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目的复制一种理想的多囊卵巢综合征(PCOS)动物模型,并检测LHR、INSR和AR基因DNA甲基化状态。方法给模型组24日龄大鼠皮下埋植左旋18-甲基炔诺酮硅胶棒3mm/只,3d后每日2次皮下注射人绒毛膜促性腺激素1.5IU。给对照组皮下注射等体积生理盐水。注射9d后观察大鼠卵巢形态学(HE染色),放射免疫法测定性激素和空腹胰岛素水平,己糖激酶法测定空腹血糖水平,计算胰岛素抵抗(HOMA)指数。用甲基化特异性PCR检测LHR、INSR和AR基因的DNA甲基化状态。结果模型组大鼠卵巢重量和体积均显著高于对照组(P<0.001)。模型组卵巢各级发育期卵泡及黄体少见,卵泡多呈囊性扩张。模型组大鼠血清孕激素、睾酮、促黄体生成激素(LH)、空腹胰岛素、空腹血糖水平均显著高于对照组(P<0.05);LH/促卵泡生长激素(FSH)比值和HOMA指数也均显著高于对照组(P<0.001)。模型组INSR基因甲基化率为76.7%,显著高于对照组(20.0%)(P<0.001)。结论DNA甲基化介导的胰岛素受体基因转录抑制是PCOS胰岛素抵抗发生机制之一。
Objective To clone an ideal animal model of polycystic ovary syndrome (PCOS) and to detect the DNA methylation status of LHR, INSR and AR genes. Methods 24-day-old rats were subcutaneously implanted with 3-mm L-18-norethindrone gel in a model group. Subcutaneous injection of human chorionic gonadotropin 1.5 IU twice a day for 3 days. The control group was injected subcutaneously with equal volume of normal saline. The morphological changes of ovaries were observed 9 days after injection. The levels of sex hormones and fasting insulin were determined by radioimmunoassay. Fasting plasma glucose was measured by hexokinase method and the index of insulin resistance (HOMA) was calculated. Methylation-specific PCR was used to detect DNA methylation status of LHR, INSR and AR genes. Results The weight and volume of ovary in model group were significantly higher than those in control group (P <0.001). Ovarian follicles and corpus luteum at developmental stage were rare in the model group, and follicles were mostly cystic dilatation. The levels of serum progesterone, testosterone, luteinizing hormone (LH), fasting insulin and fasting blood glucose in the model group were significantly higher than those in the control group (P <0.05). The LH / FSH ratio and HOMA index Were significantly higher than the control group (P <0.001). The methylation rate of INSR gene in model group was 76.7%, which was significantly higher than that in control group (20.0%) (P <0.001). Conclusion DNA methylation-mediated inhibition of insulin receptor gene transcription is one of the mechanisms of PCOS insulin resistance.