DNA甲基转移酶抑制剂5-Aza-CdR对AID基因修饰的牛胎儿成纤维细胞的作用

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.016

摘要/Abstract

摘要： 以转AID基因细胞和AID基因敲减细胞为研究对象,旨在探讨5-氮-2'-脱氧胞苷（5-Aza-2'-deoxycytidine,5-Aza-CdR）对其细胞形态、细胞周期、相关基因表达及其基因启动子区甲基化状态变化的影响。此外,还分析了整体基因组去甲基化和位点特异性去甲基化之间存在的差别,探讨提高体细胞重编程效率的方法及其作用机制。通过Real-time PCR、BSP（Bisulfite Sequencing PCR）、Western blotting和流式细胞术等技术分析了5-Aza-CdR对转AID基因细胞和AID基因敲减细胞的影响。结果表明,5-Aza-CdR对转AID基因细胞的影响存在明显的剂量效应,浓度为4 µmol/L时,具有明显的细胞毒性,大量细胞死亡（P<0.05）。当使用处理浓度为1-3 µmol/L时,细胞形态发生改变,细胞增殖被抑制。核型分析显示,3 µmol/L浓度组处理就可以导致转AID基因细胞出现异常核型。使用1 µmol/L浓度处理细胞,明显增加了表达报告基因细胞的数量,细胞AID和SOX2的表达量都有所提高,并且SOX2基因启动子区的DNA甲基化水平也有所下降。5-Aza-CdR处理AID基因敲减细胞后,OCT4和SOX2的表达量较对照组明显升高,而NANOG却没有发生变化。结果显示,5-Aza-CdR处理转AID基因细胞可改变细胞形态、细胞周期和报告基因的表达,5-Aza-CdR处理后基因组整体去甲基化和AID基因的位点特异性去甲基化协同作用于体细胞重编程。

关键词: 5-Aza-CdR, AID基因, DNA去甲基化, 体细胞核移植

Abstract: In order to improve the efficiency of somatic cell nuclear transfer,the researchers used drugs which can alter DNA methylation or histone modifications to benefit somatic cell nuclear transfer. Among them,5-aza-2'-deoxycytidine（5-Aza-CdR）blocked DNA methylation by inhibiting methyl group transfer to adenine or cytosine,and ultimately reduced genomic methylation. In this study,the AID-overexpressing and AID-knocked-down cells were used to study the effects of 5-Aza-CdR（1,2,3,4,and 5 µmol/L）on cell morphology,cell cycle,related gene expression and the changes of methylation status in the gene promoter region. Additionally,the differences between genomic DNA demethylation and site-specific DNA demethylation was analyzed,the methods and its action mechanism of increasing the reprogramming efficiency of somatic cells were discussed. Real-time PCR,BSP（Bisulfite Sequencing PCR）,Western blotting,and flow cytometry were employed to analyze the effects of 5-Aza-CdR on the AID-transgenic and AID-knocked-down cells. The results showed that 5-Aza-CdR presented dose-dependent effect on AID-transgenic cells,while the 5-Aza-CdR concentration was 4 µmol/L,obvious toxicity was observed as a lot of cells were dead（P<0.05）. When treated by 1-3 µmol/L,the cell’s morphology changed,and proliferation of cells was inhibited. Karyotype analysis showed that 3 µmol/L treatments resulted in the emergence of a small proportion of aneuploidy cells,indicating that high concentrations of 5-Aza-CdR increased the rate of aneuploidy. The number of cells which expressed red fluorescent protein（DsRed）significantly increased after 1 µmol/L 5-Aza-CdR treatment,compared to the control group,the expression of AID and SOX2 were also increased,and the methylation levels in SOX2 promoter region were somehow decreased. After treated by 5-Aza-CdR,the expression of the OCT4 and SOX2genes increased in AID-knocked-down cells,but the expression of the NANOG did not change. The above results revealed that 5-Aza-CdR treatment affected the cell morphology,cell cycle and reporter gene expression in bovine transgenic cells. In conclusion,while treated by 5-Aza-CdR,the genomic demethylation and loci-specific demethylation of AID act synergistically in somatic cell reprogramming.

Key words: 5-Aza-CdR, AID, DNA demethylation, somatic cell nuclear transfer

奥旭东,萨如拉,王杰,王会敏,于海泉. DNA甲基转移酶抑制剂5-Aza-CdR对AID基因修饰的牛胎儿成纤维细胞的作用[J]. 生物技术通报, 2016, 32(8): 103-112.

AO Xu-dong ,SA Ru-la, WANG Jie, WANG Hui-min ,YU Hai-quan. The Effect of DNA Methyltransferase Inhibitor 5-Aza-CdR on AID Gene-modified Bovine Fetal Fibroblasts[J]. Biotechnology Bulletin, 2016, 32(8): 103-112.

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