Name: an in vivo estrogen deficiency mouse model for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause
Uploaded: 2019-08-13T14:00:00
Description: Watch this Scientific Journal Video about An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause at JoVE.com

这项工作得到了国家自然科学基金(81202526至J.X.)、中国国家自然科学基金(81302769至B.S.)、北京市自然科学基金(47144226至B.S.)的支持。科学基金会(201110490325 至 J.X.)和中国教育部博士计划基金会 (201211106120031 至 B.S.)。

所有动物护理和实验规程均经中国医学科学院动物护理与使用委员会和北京协和医学院批准(许可号:SYXK(北京)2013-0023)。apoE-/-小鼠的起源是C57BL/6J9,10。
1. 通过 apoE 中的双侧切口进行双边卵巢切除术-/-小鼠
<ol><li>在产间(年龄28天),用避免素(三溴二苯醚乙醇;200毫克/千克;腹内)麻醉雌性apoE-/-C57BL/6J小鼠。 <strong...

<ol>
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这里描述的方法类似于在更年期妇女看到的脂质紊乱和动脉粥样硬化的小鼠模型。有充分证据表明,绝经后妇女的雌激素缺乏会加重先前存在或持续的高胆固醇血症的发病率,并逐渐复杂和广泛地出现动脉硬化病变1。为了模拟临床上容易发生动脉粥样硬化的情况,应用了缺乏动脉硬化的小鼠,这是一种可重复和方便的动物来源,用于研究23、24、25。<sup class="xr...

流行病学和临床研究显示,绝经后妇女患心血管疾病的风险比绝经前妇女1、2要大得多。激素替代疗法(HRT)可降低心血管疾病的相对风险至0.37-0.79 3。在其他并发症中,由心血管疾病引起的动脉粥样硬化是全世界4.然而,缺乏涉及雌激素缺乏小鼠出现动脉粥样硬化倾向状态的实验室模型。该协议提供了一个体内雌激素缺乏小鼠模型,用于筛查绝经后心血管功能障碍的外源性雌激素治疗。
先前的研究表明,在高胆固醇饮食中应用OVX在动脉粥样硬化啮齿动物中可以模仿绝经后妇女患有动脉粥样硬化5、6、7、8。一种类似更年期女性动脉粥样硬化状态的可重复和方便的动物模型是外源雌激素研究的基础。在这里,双侧侧切口的双边卵巢切除术被应用于动脉粥样硬化易脂蛋白E敲除(apoE-/-)小鼠9,10。与中腹或背侧切口相比,双背侧切口是一种更容易、更省时的方法,可以避免严重的腹腔粘附和炎症。通过薄荷传播(见材料表)的渗透管理是非侵入性和方便的,使其广泛适用作为一个长期管理模式11。慢释放颗粒植入也很受欢迎6。然而,植入物可能会加重感染的发生率,特别是在接受OVX的小鼠中。其他非侵入性管理模式,如口腔口和供水管理,也有许多缺点。口腔食道通常对小鼠造成压力,并可能导致食管损伤。通过饮用水管理激素是非常有益的;然而,添加DMSO作为乳化剂是不可避免的,因为外源雌激素不溶于水。在这里,我们选择通过长期给政用的薄荷替代17°-雌二醇或植物雌激素激素。
最近,HRT对绝经后妇女心血管系统的有益作用在妇女健康倡议(WHI)试验12中受到质疑。一方面,仅外源雌激素对心血管系统有有益的影响;另一方面,它可以与醋酸甲酰乙酰丙酮结合,增加心血管事件的风险。更严重的是,HRT可能导致乳房和子宫肿瘤进展,这种效果已经明显限制了其使用13,14。更多的兴趣集中在肿瘤细胞15、16、17中缺乏线粒体活性的外源雌激素的心血管保护作用上。对人类和动物的多项研究表明,具有与雌激素结构相似的植物雌激素在心血管保护方面可以发挥有益的作用。
因此,本工作的目标是(i) 通过双背侧切口在 apoE-/-小鼠中通过双边卵巢切除术建立体内雌激素缺乏小鼠模型,以及 (ii) 比较血管保护作用施用17°-雌二醇和伪丙二醇(PPD),通过薄荷传播。17°-雌二醇是一种外源雌激素,属于女性性激素6,11,19。PPD,一种来自迪奥斯科卡植物的类固醇皂苷和植物雌激素,以前曾被报道具有抗肿瘤的特性20。

