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目的研究微小RNA-29b(miR-29b)在植入前小鼠胚胎中的表达模式,探讨其在小鼠植入前胚胎发育中的生物学作用。方法采用实时定量PCR法检测小鼠植入前胚胎发育过程中miR-29b及其下游DNA甲基转移酶3a/3b(Dnmt3a/3b)的表达模式。采用显微注射商业化的miR-29b抑制剂的实验方法探讨其在早期胚胎中的功能。结果 miR-29b在小鼠二细胞阶段高表达,四细胞阶段开始表达下降,并在植入前维持较低的表达水平。Dnmt3a和Dnmt3b均在小鼠四细胞阶段表达量升高;其中,Dnmt3a在囊胚期表达降低并维持在较低的水平,而Dnmt3b在植入前均维持在较高的水平。miR-29b抑制剂抑制miR-29b功能后,小鼠胚胎从桑葚期到囊胚期发育明显阻滞。结论 miR-29b在小鼠植入前胚胎各阶段均有表达,呈现二细胞特异性高表达模式;Dnmt3a/3b在小鼠植入前胚胎中均有表达,且与miR-29b的表达呈现一定负相关性;miR-29b功能下调,引起植入前小鼠胚胎发育阻滞。
Objective To study the expression pattern of microRNA-29b (miR-29b) in preimplantation mouse embryos and to explore the biological role of miR-29b in preimplantation embryo development in mice. Methods Real-time quantitative PCR was used to detect the expression pattern of miR-29b and its downstream DNA methyltransferase 3a / 3b (Dnmt3a / 3b) in mouse preimplantation embryos. Experiments with microinjection of commercial miR-29b inhibitors were used to investigate their function in early embryos. Results miR-29b was highly expressed in the two-cell stage of the mouse and decreased at the beginning of the four-cell stage. The expression of miR-29b was maintained at a low level before implantation. Dnmt3a and Dnmt3b were all increased in the four-cell stage of the mouse; Dnmt3a expression at the blastocyst stage decreased and remained at a low level, while Dnmt3b maintained at a higher level before implantation. After miR-29b inhibitor inhibits the function of miR-29b, the development of mouse embryos from the mulberry stage to the blastocyst stage is obviously blocked. Conclusions miR-29b is expressed in all stages of mouse preimplantation embryos and presents a two-cell specific high expression pattern. Dnmt3a / 3b is expressed in preimplantation embryos of mice and shows a certain expression with miR-29b Negative correlation; miR-29b function down, causing preimplantation mouse embryonic development block.