长期慢性<em>铜绿假单胞菌</em>在呼吸道感染小鼠
Summary
我们所描述的琼脂珠的方法建立持久的长期慢性铜绿假单胞菌呼吸道感染的小鼠模型。
Abstract
慢性呼吸道感染的小鼠模型是在囊性纤维化（CF）的研究的重要资产，虽然有许多关于模型本身的担忧。炎症和感染的早期阶段，已被广泛研究使用铜绿假单胞菌琼脂珠的小鼠模型，而只有少数报道都集中在体内长期慢性感染。长期慢性感染的主要挑战仍然是低细菌的负担由P.假单胞菌和鼠周激发后感染的比例较低，表明细菌细胞被宿主逐步清除。
本文提出了获得有效的长期慢性感染小鼠的方法。此方法是基于P的嵌入绿脓杆菌临床分离株中的琼脂珠在体外 ，然后在C57Bl/6NCrl小鼠气管内滴注。双侧肺部感染与相关SEVERAL可测量读奏包括体重减轻，死亡率，慢性感染和炎性反应。该P.铜绿假单胞菌 RP73临床菌株优于PAO1参考实验室菌株，因为它导致了一个相对较低的死亡率，更严重的病变，以及更高的慢性感染。 体育铜绿假单胞菌定植可能会持续在肺超过三个月。鼠肺组织病理类似于CF治疗晚期慢性肺疾病。
此小鼠模型中最密切模仿人类疾病的过程中，都对发病机理的研究和对新疗法的评价可以使用。
Introduction
囊性纤维化（CF）是在囊性纤维化跨膜电导调节器（CFTR）基因突变引起的遗传性疾病。这个基因编码表达于大多数上皮细胞的膜中的氯离子通道。支气管扩张症，粘液堵塞和脑实质的破坏主要是绿脓杆菌引起的感染，逐步导致严重的肺部疾病和死亡率在大多数的CF患者1。了解CF的发病机制和新疗法的进一步发展依赖于动物模型与CF的特征。一些小鼠，转基因的CFTR基因，已经产生，但在这些物种的复述CF状肺病和见于CF患者几个其他器官异常的能力的限制已被广泛记载2。
感染的发展是在CF动物模型的主要挑战之一。文献CL早表明慢性感染持久一个月以上，可以实现仅将小鼠接种菌嵌在固定剂如琼脂，琼脂糖，或海藻藻3-5。这些固定剂提供在微需氧/厌氧条件下，使细菌的生长在小菌落的形状，类似于在CF患者6的粘液的生长。慢性感染的这个模型导致细菌在引起气道炎症和损伤7肺内的持久性。但是，根据所使用的方法，所述细菌菌株并接种在肺剂量，慢性感染小鼠在不同时间点恢复在肺的百分比和细菌负荷可以相差很大。特别是，对于长期慢性感染的主要挑战仍然是低细菌的负担由P.铜绿假单胞菌和鼠周挑战后感染的比例较低，说明THA吨的细菌细胞被宿主逐步清除。通过选择P。铜绿假单胞菌 RP73从CF卡的集合临床分离菌株8，我们成功获得低死亡率，更严重的病变，慢性感染的比例较高与稳定的细菌负荷长达一个月的C57Bl/6NCrl小鼠。
本文详细介绍了嵌入P的方法在铜绿假单胞菌琼脂珠;我们已经感染小鼠气管内滴入，测得的细菌负荷和细胞因子在肺，收集支气管肺泡灌洗液和进行组织学检查。总体而言，这一协议将有助于研究人员在处理上发病8,9极其重要的问题和对体育测试新疗法铜绿假单胞菌慢性感染10,11。
Protocol
Representative Results
Discussion
在P的关键步骤铜绿假单胞菌珠准备和鼠标的挑战，现报道如下。
用于小鼠的挑战的铜绿假单胞菌菌株是至关重要的。死亡率，慢性感染或间隙可能有所不同显著取决于用于挑战的细菌菌株。该P.铜绿假单胞菌 RP73临床菌株优于PAO1参考实验室菌株，因为它导致了一个相对较低的死亡率，更严重的病变，以及更高的慢性感染。在临床前研究的药物测试?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
研究Bragonzi的实验室已经由意大利囊性纤维化基金会（CFaCore）和欧盟-F7-2009-223670。这项工作的一部分进行了在蒸馏器，一个先进的显微镜实验室，并在病理解剖学（圣Raffaele科学研究所）的单位，进行组织病理学鼠标。
Materials
| Bacto Tryptic Soy Broth | Becton Dickinson | 211823 | |
| Difco Agar, granulated | Becton Dickinson | 214510 | |
| Heavy mineral oil | Sigma-Aldrich | 330760-1L | |
| S-(+)-Ketamine hydrochloride | Sigma-Aldrich | K1884 | |
| Xylazine hydrochloride | Sigma-Aldrich | X1251 | |
| 1 ml syringe 25 G 5/8'' 0.5 x 16 mm | PIC | 3071250300350 | |
| Catheter 22GA 0.9 x 25 mm | Becton Dickinson | 381223 | |
| Graefe Forceps – 0.5 mm Tips Curved | Fine Science Tools | 11152-10 | |
| Scissors, Iris, 11 cm, straight | World Precision Instruments | 501758 | |
| Suture clips | Fine Science Tools | 12040-01 | |
| Suture thread | Fine Science Tools | 18020-40 | |
| RPMI 1640 | Lonza | BE12-167F | |
| Complete protease inhibitor cocktail | Roche | 11836145001 | |
| Fast-Read 102 Burker disposable chamber | Biosigma | 390497 | |
| Tuerk solution | Fluka | 93770 | |
| RBC lysis buffer | Biolegend | 420301 | |
| Fetal bovine serum | Lonza | DE14-801F | |
| EZ cytofunnel | Thermo Scientific | A78710021 | |
| Superfrost ultra plus microscope slides | Thermo Scientific | J3800AMNZ | |
| Diff-Quik Romanowsky staining set | Medion Diagnostics | 130832 | |
| Hexadecyltrimethylammonium chloride | Sigma-Aldrich | 52366-10G | |
| 96-well EIA/RIA plate | Costar | 3590 | |
| 3,3’,5,5’- tetramethylbenzidine | Sigma-Aldrich | T8665-1L | |
| Sulfuric acid | Sigma-Aldrich | 320501-1L | |
| 10% neutral buffered formalin | Bio-optica | 05-01005Q | |
| Harris haematoxylin non Papanicolau | Bio-optica | 05-M06004 | |
| Eosin plus alcoholic solution | Bio-optica | 05-M11007 | |
| [header] | |||
| Equipment | |||
| Shaking incubator | Amerex Instruments | Steady Shake 757 | |
| Water bath | Grant | SUB14 | |
| Homogenizer | Ystral | ||
| Precision balance | KERN | 440-47N | |
| Cytocentrifuge | Thermo Scientific | A78300003 | |
| Low Cost Heating Pad | 2biol | LCHP | |
| Homogenization probe | Ystral | 2366931(great) | |
| 2366925(small) | |||
| Inverted optical microscope | Zeiss | Axioplan2 | |
| Camera (microscope) | Zeiss | Axiocam MRc5 | |
| Rotary microtome | Leica | RM2255 |
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