平行板灌注系统的体外模型研究了细菌对移植组织的粘附性
1Cardiovascular Developmental Biology, Department of Cardiovascular Sciences,KU Leuven, 2Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences,KU Leuven, 3Department of Biohybrid & Medical Textiles, AME – Helmholtz Institute for Biomedical Engineering,RWTH Aachen University, 4European Homograft Bank,Saint Jean Clinique, 5Division of Clinical Cardiac Surgery, Department of Cardiovascular Sciences,KU Leuven
Summary
我们描述了一个内部设计的体外流室模型, 它允许研究细菌对移植组织的粘附。
Abstract
各种瓣膜导管和支架安装的瓣膜用于先天性心脏病患者的右心室流出道 (rvot) 瓣膜置换术。然而, 当使用假肢材料时, 这些移植物容易受到细菌感染和各种宿主反应。
识别在微生物血管内粘连中发挥重要作用的细菌和宿主因素对于更好地了解感染性心内膜炎 (ie) 等感染的病理生理学和开展预防工作具有重要意义策略。因此, 有必要建立有效的模型来研究细菌在生理剪切条件下的粘附情况。在这里, 我们描述了一个新设计的基于平行板的体外灌注室的使用情况, 该室允许研究细菌对不同器官移植组织的粘附性, 如暴露的细胞外基质、内皮细胞和惰性区域.该方法结合成菌区量值, 可用于评价移植物材料在流动条件下对细菌粘附的倾向。此外, 流室系统还可用于研究血液成分在剪切条件下细菌粘附中的作用。我们证明, 组织的来源, 其表面形态和细菌物种特异性不是主要的决定细菌坚持到移植组织的因素, 使用我们的内部设计的体外灌注模型。
Introduction
金黄色葡萄球菌(金黄色葡萄球菌) 采用了各种毒力策略来规避宿主免疫防御系统, 使植入人体循环的生物或非生物表面殖民, 从而导致严重的血管内感染, 如败血症和ie1,2,3,4,5。ie 仍然是一个重要的治疗相关并发症的患者植入假体心脏瓣膜后, 而个别因素的原因, 导致 ie7 的发病还没有完全了解 6,7。在流动条件下, 细菌会遇到剪切力, 为了粘附在容器壁8 上, 它们需要克服剪切力。模型可以研究细菌和假体瓣膜组织或内皮在流动中的相互作用, 因为它们更多地反映了体内的情况。
几种特殊的机制促进细菌粘附到内皮细胞 (ecs) 和暴露的内皮细胞下基质 (ecm), 导致组织定植和植物成熟, 是 ie9中必不可少的早期步骤。各种葡萄球菌表面蛋白或 mscram (识别粘附基质分子的微生物表面成分) 被描述为通过与纤维连接蛋白等分子相互作用而与宿主细胞和 ecm 蛋白粘附的介质,纤维蛋白原、胶原蛋白和冯·威利布兰德因子 (vwf)8、10、11。然而, 鉴于分子内折叠的一些毒力因素, 大多在静态条件下研究, 其中许多相互作用可能有不同的相关性, 在循环血液的血管内感染。
因此, 我们提出了一个内部设计的体外平行板流动室模型, 允许评估细菌在组织移植中植入 rvot 位置时对 ecm 和 ecs 不同成分的粘附情况。本研究中描述的方法的总体目的是研究细菌与潜在血管内组织在流动条件下的相互作用机制, 这些机制与血液病原体的体内环境密切相关, 如s. 金牛座.这种新的方法侧重于移植组织表面对细菌粘附的敏感性, 以确定影响 ie 发展的潜在危险因素。
Protocol
1. 为体外研究准备移植组织 注: 使用了三种类型的组织: 牛心包贴片 (bp)、冷冻保留同种异体移植 (ch) 和牛颈静脉移植 (bjv)。在 bjv 导管和 ch (由欧洲同种异体移植银行 (ehb) 处理并在使用前储存在液氮中的组织) 的情况下, 使用壁面和瓣膜小叶。从制造商处购买了 bp 贴片和 bjv 管道。使用前, 按照 ehb 指令12 解冻 ch。 使用前使用0.9% 的氯化钠冲洗所…
Representative Results
为了更好地了解 ie 发育背后的机制, 该模型能够评估感染发病体内存在的细菌和组织相关因素。 在详细的, 新的体外方法允许量化细菌在流动条件下对不同的移植组织的粘附, 在施加剪切应力的组织上灌注荧光标记的细菌, 在3-10 的生理范围内施加剪切应力/cm2为 rvot。在这项工作中, 我们使用了 4 mL/mi…
Discussion
最近的临床观察使 ie 作为一种并发症的患者接受瓣膜置换术 rvot6,13。ie 中植入瓣膜功能障碍是细菌与血管内移植物相互作用的结果, 导致广泛的炎症和凝血反应1,14。提出的新的体外模型使我们能够研究组织结构和细菌因素的差异是否可能调节体内使用的移植物15对感染的易感性.</sup…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究是由向生殖健康提供的研究基金 ku leuven (ot/14/197) 的赠款赞助的。trv 是佛兰德研究基金会的博士后研究员 (比利时;grant 编号-12k0916n) 和 rh 由 uz leuven 的临床研究基金提供支助。
Materials
| Bovine Pericardium (BP) patch, Supple Peri-Guard Pericardium | Synovis Surgical Innovations, USA | PC-0404SN | |
