Name: tobacco hornworm as an insect model system for cannabinoid pre-clinical studies
Uploaded: 2021-12-29T15:00:00
Description: Watch this Scientific Journal Video about Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies at JoVE.com

这项研究得到了科罗拉多州立大学普韦布洛分校大麻研究所、科学与信息通信技术部（2021-DD-UP-0379）和春川市（大麻研发和工业化，2020-2021）的支持。

1. 角虫制备及大麻二酚处理
<ol><li>获得150-200可行 M。 六角 蛋和小麦胚芽为基础的人造饮食（见 材料表）。</li>
	<li>将角虫卵放入具有小麦胚芽人造饮食（AD）层的聚苯乙烯培养皿中，并将卵转移到保持25°C且相对湿度为40%-60%的昆虫饲养室（ 见材料表）。</li>
	<li>让烟草角虫卵在相对湿度保持在25°C且相对湿度为40%-60%的昆虫饲养室内孵化1-3天。<...

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喂养研究表明，高剂量的CBD（2 mM）抑制了昆虫的生长并增加了死亡率31。昆虫模型还显示出对乙醇的敏感性;然而，CBD有效地解毒了乙醇毒性，将其存活率，饮食消耗和食物搜索行为提高到与对照组相似的水平（图3A，B）31。所描述的昆虫模型系统由三个关键步骤组成：（1）确保 M. sexta的卵在大小和时间上均匀孵化，（2...

