5-Aza-CdR联合TSA对牛核移植胚胎体外发育及表观遗传状态的影响

Effect of 5-Aza-CdR and TSA on the in vitro development of bovine cloned embryos and epigenetic status

【目的】探讨5-氮杂-2′-脱氧胞苷(5-Aza-2-′Deoxycytidine,5-Aza-CdR)联合曲古抑菌素A(Trichosta-tin A,TSA)对牛核移植胚胎体外发育及其表观遗传状态的影响。【方法】以体外受精胚胎作为对照组,将部分体细胞、核移植胚胎以及体细胞联合核移植胚胎用5-Aza-CdR+TSA(5-Aza-CdR:20 nmol/L,72 h;TSA:50 nmol/L,12 h)进行处理,统计核移植胚胎体外发育率,并对2-细胞、8-细胞和囊胚阶段胚胎进行DNA甲基化和组蛋白乙酰化检测,研究5-Aza-CdR+TSA处理对核移植胚胎体外发育及其表观遗传状态的影响。【结果】与对照组相比,5-Aza-CdR+TSA单独处理供体细胞和核移植胚胎均可以显著提高克隆胚胎的囊胚发育率和囊胚细胞数(P<0.05);供体细胞和核移植胚胎均用5-Aza-CdR+TSA处理后,核移植胚胎体外发育能力进一步提高,囊胚发育率显著高于单独处理供体细胞或核移植胚胎组(P<0.05)。免疫荧光检测发现,体细胞和核移植胚胎均用5-Aza-CdR+TSA处理,不但显著降低了2-细胞和8-细胞阶段DNA甲基化水平,而且也显著增加了组蛋白乙酰化水平(P<0.05),使核移植胚胎附植前各发育阶段的表观遗传状态更接近于体外受精胚胎。【结论】5-Aza-CdR联合TSA处理供体细胞和核移植胚胎可以更明显地提高克隆胚胎的体外发育能力,改变核移植胚胎的DNA甲基化和组蛋白乙酰化水平,使它们的表观遗传状态及体外发育率更接近于体外受精胚胎。

[Objective] The objective was to study the effect of 5-Aza 2＇-Deoxycytidine （5-Aza-CdR） and Trichostatin A （TSA） on the development of bovine cloned embryos and epigenetic status. [Method] Relative to untreated controls, treatment of donor cells, cloned embryos, and continuous treatment of both donor cells and cloned embryos were performed with 5-Aza-CdR and TSA （5 Aza-CdR： 20 nmol/L, 72 h; TSA ： 50 nmol/L, 12 h）, in vitro developmental rates of nuclear transfer embryos calculated, DNA methyla- tion and Histone acetylation status of 2-cell stage,8-cell stage and blastocyst stage embryos detected,effect of 5-Aza-CdR and TSA on the development of bovine cloned embryos and epigenetic status studied. [Result] Relative to untreated controls, sole treatment of donor cells, cloned embryos significantly increased the blastocyst rate and cell number of blastocyst of cloned embryos （P〈0.05） ;after continuous treatment of both donor cells and cloned embryos, in vitro developmental ability of cloned embryos further increased, and the blastocyst rate was significantly higher than the sole treated donor cells group and the sole treated embryos group（P〈0.05）. Immunofluorescence assay found that donor cells and cloned embryos both treated by 5-Aza-CdR and TSA not only significantly decreased DNA methylation status of 2-cell and 8-cell stage embryos,but also significantly increased their Histone acetylation status（P〈0.05）. [Conclusion] The development of cloned bovine embryos was enhanced by 5-Aza CdR and TSA;furthermore,the combination was more effective than either one alone, combined treatment could change DNA methylation and Histone acetylation status of nuclear transfer embryos,make their epigenetic status and in vitro developmental rates closer to IVF embryos.