Name: using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities
Uploaded: 2021-03-21T15:00:00
Description: Watch this Scientific Journal Video about Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities at JoVE.com

这项工作得到了中国国家自然科学基金委员会的支持（91643203号，91543208，81502835）。

所述的所有程序均由青岛大学动物保护与使用委员会（IACUC）批准。在我们的实验室里，卵子被孵育在两个孵化器里。鸡蛋直立在孵化器里，随机放在货架上。卵子的孵化条件如下：孵化温度从37.9°C开始，随着孵化的进行逐渐下降到37.1°C：湿度从 50% 开始，逐渐增加到 70%。
1. 曝光方法
注：通过多种方式将环境污染物暴露在鸡胚胎中。在本节中，详细描述?...

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鸡胚胎在发育研究中一直是200多年的经典模型。本手稿中介绍的方法已用于评估多个环境污染物，包括全氟异丙酸、颗粒物和柴油废气，成功5、7、8、9、10、11、12。通过这些方法，发展性心毒性得到了经...

鸡胚胎是一种经典的发育模式，已使用两百多年。与传统模型相比，鸡胚胎模型具有多种优点。首先，早在70多年前，鸡胚胎的正常发育就已在汉堡-哈密尔顿分期指南2中得到了非常清晰的说明，其中共确定了鸡胚胎发育过程中的46个阶段，具有精确的时间和形态特征，便于检测异常发育。此外，鸡胚胎模型还有其他特点，如成本相对较低，数量多余，暴露剂量控制相对准确，壳内有一个独立的封闭系统，以及容易操作发育中的胚胎，所有这些都保证了其作为强大的毒理学评估模型的潜力。 
在心毒性方面，鸡胚胎具有四室心脏，类似于哺乳动物心脏，但壁较厚，更容易进行形态评估。此外，鸡胚胎允许发育吸入暴露，这在哺乳动物模型中是不可能的：在发育的后期阶段，鸡胚胎将从内部呼吸过渡到外部呼吸（通过肺部获得氧气）：后者要求胚胎用嘴穿透空气细胞膜，并开始呼吸空气3，使空气细胞成为一个小型吸入室。利用这一现象，可以评估气体污染物对心脏（和其他器官）的毒理作用，而无需专门的吸入室仪器。 
在这篇手稿中，介绍了几种暴露/终点评估方法，所有这些方法都有助于使鸡胚胎成为评估接触环境污染物后发展性心毒性的有力工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% phosphate buffered formaldehydefixative</td>
			<td>Biosharp, Hefei, China</td>
			<td>REF: BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Guoyao,Shanghai,China</td>
			<td>CAS:64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosignaling monitor BL-420E+</td>
			<td>Taimeng, Chengdu, China</td>
			<td>BL-420E+</td>
			<td></td>
		</tr>
		<tr>
			<td>Candling lamp</td>
			<td>Zhenwei, Dezhou, China</td>
			<td>WZ-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringe</td>
			<td>Zhiyu, Jiangsu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Keyu,Dezhou, China</td>
			<td>KFX</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical balance</td>
			<td>OHAUS, Shanghai, China</td>
			<td>AR 224CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro-thermal incubator</td>
			<td>Shenxian, Shanghai, China</td>
			<td>DHP-9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Guoyao,Shanghai,China</td>
			<td>CAS:64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertile chicken egg</td>
			<td>Jianuo, Jining, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Staining Kit</td>
			<td>Beyotime, Bejing, China</td>
			<td>C0105</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology paraffin</td>
			<td>Aladdin, Shanghai, China</td>
			<td>P100928-500g</td>
			<td>Melt point 52~54&#176;C</td>
		</tr>
		<tr>
			<td>Histology paraffin</td>
			<td>Aladdin, Shanghai, China</td>
			<td>P100936-500g</td>
			<td>Melt point 62~64&#176;C</td>
		</tr>
		<tr>
			<td>IV catheter</td>
			<td>KDL, Zhejiang, China</td>
			<td></td>
			<td>The catheters have to be soft, plastic ones.</td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>Genechem, Shanghai, China</td>
			<td></td>
			<td>The lentivirus were individually designed/synthesized by Genechem.</td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome staining kit</td>
			<td>Solarbio, Beijing, China</td>
			<td>G1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal probe</td>
			<td>Jinuotai, Beijing, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector (5 uL)</td>
			<td>Anting,Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>CAIKON, Shanghai, China</td>
			<td>XSP-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Germany</td>
			<td>HistoCore BIOCUT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome blade</td>
			<td>Leica,Germany</td>
			<td>Leica 819</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbitual sodium</td>
			<td>Yitai Technology Co. Ltd.,&#160; Wuhan, China</td>
			<td>CAS: 57-33-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter(10ul)</td>
			<td>Sartorius, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodide</td>
			<td>Longyuquan, Taian, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Anqisheng,Suzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Kelun,Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sunflower oil</td>
			<td>Mighty Jiage, Jiangsu, China</td>
			<td></td>
			<td>Any commerical sunflower oil for human consumption should work</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>M&#38;G, Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tedlar PVF Bag (5L)</td>
			<td>Delin, Dalian, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>SCILOGEX, Rocky Hill, CT, US</td>
			<td>MX-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Guoyao,Shanghai,China</td>
			<td>CAS:1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

using chicken embryo as a powerful tool in assessment of developmental cardiotoxicities

