通过反复吸入结晶二氧化硅粉尘建立的矽肺小鼠模型
1Key Laboratory of Industrial Dust Control and Occupational Health of the Ministry of Education,Anhui University of Science and Technology, 2Key Laboratory of Industrial Dust Deep Reduction and Occupational Health and Safety of Anhui Higher Education Institutes,Anhui University of Science and Technology, 3Anhui Province Engineering Laboratory of Occupational Health and Safety, 4School of Medicine, Department of Medical Frontier Experimental Center,Anhui University of Science and Technology
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 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?…
Representative Results
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 ?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.5 mL tube | Biosharp | BS-05-M | |
| 10% formalin neutral fixative | Nanchang Yulu Experimental Equipment Co. | NA | |
| Adobe Illustrator | Adobe | NA | |
| Alcohol disinfectant | Xintai Kanyuan Disinfection Products Co. | NA | |
| CD68 | Abcam | ab125212 | |
| Citrate antigen retrieval solution | biosharp life science | BL619A | |
| DAB chromogenic kit | NJJCBio | W026-1-1 | |
| Dimethyl benzene | West Asia Chemical Technology (Shandong) Co | NA | |
| Enhanced BCA protein assay kit | Beyotime Biotechnology | P0009 | |
| Hematoxylin and Eosin (H&E) | Beyotime Biotechnology | C0105S | |
| HRP substrate | Millipore Corporation | P90720 | |
| HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) | Proteintech | Sa00001-2 | |
| Iceacetic acid | West Asia Chemical Technology (Shandong) Co | NA | |
| ImageJ | NIH | NA | |
| Isoflurane | RWD Life Science | R510-22 | |
| Masson's Trichrome stain kit | Solarbio | G1340 | |
| Methanol | Macklin | NA | |
| Microtubes | Millipore | AXYMCT150CS | |
| NF-κB p65 | Cell Signaling Technology | 8242S | |
| Oscillatory thermostatic metal bath | Abson | NA | |
| Paraffin embedding machine | Precision (Changzhou) Medical Equipment Co. | PBM-A | |
| Paraffin Slicer | Jinhua Kratai Instruments Co. | NA | |
| Phosphate buffer (PBS) | Biosharp | BL601A | |
| Physiological saline | The First People's Hospital of Huainan City | NA | |
| Pipettes | Eppendorf | NA | |
| PMSF | Beyotime Biotechnological | ST505 | |
| Polarized light microscope | Olympus | BX51 | |
| Precision balance | Acculab | ALC-110.4 | |
| Prism7.0 | GraphPad | Version 7.0 | |
| PVDF membranes | Millipore | 3010040001 | |
| RIPA lysis buffer | Beyotime Biotechnology | P0013B | |
| RODI IOT intelligent multifunctional water purification system | RSJ | RODI-220BN | |
| Scilogex SK-D1807-E 3D Shaker | Scilogex | NA | |
| SDS-PAGE gel preparation kit | Beyotime Biotechnology | P0012A | |
| Silicon dioxid | Sigma | #BCBV6865 | |
| Sirius red staining | Nanjing SenBeiJia Biological Technology Co., Ltd. | 181012 | |
| Small animal anesthesia machine | Anhui Yaokun Biotech Co., Ltd. | ZL-04A | |
| Universal Pipette Tips (0.1–10 µL) | KIRGEN | KG1011 | |
| Universal Pipette Tips (100–1000 µL) | KIRGEN | KG1313 | |
| Universal Pipette Tips (1–200 µL) | KIRGEN | KG1212 | |
| Vortex mixer | VWR | NA | |
| ZEISS GeminiSEM 500 | Zeiss Germany | SEM 500 | |
| β-actin | Bioss | bs-0061R |
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Citar este artigo
Cao, H., Li, B., Chen, H., Zhao, Y., Zou, Y., Liu, Y., Mu, M., Tao, X. A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust. J. Vis. Exp. (191), e64862, doi:10.3791/64862 (2023).