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Introduction
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Biologia do Desenvolvimento

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

Published: October 02, 2018
doi:
10.3791/58379
, , , , , ,
1Department of Pediatrics,Johns Hopkins University School of Medicine, 2Department of Health,Beijing Military General Hospital, 3Southern Medical University, 4Department of Molecular and Cellular Physiology,Johns Hopkins University School of Medicine, 5Department of Gynecology and Obstetrics,Johns Hopkins University School of Medicine

Summary

我们描述了一个瘦的 pcos 样小鼠模型的发展与 dihydrotestosterone 丸研究 pcos 的病理生理学和这些 pcos 样水坝的后代。

Abstract

Hyperandrogenemia 在女性生殖和代谢功能中起着至关重要的作用, 是多囊卵巢综合征的标志。开发一种瘦的 pcos 样小鼠模型, 模仿妇女与 PCOS 是临床意义。在本协议中, 我们描述了这样一个模型。通过插入4毫米的氢睾酮 (dihydrotestosterone) 晶体粉末颗粒 (总长度的颗粒是8毫米), 并在每月更换, 我们能够产生一个 PCOS 样的小鼠模型与血清氢睾酮水平2倍高于未植入 dht (无氢睾酮) 的小鼠。我们观察到生殖和代谢功能障碍, 而不改变体重和身体组成。在表现出高度不孕症的同时, 这些 PCOS 样雌性小鼠的一小部分可以怀孕, 他们的后代显示延迟青春期和增加睾酮作为成人。这种 pcos 样瘦老鼠模型是一个有用的工具, 研究 pcos 的病理生理学和这些 pcos 样水坝的后代。

Introduction

Hyperandrogenism 是多囊卵巢综合征 (PCOS) 的标志, 根据 NIH 标准和雄激素过剩和 pcos (AE pcos) 社会。患有 PCOS 的妇女很难怀孕, 并增加了妊娠并发症的风险1。即使他们怀孕了, 他们的雌性后代也会有不良的健康结局2,3。动物模型已经开发使用各种策略4,5,6,7,8,9,10,11,12并表现出多囊卵巢综合征 (停止排卵、葡萄糖和胰岛素耐受) 的特点, 体重增加和肥胖与脂肪细胞体积增大和脂肪细胞重量增加有关。生产用于研究 PCOS 的动物模型有两种主要的策略。一是治疗与高水平的雄激素直接 (外源性雄激素注射/插入) 或间接 (如阻断雄激素转化为雌激素与芳香化酶抑制剂) 出生后13。另一种是由胎儿 hyperexposure 的雄激素在妊娠期14,15学习的后代。例如, 恒河猴16、17、绵羊18的雌性后代和在宫内期间接触雄性雄激素水平的啮齿类动物, 在以后的生活中发育成 PCOS 样特征。这些模型大大增强了我们对雄激素效应的认识, 以及胎儿的编程和子宫环境的影响。然而, 这些模型有自己的局限性: 1) 动物发育肥胖, 因此很难将 hyperandrogenemia 的影响与肥胖引起的生殖和代谢功能障碍分开;2) 在怀孕前, 有 PCOS 的妇女已经表现出高水平的雄激素, 因此卵母细胞在受精前暴露在雄激素过剩的水平;3) 出生后或妊娠期间使用的睾酮 (T) 或 dihydrotestosterone (DHT) 的药理剂量可能不能反映 PCOS 的雄激素环境。在卵巢滤泡液和/或血清中测定睾酮和 dht 水平, 睾酮和 dht 水平在女性 PCOS5、19、20、21 的妇女中高出1.5 至3.9 倍. ,22,23与未受影响的妇女相比。我们创建了一个成年小鼠模型23,24,25 , 发展生殖和代谢功能障碍在两周内启动慢性双氢睾酮暴露从插入一个球团与4mm 长度的水晶 DHT 粉 (球团总长度为 8mm)。该模型产生的血清氢睾酮水平约2 倍以上 (称为 2xDHT) 比控制小鼠没有 DHT 治疗。2xDHT 小鼠不表现基底血清雌二醇、睾酮、LH 和不发育肥胖的变化, 并显示类似的卵巢重量, 血清胆固醇水平, 游离脂肪酸, 瘦素, TNFα和 IL-623,24, 25相对于控制, 甚至3.5 月后, DHT 插入23,24,25。此外, 通过交配雌性已经开发出 PCOS 的特点, 我们可以研究 hyperandrogenic 的产妇环境对后代的新陈代谢和生殖健康的影响15。

