Name: a silicosis mouse model established by repeated inhalation of crystalline silica dust
Uploaded: 2023-01-06T14:40:00
Description: Watch this Scientific Journal Video about A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust at JoVE.com

本研究由安徽省高校协同创新计划（GXXT-2021-077）和安徽理工大学研究生创新基金（2021CX2120）资助。

所有程序均遵循美国国立卫生研究院《实验动物护理和使用指南》（NIH 出版物第 8023 号，1978 年修订）的指南，并得到安徽理工大学医学院机构动物护理和使用委员会的批准。
1. 管理和喂养小鼠
<ol><li>将20只健康的C57BL / 6雄性小鼠以1：1的比例分配到实验组或载体组。使小鼠适应新环境1周。</li>
	<li>提供每天 12 小时的恒定光照时间。使用时间控制开关进行精确?...

<ol>
	<li>Olsson, A., Kromhout, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occupational+cancer+burden:+the+contribution+of+exposure+to+process-generated+substances+at+the+workplace.">Occupational cancer burden: the contribution of exposure to process-generated substances at the workplace.</a> Molecular Oncology. 15 (3), 753-763 (2021).</li><li>The Lancet Respiratory. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+world+is+failing+on+silicosis.">The world is failing on silicosis.</a> The Lancet. Respiratory Medicine. 7 (4), 283 (2019).</li><li>Weissman, D. 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矽肺小鼠模型对于研究矽肺病的发病机制和治疗至关重要。该协议描述了一种通过重复鼻腔暴露制备小鼠矽肺病模型的方法。该方法可以研究不同暴露时间引起的矽肺病的病理特征。小鼠在呼吸机上麻醉，并监测它们的呼吸频率。随着时间的流逝，最初的呼吸频率逐渐减慢和加深。麻醉使小鼠的肌肉放松，导致深呼吸，并允许它们在缓慢、深呼吸的时间窗口内吸入CSD。在此过程中，操作者用拇指?...

