-中国妇产科医院联盟
快速导航>>
 妊娠期糖尿病患者不同治疗时机不同对母婴预后的影响
 高龄经产妇围生期临床特点与妊娠结局的临床分析
 中西医结合治疗卵巢功能减退成功妊娠1例分析
 四价HPV疫苗不会增加多发性硬化症风险
 宫颈腺癌治疗进展
 他汀类药物的使用与子宫内膜癌生存率的相关性
 联合检查是子宫颈癌筛查的最优方法:三个中国大样本
 虚弱指数可预测妇科肿瘤患者严重并发症

更多内容...

第一内含子DNA甲基化在子宫内膜异位症中对SF-1的上调作用  
 

第一内含子DNA甲基化在子宫内膜异位症中对SF-1

的上调作用

薛晴1,徐阳1,左文莉1Serdar E. Bulun2,张蕾,尚鶄

1妇产科,北京大学第一医院,北京市西城区西安门大街一号100034

2 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University

 

前言:子宫内膜异位症的病因目前尚不清楚,但为大家所公认的是它是一种雌激素依赖性疾病。芳香化酶是最后一步的合成酶,把C19 类固醇转化成雌激素。芳香化酶受类固醇基因因子-1 steroidogenic factor-1 (SF-1)的调节。SF-1基因的第一内含子,有一个长度约为400bpCpG岛,我们率先对此处的甲基化进行研究

 

1  SF-1基因第一内含子CpG岛示意图

 

 

目的:研究第一内含子CpG岛甲基化在子宫内膜异位症中对SF-1的调节作用

 

方法:子宫内膜异位症患者行手术剥除卵巢异位囊肿,并取子宫内膜作为对照。组织取出后,立即做细胞原代培养,分离出间质细胞,并进行传代。在位内膜组8例,异位内膜组8例。Real Time PCR 定量分析检测SF-1基因mRNA表达量。将在位内膜、卵巢内异症病灶(异位内膜组织)进行石蜡切片,免疫组化Envision二步法法检测SF-1蛋白的表达。PCR、克隆、Busulfite测序方法(每例随机取68个克隆进行测序)研究SF-1基因内含子区域的CpG岛的甲基化程度。运用相关分析了解内含子区域的CpG岛的甲基化机制与SF-1基因表达的相关性。

 

结果:

1Real Time PCR 定量分析显示SF-1mRNA在异位病灶的间质细胞中高度表达,但是在位内膜的间质细胞中没有表达,差异达9000多倍,P0.001

 

2.在位内膜的腺体和间质细胞中均无SF-1蛋白表达;卵巢内异症病灶的间质细胞核SF-1蛋白的阳性表达率为82%14/17, P0.001

2  SF-1在卵巢内异症囊肿病灶中的表达,卵巢内异症囊肿病灶间质细胞核出现黄染。DAB  ×200, 在位内膜的腺体细胞和间质细胞核均未见黄染。DAB ×200

 

3DNABisulfite试剂处理后,PCR扩增225bp片段DNA,克隆,每例随机取68个克隆进行测序,(共18CG位点)显示第一内含子的CpG岛在在位内膜中低度甲基化,在异位内膜中高度甲基化,有显著性差异,P0.001

3  在位和异位内膜间质细胞SF-1基因CpG岛甲基化百分率

 

4DNA甲基化程度和mRNA表达有相关性, spearman correlation coefficient相关系数为0.96P<0.0001

4  异位内膜间质细胞SF-1基因CpG岛甲基化与mRNA表达有相关性

 


结论SF-1基因的第一内含子的CpG岛的甲基化在子宫内膜异位症中对SF-1表达有调节作用

因为SF-1可以促进芳香化酶和局部雌激素的合成,因此我们的研究对了解子宫内膜异位症的病因,以及为找到更有效的治疗方法提供实验依据。

 

本研究获国家自然科学基金面上项目309731832009-2012年)资助。

 

 

    

 

 

 

 

 

Distinct epigenetic changes of steroidogenic factor-1 in the stromal cells of endometriosis

XUE Qing, XU Yang, ZUO Wenli, Serdar E Bulun, ZHANG Lei, SHANG Jing

Department of Obstetrics and Gynecology, Peking University First Hospital, 100034  Beijing, China

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, USA

 

Here, we identified a CpG islands in SF-1 gene, which is located at the first intron region.

Methods Eutopic endometrium and ectoic endometrium from the walls of ovarian endometriomas (containing a dense brown chocolate-like fluid) (n=8) were obtained immediately after surgery. Stromal cells were isolated from these two types of tissues. DNA was treated with sodium bisulfite following the manufacturer’s protocol. PCR were cloned into the pGEM-Teasy vector (Promega, Madison, WI). Following transformation, 6 to 8 clones with the right insert were randomly picked from each PCR and sequenced on an Applied Biosystems 377 instrument. Percent methylation of each clone obtained from each of the 8 patients in each group was treated as a single value for the statistical analysis of bisulfite sequencing. The data were analyzed using Student’s t-test with statistical significance at the level of P<0.05 when comparing percent methylation between the two groups of cells. spearman correlation coefficients were calculated for the correlation between SF-1 mRNA levels and percent methylation.

Results SF-1 mRNA and protein levels in endometriotic stromal cells were significantly higher than those in endometrial stromal cells (P< 0.001). No immunoreactivity of SF-1 was observed in the eutopic endometrium,  Weak to strong immunoreactivity of SF-1 was observed in the endometrial stromal cells of ovarian endometriosisand it was expressed in 14 of 17 cases (82%). SF-1 protein was not expressed in the endometrial epithelial cells of ovarian endometriosis. The endometriotic stromal cells that express high levels of SF-1, showed a dense methylation pattern at this intron region of SF-1 gene. In contrast, the majority of the CpG sites were not methylated in SF-1-negtive endometrial stromal cells. There was a significant difference (P<0.001, Student’s t-test) in methylation status between the two groups of cells (Fig 3). As shown in Figure 4, strong correlations between RNA expression and percent methylation in intron I regions are observed. Specifically, the spearman correlation coefficient is0.96P<0.0001.

Conclusions. This is the first demonstration of intron methylation-dependent regulation of SF-1 in any mammalian tissue. Specifically, hypermethylation of this intron region upregulates SF-1 RNA expression, which is totally different from that hypermethylation of the 5' regulatory region silences gene expression.

 

 

 

 

 

 

 

·上一条:补肾滋肝调经汤改善高雄激素雌鼠子宫内膜容受性及胎盘功能
·下一条:大鼠骨髓及人脐带间充质干细胞对模型鼠尿失禁的修复研究

关于我们 | 活动影集 | 在线咨询 | 新闻动态 | 联系我们

Copyright 2011© 中国妇产科医院联盟 All Rights Reserved
咨询电话:电话:010-65735765  传真:010-65735695   地址:北京市朝阳区朝阳路8号朗廷大厦B座1007
Email:cpam2007@sohu.com  备案许可证 号:京ICP035号   技术支持:世瑞博网络