小鼠肺损伤的诱导,通过博霉素内切注射
1Servizio di Allevamento e Sperimentazione Animale, Polo ScientificoTecnologico,Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS)-Istituto Nazionale Ricovero e Cura Anziani (INRCA), 2Dipartimento di Scienze Cliniche e Molecolari,Università Politecnica delle Marche, 3UOS Centro di Terapia Cellulare “G. Lanzani”,Azienda Socio Sanitaria Territoriale (ASST) Papa Giovanni XXIII
Summary
在这里,我们提出了一种有效的方法,以研究静脉注射人类中发性血质细胞的抗纤维化活性,从整个脐带获得后,通过在C57BL的内切治疗注射注射肺损伤/6 小鼠。该协议可以很容易地扩展到其他疗法的临床前测试。
Abstract
肺纤维化是几种人类肺病的标志,病因不同。由于目前的疗法相当有限,小鼠模型仍然是开发新的抗纤维化策略的重要工具。在这里,我们提供了一种有效的方法,以研究从全脐带(hUC-MSC)获得的人类中位血质基质细胞在体内抗纤维化活性,以减轻博霉素引起的肺损伤。C57BL/6小鼠接受单次内切注射布洛霉素(1.5 U/kg体重),然后两次向尾静脉注入hUC-MSC(2.5 x 10 5),在布洛霉素给予后24小时和7天。在第8天、第14天或第21天牺牲时,将分析炎症和纤维化变化、胶原蛋白含量和hUC-MSC在外植肺组织中的存在。将小霉素注射到小鼠气管中,可直接瞄准肺部,导致广泛的肺部炎症和纤维化。双剂量hUC-MSC的系统施用导致博霉素引起的肺损伤的早期钝化。静脉注射hUC-MSC被暂时移植到小鼠肺部,在那里他们发挥抗炎和抗纤维化活性。最后,该方案已成功应用于人类肺纤维化实验小鼠模型hUC-MSC的临床前测试。然而,这种技术可以很容易地扩展,既研究不同内切施用的物质对肺部病理生理学的影响,也验证新的抗炎和抗纤维化全身疗法。
Introduction
肺纤维化是一种渐进的病理过程,其特点是细胞外基质成分(主要是I型胶原蛋白)在肺间质内沉积过多,导致肺功能受损。它是几种具有不同病因的人类肺部疾病的标志,是临床预后不良因素。由于目前的疗法相当有限1,小鼠模型仍然是一个重要工具,以进一步调查影响疾病发病和进展的致病机制,以及开发新的抗纤维化药物策略2,3.
迄今为止,博洛霉素的施用一直是实验诱发肺纤维化最常见的应用模型。除了多种分娩方法(包括静脉注射、腹管内、皮下和吸入外,宫内注射的博洛霉素已成为最常用的途径4、5。本文所描述的方法已经开发出来,以避免霉素对气管粘管的烫伤作用。事实上,通过将气管外在并通过操作显微镜进行可视化,可以实现将整个体积的博洛霉素溶液直接注入下气道,而不会在上气道中溢出。当有所需的外科专业知识和仪器可用时,此方法允许安全、可靠和可重复的诱导肺部炎症和纤维化,如下所述。
Protocol
所有动物护理和实验程序均获得意大利卫生部的批准(授权书456/2016-PR),并根据《赫尔辛基公约宣言》执行。 1. 老鼠 购买后,让小鼠在注射前至少7天适应。注:小鼠在无病原体条件下被安置在动物设施中,在12小时光/暗循环的恒定温度和湿度下保持,并免费获得水和标准颗粒食物。 使用雌性C57BL/6小鼠,在12至16周大时注射。 2. 内切治疗注射?…
Representative Results
肺损伤是由在100μL无菌盐水中单次注射1.5 U/kg的硫酸洋霉素体重引起的。对照动物接受相同量盐水的内切注射。两针hUC-MSC(2.5 x 105在200 μL无菌盐水)注入小鼠尾静脉,24小时和7天后,博洛霉素。对照动物接受同等体积的无菌盐水的静脉注射。小鼠在博霉素给给后的第8天、第14天和第21天被牺牲用于肺外植和组织处理(图1)。 <p class="jove_content" fo:ke…
Discussion
内切管管理是向肺部输送外源性药物的优先途径。几年来,将肌霉素直接注射到气管中已被广泛用于诱发肺纤维化13,最近,已经开发出更先进的非侵入性技术,以实现这14,15 ,16.
此处描述的方法提供了一些有意义的好处,以超过一些潜在的限制。气管的注射需要手术干预,携带手术本身引起的并?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了意大利部长德拉·萨洛(阿曼多·加布里埃利)的RF-2011-02352331赠款的支持。
Materials
| C57BL/6 mice | Charles River | Jax Mice Stock n. 000664 | |
| 2,2,2-Tribromoethanol (Avertin) | Sigma-Aldrich | T48402 | |
| Barraquer Micro Needle Holder | Lawton | 62-3755 | |
| Bleomycin sulfate | Sigma-Aldrich | B1141000 | |
| Bürker chamber | Brand | 718905 | |
| Culture Flasks | EuroClone | ET7076 | |
| Disposable razors | Unigloves | 4080 | |
| Dissecting Forceps | Aesculap Surgical Instruments | BD311R | |
| DPBS | Gibco | 14190-144 | |
| Heating pad | 2Biological Instruments | 557023 | |
| Isoflurane Vet | Merial Italia | N01AB06 | |
| Operating Microscope | Carl Zeiss | Model OPM 16 | |
| TrypLE Select Enzyme | Gibco | 12563-029 | |
| Vannas Micro Scissors | Aesculap Surgical Instruments | OC498R | |
| Vicryl Plus 4/0 Absorbable Suture, FS-2 needle 19 mm | Ethicon | VCP392ZH |
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Cite This Article
Orlando, F., Paolini, C., Agarbati, S., Tonnini, C., Grieco, A., Capelli, C., Introna, M., Provinciali, M., Gabrielli, A., Moroncini, G. Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin. J. Vis. Exp. (146), e58922, doi:10.3791/58922 (2019).