| Bovine Jugular Vein conduits (BJV) | Contegra conduit; Medtronic Inc, USA | M333105D001 | |
| CH cryopreserved homograft | European Homograft Bank (EHB) | – | |
| Acu-Punch | Acuderm Inc, USA | P850 (8 mm); P1050 (10 mm) | |
| human Albumin | Flexbumin; Baxter, Belgium | BE171464 LOT:16G12C |
|
| Tryptic soy broth (TSB) | Fluka, Steinheim, Germany | 22092-500G | |
| Heart infusion broth (BHI) | Fluka | 53286-500G | |
| Phosphate buffered saline (PBS). | Gibco | 14190-094 | |
| 5(6)-Carboxyfluorescein N-hydroxysuccinimide ester (CF) | Sigma-Aldrich, Germany | 21878-100MG-F | |
| Peristaltic pump (MODEL ISM444B) | Ismatec BVP-Z Standard; Cole Parmer, Wertheim, Germany | 631942-2 | |
| Sonication bath | VWR Ultrasonic Cleaner; VWR, Radnor, Pa | 142-6044 | 230V/50 -60Hz 60VA; HF45kHz, 30W |
| ProLong Gold Antifade Mountant | Invitrogen by ThermoFisher | P36930 | |
| InCell Analyzer 2000 (fluorescence scanner) | GE Healthcare Life Sciences, Pittsburgh, Pa | 29027886 | |
| Arium Pro VF – ultrapure water – H2O MilliQ | Millipore | 87206462 | |
| Microscopic slides – Tissue Culture Chambers (1-well) | Sarstedt | 94.6140.102 | |
| 1-well on Lumox detachable | Sarstedt | 94.6150.101 | |
| Stainless Steel – surgical Blades | Swann-Morton | 311 | |
| Tygon Silicone Tubing, 1/8"ID x 1/4"OD | Cole-Parmer | EW-95702-06 | Temperature range: –80 to 200°C Sterilize: With ethylene oxide, gamma irradiation, or autoclave for 30 min, 15 psi of pressure |
| PharMed BPT Tubing | Saint-Gobain | AY242012 | Autoclavable 30 min at 121°C |
| Tygon LMT-55 Tubing | Saint Gobain Performance Plastics™ | 15312022 | |
| Thermostat | BMG BIOMEDIZINTECHNIK | 300-0042 | 230V, 90VA, 50Hz |
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Tags
In Vitro ModelParallel-plate Perfusion SystemBacterial AdherenceInfective EndocarditisPathogenesisBacteriaBlood CellsEndothelial CellsFlow ConditionsVegetation FormationVascular PermeabilityCell MobilityGene ExpressionBacterial SuspensionShear StressPeristaltic PumpFluorescently-labeled Colony Forming UnitsPhosphate Buffered Saline
Citazione di questo articolo
Ditkowski, B., Veloso, T. R., Bezulska-Ditkowska, M., Lubig, A., Jockenhoevel, S., Mela, P., Jashari, R., Gewillig, M., Meyns, B., Hoylaerts, M. F., Heying, R. An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues. J. Vis. Exp. (143), e58476, doi:10.3791/58476 (2019).