【摘要】 目的探讨非肌肉肌球蛋白Ⅱ(non-muscle myosinⅡ,NMⅡ)ATP酶抑制剂(-)-Blebbistatin对小鼠早期胚胎体外发育过程及其干性相关基因表达的影响。方法采用SPF级ICR品系小鼠,对10只雌鼠采用孕马血清促性腺激素和人绒毛膜促性腺激素进行诱导超数排卵,雌鼠与雄鼠1︰1合笼。分别于E 0.5、E 1.5、E 2.5(受孕0.5、1.5、2.5天)收集胚胎。桑椹胚随机分为对照组和实验组,对照组液滴为20μl Ksom培养基,实验组液滴为加入25μM(-)-Blebbistatin的20μl Ksom培养基,体外培养24小时。进行免疫荧光标记,观察小鼠胚胎中微丝骨架的分布情况,以及桑椹胚中nanog、Oct4、sox2、ssea1和estrogen等5个干性相关基因的表达情况。结果小鼠早期胚胎的发育经历细胞分裂后裂球数目增加、桑椹胚致密化、囊胚期囊胚腔的扩张过程。(-)-Blebbistatin组胚胎则无法形成囊胚腔进入囊胚时期,大多数胚胎裂球松散,整体呈扁塌状。实验共重复3次,实验组胚胎共62枚,均未能形成囊胚;对照组胚胎共60枚,全部形成囊胚。(-)-Blebbistatin抑制NMⅡ后小鼠胚胎干性相关基因nanog,Oct4,sox2,ssea1,estrogen的表达均明显低于对照组(P值均﹤0.05)。结论抑制NMⅡATP酶活性会阻滞小鼠囊胚的体外发育,并降低干性相关基因的表达。更多还原

【Abstract】 Objective To explore the influence of ATP enzyme inhibitors(-)-Blebbistatin of nonmuscle myosin Ⅱ(NM Ⅱ)on the blastocyst formation of mouse in vitro during early embryos and on the expression of pluripotent genes. Methods Ten of SPF class ICR strain female mice were induced by pregnant mare serum gonadotropin and human chorionic gonadotropin for superovulation. Female mice and male mice were caged with 1 ︰ 1.The morulae were taken from the uterus of the mice that have been pregnant for 0.5, 1.5, 2.5 days. Then the morulae were randomly distributed into the control group and the experimental group; drop culture of control group was 20 μl Ksom; that of experimental group was 20 μl Ksom added 25 μM(-)-Blebbistatin. After culturing in vitro for 24 hours, the morulae were labeled by immunofluorescence. The development of blastocysts was observed, and the expression of stemness genes of nanog, Oct4, sox2, ssea1 and estrogen was assessed. Results The development of early embryo in mice experienced increased number of blastomere after cell division, densification of morula and expansion of the blastocoel of blastula period. Embryo of(-)-Blebbistatin group could not formed into the blastocyst period, most of the embryo blastomere loosed and saddle shaped as a whole. The experiment was repeated three times. There were 62 embryos in the experimental group and all the blastocoel could not be formed. There were 60 embryos in the control group and all the blastocoel could be formed. After being treated by(-)-Blebbistatin, pharmacological inhibition of NM II, the gene expression of stemness genes of nanog, Oct4, sox2, ssea1 and estrogen, was significantly lower than that of the control group(P ﹤ 0.05). ConclusionInhibiting NM Ⅱ blocks the mouse blastocysts development and reduces the expression of pluripotent genes.更多还原