SAHA处理对猪手工克隆胚胎体外发育潜能的影响

Effects of SAHA on the developmental potential of porcine handmade cloned embryos in vitro

为了探讨组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸（SAHA）对德保猪手工克隆胚胎（HMC）发育潜能的影响,试验摸索SAHA的适宜处理浓度[0（对照）,1.0,2.5,5.0,7.5,10.0μmol/L]和时间（0,6,12,24 h）;之后分为4组,SAHA组、体外受精（IVF）组、孤雌激活（PA）组、对照（HMCC）组,分别在体外发育的1细胞期、2细胞期、4细胞期、囊胚期收集胚胎,在相同时期下比较各组胚胎组蛋白H4K8乙酰化（Ac H4K8）水平差异和相关基因（HDAC1、HAT1、ASF1A、OCT-4）相对表达量。结果表明：7.5μmol/L SAHA处理囊胚率显著高于对照（P〈0.05）,12 h囊胚率显著高于0 h（P〈0.05）;所以适宜处理浓度为7.5μmol/L,适宜处理时间为12 h。在1细胞期、2细胞期、囊胚期,SAHA组Ac H4K8水平接近IVF组水平（P〉0.05）。在囊胚期,SAHA组HDAC1基因相对表达量接近IVF组（P〉0.05）;在囊胚期,SAHA组OCT-4基因相对表达量接近IVF组（P〉0.05）。说明SAHA可以使HMC胚胎Ac H4K8水平接近IVF水平,并纠正克隆胚胎乙酰化的异常,从而提高克隆胚胎发育潜能。

The aim of the present study was to explore the effects of histone deacetylase inhibitor SAHA on the development potential of Debao porcine handmade cloning（HMC） embryos.After exploring the proper treatment concentration（0 as control,1.0,2.5,5.0,7.5 and10.0 μmol/L） and time（0,6,12 and 24 h） of SAHA,the experiment was divided into 4 groups： SAHA group,in vitro fertilization（IVF）group,parthenogenetic activation（PA） group and control group（HMC-C group）.The embryos in 4 groups were collected in 1,2 and 4 cells stage and blastocyst stage.Then the acetylation of histone H4K8（AcH4K8） levels and related genes（HDAC1,HAT1,ASF1 A,OCT-4） expression changes were compared at the same time.The results showed that the optimum concentration of SAHA was 7.5 μmol/L,which was significantly higher than that of control group（P〈0.05）.The optimal treatment time of SAHA was 12 h,which was significantly higher than that of 0 h（P〈0.05）.AcH4K8 expression changes showed that the AcH4K8 level of SAHA group in 1,2 cell stage and blastocyst stage was closed to that of IVF group（P〈0 05）.The expression of HDAC1 gene in SAHA group was similar to that of group IVF（P〉0.05） in blastocyst stage.The relative expression of OCT-4 in SAHA group was not different from IVF group（P〉0.05）.The results indicated that SAHA could make the AcH4K8 level of HMC embryos close to IVF level,and correct the abnormal acetylation of cloned embryos,so as to improve the developmental potential of cloned embryos.