Name: generation of a mouse artificial decidualization model with ovariectomy for endometrial decidualization research
Uploaded: 2022-07-27T15:00:00
Description: Watch this Scientific Journal Video about Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research at JoVE.com

笔者希望感谢国家自然科学基金（82001629，XQS）、江苏省自然科学基金青年计划（BK20200116，XQS）和江苏省博士后科研基金（2021K277B，XQS）的支持。

所有描述的动物实验均由南京大学医学院附属鼓楼医院动物使用与护理委员会（第20171202号）批准。所有操作均遵循适当的动物护理和使用机构以及国家准则。
注意：小鼠在特定的无病原体（SPF）环境中饲养，温度为22°C±1°C，相对湿度为50%±1%，光照/黑暗循环为12小时/ 12小时，并且可以自由获取食物和水。
1.小鼠卵巢切除术
<ol><li>手术前1?...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterine+glands+coordinate+on-time+embryo+implantation+and+impact+endometrial+decidualization+for+pregnancy+success.">Uterine glands coordinate on-time embryo implantation and impact endometrial decidualization for pregnancy success.</a> Nature Communications. 9 (1), 2435 (2018).</li><li>Tian, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+monoamine+oxidase+a+impairs+endometrial+receptivity+in+women+with+adenomyosis+via+downregulation+of+FOXO1dagger.">Attenuated monoamine oxidase a impairs endometrial receptivity in women with adenomyosis via downregulation of FOXO1dagger.</a> Biology of Reproduction. 105 (6), 1443-1457 (2021).</li><li>Large, M. J., DeMayo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+embryo+implantation+and+endometrial+decidualization+by+progesterone+receptor+signaling.">The regulation of embryo implantation and endometrial decidualization by progesterone receptor signaling.</a> Molecular and Cellular Endocrinology. 358 (2), 155-165 (2012).</li><li>Dunn, C. L., Kelly, R. W., Critchley, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decidualization+of+the+human+endometrial+stromal+cell:+an+enigmatic+transformation.">Decidualization of the human endometrial stromal cell: an enigmatic transformation.</a> Reproductive BioMedicine Online. 7 (2), 151-161 (2003).</li><li>Zhu, H., Hou, C. C., Luo, L. F., Hu, Y. J., Yang, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+stromal+cells+and+decidualized+stromal+cells:+Origins,+transformation+and+functions.">Endometrial stromal cells and decidualized stromal cells: Origins, transformation and functions.</a> Gene. 551 (1), 1-14 (2014).</li><li>Jose, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+and+decidual+stromal+precursors+show+a+different+decidualization+capacity.">Endometrial and decidual stromal precursors show a different decidualization capacity.</a> Reproduction. 160 (1), 83-91 (2020).</li><li>Pan-Castillo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphophysical+dynamics+of+human+endometrial+cells+during+decidualization.">Morphophysical dynamics of human endometrial cells during decidualization.</a> Nanomedicine. 14 (7), 2235-2245 (2018).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+mouse+decidualization+models+at+the+molecular+level.">Comparative analysis of mouse decidualization models at the molecular level.</a> Genes. 11 (8), 935 (2020).</li><li>De Clercq, K., Hennes, A., Vriens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+endometrial+epithelial+and+stromal+cells+for+in+vitro+decidualization.">Isolation of mouse endometrial epithelial and stromal cells for in vitro decidualization.</a> Journal of Visualized Experiments. 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小鼠的蜕膜化是一个自发的过程，取决于胚胎的存在，这与人类不同。然而，已经发现人工刺激，如子宫注射玻璃珠和子宫撕裂，可以诱导子宫内膜而不是胚胎的蜕膜化。此外，研究人员发现，许多因素可以诱导蜕膜或参与蜕膜化，例如将类固醇激素，前列腺素和生长抑制因子注射到子宫腔中21。与上述建模方法相比，这里详述的卵巢切除术小鼠模型，使用芝麻油作为兴奋剂，?...