工人不可避免地会接触到不规则的结晶二氧化硅粉尘 （CSD），这些粉尘可以被吸入，并且在许多职业环境中毒性更大，包括采矿、陶器、玻璃、石英加工和混凝土 1,2。一种称为矽肺的慢性粉尘吸入病症会导致进行性肺纤维化3。根据流行病学数据，在过去的几十年里，全球矽肺病的发病率一直在下降，但近年来，它一直在增加并影响年轻人4,5,6。矽肺病的潜在机制因其隐匿的发病和漫长的潜伏期而对科学研究提出了重大挑战。目前尚不清楚矽肺病是如何发展的。此外，目前没有任何药物可以阻止矽肺病的进展和逆转肺纤维化。
目前用于矽肺病的小鼠模型涉及气管摄入混合 CSD 悬浮液。例如，通过在麻醉后采用颈气管创伤将 CSD 施用于肺部不符合人体反复暴露于染料粉尘7.暴露于环境粉尘对个人的影响可以通过以气溶胶的形式暴露于CSD来研究，这更准确地反映了这种有毒物质的环境浓度8。然而，由于小鼠鼻子的独特生理结构，环境性CSD不能简单地直接吸入肺部9。此外，与该技术相关的设备价格昂贵，这导致研究人员重新评估小鼠矽肺病模型10。通过在 2 周内通过鼻滴吸入 CSD 悬浮液 5 次，可以建立矽肺病的动态模型。该模型一致且安全，同时易于使用。需要注意的是，这项研究允许小鼠重复吸入CSD。通过该程序创建的小鼠矽肺病模型有望更有利于研究要求。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mL tube</td>
			<td>Biosharp</td>
			<td>BS-05-M</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin neutral fixative</td>
			<td>Nanchang Yulu Experimental Equipment Co.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe&#160;</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol disinfectant</td>
			<td>Xintai Kanyuan Disinfection Products Co.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>CD68</td>
			<td>Abcam</td>
			<td>ab125212</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate antigen retrieval solution</td>
			<td>biosharp life science</td>
			<td>BL619A</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB chromogenic kit</td>
			<td>NJJCBio</td>
			<td>W026-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl benzene</td>
			<td>West Asia Chemical Technology (Shandong) Co</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced BCA protein assay kit</td>
			<td>Beyotime Biotechnology</td>
			<td>P0009</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin (H&#38;E)</td>
			<td>Beyotime Biotechnology</td>
			<td>C0105S</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP substrate</td>
			<td>Millipore Corporation</td>
			<td>P90720</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)</td>
			<td>Proteintech</td>
			<td>Sa00001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Iceacetic acid</td>
			<td>West Asia Chemical Technology (Shandong) Co</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science</td>
			<td>R510-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s Trichrome stain kit</td>
			<td>Solarbio</td>
			<td>G1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Macklin</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Millipore</td>
			<td>AXYMCT150CS</td>
			<td></td>
		</tr>
		<tr>
			<td>NF-&#954;B p65</td>
			<td>Cell Signaling Technology</td>
			<td>8242S</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscillatory thermostatic metal bath</td>
			<td>Abson</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin embedding machine</td>
			<td>Precision (Changzhou) Medical Equipment Co.</td>
			<td>PBM-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Slicer</td>
			<td>Jinhua Kratai Instruments Co.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer (PBS)&#160;</td>
			<td>Biosharp</td>
			<td>BL601A</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological saline&#160;</td>
			<td>The First People&#39;s Hospital of Huainan City</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Beyotime Biotechnological</td>
			<td>ST505</td>
			<td></td>
		</tr>
		<tr>
			<td>Polarized light microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance</td>
			<td>Acculab</td>
			<td>ALC-110.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism7.0</td>
			<td>GraphPad&#160;</td>
			<td>Version 7.0</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membranes</td>
			<td>Millipore</td>
			<td>3010040001</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Beyotime Biotechnology</td>
			<td>P0013B</td>
			<td></td>
		</tr>
		<tr>
			<td>RODI IOT intelligent multifunctional water purification system</td>
			<td>RSJ</td>
			<td>RODI-220BN</td>
			<td></td>
		</tr>
		<tr>
			<td>Scilogex SK-D1807-E 3D Shaker</td>
			<td>Scilogex</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE gel preparation kit</td>
			<td>Beyotime Biotechnology</td>
			<td>P0012A</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon dioxid</td>
			<td>Sigma</td>
			<td>#BCBV6865</td>
			<td></td>
		</tr>
		<tr>
			<td>Sirius red staining</td>
			<td>Nanjing SenBeiJia Biological Technology Co., Ltd.</td>
			<td>181012</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>Anhui Yaokun Biotech Co., Ltd.</td>
			<td>ZL-04A</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipette Tips (0.1&#8211;10 &#181;L)</td>
			<td>KIRGEN</td>
			<td>KG1011</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipette Tips (100&#8211;1000 &#181;L)</td>
			<td>KIRGEN</td>
			<td>KG1313</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Pipette Tips (1&#8211;200 &#181;L)</td>
			<td>KIRGEN</td>
			<td>KG1212</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex&#160;mixer&#160;</td>
			<td>VWR</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS GeminiSEM 500</td>
			<td>Zeiss Germany</td>
			<td>SEM 500</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Bioss</td>
			<td>bs-0061R</td>
			<td></td>
		</tr>
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a silicosis mouse model established by repeated inhalation of crystalline silica dust

矽肺可由在工业环境中暴露于呼吸性结晶二氧化硅粉尘 （CSD） 引起。人类矽肺病的病理生理学、筛查和治疗都已使用小鼠矽肺病模型进行了广泛研究。通过反复让小鼠将CSD吸入肺部，小鼠可以模仿人类矽肺病的临床症状。这种方法在时间和产出方面是实用和有效的，并且不会因手术而对上呼吸道造成机械损伤。此外，该模型可以成功地模拟矽肺病的急性/慢性转化过程。主要程序如下。将灭菌的1-5μmCSD粉末充分研磨，悬浮在盐水中，并在超声水浴中分散30 min。在异氟醚诱导的麻醉下，小鼠从浅快速呼吸切换到深而缓慢的抽吸约2秒。将鼠标放在手掌中，拇指尖轻轻接触鼠标下颌的唇缘，使气道变直。每次呼气后，小鼠通过一个鼻孔一滴一滴地吸入二氧化硅悬浮液，在4-8秒内完成该过程。在小鼠的呼吸稳定后，抚摸和抚摸它们的胸部，以防止吸入的CSD被咳出。然后将小鼠放回笼子里。总之，该模型可以量化 CSD 沿着微小颗粒进入肺部的典型生理通道，从上呼吸道到末端细支气管和肺泡。它还可以复制员工因工作而经常暴露的情况。该模型可以由一个人执行，不需要昂贵的设备。它方便有效地模拟了人体矽肺病的疾病特征，重复性高。

