Zebularine和Scriptaid对绵羊卵丘细胞重编程的影响

Effect of Zebularine and Scriptaid on cumulus cells reprogramming in sheep

为研究组蛋白去乙酰化酶抑制剂Scriptaid和DNA甲基转移酶抑制剂Zebularine对卵丘细胞重编程的影响,试验分别采用5 nmol/L Zebularine、0.2μmol/L Scriptaid单独或联合处理绵羊卵丘细胞12 h,用MTT法检测两种药物对卵丘细胞增殖的影响,用荧光定量PCR法检测Oct4、Sox2、GDF9、BMP15和DNMT1基因的表达情况。结果表明:Zebularine和Scriptaid联合处理和单独处理卵丘细胞后增殖率差异不显著(P>0.05);单独Zebularine或Scriptaid处理卵丘细胞后Oct4表达量显著增加(P<0.05),联合处理对Oct4表达量无影响(P>0.05);而对于转化生长因子BMP15、GDF9和多潜能基因Sox2,除Zebularine单独处理的卵丘细胞GDF9表达量显著增加(P<0.05)外,其他处理间均差异不显著(P>0.05);两种药物单独及联合处理后卵丘细胞DNMT1表达量均显著降低(P<0.05)。说明Zebularine和Scriptaid单独及联合处理绵羊卵丘细胞对细胞增殖无显著影响(P>0.05),但可提高多潜能基因(Oct4、Sox2)、细胞分化相关基因(BMP15、GDF9)表达量,降低DNMT1基因表达量。

To research the effects of histone deacetylase inhibitor Scriptaid and DNA methyltransferase inhibitor Zebularine on cumulus cells reprogramming,this study used 5 nmol/L Zebularine and 0.2μmol/L Scriptaid separate and joint processing sheep cumulus cells.MTT method was used to detect the effects of the two drugs on the proliferation of cumulus cells,and the expression of Oct4,Sox2,GDF9,BMP15 and DNMT1 were detected by fluorescence quantitative PCR.Results showed that there was no significant difference in the proliferation rate of cumulus cells between the combined treatment of Zebularine and Scriptaid and the treatment with Zebularine or Scriptaid alone(P>0.05).After treatment with Zebularine alone or Scriptaid alone,Oct4 expression was significantly increased(P<0.05),Combined treatment had no effect on Oct4 expression(P>0.05).For the transformed growth factor BMP15,GDF9 and the pluripotent gene Sox2,except for the significantly increased expression of GDF9 in the cumulus cells of the Zebularine group(P<0.05),the expression differences in other groups were not significant(P>0.05).The expression of DNMT1 was significantly decreased after treatment with the two drugs alone and in combination(P<0.05).It indicated that the cumulus cells of sheep were treated with Zebularine and Scriptaid respectively and combined,had no significant effect on cell proliferation,but could increase the expression levels of pluripotent gene(Oct4、Sox2)and cell differentiation related gene(BMP15、GDF9),and reduce the expression levels of DNA methylation related genes(DNMT1).