为探究山羊肌内前体脂肪细胞分化过程中中胚层特异转录本(mesoderm specific transcript,MEST)基因的转录及其启动子区DNA甲基化水平的调节机制。本试验通过分离并培养山羊肌内前体脂肪细胞作为试验材料,采用实时荧光定量PCR(RT-qPCR)...为探究山羊肌内前体脂肪细胞分化过程中中胚层特异转录本(mesoderm specific transcript,MEST)基因的转录及其启动子区DNA甲基化水平的调节机制。本试验通过分离并培养山羊肌内前体脂肪细胞作为试验材料,采用实时荧光定量PCR(RT-qPCR)技术检测MEST基因在细胞分化过程中的表达量变化;运用生物信息学工具预测MEST基因启动子区的CpG岛,针对CpG岛应用亚硫酸氢盐测序PCR(BSP)方法分析其甲基化状态。结果显示,MEST基因在细胞分化前的mRNA表达水平显著高于分化后(P<0.01);在MEST基因的启动子区识别到两个CpG岛。对比分化第0天和第4天,发现MEST基因启动子区的目标片段甲基化率从79.2%显著上升至88%(P<0.05)。进一步分析显示,MEST基因mRNA表达水平与启动子区甲基化水平之间存在显著的负相关性(r=-0.997,P<0.0001)。在山羊肌内前体脂肪细胞分化前后,MEST基因的表达量及其启动子区的甲基化水平均发生显著变化,且MEST基因的表达受到启动子区甲基化的负向调控。展开更多
The Mest （mesoderm-specific transcript） gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this...The Mest （mesoderm-specific transcript） gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this gene, the cDNA of full length Mest gene was obtained using 5＇- and 3＇-RACE from the Chinese Large Toad （Bufo gargarizans）. The transcript is 1 325bp in length which contains a complete open reading frame （ORF） encoding a polypeptide of 326 amino acids （GenBank accession number： ABQ10905）. There is a typical 0./13 hydrolase fold domain in the putative gene product, and it shows high similarity to sequence of homologous protein of Xenopus tropicali （86%）, mammlian （70% - 80%）. RT-PCR （reverse transcriptase-polymerase chain reaction） analysis demonstrated that the Bufo gargarizans Mest （BgMest） gene is expressed widely in testis, ovary, liver, kidney, spleen, brain, stomach and lung. The conservation of the BgMest gene sequences, protein secondary structure of the BgMest protein, in addition to the expression pattern of the BgMest gene, suggested that the function of BgMest was conserved in amphibians. However, the phylogenetic tree of the imprinting gene of the mammals and other vertebrates examined in this study indicated their divergent origins.展开更多
目的:探讨正常人的前列腺移行带(PZ)和外周带(TZ)基质成纤维细胞中MEST(mesoderm specific transcript)基因的甲基化水平变化和表达差异,及DNA甲基化抑制剂(5-Aza-CdR)对其表达水平的影响。方法:用硫化测序PCR(bisulfite sequencing PCR...目的:探讨正常人的前列腺移行带(PZ)和外周带(TZ)基质成纤维细胞中MEST(mesoderm specific transcript)基因的甲基化水平变化和表达差异,及DNA甲基化抑制剂(5-Aza-CdR)对其表达水平的影响。方法:用硫化测序PCR(bisulfite sequencing PCR,BSP)和实时荧光定量PCR方法分别检测5-Aza-CdR处理前、后在前列腺PZ和TZ原代成纤维细胞中MEST基因的甲基化水平和相应的mRNA表达。结果:MEST基因在PZ成纤维细胞中为低甲基化高表达,在TZ成纤维细胞中为高甲基化低表达。加入5-Aza-CdR后,MEST在PZ和TZ成纤维细胞中都为去甲基化,而表达水平上升,但是TZ成纤维细胞表达水平的改变比PZ成纤维细胞大。结论:MEST基因的DNA甲基化差异可能是前列腺外周带和移行带成纤维细胞生物学行为差异的分子基础。展开更多
The theory of MeSH thesaurus method is analyzed.MeSH thesaurus is highly conceptual and coordinative.It expresses the content of a document through MeSH descriptor concept.