采用所提方法研究了小鼠矽肺病的潜在发病机制。我们发现，实验组小鼠的体重相对于对照组明显下降，停止暴露后体重恢复缓慢。由于这里使用的优化剂量，在本实验中，在暴露于二氧化硅的小鼠中没有观察到死亡率。反复滴鼻到CSD的技术路线图如图1所示。前面描述的程序包括 CSD 悬吊准备、异氟醚诱导麻醉、鼻滴和胸腔按摩12。我们证明了静态喂养 4 ?...

Watch this Scientific Journal Video about A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust at JoVE.com

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

该方案描述了一种通过鼻滴反复暴露于二氧化硅悬浮液来建立矽肺小鼠模型的方法。该模型可以高效、方便、灵活地模拟人体矽肺病的病理过程，重复性和经济性高。

通过反复吸入结晶二氧化硅粉尘建立的矽肺小鼠模型

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

在所描述的晶体二氧化硅粉尘激光内输注产生的小鼠矽肺病模型中，所有CSD悬浮液多次模拟了人类矽肺病的发生和发展。这种方法既省时又省级，实用有效。此外，不会因手术而对上呼吸道造成机械损伤。该方法还可用于制备病毒、细菌等的疾病模型。演示该程序的将是我实验室的硕士生曹航兵，他擅长制备矽肺小鼠模型。在进行鼻滴之前至少一天，首先准备结晶二氧化硅粉或 CSD。为此，在玛瑙研钵中研磨二氧化硅半小时。为了检查颗粒大小和形状，使用导电胶带准备样品进行扫描电子显微镜或SEM以结合硅颗粒。使用吹风机轻轻吹走未牢固粘合的颗粒，然后再继续拍摄图像。接下来，使用超声波振荡器将磨碎的二氧化硅和无菌盐水以每毫升 20 毫克的浓度混合。最后，涡旋准备好的CSD悬浮液10秒钟。通过鼻滴给麻醉的小鼠施用 50 微升 CSD，持续 4 至 8 秒。用等量的生理盐水处理对照小鼠，然后用手掌握住小鼠的身体，用拇指和食指捏住脖子后面的皮肤，同时用其他手指固定小鼠的后肢。用另一根食指轻轻按压鼠标的心脏跳动区域 5 到 10 次，持续 5 秒钟。在60摄氏度的热板上加热石蜡包埋的肺组织四个多小时。为了对石蜡切片进行脱蜡和水合，将载玻片在二甲苯中浸泡两次30分钟，然后依次将载玻片浸入无水95%85%和75%乙醇中各浸泡5分钟，最后浸入去离子水中，然后将载玻片放入苏木精染色桶中10分钟，然后在流水下轻轻冲洗5分钟。将其浸入伊红染色桶中 10 秒钟。将样品在75%85%95%和无水乙醇中各脱水5分钟后，将其浸入二甲苯中5分钟以清除组织切片，并用约60微升中性树脂滴密封。小心地将盖玻片放在该部分上。对于Masson染色，用新鲜制备的50%Weigert苏木精对脱蜡和水合的石蜡样品染色10分钟，然后在将组织浸泡在酸性乙醇液化中10秒钟后，用流水轻轻冲洗载玻片以使细胞核变蓝。接下来，用红色染色液滴对样品染色7分钟，并用30%盐酸工作溶液洗涤1分钟。将载玻片浸入 95% 酒精中 20 秒后，用无水乙醇将样品脱水 1 至 3 秒两次，如前所述，清除并密封样品。对于天狼星红染色，用天狼星红染色溶液浸润脱蜡和水合部分一小时，然后用Mayer苏血染色溶液对细胞核染色8至10分钟。在流水下轻轻冲洗 10 分钟，然后脱水、清除和密封载玻片。用 30 毫升每毫升 3 毫克的 EDTA 抗原修复溶液浸润脱蜡和水合石蜡样品。将样品煮沸 20 至 30 分钟，然后用去离子水洗涤并在 PBST 中孵育 5 分钟。用新鲜制备的0.3%过氧化氢溶液将样品浸泡15分钟，并用PBST洗涤3次，每次5分钟。用 0.3% Triton 100 溶液透化标本膜 15 分钟，并用 30 至 40 μL 5% 牛血清白蛋白或 BSA 封闭 1 小时。除去封闭液后，加入适当稀释的一抗NF-kappa B和CD-68，并将标本在显微镜载玻片IHC湿箱中以2至8摄氏度孵育过夜。第二天，将样品转移到室温下一小时后，用PBST洗涤3次，每次5分钟。