河南农业科学
河南農業科學
하남농업과학
JOURNAL OF HENAN AGRICULTURAL SCIENCES
2014年
11期
126-131
,共6页
边艳杰%王杰%王春梅%李庆章
邊豔傑%王傑%王春梅%李慶章
변염걸%왕걸%왕춘매%리경장
5 氮杂 2′-脱氧胞苷%奶牛乳腺上皮细胞%PPARγ基因%DNA甲基化
5 氮雜 2′-脫氧胞苷%奶牛乳腺上皮細胞%PPARγ基因%DNA甲基化
5 담잡 2′-탈양포감%내우유선상피세포%PPARγ기인%DNA갑기화
5-aza-2′-deoxycytidine%daily cows mammary epithelial cells%PPARγ gene%methylation
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EpiQuikTM DNA 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96 h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。
為闡明DNA甲基化對奶牛乳腺泌乳功能調控的機製，研究瞭甲基化抑製劑5氮雜2′脫氧胞苷（5 Aza dC）對奶牛乳腺上皮細胞中PPARγ基因啟動子甲基化狀態及其錶達的影響，以體外培養的奶牛乳腺上皮細胞為模型，採用0.1、0.5、1.0、5.0μmol／L的5 Aza dC對奶牛乳腺上皮細胞進行處理，用EpiQuikTM DNA methyltransferase assay試劑盒進行甲基轉移酶活性檢測，採用CASY細胞分析儀檢測細胞活力和增殖能力，利用亞硫痠氫鹽測序（BSP ）技術檢測瞭PPARγ啟動子的甲基化特徵，qRT PCR法檢測PPARγmRNA錶達的變化，Western blot檢測PPARγ蛋白錶達的變化。結果顯示：與空白對照組相比，0.1μmol／L的5 Aza dC對奶牛乳腺細胞生長無毒性，處理96 h甲基轉移酶活性極顯著降低，奶牛乳腺細胞的PPARγ基因啟動子甲基化程度降低，PPARγ錶達升高。錶明5 Aza dC可降低奶牛乳腺細胞中PPARγ啟動子區域的甲基化程度，促進PPARγ錶達。
위천명DNA갑기화대내우유선비유공능조공적궤제，연구료갑기화억제제5담잡2′탈양포감（5 Aza dC）대내우유선상피세포중PPARγ기인계동자갑기화상태급기표체적영향，이체외배양적내우유선상피세포위모형，채용0.1、0.5、1.0、5.0μmol／L적5 Aza dC대내우유선상피세포진행처리，용EpiQuikTM DNA methyltransferase assay시제합진행갑기전이매활성검측，채용CASY세포분석의검측세포활력화증식능력，이용아류산경염측서（BSP ）기술검측료PPARγ계동자적갑기화특정，qRT PCR법검측PPARγmRNA표체적변화，Western blot검측PPARγ단백표체적변화。결과현시：여공백대조조상비，0.1μmol／L적5 Aza dC대내우유선세포생장무독성，처리96 h갑기전이매활성겁현저강저，내우유선세포적PPARγ기인계동자갑기화정도강저，PPARγ표체승고。표명5 Aza dC가강저내우유선세포중PPARγ계동자구역적갑기화정도，촉진PPARγ표체。
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 of 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.