在过去几年中，公众的注意力一直集中在大麻素上，因为它们具有治疗潜力，包括治疗癫痫1，帕金森病2，多发性硬化症3以及各种形式的癌症4，5，6 与大麻二酚（CBD）。由于 大麻 在2018年的"农业改进法"中作为农业商品合法化，公法115-334（2018年农业法案）， 大麻 及其大麻素衍生物在食品，化妆品和制药行业中呈指数级增长。此外，单一大麻素和大麻素混合物的临床级分离株已在人类受试者7、细胞系5、8和各种动物模型系统中成功测试9，10。
临床试验是验证大麻素对特定疾病的疗效和不良反应的理想选择。然而，临床试验中存在许多挑战，包括伦理/IRB批准、招募和保留受试者11。为了克服这些障碍，使用了各种人类细胞系，因为人源性细胞系具有成本效益，易于处理，可以绕过伦理问题，并提供一致且可重复的结果，因为细胞系是"纯细胞群，没有其他细胞和化学物质的交叉污染"12。
Alves等人（2021）13 在胎盘滋养层中以剂量依赖性方式测试CBD，胎盘滋养层细胞是胎盘的特化细胞，在胚胎植入和与蜕膜化母体子宫的相互作用中起着至关重要的作用14。他们的结果表明，CBD导致细胞活力丧失，细胞周期进展破坏和细胞凋亡诱导。这些观察结果表明了孕妇使用 大麻 的潜在负面影响13。同样，一系列细胞系也用于检查CBD在人类疾病中的药理作用，特别是各种形式的癌症。 体外 研究成功地证明了胰腺癌细胞15、breast8和结直肠癌细胞16的抗癌作用。然而，虽然具有广泛可用且易于处理的特定细胞系，但由于其生长条件或处理的改变，HeLa，HEK293等特定细胞系容易发生遗传和表型变化17。
在大麻研究中，各种动物模型系统，从小鼠18，豚鼠19和兔子19等小动物到犬20，仔猪21，猴子22，马23等大型动物，已被用于探索未知的治疗效果。小鼠一直是大麻素研究的首选动物模型系统，因为它们的解剖学，生理学和遗传相似性与人类24。最重要的是，小鼠的神经系统中有CB1 / 2受体，这些受体存在于人类中。它们的生命周期也比人类受试者短，更容易维护和丰富的遗传资源，因此更容易在整个生命周期中监测大麻素的影响。哺乳动物系统被广泛使用，并已成功证明CBD可缓解癫痫发作1，创伤后应激障碍9，口腔溃疡25和痴呆样症状10。小鼠模型还使社区内个体的社会互动研究成为可能，这在大型动物和人类中非常困难26。
尽管动物模型系统具有所有优点，但它仍然成本高昂，并且在药物管理和数据收集期间需要重症监护。此外，由于实验设计和严谨性的局限性，在研究中使用小鼠的不可重复性和对人类条件的概括性差，因此存在审查27。
随着对大麻素的医学/临床前研究的需求不断增加，需要一个非哺乳动物模型系统。传统上，无脊椎动物模型比脊椎动物模型具有独特的优势。显著的好处包括饲养许多标本的便利性和低成本，并使研究人员能够监测多代遗传同质种群28。最近的一项研究证明，果蝇 黑腹果 蝇是一种有效的昆虫模型系统，用于研究大麻素在调节进食行为中的药理功能29。在昆虫模型系统中，作者专注于烟草角虫 Manduca sexta，也称为卡罗来纳狮身人面像蛾或鹰蛾，作为大麻素研究的新型昆虫模型系统。
Manduca sexta 属于Sphingidae家族。这种昆虫是美国南部最常见的植物害虫，它们以茄科植物为食。昆虫模型在昆虫生理学，生物化学，神经生物学和药物相互作用研究方面有着悠久的历史。 Manduca sexta的研究组合包括一个基因组序列草案，允许对基本细胞过程进行分子水平的理解30。该模型系统的另一个关键好处是其大尺寸，在幼虫发育的18-25天内长度超过100毫米，重量达到10克以上。大尺寸使研究人员能够轻松实时监测形态和行为变化，以响应CBD治疗。此外，由于大小，用腹部神经系统检查电生理反应，包括在没有高分辨率显微镜设置的情况下从幼虫上解剖的神经节。独特的功能使研究人员能够轻松调查对施用大麻素的急性和长期反应。
尽管有这种多功能性， 但M. sexta 直到最近才被探索，因为它适合作为 大麻 和大麻素研究的实验模型。2019年，作者首次使用昆虫模型系统来解决 大麻 已经进化到产生大麻二酚以保护自己免受昆虫食草动物侵害的假设30，31。结果清楚地表明，这些植物利用CBD作为喂养威慑，并抑制了害虫 M. sexta caterpillar的生长，并导致死亡率增加31。该研究还证明了CBD对中毒乙醇幼虫的拯救作用，确定了乙醇作为CBD载体的潜在载体效应。如图所示，昆虫模型系统在3-4周内有效地研究了大麻素的治疗效果，并且比其他动物系统更少的劳动力和成本。虽然昆虫模型缺乏大麻素受体（即没有CB1/2受体），但模型系统提供了一个有价值的工具，通过大麻素受体无关的方式了解大麻素的药理作用。
本研究的作者以前曾使用烟草角虫作为大麻素研究的模型系统31。在仔细考虑了使用 M. sexta的益处和风险之后，我们提供了一种方法，涉及适当的护理和准备临床前试验的饮食，为未来的临床前实验室使用提供了机会。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analytic balance</td>
			<td>Mettler Instrument Corp.</td>
			<td>AE100S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannabidiol isolate (&#62;99.4%)</td>
			<td>Lilu&#39;s Garden</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td>VWR INTERNATIONAL</td>
			<td>470150-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 50mL clear polypropylene (PP) centrifuge tubes</td>
			<td>VWR</td>
			<td>89093-192</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol, 200 Proof</td>
			<td>Sigma-Aldrich</td>
			<td>EX0276-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fear conditioning chamber</td>
			<td>Coulbourn Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect rearing chamber</td>
			<td>Darwin Chambers</td>
			<td>INR034</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium chain triglycerides (MCT) oil</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Motion detection software (Actimetrics)</td>
			<td>Coulbourn Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene petri dish (120 mm x 120 mm x 17mm)</td>
			<td>VWR INTERNATIONAL</td>
			<td>688161</td>
			<td></td>
		</tr>
		<tr>
			<td>Tobacco hormworm artificial diet</td>
			<td>Carolina Biological Supply Company</td>
			<td>Item # 143908</td>
			<td>Ready-To-Use-Hornworm-Diet</td>
		</tr>
		<tr>
			<td>Tobacco hormworm eggs</td>
			<td>Carolina Biological Supply Company</td>
			<td>Item # 143880</td>
			<td>Unit of 30-50</td>
		</tr>
	</tbody>
</table>

tobacco hornworm as an insect model system for cannabinoid pre-clinical studies

随着医学中对大麻素的日益关注，几种哺乳动物模式生物已被用于阐明其未知的药物功能。然而，哺乳动物研究仍然存在许多困难，这需要开发用于大麻素研究的非哺乳动物模式生物。作者建议将烟草角虫 Manduca sexta 作为一种新的昆虫模型系统。该协议提供了有关用不同量的大麻二酚（CBD）制备人造饮食的信息，建立培养环境，并监测其对CBD治疗的生理和行为变化。简而言之，在接受角虫卵后，在25°C下以12：12的明暗循环允许卵1-3天孵化，然后随机分配到对照组（基于小麦胚芽的人造饮食;AD），载体（AD + 0.1%中链甘油三酯油;MCT油）和治疗组（AD + 0.1%MCT + 1 mM或2mM CBD）。一旦培养基准备好，将1龄 幼虫单独放入带有木制烤串棒的50 mL试管中，然后用干酪布覆盖试管。以2天的间隔进行测量，以对CBD管理的生理和行为反应。这种简单的培养程序使研究人员能够在给定的实验中测试大型标本。此外，相对较短的生命周期使研究人员能够研究大麻素治疗对同质种群多代的影响，从而允许数据支持高等哺乳动物模式生物的实验设计。

