抑癌基因RB1在小鼠不同发育阶段卵母细胞中的表达及甲基化状态的研究

摘要

Objective: To study mRNA expression of tumor suppressor gene RBI and epigenetic DNA methylation status in different stages oocytes of mice for exploring the role of RBI gene in the oocyte growth and development and providing theoretical basis in the diagnosis of cancer before embryo implantation.rnMethods: The mRNA expressions of RBI in different stages of oocytes including GV stage (germinal vesicle), MI stage(metaphase Ⅰ), and MII stage(metaphase Ⅱ)were detected by the improved single-cell RT-PCR method. The method for determining the relative expression level of RBI mRNA was direct reverse transcription after cell lysis, whole transcriptome amplification, and fluorescence quantitative PCR. The cells were lysed and treated by bisulfite. The DNA modified was purified. Then the target DNA fragments were amplified by semi-nested PCR. Finally the target DNA fragments were TA cloned and sequenced methylation state in different stages of oocytes.rnRemits: The expression levels of RBI mRNA were 0.054 ± 0.004, 0.080 ±0.005 and 0.101 ± 0.005 in different stages respectively. The expression of imprinted RBI gene in MII stage was two times higher than that in the GV stage(P<0. 05). The DNA methylation status in MI and MII stages was completely non-methylated state in all 24 CpG sites, but there was 1-2 CpG sites methylation in GV stage occasionally.rnConclusions: RBI mRNA expression was increased during the oocyte growth and development. The methylation of RBI DNA in oocytes(GV-MII)had not yet occurred.%目的 研究抑癌基因RB1在小鼠不同阶段卵母细胞中的mRNA表达及DNA甲基化的状态,旨在探讨RB1基因在卵母细胞生长发育中的作用,为胚胎植入前肿瘤的诊断提供理论基础. 方法 采用改良的逆转录-聚合酶链式反应(RT-PCR)方法检测RB1在不同发育阶段卵母细胞[GⅤ期(生殖泡期)、MⅠ期(第一次减数分裂中期)、MⅡ期(第二次减数分裂中期)]中的mRNA表达,即细胞裂解后直接逆转录+全转录扩增cDNA+荧光定量PCR扩增方法检测不同发育阶段卵母细胞的相对表达量.同时还进行DNA甲基化的研究:细胞裂解后直接进行亚硫酸盐处理,再进行半巢式PCR扩增出目的片段,把目的片段进行T载体(TA)克隆测序RB1在不同阶段卵母细胞中的甲基化状态. 结果 RB1在小鼠GⅤ期、MⅠ期、MⅡ期卵母细胞的表达量分别是:(0.054±0.004)、(0.080±-0.005)、(0.101±0.005),RB1在MⅡ期中的表达量约是GⅤ期的2倍,差别具有统计学意义(P＜0.05).RB1甲基化:MⅠ期和MⅡ期卵母细胞的24个CpG位点C完全变成T,所有CpG位点完全非甲基化.GⅤ期偶见1～2个CpG位点甲基化,所有CpG位点基本完全非甲基化. 结论 RB1 mRNA随着卵母细胞的生长发育表达量不断增加,RB1 DNA在卵母细胞(GⅤ～MⅡ)中尚未发生甲基化.