文摘为探究山羊肌内前体脂肪细胞分化过程中中胚层特异转录本(mesoderm specific transcript,MEST)基因的转录及其启动子区DNA甲基化水平的调节机制。本试验通过分离并培养山羊肌内前体脂肪细胞作为试验材料,采用实时荧光定量PCR(RT-qPCR)技术检测MEST基因在细胞分化过程中的表达量变化;运用生物信息学工具预测MEST基因启动子区的CpG岛,针对CpG岛应用亚硫酸氢盐测序PCR(BSP)方法分析其甲基化状态。结果显示,MEST基因在细胞分化前的mRNA表达水平显著高于分化后(P<0.01);在MEST基因的启动子区识别到两个CpG岛。对比分化第0天和第4天,发现MEST基因启动子区的目标片段甲基化率从79.2%显著上升至88%(P<0.05)。进一步分析显示,MEST基因mRNA表达水平与启动子区甲基化水平之间存在显著的负相关性(r=-0.997,P<0.0001)。在山羊肌内前体脂肪细胞分化前后,MEST基因的表达量及其启动子区的甲基化水平均发生显著变化,且MEST基因的表达受到启动子区甲基化的负向调控。
基金Supported by the National Natural Science Foundation of China (No.30770296)the Natural and Science Key Project of Anhui Educational Department (KJ2007A022)the Key Lab Project of Biotic Environment and Ecology Safety in Anhui Province (2006)
文摘The Mest （mesoderm-specific transcript） gene has been considered an imprinting gene in human and mouse, and was also confirmed in other mammals and flowering plants. To investigate the function and evolution of this gene, the cDNA of full length Mest gene was obtained using 5＇- and 3＇-RACE from the Chinese Large Toad （Bufo gargarizans）. The transcript is 1 325bp in length which contains a complete open reading frame （ORF） encoding a polypeptide of 326 amino acids （GenBank accession number： ABQ10905）. There is a typical 0./13 hydrolase fold domain in the putative gene product, and it shows high similarity to sequence of homologous protein of Xenopus tropicali （86%）, mammlian （70% - 80%）. RT-PCR （reverse transcriptase-polymerase chain reaction） analysis demonstrated that the Bufo gargarizans Mest （BgMest） gene is expressed widely in testis, ovary, liver, kidney, spleen, brain, stomach and lung. The conservation of the BgMest gene sequences, protein secondary structure of the BgMest protein, in addition to the expression pattern of the BgMest gene, suggested that the function of BgMest was conserved in amphibians. However, the phylogenetic tree of the imprinting gene of the mammals and other vertebrates examined in this study indicated their divergent origins.
文摘目的:探讨正常人的前列腺移行带(PZ)和外周带(TZ)基质成纤维细胞中MEST(mesoderm specific transcript)基因的甲基化水平变化和表达差异,及DNA甲基化抑制剂(5-Aza-CdR)对其表达水平的影响。方法:用硫化测序PCR(bisulfite sequencing PCR,BSP)和实时荧光定量PCR方法分别检测5-Aza-CdR处理前、后在前列腺PZ和TZ原代成纤维细胞中MEST基因的甲基化水平和相应的mRNA表达。结果:MEST基因在PZ成纤维细胞中为低甲基化高表达,在TZ成纤维细胞中为高甲基化低表达。加入5-Aza-CdR后,MEST在PZ和TZ成纤维细胞中都为去甲基化,而表达水平上升,但是TZ成纤维细胞表达水平的改变比PZ成纤维细胞大。结论:MEST基因的DNA甲基化差异可能是前列腺外周带和移行带成纤维细胞生物学行为差异的分子基础。
文摘The theory of MeSH thesaurus method is analyzed.MeSH thesaurus is highly conceptual and coordinative.It expresses the content of a document through MeSH descriptor concept.
