使用鸡胚胎作为评估发育性心脏病的有力工具
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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?…
Representative Results
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）。 ?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| 4% phosphate buffered formaldehydefixative | Biosharp, Hefei, China | REF: BL539A | |
| 75% ethanol | Guoyao,Shanghai,China | CAS:64-17-5 | |
| Biosignaling monitor BL-420E+ | Taimeng, Chengdu, China | BL-420E+ | |
| Candling lamp | Zhenwei, Dezhou, China | WZ-001 | |
| Disposable syringe | Zhiyu, Jiangsu, China | ||
| Egg incubator | Keyu,Dezhou, China | KFX | |
| Electrical balance | OHAUS, Shanghai, China | AR 224CN | |
| Electro-thermal incubator | Shenxian, Shanghai, China | DHP-9022 | |
| Ethanol absolute | Guoyao,Shanghai,China | CAS:64-17-5 | |
| Fertile chicken egg | Jianuo, Jining, China | ||
| Hematoxylin and Eosin Staining Kit | Beyotime, Bejing, China | C0105 | |
| Histology paraffin | Aladdin, Shanghai, China | P100928-500g | Melt point 52~54°C |
| Histology paraffin | Aladdin, Shanghai, China | P100936-500g | Melt point 62~64°C |
| IV catheter | KDL, Zhejiang, China | The catheters have to be soft, plastic ones. | |
| Lentivirus | Genechem, Shanghai, China | The lentivirus were individually designed/synthesized by Genechem. | |
| Masson's trichrome staining kit | Solarbio, Beijing, China | G1340 | |
| Metal probe | Jinuotai, Beijing, China | ||
| Microinjector (5 uL) | Anting,Shanghai, China | ||
| Microscope | CAIKON, Shanghai, China | XSP-500 | |
| Microtome | Leica, Germany | HistoCore BIOCUT | |
| Microtome blade | Leica,Germany | Leica 819 | |
| Pentobarbitual sodium | Yitai Technology Co. Ltd., Wuhan, China | CAS: 57-33-0 | |
| Pipetter(10ul) | Sartorius, Germany | ||
| Povidone iodide | Longyuquan, Taian, China | ||
| Scissor | Anqisheng,Suzhou, China | ||
| Sterile saline | Kelun,Chengdu, China | ||
| Sunflower oil | Mighty Jiage, Jiangsu, China | Any commerical sunflower oil for human consumption should work | |
| Tape | M&G, Shanghai, China | ||
| Tedlar PVF Bag (5L) | Delin, Dalian, China | ||
| Vortex mixer | SCILOGEX, Rocky Hill, CT, US | MX-F | |
| Xylene | Guoyao,Shanghai,China | CAS:1330-20-7 |
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Cite This Article
Jiang, Q., Xu, X., DeWitt, J. C., Zheng, Y. Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities. J. Vis. Exp. (169), e62189, doi:10.3791/62189 (2021).