解剖学报

解剖学报 ›› 2020, Vol. 51 ›› Issue (3): 367-372.doi: 10.16098/j.issn.0529-1356.2020.03.009

• 细胞和分子生物学 • 上一篇    下一篇

双氢睾酮对大鼠原代卵泡颗粒细胞抗苗勒管激素表达的影响

王美玲1 董玉婷1 叶晓锋2 黑常春1 蔡玉芳1 孔斌1 赵承军1 常青1*    

  1. 1.宁夏医科大学基础医学院人体解剖学与组织学胚胎学系, 生育力保持教育部重点实验室，银川 750004; 2.宁夏医科大学总医院肿瘤医院肿瘤外二科，银川 750004
  • 收稿日期:2019-09-27 修回日期:2019-11-28 出版日期:2020-06-06 发布日期:2020-06-06
  • 通讯作者: 常青 E-mail:seryin55@163.com
  • 基金资助:
    黄体生成素对多囊卵巢综合症卵巢缝隙连接蛋白表达调节的研究。;雄激素受体激活Wnt信号通路在胃癌细胞增殖、EMT和侵袭中的研究

Effects of dihydrotestosterone on the expression of anti-Müllerian hormone in primary follicular granulosa cells in rats

WANG Mei-ling1 DONG Yu-ting1 YE Xiao-feng2 HEI Chang-chun1 CAI Yu-fang1 KONG Bin1 ZHAO Cheng-jun1 CHANG Qing1*   

  1. 1. Department of Human Anatomy, Histology and Embryology, School of Basic Medicine, Ningxia Medical University, Fertility Conservation Key Laboratory of Ministry of Education, Yinchuan 750004, China; 2. Second Department of Oncology, Cancer Hospital of  General Hospital of Ningxia Medical University, Yinchuan 750004, China
  • Received:2019-09-27 Revised:2019-11-28 Online:2020-06-06 Published:2020-06-06
  • Contact: CHANG Qing E-mail:seryin55@163.com

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摘要：

目的  探讨双氢睾酮（DHT）对大鼠原代卵泡颗粒细胞抗苗勒管激素（AMH）表达的影响。  方法  从95只21 d SD雌性大鼠中提取颗粒细胞原代培养48 h后，先用HE染色检测细胞形态，卵泡刺激素受体（FSHR）细胞免疫荧光检测细胞纯度；然后根据干预方法和实验的不同，随机将细胞分为对照组（无药物干预）、10-8mol/L DHT组、10-5mol/L DHT组，2×10-5mol/ L蛋白激酶B(Akt)抑制剂（MK-2206 2Hcl）组和10-8mol/L DHT+2×10-5mol/L MK-2206 2Hcl组，分别使用相应浓度的试剂干预细胞3 h。干预完成后，细胞免疫荧光染色观察AMH表达的部位和变化；Western blotting测定不同组的AMH、Akt、p-Akt蛋白表达量。  结果 大鼠原代卵泡颗粒细胞的纯度为93.33%±3.09%，AMH表达在大鼠原代卵泡颗粒细胞膜和细胞质中；与对照组相比，10-8 mol/L DHT和10-5 mol/L DHT均可使颗粒细胞AMH表达增加（P<0.05），但这两个浓度的DHT对颗粒细胞AMH的表达影响差异无显著性（P>0.05）；与对照组相比，10-8 mol/L DHT可以使p-Akt、AMH表达升高，2×10-5 MK-2206 2Hcl使AMH表达降低（P<0.05）；与2×10-5MK-2206 2Hcl相比，10-8mol/L DHT+2×10-5 MK-2206 2Hcl处理组AMH表达升高（P<0.05）。  结论  DHT可以上调大鼠原代卵泡颗粒细胞AMH的表达，这种作用可能与Akt信号通路有关。

关键词: 双氢睾酮, 抗苗勒管激素, 卵泡颗粒细胞, 免疫荧光, 免疫印迹法, 大鼠

Abstract:

Objective To explore how dihydrotestosterone (DHT) affects the expression of anti-Müllerian hormone (AMH) in primary rat follicular granulosa cells, and then provides a new experimental basis for the role of DHT and AMH in follicular development.  Methods  Granulosa cells was extracted from the ovaries of 95 21 days SD female rats for primary culture. Firstly, cell morphology were detected by HE staining and follicke stimulating hormone receptor(FSHR) immunofluorescence detected cell purity after 48 hours of cell culture. Then, cells were randomly divided into control group (without drug intervention), 10-8mol/L DHT group, 10-5mol/L DHT group, 2×10-5 protein kinase B(Akt) inhibitor (MK-2206 2Hcl) group and 10-8mol/L DHT+ 2×10-5 mol/L MK-2206 2Hcl group according to different intervention method  and experiments，corresponding concentrations were used in each group to intervene cells for 3 hours. After intervention, sites and expression changes of AMH were detected by immunofluorescence. Changes of AMH, Akt and p-Akt were detected by Western blotting.   Results  Immunofluorescence showed that the purity of primary follicular granule cells in rats was 93.33%±3.09%,and AMH was expressed in the membrane and cytoplasm of primary follicular granule cells in rats; Compared with the control group, both 10-8 mol/L DHT and 10-5 mol/L DHT increased the expression of AMH in granulosa cells (P<0.05),but there was no difference in the effect of these two concentrations of DHT on the expression of AMH in granulosa cells (P>0.05); Compared with the control group, 10-8 mol/L DHT increased p-Akt and AMH expression, and 2×10-5MK-2206 2Hcl decreased AMH expression (P<0.05); Compared with the 2×10-5 MK-2206 2Hcl, AMH expression was increased in the 10-8 mol/L DHT+2×10-5 MK-2206 2Hcl treatment group (P<0.05).   Conclusion  DHT can up-regulate the expression of AMH in primary follicular granulosa cells in rats, and this effect may be related to the Akt signaling pathway.

Key words: Dihydrotestosterone, Anti-Müllerian hormone, Follicular granulosa cell, Immunofluorescence, Western blotting, Rat

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