst cells in the medium dose group was more than that in the conventional group(P<0.05).④Compared with the conventional group,there was no difference in the positive ratio of microfilaments of reconstructed mice eggs in low-dose,medium-dose,high-dose groups at different time points(P>0.05).The average optical density values of microfilaments in different time groups were different(P<0.05),there were significant differences between time points(P<0.05),and the time variation trend was different among groups(P<0.05).That is,different concentrations of CB and different observation time had significant effects on the recovery of reconstructed egg cytoskeleton in mice,and there were significant differences.Conclusion In the concentration range of CB selected in this study,the medium dose group was better than the conventional group.