雌二醇诱致大鼠垂体催乳素瘤细胞中催乳素基因第1、2、4外显子的低甲基化  

Hypomethylation of CpG Sites in Exon 1,2 and 4 of PRL Gene of Pituitary Prolactionoma Induced by Estradiol in S D Rat

作　　者：狄安稞[1] 周远征[1] 单惠敏[1] 黄曼影[1] 吴雪梅[1] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础医学院生理研究室

出　　处：《基础医学与临床》1997年第6期30-35,共6页Basic and Clinical Medicine

摘　　要：本工作探讨了雌二醇（Ｅ２）诱致的雄性ＳＤ（Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ）大鼠垂体催乳素（ｐｒｏ－ｌａｃｔｉｎ，ＰＲＬ）瘤中ＰＲＬ基因含ＣＣＧＧ位点的第１、２、４外显子中ＣＣＧＧ位点甲基化状态的改变。提取基因组ＤＮＡ并分别用对非甲基化ＣＣＧＧ位点不敏感而对甲基化ＣＣＧＧ敏感的ＨｐａⅡ及对甲基化和非甲基化ＣＣＧＧ位点均不敏感的ＭｓｐⅠ进行完全消化后，利用聚合酶链反应（Ｐｏｌｙｍｅｒｓａｓｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）方法，对含有ＣＣＧＧ位点的ＰＲＬ基因第１、第２和第４外显子的部分序列进行扩增。结果：用ＨｐａⅡ消化后，正常大鼠基因组ＤＮＡ用三对不同引物经ＰＣＲ扩增，均得到预期的含ＣＣＧＧ位点的特异片段（分别为１６３９ｂｐ、２６８ｂｐ和１３３ｂｐ），而Ｅ２致瘤组则均未能扩增出相应的片段，提示正常大鼠ＰＲＬ基因第１、２、４外显子ＣＣＧＧ位点均成甲基化状态，而Ｅ２致瘤后，则均成非甲基化状态。不管是正常对照组，还是Ｅ２致瘤组，经ＭｓｐⅠ消化后，均未能扩增出预期的ＰＲＬ基因片段，而未经ＭｓｐⅠ、ＨｐａⅡ消化组，均扩增出了预期的片段。以上结果表明，Ｅ２所致的大鼠垂体ＰＲＬ瘤，其ＰＲＬ基因外显子中的ＣＣＧＧ位点均呈低甲基?he methylation status of CCGG sites in exon 1,2 and 4 of prolactin (PRL) gene of prolactinoma induced by estradiol (E2) was analyzed in S.D. (SpragueDawley) rats. Genomic DNA isolated from prolactinoma and normal anterior pituitary tissues is subjected to digestion to completion with the specified restriction endonuclease MspⅠ (not sensitive to methylated CCGG site) and HpaⅡ (sensitive to methylated CCGG site) under conditions specified by the supplier (Promega CO., U.S.A.).A PCR (Polymerase chain reaction)based Methylation Assay was applied. This assay relied on the ability of MspⅠ to cut both the mehtylated and demethylated CCGG sites and the ability of Hpa[KG*4〗Ⅱ to cut only the demethyletted CCGG sites in PRL gene.The digested DNA were amplified with primers flanking the CCGG sites in exon 1,2 and 4 of PRL gene. The PCR results indicated that all the CCGG sites in PRL gene of prolactinoma were demethylated,but they were methylated in normal anterior pituitary. The hypomethylation of PRL gene may be the partial cause of prolactinoma and hyperprolatinemia induced by estradiol.

关 键 词：雌二醇 催乳素基因 外显子 垂体催乳素瘤 PCR 

分 类 号：R736.4[医药卫生—肿瘤] R73-3[医药卫生—临床医学]