PD0325901和CHIR99021促进牛体细胞克隆胚胎体外发育的研究

Promotion of invitrodevelopment of bovine somatic nuclear transfer embryos by PD0325901 and CHIR99021

【目的】选取MAP2K和GSK3的抑制剂PD0325901、CHIR99021(简称2i)对牛体细胞核移植(SCNT)胚胎进行处理,研究这2种小分子物质对胚胎发育及其多能性基因(OCT4、SOX2、KLF4、DPPA3、CDX2、GATA4、NANOG)表达的影响。【方法】以牛皮肤成纤维细胞为供体,牛MⅡ期卵母细胞为受体,构建牛SCNT胚胎,采用添加2i(PD0325901 0.4μmol/L,CHIR99021 3μmol/L)的胚胎培养液体外培养SCNT胚胎72h,以mSOF+DMSO培养液处理的SCNT胚胎及体外受精(IVF)胚胎为对照,统计核移植胚胎体外发育率,并对胚胎多能性相关基因的表达进行实时定量PCR检测。【结果】与对照相比,2i处理能使SCNT胚胎囊胚率和囊胚细胞总数显著增加,凋亡细胞率显著下降,多能性相关基因NANOG和SOX2表达量上调,而细胞分化相关基因GATA4的表达量下调,其他基因表达量变化不显著,从而使SCNT胚胎的发育状态更接近于IVF胚胎。【结论】2i共处理72h可优化SCNT胚胎发育状态。

[Objective] The objective was to investigate the effect of MAP2K and GSK3 pathway inhib- itors PD0325901 and CHIR99021 on development capacity and expression of pluripotency genes （OCT4, SOX2,KLF4,DPPA3 ,CDX2,GATA4,and NANOG） in vitro of bovine somatic cell nuclear transfer em- bryos. [Method] Using bovine skin fibroblasts as donors and MII oocytes as receptors, SCNT embryos were constructed and treated with PD0325901 ＋CHIR99021 （PD0325901 0. 4 μmol/L, CHIR99021 3 μmol/L） as tests and mSOF＋DMSO as control for 72 h in vitro. In addition,embryos were counted and development rates and embryonic development related genes were analyzed by real-time PCR. [Result] Compared to untreated control,the 2i treatment increased the blastocyst rate and total blastocyst cell num-ber while decreased apoptosis rate markedly. The expression of pluripotency related genes was upgraded. Expression of NANOG and SOX2 was significantly increased and that of GATA4 decreased while the rest genes were not significantly changed. SCNT embryo pluripotent expression related genes were closer to IVF embryo. [Conclusion] Treatment with 2i can optimize SCNT embryonic development state.