这种新范式 (与 NIH 和 AE-PCOS 社会标准有关) 通过生产相对较不受影响的妇女的雄激素水平与 2-到3倍高的睾酮或 DHT 水平较高的男性荷尔蒙的比例来模拟这种疾病。然而, 这一模型是由连续外源双氢睾酮, 而不是从编程内源 hyperandrogenism 一旦 DHT 被撤回。本文的总体目标是集中于 1) 如何使 DHT 颗粒;2) 如何生成像小鼠模型这样的瘦 PCOS;3) 评估来自这些水坝的雌性后代的战略。对表型的其他测量和评估在本手稿中没有提到, 但可以在5、15、23、24、25、26中找到。

Protocol

在这里, 我们提出了氢睾酮颗粒的准备和插入, 以及生殖和新陈代谢测试的详细协议。在这项研究中使用的老鼠是一个混合的背景 (C57/B6, CD1, 129Sv), 并保持与食品和水广告随意在14/10 小时/暗循环24°c 在百老汇研究建筑动物设施在约翰霍普金斯大学学校医学。所有程序均经约翰霍普金斯大学动物保育和使用委员会批准。 1. 创建类似 PCOS 的小鼠模型 氢睾酮颗?…

Representative Results

血清双氢睾酮水平与葡萄糖耐受试验 用 ELISA 法和 LC-MS 根据1.24–1.25 和2.9、3.0 测定血清中的双氢睾酮水平。双氢睾酮的绝对值在质谱和 ELISA 上是不同的, 但是, dht 的相对折叠 (大约2倍) 与无氢睾酮的插入是相似的从测试和横跨实验15,23,24 (图 2A<…

Discussion

Hyperandrogenism 是 PCOS 的一个重要特征。本协议所用的血清氢睾酮水平 (dht 小鼠的两倍以上) 比以前的研究中其他研究者报告的要低, 并且被校准成比例模拟的妇女与 PCOS5,19, 20,21。与其他模型不同的是, 这种2倍的 dht 模型不会改变体重和全身成分, 与无氢睾酮的小鼠相比, 3.5 月后, dht 插入23</s…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家卫生研究院 (赠款 R00-HD068130 白雪) 和巴尔的摩糖尿病研究中心的支持: 飞行员和可行性赠款 (白雪)。

Materials

Crystalline 5α-DHT powder   Sigma-Aldrich A8380-1G
Dow Corning Silastic tubing Fisher Scientific 11-189-15D 0.04in/1mm inner diameter x0.085in/2.15mm outer diameter
Medical adhesive silicone  Factor II, InC.  A-100
Goggles, lab coats, gloves and masks.
 10 µL pipette tips without filter USA Scientific 11113700
Microscope slide for smear Fisher Scientific 12-550-003
Diff Quik for staining cells Fisher Scientific NC9979740
  Lancet Fisher Scientific NC9416572
3 mL Syring  Becton, Dickinson and Company (BD), 30985
 attached needle: 20G BD 305176
 Ruler: any length than 10cm with milimeter scale. 
Xylazine  Vet one AnnSeA LA, MWI, Boise NDC13985-704-10 100mg/ml
Ketamine Hydrochloride Hospira, Inc NDC 0409-2051-05 100mg/ml
 Surgical staple  AutoClip® System, Fine Science Tool 12020-00
 Insulin syringe BD 329461 1/2 CC, low dose U-100 insulin syringe
 Trochar  Innovative Research of America MP-182
Microscope Carl Zeiss Primo Star 415500-0010-001 Germany
Ear punch Fisher Scientific 13-812-201
Testosterone rat/mouse ELISA kit IBL B79174
DHT ELISA kit Alpha Diagnostic International 1940
One touch ultra glucometer Life Scan, Inc.
One touch ultra test stripes Life Scan, Inc.
Eppendorf tube Fisher Scientific 05-402-18
Razor blade Fisher Scientific 12-640
Clidox Fisher Scientific NC0089321
surgical underpad Fisher Scientific 50587953 Supplier Diversity Partner
Manufacturer:  Andwin Scientific 56616018
Betadine Antiseptic Solution Walgreens
3M Vetbond (n-butyl cyanoacrylate) 3M Science. Applied to Life
Animal tattoo ink paste Ketchum manufacturing Inc. Brockville, Ontario, Canada
Scale Ohaus Corporation  HH120D Pine Brook, NJ
Electronic digital caliper NEIKO Tools USA 01407A available from Amazon
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Tags

Keywords: Polycystic Ovary SyndromePCOSHyperandrogenismDihydrotestosteroneDHTMouse ModelSilicone TubingPellet ImplantationSurgical ProcedureAndrogen Exposure
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Xue, P., Wang, Z., Fu, X., Wang, J., Punchhi, G., Wolfe, A., Wu, S. A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome. J. Vis. Exp. (140), e58379, doi:10.3791/58379 (2018).
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