<table>
	<tbody>
		<tr>
			<td>17&#946;-estradiol, &#62;98%</td>
			<td>Sigma-Aldrich</td>
			<td>E8875-250MG</td>
			<td>Estrogen</td>
		</tr>
		<tr>
			<td>Disposable syringes (with 25G needles)</td>
			<td>Hunan Luzhou Huikang Development Co., Ltd</td>
			<td>0.5*19TWLB</td>
			<td>Cardiac bleeding</td>
		</tr>
		<tr>
			<td>High-cholesterol mouse diet</td>
			<td>Huafukang Bio-Technology</td>
			<td>N/A</td>
			<td>1.25% cholesterol, 0% cholate</td>
		</tr>
		<tr>
			<td>High-Resolution In Vivo Micro-Imaging System</td>
			<td>VisualSonics</td>
			<td>Vevo&#174;770</td>
			<td>Measurements of intima-media thickness and cardiac dysfunction</td>
		</tr>
		<tr>
			<td>2-Methyl-2-butanol</td>
			<td>Sigma-Aldrich</td>
			<td>152463-250ML</td>
			<td>Preparation of avertin</td>
		</tr>
		<tr>
			<td>Micro Dissecting forceps, Curved 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Micro Dissecting forceps, Straight 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, Curved/Sharp 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, Straight/Sharp 8mm</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Monofilament suture 4-0 1/2 5*12 19mm</td>
			<td>Shanghai Pudong Jinhuan Medical Supplies Co., Ltd</td>
			<td>R413</td>
			<td>Suture and ligation of the tissues</td>
		</tr>
		<tr>
			<td>Nut cream (Nutella)</td>
			<td>Ferrero</td>
			<td>N/A</td>
			<td>Medium for peroral 17&#946;-estradiol or PPD</td>
		</tr>
		<tr>
			<td>OptiVisor optical glass binocular magnifier</td>
			<td>Dohegan Optical Company Inc.</td>
			<td>N/A</td>
			<td>Assistant of identifying the tissues during ovariectomy</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline at pH 7.4</td>
			<td>SIGMA</td>
			<td>P3813</td>
			<td>Preparing 1 L saline</td>
		</tr>
		<tr>
			<td>Pro MultiLabel Microplate Reader</td>
			<td>Tecan</td>
			<td>Infinite M1000</td>
			<td>Plasma TC and TG determination</td>
		</tr>
		<tr>
			<td>Pseudoprotodioscin</td>
			<td>Shanghai Winherb Medical S &#38; T Development</td>
			<td>W-0427</td>
			<td>CAS registry no. 102115-79-7</td>
		</tr>
		<tr>
			<td>Rimadyl, 50 mg/mL</td>
			<td>Pfizer Pharma GmbH</td>
			<td>462986</td>
			<td>Postoperative analgesia after ovariectomy</td>
		</tr>
		<tr>
			<td>Sesame oil</td>
			<td>Sigma-Aldrich</td>
			<td>S3547-1L</td>
			<td>Dissolving the 17&#946;-estradiol or PPD</td>
		</tr>
		<tr>
			<td>Solcoseryl Eye-Gel</td>
			<td>Menarini, Solco Basle Ltd.</td>
			<td></td>
			<td>Eye protection during anesthesia</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>MCALON</td>
			<td>MCL-6STV</td>
			<td>Image of the intimal region of aorta</td>
		</tr>
		<tr>
			<td>Table model high speed centrifuge</td>
			<td>SIGMA</td>
			<td>1-14K</td>
			<td>Preparation of plasma</td>
		</tr>
		<tr>
			<td>Scissors, slight Curve (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Scissors, straight Flat (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Tissue forceps, serrated, slight Curve (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Tissue forceps, serrated, straight Flat (14cm)</td>
			<td>Kanghua Medical Equipment Co., Ltd</td>
			<td></td>
			<td>Surgical tools</td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-5G</td>
			<td>Preparation of avertin</td>
		</tr>
		<tr>
			<td>Triglycerides (TG) assay kit</td>
			<td>Institute of Nanjing Jiancheng Biology Engineering</td>
			<td>A110-1</td>
			<td>Plasma TG determination</td>
		</tr>
		<tr>
			<td>Total cholesterols (TC) assay kit</td>
			<td>Institute of Nanjing Jiancheng Biology Engineering</td>
			<td>A111-1</td>
			<td>Plasma TC determination</td>
		</tr>
	</tbody>
</table>