曼杜卡六分仪 作为检查大麻素毒性的模型系统 图1 描述了使用烟草角虫 Manduca sexta的CBD实验的关键组成部分。大量昆虫（>20）在25°C下以12小时：12小时=光：暗循环单独饲养。每隔2天测量昆虫的大小，体重和死亡率，以监测高剂量CBD（2mM）治疗后的短期和长期反应。
图2 显示了CBD对昆虫生长...

Watch this Scientific Journal Video about Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies at JoVE.com

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies

本方案为在大麻素研究中使用烟草角虫 Manduca sexta 提供了教学信息。 这里描述的方法包括所有必要的用品和方案，以监测昆虫模型的生理和行为变化，以响应大麻二酚（CBD）治疗。

烟草角虫作为大麻素临床前研究的昆虫模型系统

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical ...

该协议建议使用烟草角虫作为大麻素临床前研究的昆虫模型系统。该系统使研究人员能够轻松研究对大麻素的急性和长期反应。昆虫模型系统使研究人员能够在三到四周内研究大麻素在大量人群中的治疗效果，而劳动力和成本比其他动物系统少。获得150至200个可行的M.sexta鸡蛋和基于小麦胚芽的人造日粮。将角虫卵放入具有小麦胚芽人造饮食层的聚苯乙烯培养皿中，并将卵转移到保持25摄氏度且相对湿度为40%至60%的昆虫饲养室中。让卵在昆虫饲养室内孵化一到三天。为了制备200毫摩尔大麻二酚储备溶液，在20毫升乙醇或100%中链甘油三酯油中加入1.26克大于98%纯度的大麻二酚分离物。将5毫升和10毫升的200毫摩尔大麻二酚储备溶液分别添加到1， 000克人工饮食中，分别用于一毫摩尔和两毫摩尔大麻二酚。将20克对照载体和含大麻二酚的培养基分配到50毫升管的底部。将大约两毫米长的首龄幼虫随机分布在50毫升的试管中，并用穿孔的盖子或干酪布盖住。将它们生长在保持25摄氏度的昆虫饲养室中，具有12小时的光暗循环。为了测量幼虫的生长，分别用分析天平和标尺记录每个组的初始质量和大小，并将幼虫引入各自的饮食中。在每次测量时，以两天的间隔从初始质量中减去幼虫的质量，以确定幼虫发育阶段之间的质量增益。此外，记录幼虫发育阶段之间的天数，以了解化蛹前发育时间框架的差异。为了测量饮食消耗，在实验开始时记录饮食的初始克数，并在幼虫进入完全化蛹阶段时从剩余的饮食量中减去它。对于幼虫活动性测量，让受试昆虫适应恐惧调节室至少五分钟，并跟踪三组五龄昆虫行进五分钟的距离。然后，运行运动检测软件以分析移动响应。在人工饮食中饲养的昆虫显示出最佳的生长性能。使用0.1%中链甘油三酯油作为CBD分离物溶解剂的载体对照组也显示出正常生长，没有任何不利影响。然而，两毫米的高剂量CBD诱导了体重减轻，并导致死亡率高于对照组和车辆组。在AD上饲养的M.sexta幼虫，用一毫米CBD消耗的饮食量至少是乙醇添加饮食的3.1倍。然而，在两毫摩尔CBD添加培养基上饲养的昆虫的饮食消耗与仅用乙醇饮食饲养的幼虫没有显着差异。不同条件下饲养的M.sexta幼虫的移动指数表明，1%乙醇和1毫摩尔CBD处理的幼虫不影响活动性。2%乙醇处理被证明是致命的。在含有2%乙醇的AD中添加高剂量的CBD可恢复低流动性。一旦CBD被添加到饮食中，就将饮食CBD混合物完全同质化，并在保持25摄氏度，湿度为40%至60%的昆虫饲养室中生长昆虫，以避免真菌爆发。电生理反应也可以通过从Manduca sexta解剖的腹链神经节进行监测，以确定CBD是否影响烟草角虫的中枢神经系统。昆虫模型系统将使研究人员能够通过多代测试来探索其他主要和次要大麻素的药物作用，从而允许在高等哺乳动物中进行实验设计。

Research

JoVE Journal

Biology

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

科研

生物学

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

表征草食动物的耐药机制：Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Establishment of an Extracellular Acidic pH Culture System

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...