5-Aza-dC对牛体细胞核移植胚早期发育及其甲基化水平的影响
摘要
自1996年体细胞克隆绵羊“多莉”成功诞生以来,多种哺乳动物都成功的得到体细胞克隆后代。但体细胞核移植的效率却一直没有显著的提高,主要原因是供体细胞核在受体卵胞质中不能完全被重编程。DNA甲基化是哺乳动物主要的表观遗传修饰形式,是调节基因组功能的重要手段。在体细胞核移植胚胎早期发育中,8-细胞前DNA甲基化程度普遍高于正常受精胚胎。这种甲基化水平的异常可能是导致体细胞克隆发育异常的一个重要原因。本论文系统研究了牛体细胞克隆胚早期发育过程中DNA甲基化模式,同时研究了用DNA甲基化酶抑制剂(5-Aza-dC)处理供体细胞和(或)早期克隆胚,降低DNA甲基化水平对克隆胚发育及其甲基化水平的影响。
1.检测了牛SCNT胚胎附植前各个发育阶段的DNA甲基化水平,对比了克隆胚胎在DNA甲基化水平上与IVF胚胎的差异。结果显示,克隆胚胎各个发育阶段DNA甲基化水平均高于体外受精胚胎,其中2-细胞到桑椹胚阶段甲基化水平显著高于IVF胚胎(P<0.05)。体外受精胚胎甲基化水平随着胚胎发育逐渐降低,到8-细胞阶段降到最低水平,从头甲基化发生在8-细胞到桑椹胚之间。而克隆胚胎单细胞核甲基化水平降低到4-细胞阶段,从头甲基化发生在4-细胞到8-细胞阶段。
2.优化了5-Aza-dC处理方法,研究了DNA甲基化水平变化与克隆胚胎体外发育之间的关系。结果表明,低浓度的5-Aza-dC (20nM,72h)单独处理供体细胞或者重构胚不能显著提高克隆胚胎的体外发育率,对克隆胚的DNA甲基化水平没有明显影响;而供体细胞和重构胚均用低浓度5-Aza-dC (20nM,72h)处理可以显著提高囊胚发育率,2-细胞阶段DNA甲基化水平降低,8-细胞阶段的DNA甲基化水平显著降低且与IVF胚胎相比差异不再显著,说明克隆胚胎DNA甲基化水平影响其体外发育,降低2-细胞和8-细胞阶段的DNA甲基化水平可以提高克隆胚胎的体外发育能力。
3.亚硫酸氢盐测序法研究5-Aza-dC (20nM,72h)同时处理供体细胞和重构胚后的8-细胞阶段克隆胚的Dnmt3a基因DNA甲基化水平。结果显示,5-Aza-dC处理过的克隆胚的Dnmt3a甲基化程度(38.9%)更接近于体外受精胚(43.3%),而未处理的克隆胚的Dnmt3a甲基化程度较低(25.6%)。
Since the birth of the somatic cloned sheep“Dolly”in 1996, there were more than ten mammalian species which had been cloned. However, SCNT efficiency hasn’t improved obviously since then, imcomplete reprogramming of donor cell nucleus by recipient oocytes may be one of the important reseasons. Methylation of DNA is a main epigenetic modification for regulating gene functions. Level of DNA methylation in somatic cloned embryos is higher than fertilized embryos prior to 8-cell stage. Abnormal level of DNA methylation in somatic cloned embryos may result in development abnormality. In the present study, model of DNA methylation in bovine SCNT embryos was studied systemically, the effects of 5-Aza-dC, a inhibitor of methylation enzyme, on the developmental competence and methylation level of DNA were also studied.
1.The DNA methylation levels of IVF and SCNT embryos were analysed by immunodetection method, and the differences between two kinds of embryos were investigated. The results showed that, the DNA methylation level of cloned embryos were higher than that of IVF embryos, wich was significant from 2-cell to blastocyst stage, and the precocious de novo methylation was found in cloned embryos. The results revealed that the DNA methylation status were abnormal in cloned embryos.
2.The epigenetic modification status of cloned embryos were analysed after donor cells and cloned embryos were treated with 5-Aza-dC, and the relationship between DNA methylation and the development of cloned embryos were investigated. The results showed that, the in vitro development potential and epigenetic modification status of cloned embryos were not changed after the donor cells or cloned embryos were treated with low concentration of 5-Aza-dC (20nM, 72h); when both donor cells and cloned embryos were treated with 5-Aza-dC (20nM, 72h), the blastocyst rate and blastocyst cell number were significantly increased, and the DNA methylation level of 2-cell embryos was reduced, which became significant at 8-cell stage.