an in vivo estrogen deficiency mouse model for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause

绝经后妇女患心血管疾病的风险比绝经前妇女大。与卵巢功能完整的雌性小鼠相比,雌性小鼠在失异时出现卵巢切除术(OVX)增加了主动脉动脉粥样硬化的病变。然而,缺乏涉及雌激素缺乏小鼠的实验室模型,这些小鼠具有动脉粥样硬化的易发性状态。这种缺陷是至关重要的,因为更年期妇女的临床雌激素缺乏可能加剧先前存在的或持续的脂质紊乱和动脉粥样硬化的发生率。在这项研究中,我们通过双背侧切口在脂蛋白E(apoE)-/-小鼠中通过双边卵巢切除术建立了体内雌激素缺乏小鼠模型。然后,我们比较17°-雌二醇和伪原蛋白素(PPD)(植物雌激素)通过薄杉醇传播的内给的影响。我们发现,虽然PPD对降低OVX apoE-/-小鼠的最终体重和血浆TG有一定作用,但它具有抗动脉粥样硬化和心脏保护能力,与17°雌二醇相当。PPD是一种植物雌激素,已报告发挥抗肿瘤的特性。因此,该方法适用于通过口服给雌激素筛选植物雌激素,以取代绝经后妇女的传统激素替代疗法,据报告,这种疗法具有潜在的有害肿瘤遗传能力。通过木耳传播的口服给政是无创的,使其广泛适用于许多患者。本文包含通过 apoE-/-小鼠和 Peroral 17°-雌二醇或植物雌激素激素通过薄荷传播的双背侧切口进行双边卵巢切除术的逐步演示。血浆脂质和心血管功能分析使用超声心动图跟踪。

本研究中使用的一种典型的实验治疗方案如图1所示。在产间(年龄28天),雌性apoE-/-C57BL/6J小鼠用非他林(三溴二苯醚乙醇;200毫克/千克;腹内)进行麻醉。小鼠通过1厘米背切口进行双边OVX或假手术。在双边OVX一周后,小鼠被喂食高胆固醇饮食(1.25%胆固醇,0%乔拉)12周。17°-雌二醇(0.1毫克+千克-1)或PPD(0.5毫克+kg-1)在治疗的最后9周内通过薄桃传播?...

Watch this Scientific Journal Video about An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause at JoVE.com

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

临床上,更年期妇女雌激素缺乏可能加重脂质紊乱和动脉粥样硬化的发生率。我们通过在apoE-/-小鼠的双背侧切口,通过双边卵巢切除术建立了体内雌激素缺乏模型。小鼠模型适用于绝经后心血管功能障碍的外源雌激素治疗。

