小鼠粪便分离和微生物群移植
1Division of Clinical Pharmacology, Department of Medicine Vanderbilt University Medical Center and Department of Molecular Physiology and Biophysics,Vanderbilt University, 2Division of Pediatric Nephrology Department of Pediatrics,Vanderbilt University Medical Center, 3Department of Pathology, Microbiology, and Immunology,Vanderbilt University Medical Center
Summary
这里的目标是概述一个方案，以研究心血管疾病中生态失调的机制。本文讨论了如何无菌收集和移植鼠粪便样本，分离肠道，并使用“Swiss-roll”方法，然后使用免疫染色技术来询问胃肠道的变化。
Abstract
肠道微生物群失调在心血管和代谢紊乱的病理生理学中起作用，但其机制尚不清楚。粪便微生物群移植（FMT）是描述总微生物群或分离物种在疾病病理生理学中的直接作用的宝贵方法。对于复发性 艰难梭菌 感染患者来说，这是一种安全的治疗选择。临床前研究表明，操纵肠道微生物群是研究生态失调与疾病之间机制联系的有用工具。粪便微生物群移植可能有助于阐明用于管理和治疗心脏代谢疾病的新型肠道微生物群靶向疗法。尽管啮齿动物的成功率很高，但仍存在与移植相关的翻译变化。这里的目标是为研究肠道微生物组在实验性心血管疾病中的作用提供指导。在本研究中，描述了小鼠研究中粪便微生物群的收集、处理、处理和移植的详细方案。描述了人类和啮齿动物捐赠者的收集和处理步骤。最后，我们描述了使用瑞士滚动和免疫染色技术的组合来评估心血管疾病的肠道特异性形态和完整性变化以及相关的肠道微生物群机制。
Introduction
心脏代谢紊乱，包括心脏病和中风，是全球主要死亡原因1。缺乏身体活动、营养不良、年龄增长和遗传因素调节这些疾病的病理生理学。越来越多的证据支持肠道微生物群影响心血管和代谢紊乱的概念，包括2型糖尿病2，肥胖3和高血压4，这可能是开发这些疾病新治疗方法的关键。
微生物群引起疾病的确切机制仍然未知，目前的研究差异很大，部分原因是方法学差异。粪便微生物群移植（FMT）是描述总微生物群或分离物种在疾病病理生理学中的直接作用的宝贵方法。FMT广泛用于动物研究以诱导或抑制表型。例如，热量摄入和葡萄糖代谢可以通过将粪便从生病的供体转移到健康的受体来调节5，6。在人类中，FMT 已被证明是复发性艰难梭菌感染患者的安全治疗选择7。支持其在心血管疾病管理中使用的证据正在出现;例如，从瘦到代谢综合征患者的FMT可提高胰岛素敏感性8。在人类和啮齿动物研究中，肠道生态失调也与高血压有关9，10，11。从喂食高盐饮食的小鼠到无菌小鼠的FMT使受体容易发炎和高血压12。
尽管FMT在啮齿动物中的成功率很高，但翻译挑战仍然存在。使用FMT治疗肥胖和代谢综合征的临床试验表明，对这些疾病的影响很小或没有影响13，14，15。因此，需要更多的研究来确定针对肠道微生物群治疗心脏代谢紊乱的其他治疗途径。大多数关于肠道微生物群和心血管疾病的现有证据都是相关的。所描述的协议讨论了如何利用FMT和瑞士滚动技术的组合来显示疾病与肠道微生物群之间的关联，并直接评估肠道所有部分的完整性16，17，18。
该方法的总体目标是为研究肠道微生物组在实验性心血管疾病中的作用提供指导。该协议在实验设计中提供了更多细节和关键考虑因素，以促进生理翻译并提高研究结果的严谨性和可重复性。
Protocol
范德比尔特大学的机构动物护理和使用委员会批准了本手稿中描述的所有程序。从杰克逊实验室购买的3个月大的C57B1 / 6雄性小鼠根据实验动物护理和使用指南进行饲养和护理。 1. 人类粪便样本的收集、储存和处理 收集粪便样本，如果受试者在诊所，请使用无菌容器。在收集后36小时内将粪便样品冷藏在4°C，直到准备好处理。或者，使用市售工具收集粪便…
Representative Results
图 1 总结了上述步骤。将小鼠盲肠内容物或人粪便重悬于无菌盐水中以制备浆液，通过管饲法给予无菌小鼠（100μL），首先连续3天，然后每3天一次。在协议结束时，通过尾袖法测量血压，对小鼠实施安乐死，并收获组织以评估肠道微生物群的变化以及心血管和代谢的变化。 选择微生物群的一个关键步骤是确保感兴趣的疾病表型存在于供体中并与生态失…
Discussion
研究肠道微生物群在心血管和代谢疾病中的因果作用的一种有价值的方法是将总微生物群或选择感兴趣的物种转移到无菌小鼠中。在这里，我们描述了从人类和传统饲养的小鼠收集粪便样本到无菌小鼠中的方案，以研究肠道微生物群在高血压疾病中的作用。
