高雄激素PCOS大鼠模型中胰岛素受体基因甲基化分析

The analysis on gene methylation of insulin receptor in hyperandrogenitic model rat with because of polycystic ovary syndrome

目的:建立高雄激素诱导的多囊卵巢综合征(PCOS)大鼠模型,探讨胰岛素受体(INSR)基因启动子区域甲基化状态与卵巢和子宫内膜中的局部病理变化及PCOS发病的关系。方法:45只雌性SD幼年大鼠随机分为两组,PCOS组皮下注射脱氢表雄酮(DHEA)建立PCOS模型,对照组同期皮下注射等量生理盐水。通过血清性激素水平测定和卵巢组织HE染色病理切片评估PCOS模型;免疫组化法检测大鼠卵巢、子宫内膜组织INSR蛋白的表达与定位;通过甲基化特异性PCR(MSP)定性分析两组大鼠血液、卵巢组织insr基因启动子区域的甲基化状态。结果:血清性激素水平及卵巢HE染色病理结果提示成功建立PCOS动物模型。免疫组化检测显示INSR蛋白定位表达于细胞膜,在卵巢间质及卵泡颗粒细胞、膜细胞、卵母细胞中均阳性表达,两组卵泡及间质中的表达强度无差异(P>0.05);INSR在子宫内膜中表达,模型组子宫内膜间质及腺体中表达强度高于对照组(P<0.05)。MSP结果显示模型组与对照组血液的DNA样本均检出部分甲基化条带、无甲基化条带;模型组卵巢组织的DNA中检出部分甲基化、完全甲基化、无甲基化条带,而在对照组卵巢组织DNA中仅检出部分甲基化、无甲基化条带,两组之间血液和卵巢的甲基化率均无差异(P>0.05)。结论:INSR在子宫内膜的高表达与PCOS模型子宫内膜增生有相关性;暂未发现INSR在卵巢表达与卵泡发育障碍及卵巢间质增生具有相关性;两组卵巢组织insr基因甲基化程度不同,证明PCOS大鼠可能在表观遗传学方面发生了一定改变。

Objective： To establish the model rat with polycystic ovary syndrome （PCOS） induced by excessive androgen, and then to analysis correlation between gene methylation in the promoter region of insulin receptor （INSR） and the local histopathological changes of ovary and endometrium. Methods： 45 young female Sprague Dawley rats were divided randomly into two groups. The rats of model group （model rate with PCOS） was treated by hypodermic injection with dehydroepiandros -terone （DHEA）. And the rats of control group was injected sodium chloride instead. The model rats were assessed by the level of reproductive hormone in serum and the pathological analysis of ovarian tissue section with HE staining. Localization, distribution and expression of INSR protein in ovarian tissue and endometrial tissue was conducted by immunohistochemistry （IHC）. Qualitative analysis of INSR gene methylation of the blood or ovary tissue of the two groups was examined by specific PCR （MSP）. Results： Level of reproductive hormone and the morphology analysis of ovarian tissue indicated that model rat with PCOS was successful. INSR mainly located and expressed in the cell membrane by IHC analysis. And there were positive expression in granulosa cells of ovarian follicle, the oocyte and theca cells, especially in the granule cell, but there was no significant difference between the two groups（P〉0.05）. The expression of INSR in endometrial glandular epithelial cells and stromal cells in model group was significant higher than that of the controls group（P〈0.05）. MSP analysis showed partial methylation and unmethylated bands in the promoter region of INSR genes were found in the blood of rats in both two groups. In the rats ovary issue of model group, partially methylated, fully methylated bands and unmethylated bands were all delected, while in the control group, unmethylated and partially methylated bands were detected. There were no significant differences of methylation rate in both blood or ovary tissue of the two groups（P〉0.05）. Conclusion： Expression of INSR signifi candy in the endomdrium may be correlated with endometrial hyperplasia. There is no any significant correlation be tween expression of INSR in ovary tissue and the developmental disorders of ovary and ovarian follicle stromal hyper plasia. The different of INSR gene methylation in the rat ovary tissue between the two groups indicate that rat wilb PCOS may has been changed in epigenetics.