用于筛查绝经后心血管功能障碍的外源雌激素治疗的活体雌激素缺乏小鼠模型

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

缺乏涉及雌激素缺乏小鼠的实验室模型，这些小鼠提供动脉硬化性气孔数据。这个过程对更年期后心血管功能障碍的外源性雌激素治疗特别有帮助。该技术的主要优点是，没有双多面切口的奥瓦里切除术是一种更容易，更不耗时的方法，避免严重的腹腔粘附和炎症。演示这个程序的将是我实验室的研究生张音。要对APOE敲除小鼠进行双边曲张切除术，首先确认对踏板反射缺乏反应，然后将动物放在加热垫上的易发位置。将软膏涂抹在动物的眼睛上，用手术窗帘盖住动物。做一厘米的切口侧向中线，一厘米的侧向到成本肋骨，并使用钳子钝解皮下组织。使用解剖护目镜，识别腹腔中的白色脂肪组织。使用微型剪刀和微型钳子，通过筋膜进行0.5至1厘米的切口，直到腹腔到达，并使用微钳轻轻缩回白色脂肪组织。在腹腔内应观察用脂肪组织包裹的粉红色桑状卵巢。使用单丝缝合线将0.5至1厘米近角和子宫角缝合，并使用微型剪刀收获卵巢。将剩余的组织返回到腹腔，并使用单个单丝缝合线单独关闭肌肉和皮肤层。然后像刚才演示的，在动物的对侧一侧收获卵巢，并允许鼠标在加热垫上恢复，并监测，直到完全恢复。每周一次，从手术前一周到手术后12周，测量每个假动物和实验动物的体重平衡。对于血浆总胆固醇和甘油三酯测量，在第12周，准备腹腔面积与70%乙醇，并插入25规格的针到右心室。慢慢吸气，直到一毫升血液流入注射器，将样品放入含有10微升0.5摩尔EDTA的1.5毫升收集管中。将管完全反转几次，将EDTA混合到血液中，并立即将管子放在冰上。在采集的30分钟内，在400倍的G和4摄氏度下将样品离心20分钟。小心收集血浆上一液，在零下80摄氏度储存。为了分离大母，在第12周，将一根25量针连接到一个装满PBS的50毫升注射器上，并将针头插入实验动物的左心室。切开右中庭，避免灌注的高压，以每分钟05至08毫升PBS的速度灌注心脏组织。用实验室组织吸收灌注液，用剪刀和钳子去除肋骨和肺。打开腹腔切除内脏，以便更好地查看主动脉。用微钳抓住心脏，使大干从脊柱上分离，直到腹腔分裂。然后用4%的半形式醛将心脏和大管固定48小时，然后在室温下或2至8摄氏度的盐水中储存组织样本数小时。经过12周的高胆固醇饮食后，奥瓦里切除小鼠表明血浆总胆固醇和甘油三酯浓度显著增加。Ovari切除小鼠，每用17-β雌性二醇（PPD通过粽子传播）进行口头管理，比假操作小鼠的血浆总胆固醇浓度显著降低。血浆甘油三酯水平下降在奥瓦里切除小鼠，每口头管理与17-β二醇，但不是与PPD。虽然在奥瓦里切除小鼠中观察到体重增加的趋势，但与假操作的小鼠相比，在四组动物之间总体体重没有显著变化。与所有其他组相比，上升大动脉的最大斑块在奥瓦里切除小鼠中增加，同时在这些动物中观察到的心脏功能整体下降。此外，与整个大动脉面积相比，大动脉病变区的平均百分比在奥瓦里切除小鼠中显著增加。正如预期的那样，这些病变的存在减少与17-β雌性二醇，或PPD治疗。切除手术的主要风险是尿管结扎，导致高死亡率。还要记得在肾动脉分支附近切深，避免主动脉损伤。

Research

JoVE Journal

Biochemistry

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

热通菌膜-双磷酶抑制剂的筛选

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  ÃƒÅ½Ã‚Â² Reporter Assay

使用基于细胞的雌激素受体对植物雌激素进行筛查β记者分析

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

将新生儿小鼠心脏巨噬细胞移植到成年小鼠体内

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...