摘要
为探讨组蛋白去乙酰化酶抑制剂羟肟酸(SAHA)对体细胞和山羊核移植胚胎早期发育的影响,采用不同浓度SAHA处理山羊胎儿成纤维细胞(Goat embryonic fibroblasts,GEFs),并检测其AcH3K9水平、细胞增殖能力、细胞周期及胚胎发育情况、乙酰化水平、多能性基因的表达水平。结果显示,SAHA处理后GEFs的AcH3K9水平显著上升(P<0.05),2.5μmol/L SAHA处理浓度即可显著提高GEFs组蛋白H3K9的乙酰化水平;浓度≤2.5μmol/L时,对细胞增殖能力无明显影响;浓度为5.0μmol/L和10.0μmol/L时,细胞活力显著下降(P<0.05)。AcH3K9上调会引起G0/G1和S期的细胞比例下降(P<0.05)。因此,选择经2.5μmol/L SAHA处理的供体细胞进行核移植。试验组的胚胎卵裂率和囊胚率显著高于对照组(P<0.05),其卵裂率接近于卵胞质单精子注射(ICSI)组。试验组的囊胚率乙酰化水平显著高于对照组,且低于ICSI组(P<0.05)。与对照组相比,试验组囊胚的Oct4、Sox2和Nanog的mRNA表达水平均上升(P<0.05),且均接近于ICSI组。说明,选用2.5μmol/L SAHA处理供体细胞可提高体细胞核移植效率和胚胎品质。
To study the effect of Suberoyl anilide hydroxmatic acid （SAHA） ,a histone deacetylase inhibitor,on donor cells and nuclear transfer embryos, a series of tests on goat embryonic fibroblasts （GEFs） were conducted, which it included immunostaining of AcH3K9, cell viability assays and cell cycle analysis. Moreover, we analyzed the global AcH3K9 level of blastocysts, the characterization of in vitro development rate of goat SCNT embryo and the expression level of pluripotency genes. The results showed that the AcH3K9 level of GEFs were significantly higher than that of untreated ones （P〈0.05）, furthermore, the treatment by concentration of 2.5 μ mol/L SAHA showed obvious effect. 0-2.5 /,mol/L SAHA treatment had no influence on cell viability,but under treatment of 5.0μmol/L and 10.0 μmol/L cell viability deccreasd significantly （P〈0.05）. Up-regulating the acetylation level induced decrease of G0/G1 and the cell rate of at S stage declined （P〈0.05）. Therefore, the donor cells treated by 2.5 μmol/L SAHA were used for nuclear transfer. The rate of cleavage and the blasto-cysts of experimental group were higher than that of control group, and the rate of cleavage was close to that of the intracytoplasmic sperm injection （ICSI） group （P〈0.05）. The AcH3K9 level of blastocysts was higher than that of control group and lower than that of ICSI group （P〈0.05）. Compared with control group,the expression levels of gene Oct4 ,Sox2 and Nanog of experiment group blastocysts increased （P〈0. 05）, and the Soz2 and Nanog expression level were similar to that of ICSI group. In conclusion,donor cells under treatment of 2.5 μmol/L SAHA can improve the efficiency of nuclear transfer and the quality of embryos.
出处
《西北农业学报》
CAS
CSCD
北大核心
2015年第9期1-8,共8页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家高技术研究发展计划(863计划)(2011AA100303)
关键词
羟肟酸
H3K9乙酰化
克隆胚胎
胎儿成纤维细胞
山羊
Suberoyl anilide hydroxamic acid
H3K9 acetylation
Cloned embryos
Embryonic fibroblast
Goat