鸡胚胎是发育研究的经典模型。在鸡胚胎发育过程中，对心脏发育的时间窗口进行了明确的界定，通过多种方法实现精确、及时的暴露相对容易。此外，鸡胚胎中的心脏发育过程与哺乳动物相似，也产生了四室心脏，使其成为评估发育性心毒性的宝贵替代模型。在我们的实验室中，鸡胚胎模型经常用于评估接触各种环境污染物后的发展性心毒性，包括单氟烷基物质和多氟烷基物质（PFAS）、颗粒物（PMs）、柴油排气（DE）和纳米材料。暴露时间可以根据需要自由选择，从发育开始（胚胎日 0，ED0）一直到孵化前一天。主要的暴露方法包括空气细胞注射、直接微注射和空气细胞吸入（最初在我们的实验室中开发），目前可用的终点包括心脏功能（心电图）、形态学（组织学评估）和分子生物学评估（免疫造血化学、qRT-PCR、西式印迹等）。当然，鸡胚胎模型也有其自身的局限性，例如抗体的可用性有限。然而，随着越来越多的实验室开始利用这一模型，它可以用来为发展性心毒性的研究作出重大贡献。

曝光结果 空气电池注射 空气细胞注射可以有效地将发育中的鸡胚胎暴露在各种制剂中，随后可以在胚胎/孵化鸡的采集样本（血清、组织等）中检测到这些样本。下面是一个例子，其中全氟辛烷磺酸 （PFOA） 被注入空气细胞，然后用超性能液相色谱质量光谱法确定血清全氟辛烷磺酸浓度。血清浓度与注射剂量相对应，表明此程序的有效性（图6）。
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Watch this Scientific Journal Video about Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities at JoVE.com