随着人类辅助生殖技术的发展，目前体外受精-胚胎移植（IVF-ET）的临床妊娠率已经达到甚至超过自然妊娠。尽管如此，许多辅助生殖临床实践中的患者仍然接受多次胚胎移植，但未能如愿以偿。但其具体分子机制尚不明确，因此临床干预无效，是生殖医学面临的重大挑战之一1，2。
子宫内膜因素约占IVF失败原因的三分之二3。人类胚胎植入分为三个阶段：定位、粘附和侵袭4，5，6。母体子宫内膜经历一系列变化以满足胚胎的到来。形成植入“窗口期”为胚胎植入提供了有利条件7，8。
在大多数哺乳动物中，囊胚粘附在子宫的管腔上皮后，囊胚周围的基质细胞迅速开始增殖和分化，间充质的快速重塑改变了其形状和功能，导致胚胎着床5，9，10。部位体积和重量的快速增加使囊胚嵌入子宫基质中，这一过程称为蜕膜化11。子宫内膜基质在准备怀孕时分化和重塑，而基质细胞的过渡为蜕膜细胞提供空间和新的信号连接以执行其功能12，13。基质细胞转化为蜕膜细胞并分泌许多标志性因子，如催乳素（PRL）、胰岛素样生长因子结合蛋白1（Igfbp1）等。研究表明，蜕膜化异常是胚胎着床失败的关键原因之一，但蜕膜化异常的原因尚不清楚，需要进一步阐明1，14。
小鼠人工蜕膜化模型对于研究蜕膜化的生理过程和分子机制至关重要。人工蜕膜化（AD）主要是指通过人工方法建立的子宫内膜蜕膜化过程，以模拟怀孕或月经周期。在形态方面，妊娠蜕膜化和人工蜕膜化总体差异不大15，16。子宫腺在蜕膜形成之前存在于子宫内膜中，在蜕膜化后消失。关于基因表达，自然妊娠蜕膜化（NPD）和AD15之间仅略有差异。因此，小鼠人工蜕膜化模型可以模拟妊娠蜕膜化，探索人类生殖疾病的未知发病机制和新疗法。
NPD，AD和 体外 蜕膜化（IVD）是实现小鼠蜕膜化的三种方法。NPD模型依赖于自然怀孕，最接近母体的生理状态，包括胚胎的影响。比较植入和非植入部位之间的差异是研究蜕膜化的一种更生理、更方便的方法。AD模型是通过使用宫内注射芝麻油作为兴奋剂来诱导假怀孕雌性小鼠与输精管切除的雄性小鼠进行蜕膜化以避免胚胎的影响而开发的。NPD和AD模型在不同的研究目的中起着至关重要的作用，但它们无法避免母体激素代谢的不同活动引起的交配失败和组内差异。IVD是一种依赖于雌激素和孕酮联合在细胞水平上的治疗方法，需要更严格的实验条件和操作能力。然而， 体外 模型不能完全模拟生理条件下的蜕膜反应15。因此，我们提出了一种从传统AD改进而来的简单而改进的诱导方法，以减少内源性激素对蜕膜化的影响。在保证蜕膜化诱导成功的基础上，更接近生理状态，更适合需要排除胚胎因素的实验。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Estrogen</td>
			<td >Sigma</td>
			<td >E2758</td>
			<td >Hormone supplement</td>
		</tr>
		<tr >
			<td >Progesterone</td>
			<td >Sigma</td>
			<td >P0130</td>
			<td>Hormone supplement</td>
		</tr>
		<tr >
			<td >Sesame oil&#160;</td>
			<td >Sigma</td>
			<td>S3547</td>
			<td>Hormone supplement</td>
		</tr>
		<tr >
			<td >Sodium pentobarbital&#160;</td>
			<td >Dainippon Sumitomo Pharma Co.,Ltd.</td>
			<td ></td>
			<td>Anaesthesia</td>
		</tr>
		<tr >
			<td >Meloxicam injection</td>
			<td >Qilu Animal Health Products Co., Ltd</td>
			<td ></td>
			<td>Analgesia</td>
		</tr>
		<tr >
			<td >Alkaline phophatase stain kit(kaplow&#39;s/azo coupling method)</td>
			<td >Solarbio</td>
			<td>G1480</td>
			<td>Alkaline phophatase stain</td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td >Servicebio</td>
			<td>G1005-2</td>
			<td>HE stain</td>
		</tr>
		<tr >
			<td >Hematoxylin</td>
			<td >Servicebio</td>
			<td >G1005-1</td>
			<td>HE stain</td>
		</tr>
		<tr >
			<td >ChamQ Universal SYBR qPCR Master Mix</td>
			<td >Vazyme</td>
			<td>Q711-02</td>
			<td>qPCR</td>
		</tr>
		<tr >
			<td >70% ethanol</td>
			<td >Lircon</td>
			<td>ZH1120090</td>
			<td>Disinfect</td>
		</tr>
		<tr >
			<td >Iodophor</td>
			<td >Runzekang</td>
			<td >RZK-DF</td>
			<td>Disinfect</td>
		</tr>
		<tr >
			<td >Erythromycin Eye Ointment</td>
			<td >Guangzhou Baiyunshan</td>
			<td ></td>
			<td>Mice eyeball protect</td>
		</tr>
		<tr >
			<td >4-0 suture</td>
			<td >Ethicon</td>
			<td >W329</td>
			<td>Incision suture</td>
		</tr>
		<tr >
			<td >10% formalin</td>
			<td >Yulu</td>
			<td >L25010118</td>
			<td>Tissue fix</td>
		</tr>
		<tr >
			<td >Optimal cutting temperature compound</td>
			<td >Sakura</td>
			<td >4583</td>
			<td>Ssection</td>
		</tr>
		<tr >
			<td >Trizol reagent</td>
			<td >Ambion</td>
			<td >15596018</td>
			<td>qPCR</td>
		</tr>
	</tbody>
</table>

generation of a mouse artificial decidualization model with ovariectomy for endometrial decidualization research