3.methylation level of Dnmt3a gene in somatic cloned 8-cell embryos after both donor cells and cloned embryos were treated with 5-Aza-dC (20nM, 72h) was detected by Bisulfite Sequencing PCR. In results, level of methylation of Dnmt3a gene in 5-Aza-dC–treated somatic cloned 8-cell embryos was 38.9%, similar to that of fertilized embryos(43.3%), and significantly higher than that of non-treated cloned embryos (25.6%).
1.检测了牛SCNT胚胎附植前各个发育阶段的DNA甲基化水平,对比了克隆胚胎在DNA甲基化水平上与IVF胚胎的差异。结果显示,克隆胚胎各个发育阶段DNA甲基化水平均高于体外受精胚胎,其中2-细胞到桑椹胚阶段甲基化水平显著高于IVF胚胎(P<0.05)。体外受精胚胎甲基化水平随着胚胎发育逐渐降低,到8-细胞阶段降到最低水平,从头甲基化发生在8-细胞到桑椹胚之间。而克隆胚胎单细胞核甲基化水平降低到4-细胞阶段,从头甲基化发生在4-细胞到8-细胞阶段。
2.优化了5-Aza-dC处理方法,研究了DNA甲基化水平变化与克隆胚胎体外发育之间的关系。结果表明,低浓度的5-Aza-dC (20nM,72h)单独处理供体细胞或者重构胚不能显著提高克隆胚胎的体外发育率,对克隆胚的DNA甲基化水平没有明显影响;而供体细胞和重构胚均用低浓度5-Aza-dC (20nM,72h)处理可以显著提高囊胚发育率,2-细胞阶段DNA甲基化水平降低,8-细胞阶段的DNA甲基化水平显著降低且与IVF胚胎相比差异不再显著,说明克隆胚胎DNA甲基化水平影响其体外发育,降低2-细胞和8-细胞阶段的DNA甲基化水平可以提高克隆胚胎的体外发育能力。
3.亚硫酸氢盐测序法研究5-Aza-dC (20nM,72h)同时处理供体细胞和重构胚后的8-细胞阶段克隆胚的Dnmt3a基因DNA甲基化水平。结果显示,5-Aza-dC处理过的克隆胚的Dnmt3a甲基化程度(38.9%)更接近于体外受精胚(43.3%),而未处理的克隆胚的Dnmt3a甲基化程度较低(25.6%)。
Since the birth of the somatic cloned sheep“Dolly”in 1996, there were more than ten mammalian species which had been cloned. However, SCNT efficiency hasn’t improved obviously since then, imcomplete reprogramming of donor cell nucleus by recipient oocytes may be one of the important reseasons. Methylation of DNA is a main epigenetic modification for regulating gene functions. Level of DNA methylation in somatic cloned embryos is higher than fertilized embryos prior to 8-cell stage. Abnormal level of DNA methylation in somatic cloned embryos may result in development abnormality. In the present study, model of DNA methylation in bovine SCNT embryos was studied systemically, the effects of 5-Aza-dC, a inhibitor of methylation enzyme, on the developmental competence and methylation level of DNA were also studied.
1.The DNA methylation levels of IVF and SCNT embryos were analysed by immunodetection method, and the differences between two kinds of embryos were investigated. The results showed that, the DNA methylation level of cloned embryos were higher than that of IVF embryos, wich was significant from 2-cell to blastocyst stage, and the precocious de novo methylation was found in cloned embryos. The results revealed that the DNA methylation status were abnormal in cloned embryos.
2.The epigenetic modification status of cloned embryos were analysed after donor cells and cloned embryos were treated with 5-Aza-dC, and the relationship between DNA methylation and the development of cloned embryos were investigated. The results showed that, the in vitro development potential and epigenetic modification status of cloned embryos were not changed after the donor cells or cloned embryos were treated with low concentration of 5-Aza-dC (20nM, 72h); when both donor cells and cloned embryos were treated with 5-Aza-dC (20nM, 72h), the blastocyst rate and blastocyst cell number were significantly increased, and the DNA methylation level of 2-cell embryos was reduced, which became significant at 8-cell stage.
3.methylation level of Dnmt3a gene in somatic cloned 8-cell embryos after both donor cells and cloned embryos were treated with 5-Aza-dC (20nM, 72h) was detected by Bisulfite Sequencing PCR. In results, level of methylation of Dnmt3a gene in 5-Aza-dC–treated somatic cloned 8-cell embryos was 38.9%, similar to that of fertilized embryos(43.3%), and significantly higher than that of non-treated cloned embryos (25.6%).
引文
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