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

鸡胚胎作为一种经典的发育模型，在我们的实验室中用于评估接触各种环境污染物后的发育性心毒性。本手稿中介绍了暴露方法和建立的形态/功能评估方法。

使用鸡胚胎作为评估发育性心脏病的有力工具

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

大家好。我是青岛大学公共卫生学院毒理学系副教授蒋启晓。我将代表我的研究小组介绍使用鸡胚胎进行心毒性评估的资产。鸡胚胎是一种经典的发育模式，具有操作方便、可访问性高、封闭式独立系统等多种优点。处理多种环境污染物，如酚类晒黑酸。柴油排气，尤其是碳纳米管上的物质。我们在实验室中建立了多种暴露方法，但对于更大的原因，功能评估也定期执行。在这段视频中，我将向您展示曝光方法和形态功能评估方法的细节。如果您有兴趣，请随时与我们联系。谢谢。以下是我们可以向您介绍的暴露方法。现在，我要介绍的第一种方法是空气细胞注射，在那里你将直接注射到空气细胞。用多碘化物溶液清洁鸡蛋表面。这是市售的多碘化物溶液，用去离子水稀释，1/5。然后，我用纸巾擦干蛋壳，不擦洗。这很重要，因为擦洗会打破外壳的保护层涂层。用铅笔清洁黑暗房间里所有的鸡蛋和空气电池的标记。排除壳上有裂缝的鸡蛋。也排除有空气细胞的鸡蛋在一边。如果这有一个钝尖，这些都是极不可能正常孵化。用 75% 的酒精对空气电池区域以及金属探头进行消毒。擦干它们，然后用探头在空气电池区域的中心钻一个小洞。不要将探针深入空气电池。相反，只需用探头的尖端打破外壳。如果孔仍然不够大，请在现有孔附近再次打破外壳，直到它足够大，允许您插入尖端。将移液器尖端插入孔中，尖端接触内膜。慢慢地，注射混合物，并在那里举行至少10秒。这是为了让已分配的石油得到充分分配。然后取下提示。现在，用刷子和一滴融化的石蜡封住这个洞。小心不要将融化的石蜡滴到内记忆中。一旦密封，将卵子放在孵化器中，直到它们达到所需的胚胎阶段。第二，我要介绍的方法是微注射。首先，鸡蛋需要按照空气注入部分的描述进行清洁和处理。此程序需要在绝育罩中执行。用丙酮消毒空气细胞区和仪器。用金属探头在空气电池区域中心钻一个小洞。不要将探针深入到空气单元中，否则内记忆可能会受损。然后，使用相同的过程小心地放大孔，直到直径约为两毫米。这可以允许内记忆的比例确认。然后将溶液装入微型喷油器中，小心地将针头通过孔插入内膜，总长约三毫米。轻轻分配溶液，然后取出针头。保持针头尽可能垂直。用一小块胶带把洞封住。完全覆盖孔，以防止胚胎脱水和死亡在随后的孵化。我要介绍的第三种方法是空气细胞输液。将气体污染物灌输到空气细胞中。清洁和处理空气细胞注射部分所述的鸡蛋。然后你可以读鸡蛋直到胚胎第17天。在胚胎日17时处理卵子，以标记空气细胞区域。在胚胎18日，从孵化器中取出卵子，用丙酮对空气细胞区和仪器进行消毒。然后小心地在空气电池的两侧钻两个小孔。一种是用于注入气体或气溶胶。另一个是扩大我们的空气。小心地控制孔的大小，使注射孔的侧面足以插入导管针，而膨胀孔的直径应稍大一些。直径约一毫米。现在，这是我们用来容纳目标气体或气溶胶的样品袋。您可以用您使用的任何设备替换。轻轻将 10 mL 的气体或气溶胶倒入注射器中。然后，小心地将注射器与内针遥控连接到导管针上，使其柔软。轻轻地将导管针插入输液孔。一旦它在那里，对导管针施加压力。这是为了尽量减少注射部位的泄漏。另一个手指可以放在周围与驱逐孔为您的输液确认。如果你轻轻地输液，应该很容易感觉到从驱逐孔流出的空气流。那是被驱逐的空气室里的原始空气。输液完成后，立即密封两个孔。这些磁带和返回鸡蛋到您的孵化器。我可以介绍我们的端点评估流程。用五巴比妥33毫克/千克完全麻醉动物。通过打开测试的 IP 喷射。然后把鸡放在手术台上。用乙醇消毒腹部区域和电极。然后，连续从腹部子的两侧插入两个针电极。通过抬起皮肤一点点，确保针头不会进入腹腔。并从那里插入针头。插入后，小心地向前推针，直到针头到达胸腔的一侧。确保针头不会深入身体或从皮肤伸出来。然后用心电图仪进行测量。其他能够进行电子政务的神学院仪器也可使用。这是我们的第二个终点评估方法是为莫特林绘制地图。这是 HME 方式中心脏的横截面。这是我们要测量的正确的心室。为此，我们使用两个统治者。标尺 1 和标尺 2。现在，我们只是复制尺子一到屏幕，我们调整到适当的大小，然后旋转。我们移动它，使水平线的两端休息在3右心室墙壁的两端。现在我们有七条测量线穿过我们的三个右心室壁。现在我们也把次要的尺子复制到图片上。我们还调整了一点大小，并旋转和更换，以便它将被放置一样。此水平线与右心室的外壁平行。垂直的一个会穿过点，统治者的白质线满足内壁或右心室。然后我们制作测量点，以便以后可以用图像 J 进行测量，我们每个样品重复这个过程 7 次，以便我们代表测量右心室。我给你看一份目前结果的摘要。这是孵化前向卵子注射的全氟辛烷磺酸，你可以看到，随着注射的增加，血清中的血清浓度 LPF 会有效增加。根据示范，效果，微注射。你可以看到，A是控制，而B，是注射大荧光q蛋白的杀毒表达的地方。你可以看到强烈的荧光。这是上皮输液有效性的证明。你可以看到，通过输液，心肌组织中观察到了巨大的光纤变化。好。你还可以看到孵化鸡0，1和2周的R-R间隔。输注柴油废气后，情况也发生了显著变化。表明您考虑了这一点，此方法。在这里，您还可以在此示例中看到上皮输注的有效性。你可以看到，心脏的右心室有效地增加后，气溶胶注入柴油排气。当然，在这里我们使用的是组织形态测量方法。我们只是告诉你这是相当有效的。好。这就是我要呈现给你的所有方法。我希望这能促进其他人的工作与鸡胚胎作为一个强大的研究工具，在发展毒理学。祝你今天愉快。

Research

JoVE Journal

Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

Microbead Implantation in the Zebrafish Embryo

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

珠嫁接到发展鸡胚四肢识别信号转导途径影响基因表达

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

In Ovo Electroporation in the Chicken Auditory Brainstem

In Ovo Electroporation in the Chicken Auditory Brainstem

在奥运电击鸡听觉脑干中

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

完整毛里丝骨骼肌的X射线衍射作为研究肌肉疾病结构基础的工具

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

使用免疫荧光检测斑马鱼胚胎心脏PM2.5诱发DNA损伤

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. ...