在小鼠中，我们使用无菌收集的盲肠内容物在需氧室中处理，而在人类中，我们收集粪便。FMT可以在样品仍然新鲜或在?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项研究得到了范德比尔特临床和转化科学奖资助UL1TR002243（又名）的支持，来自国家推进转化科学中心;美国心脏协会拨款POST903428（致J.A.I.）;国家心脏，肺和血液研究所拨款K01HL13049，R03HL155041，R01HL144941（A.K.）和NIH拨款1P01HL116263（V.K.）。 图 1 是使用 Biorender 创建的。
Materials
| Alexa Fluor 488 Tyamide SuperBoost | ThermoFisher | B40932 | |
| Anaerobic chamber | COY | 7150220 | |
| Apolipoprotein AI | Novus Biologicals | NBP2-52979 | |
| Artery Scissors – Ball Tip | Fine Science Tools | 14086-09 | |
| Bleach solution | Fisher Scientific | 14-412-53 | |
| Bovine Serum Albumin | Fisher Scientific | B14 | |
| CD3 antibody | ThermoFisher | 14-0032-82 | |
| CD68 monoclonal antibody | ThermoFisher | 14-0681-82 | |
| Centrifuge | Fisher Scientific | 75-004-221 | |
| CODA high throughput monitor | Kent Scientic Corporation | CODA-HT8 | |
| Cryogenic vials | Fisher Scientific | 10-500-26 | |
| Disposable graduate transfer pipettes | Fisher Scientific | 137119AM | |
| Disposable syringes | Fisher Scientific | 14-823-2A | |
| Ethanol | Fisher Scientific | AA33361M1 | |
| Feeding Needle | Fine Science Tools | 18061-38 | |
| Filter (30 µm) | Fisher Scientific | NC0922459 | |
| Filter paper sheet | Fisher Scientific | 09-802 | |
| Formalin (10%) | Fisher Scientific | 23-730-581 | |
| High salt diet | Teklad | TD.03142 | |
| OMNIgene.GUT | DNAgenotek | OM-200+ACP102 | |
| Osmotic mini-pumps | Alzet | MODEL 2002 | |
| PAP Pen | Millipore Sigma | Z377821-1EA | |
| Petri dish | Fisher Scientific | AS4050 | |
| Pipette tips | Fisher Scientific | 21-236-18C | |
| Pipettes | Fisher Scientific | 14-388-100 | |
| Serile Phosphate-buffered saline | Fisher Scientific | AAJ61196AP | |
| Smart spatula | Fisher Scientific | NC0133733 | |
| Stool collection device | Fisher Scientific | 50-203-7255 | |
| TBS Buffer | Fisher Scientific | R017R.0000 | |
| Triton X-100 | Millipore Sigma | 9036-19-5 |
|
| Varimix platform rocker | Fisher Scientific | 09047113Q | |
| Vortex mixer | Fisher Scientific | 02-215-41 | |
| Xylene | Fisher Scientific | 1330-20-7, 100-41-4 |
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Diesen Artikel zitieren
Ishimwe, J. A., Zhong, J., Kon, V., Kirabo, A. Murine Fecal Isolation and Microbiota Transplantation. J. Vis. Exp. (195), e64310, doi:10.3791/64310 (2023).