在室温下将样品在兔抗小鼠辣根过氧化物酶标记的二抗中孵育一小时。孵育后，再次用PBST洗涤样品三次，每次5分钟，然后将样品与酶标记抗体对应的DAB底物孵育5至20分钟。用去离子水淬灭反应后，用魏格特苏木精对样品复染 30 秒。在流水下冲洗一分钟，脱水，清除并密封组织。在进行蛋白质印迹分析之前，使用手持式电动研磨机在冰上用RIPA工作溶液匀浆组织5分钟。在冰上摇动孵育一小时，并将匀浆以 14， 800 G 离心 15 分钟，温度为 4 摄氏度。收集上清液，并用BCA蛋白测定试剂盒测定蛋白质浓度。向上清液中加入 20 微升 5X 上样缓冲液，然后在 100 摄氏度的金属浴中加热用 RIPA 制备的每微升 6 微克蛋白质储存溶液 20 分钟。在零下 80 摄氏度下将 100 微升等分试样的蛋白质溶液储存在每个试管中。对于电泳，用 RIPA 裂解物将等分试样稀释至每微升 2 至 3 微克，然后每孔加入 20 微克样品以电泳凝胶。使用湿法将蛋白质转移到用甲醇预活化的 PVDF 膜上 20 秒，电流为 400 毫安，持续 1 至 2 小时。用PBST溶液洗涤膜五次，每次5分钟后，用5%BSA或脱脂牛奶封闭一小时。将条带浸没在含有NF-κB和β肌动蛋白的一抗溶液中，并用5%BSA稀释。在2至8摄氏度下轻轻摇晃条带过夜，然后用PBST清洗。接下来，将试纸条孵育，首先在室温下在稀释的二抗中孵育一小时，轻轻摇动，然后用增强的化学发光显影剂孵育三分钟。将条带暴露于凝胶成像仪 20 秒，并通过系统软件测量条带的灰度值以评估蛋白质水平。CSD的SEM图像显示颗粒形状不规则。天狼星红染色测量胶原沉积表明，暴露于CSD一个月后，小鼠肺纤维化的进展明显加速。偏光显微镜显示三种不同类型的胶原纤维，其中红色显示的1型胶原纤维是矽肺的危险因素，但在载体组中未发现明显的纤维化。相应的纤维化评分也表明了这一点。CSD处理的小鼠肺组织的HE染色显示典型的二氧化硅结节，其特征是CSD吞噬后在中心液化坏死，周围有巨噬细胞包围。Masson染色也显示结节富集CSD并伴有纤维化。CD-68的免疫组化染色进一步揭示了肺组织中巨噬细胞的广泛存在。偏振光显微镜显示，这些巨噬细胞摄入了CSD，导致严重的肺损伤。免疫组化染色显示 NF-κ B 染色更高，表明 CSD 治疗组的炎症反应高于载体组。代表性蛋白质印迹也显示，CSD治疗的小鼠在肺NF-κB表达较高，两组间表达差异显著。为了避免呼吸道阻塞并促进呼吸恢复，必须在五秒钟后轻轻按摩小鼠的心脏区域 5 到 10 次。该模型可以探索矽肺病的一些潜在机制，并帮助治疗药物筛选，同时建立剂量和持续时间等关键参数。该技术提供了一种方便、有效和经济的方法，可以将小鼠模型暴露在颗粒物中，无论这是否会增加细菌。监测小鼠行为可以帮助确定吸入颗粒物的潜在影响。

Research

JoVE Journal

Medicine

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

自动子宫组织移植手术诱发子宫内膜异位症小鼠模型

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

小鼠模型的腔内MCAO脑梗塞评价甲酚紫染色

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

脑血管反应性通过气体吸入挑战MRI映射

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

干粉和药品的雾化吸入气雾剂递送小鼠使用鼻只曝光系统

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

反复臭氧暴露对小鼠慢性阻塞性肺疾病模型的产生

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction

氯乙烯和高脂肪饮食作为环境与肥胖相互作用的典范

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...