5-Aza-dC对奶牛乳腺上皮细胞PPARγ表达的影响

The Effect of 5-Aza-2′-Deoxycytidine on the Expression of PPARγ in Dairy Cow Mammary Epithelial Cells in vitro

为阐明DNA甲基化对奶牛乳腺泌乳功能调控的机制,研究了甲基化抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-dC)对奶牛乳腺上皮细胞中PPARγ基因启动子甲基化状态及其表达的影响,以体外培养的奶牛乳腺上皮细胞为模型,采用0.1、0.5、1.0、5.0μmol/L的5-Aza-dC对奶牛乳腺上皮细胞进行处理,用EpiQuikTMDNA methyltransferase assay试剂盒进行甲基转移酶活性检测,采用CASY细胞分析仪检测细胞活力和增殖能力,利用亚硫酸氢盐测序(BSP)技术检测了PPARγ启动子的甲基化特征,qRT-PCR法检测PPARγmRNA表达的变化,Western blot检测PPARγ蛋白表达的变化。结果显示:与空白对照组相比,0.1μmol/L的5-Aza-dC对奶牛乳腺细胞生长无毒性,处理96h甲基转移酶活性极显著降低,奶牛乳腺细胞的PPARγ基因启动子甲基化程度降低,PPARγ表达升高。表明5-Aza-dC可降低奶牛乳腺细胞中PPARγ启动子区域的甲基化程度,促进PPARγ表达。

This experiment was to explore the influences of 5-aza 2′-deoxycytidine（5-Aza-dC）on the methylation status of promoter region of PPARγgene and its expression in dairy cow mammary epithelial cells（DCMECs）.DCMECs were cultured invitro and treated with 0.1,0.5,1.0,5.0μmol/L 5-Aza-dC,methyltransferase activity was measured by EpiQuikTM DNA methyltransferase assay kit;CASY○R-technology was applied to measure cell viability and proliferation;bisulphite genomic sequencing PCR（BSP）technique was applied to detect the methylation state of the promoter region of PPARγgene in DCMECs;qRT-PCR was applied to detect PPARγmRNA expression;the protein expression level of PPARγwas determined by Western blot.Results showed that 0.1μmol/L 5-Aza-dC had no significante effect on cell viability and proliferation of DCMECs,and methyltransferase activity was significantly decreased at 9 6 h;compared with control group,methylation level of promoter region of PPARγgene in DCMECs was partially reduced after treating with 5-Aza-dC,and the mRNA and protein expression ofnbsp;PPARγwere increased significantly.Our results suggests that 5-Aza-dC can reduce methylation level of PPARγpromoter region,and promote the mRNA and protein expre s sion of PPARγgene in DCMECs.