薛晴1，徐阳1，左文莉1，Serdar E. Bulun2,张蕾，尚鶄
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。在SF-1基因的第一内含子，有一个长度约为400bp的CpG岛，我们率先对此处的甲基化进行研究。
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6到8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。
1、Real 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，P﹤0.001。
图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
4、DNA甲基化程度和mRNA表达有相关性， spearman correlation coefficient相关系数为0.96，P<0.0001。
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 endometriosis，and 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 is0.96，P<0.0001.
. 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.