子宫内膜蜕膜化是子宫内膜独特的分化过程，与月经和怀孕密切相关。蜕膜化障碍导致各种子宫内膜疾病，如不孕、复发性流产和早产。子宫内膜蜕膜化模型在生殖研究中的开发和应用长期以来一直是生殖研究者的一大亮点。鼠标已被广泛用于研究繁殖和蜕膜化。关于蜕膜化，有三种成熟的小鼠模型，即自然妊娠蜕膜化（NPD），人工蜕膜化（AD）和 体外 蜕膜化（IVD）。其中，AD被认为是小鼠蜕膜化的可靠模型，易于实现且接近NPD。本文重点介绍了一种改进的小鼠人工蜕膜化模型的生成和应用方法，以避免卵巢效应，该方法可以获得组内方差小的可重复性结果。该方法为子宫内膜蜕膜化的研究提供了良好可靠的动物模型。

小鼠蜕膜化模型指标包括子宫的一般形态、蜕膜化和非蜕膜化子宫的质量比、子宫内膜的组织学形态、蜕膜化标志物分子的表达水平。油诱导小鼠人工蜕膜子宫的一般形态更接近妊娠期子宫。子宫体变厚，子宫腔比非诱导侧小。诱导子宫角的体积和重量明显高于非诱导侧（图3A，B）。子宫组织HE染色显示，腺体消失，诱导角上的子宫内膜基质细胞分化为大、圆形...

Watch this Scientific Journal Video about Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research at JoVE.com

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

在这里，我们描述了使用卵巢切除小鼠生成人工蜕膜化模型的方法，这是子宫内膜蜕膜化研究领域的经典子宫内膜蜕膜化实验。

用于子宫内膜蜕膜化研究的卵巢切除术小鼠人工蜕膜化模型的生成

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

权力下放的损害导致子宫内膜疾病的病毒，例如死亡，复发性流产。该方法为子宫内膜分散的研究提供了良好可靠的动物模型。这种人工分散的卵巢切除术模型可以避免安吉丽娜对激素的影响，这通常可以高度可重复的结果，组内差异很小。在手术前一天开始用高温高压灭菌器对手术器械进行消毒。将保护性眼膏涂抹在眼球上，以防止麻醉期间干燥。当小鼠完全麻醉时开始手术。将小鼠置于手术表面上的俯卧位置，并在用肥皂水润湿后剃掉背部的毛发。用70%乙醇消毒皮肤。在平行于脊柱的双后肢大腿根部上缘 0.5 厘米处找到手术切口部位。以切口部位为中心，用碘伏对切口区域消毒三次。用剪刀做一个约0.5到1厘米的纵向切口。切开筋膜并用镊子被动地分开肌肉。通过薄肌肉层在靠近肾脏下极的切口区域找到一块白色脂肪垫。用止血夹夹住脂肪块，并将脂肪垫包裹的卵巢从切口中拉出。用弯曲的镊子夹住卵巢和输卵管的连接处，并用剪刀沿着卵巢蒂取出卵巢。使用电凝笔或在酒精灯上加热的一毫升注射器的针头止血。用生理盐水冲洗手术切口以防止组织粘连。使用4-0缝合线分别播种肌肉和皮肤，并再次用碘伏对手术切口进行消毒。将小鼠置于笼子中的俯卧位置，并在25摄氏度的培养箱中复苏。配备光源和通风系统约两个小时。手术后注意小鼠。将它们单独放回原来的喂食处，直到它们完全恢复并给它们足够的水和食物。皮下注射100纳克雌激素和50微升芝麻油到每只小鼠中三天，然后休息两天。在50微升芝麻油中向每只小鼠注射1毫克黄体酮和10纳克雌激素，再注射三天。操作小鼠在第三次雌激素孕酮联合注射后6小时诱导人工残留模型。找到输卵管下端的子宫角，沿子宫角缓慢注入20微升芝麻油。将组织推回，缝合切口，让小鼠恢复。用嵌入石蜡中的10%福尔马林固定三到五毫升的子宫组织并切片。用苏木精和伊红染色切片以观察形态变化。将另外三到五毫米的子宫组织嵌入最佳切割温度化合物中，在液氮中冷冻并储存在零下80摄氏度的冰箱中。在10微米内冷冻切片组织，并通过偶氮偶联方法检测子宫组织中的碱性磷酸酶活性。由油诱导的小鼠人工分散子宫的一般形态更接近妊娠期子宫。子宫体变厚，子宫腔比非诱导侧小。诱导子宫角的体积和重量明显高于非诱导侧。苏木精和子宫组织伊红染色显示，腺体消失，诱导撕裂的子宫内膜末期基质分化为大的圆形细胞质和多核蜕膜细胞，细胞边界不明确。非诱导侧切片显示正常生理状态下的子宫内膜组织形态。偶氮偶联法结果表明，油诱导侧碱性磷酸酶含量显著高于非诱导侧。QPCR结果显示，PRL-8-A-2、ALPL和IGFBP-1在诱导角中的mRNA表达显著高于非诱导角，表明油可以诱导小鼠人工分散。Homo 補充是一個漫長的過程，需要特別注意 homo 之間的污染，這是成功之鍵。在此模型的基础上，可以进行子宫角注射腺病毒，研究特定分子调控残留的相关机制。

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

A Mouse Distraction Osteogenesis Model

小鼠牵张成骨模型

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of Multicellular Human Primary Endometrial Organoids

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...