小鼠导管相关 白色念珠菌 感染模型
概要
我们建立了 白色念珠菌相关导管相关感染 （CRI） 的小鼠模型，其中在导管上形成生物膜，白色 念 珠菌与宿主之间的相互作用与临床 CRI 密切相关。该模型有助于筛选白色 念珠菌 生物膜相关CRI的治疗方法，为临床转化奠定基础。
Abstract
导管相关感染 （CRI） 是导管植入过程中由 白色念珠菌 引起的常见院内感染。通常，在导管的外表面形成生物膜并导致播散性感染，这对患者来说是致命的。诊所没有有效的预防和治疗管理。因此，迫切需要建立CRI的动物模型，以期临床前筛选其预防和治疗的新策略。在这项研究中，将一根聚乙烯导管（一种广泛使用的医用导管）插入脱毛后 BALB/c 小鼠的背部。 白假丝酵母 随后将表达增强绿色荧光蛋白的ATCC MYA-2876（SC5314）沿导管接种在皮肤表面。3天后，在荧光显微镜下在导管表面观察到强烈的荧光。通过扫描电子显微镜在导管表面发现了成熟而厚的生物膜。这些结果表明白色 念珠菌 在导管表面的粘附、定植和生物膜形成。表皮增生和皮肤标本中炎症细胞浸润表明CRI相关皮肤的组织病理学变化。综上所述，成功建立了小鼠CRI模型。该模型有望有助于白色 念珠菌 相关CRI治疗管理的研究和开发。
Introduction
近年来，随着生物医学材料的发展和应用，种植体相关感染正在成为临床难题1,2。随着医用导管在诊所的广泛应用，每年相关的感染和死亡人数都很大3,4。导管相关感染（CRI）的常见感染途径包括：（1）皮肤表面的病原体浸润到体内并粘附在导管的外表面5,6,7;（2）无菌操作不当的病原体侵入、粘附和定植在导管上;（3）血液循环中的病原体粘附在导管上并定植;（4）被病原微生物污染的药物。
念珠菌是 CRI 8,9 的第三大常见原因。在植入物表面形成生物膜后，极有可能引起血流感染和其他危及生命的侵袭性念珠菌病。预后较差，死亡率高2。据报道，在中心静脉插入后2周内在导管表面形成生物膜，几周后在导管腔内形成生物膜10,11。
在医用导管上形成的白色念珠菌（白色念珠菌）生物膜表现出由酵母、基质和菌丝体组成的双层网络12,13。 白色念珠菌生物膜的形成不仅是耐药性和免疫逃避的关键13，而且对产生播散性孢子也至关重要，这会导致进一步的血源性感染2,12，并导致更严重甚至危及生命的后果。白色念珠菌相关CRI是临床真菌血流感染的主要原因7,14，超过40%的中心静脉导管白色念珠菌感染患者会发展为菌血症15。
根据美国传染病学会的说法，念珠菌 CRI 的推荐治疗方法包括 （1） 拔除受感染的导管;（2）对患者进行14天全身抗真菌治疗8;（3） 重新植入新导管4.然而，在临床应用中，导管有时无法完全移除。一些患者只能用全身性抗生素和抗菌锁定疗法治疗，并伴有强烈的副作用16,17。
现有的白色念珠菌动物模型，如口咽念珠菌病模型、阴道念珠菌病模型和念珠菌病引起的侵袭性全身感染模型18,19 不能很好地与临床 CRI 相关。因此，在这项研究中，建立了小鼠白色念珠菌相关的CRI模型。临床常用的聚乙烯导管被用作皮下植入物20,21，并将白色念珠菌接种在皮肤表面，以模拟白色念珠菌与医用导管的粘附和生物膜的形成。
该模型已成功用于我们的实验室，以筛选不同疗法的抗生物膜作用22。此外，由于导管感染后对 白色念珠 菌的滞后检测，构建了一种含有增强绿色荧光蛋白（EGFP）的 白色念 珠菌菌株，并将其接种在小鼠体内，以利于直观观察植入导管上 白色念 珠菌的菌落和生物膜。
Protocol
实验动物，雄性BALB / c小鼠（12-16g），购自习交通大学医学中心实验动物中心。所有程序均经习交通大学机构动物伦理委员会批准，许可证号为SCXK（陕西）2021-103。 1.缓冲液和设备准备 用高表达质粒 pCaExp 转染 白色念珠菌 菌株。从ATCC购买白色念珠菌（SC5314）。通过用含有EGFP基因完整开放阅读框的高表达质粒pCaExp转染白色念珠…
Representative Results
扫描电镜可以观察到导管上的 白色念珠菌 和生物膜。如 图322所示，导管组聚乙烯导管表面光滑，未观察到粘附的病原微生物。然而，在模型组的聚乙烯导管表面可见成熟致密的 白色念 珠菌生物膜，表明在实验条件下， 白色念 珠菌能够在小鼠的导管表面成功定植并形成生物膜。此外，荧光显微镜结果进一步验证了上述结论（<strong clas…
Discussion
CRI 是临床实践中最常见的院内感染之一23.皮肤附属物中的病原体，如表皮、皮脂腺和毛囊，都是 CRI23,24 的可能原因。念珠菌是引起CRI的第三大病原体，其中白色念珠菌是最常见的生物膜感染类型25,26。因此，我们旨在建立白色念珠菌生物膜相关CRI的相关动物模型，以支?…
開示
The authors have nothing to disclose.
Acknowledgements
感谢陕西省自然科学基金（批准号：2021SF-118）和国家自然科学基金（批准号：81973409,82204631）的财政支持。
Materials
| 0.5 Mactutrius turbidibris | Shanghai Lujing Technology Co., Ltd | 5106063 | |
| 2.5% glutaraldehyde fixative solution | Xingzhi Biotechnology Co., Ltd | DF015 | |
| 4 °C refrigerator | Electrolux (China) Electric Co., Ltd | ESE6539TA | |
| Agar | Beijing Aoboxing Bio-tech Co., Ltd | 01-023 | |
| Analytical balances | Shimadzu | ATX124 | |
| Autoclaves Sterilizer | SANYO | MLS-3750 | |
| Butanol | Tianjin Chemio Reagent Co., Ltd | 200-889-7 | |
| Carbenicillin | Amresco | C0885 | |
| Eclipse Ci Nikon upright optical microscope | Nikon | Eclipse Ts2-FL | |
| Glucose | Macklin | D823520 | |
| Inoculation ring | Thermo Scientific | 251586 | |
| Isoflurane | RWD | 20210103 | |
| Paraformaldehyde | Beyotime Biotechnology | P0099 | |
| PAS dye kit | Servicebio | G1285 | |
| Peptone | Beijing Aoboxing Bio-tech Co., Ltd | 01-001 | |
| Polyethylene catheter | Shining Plastic Mall | PE100 | |
| RWD R550 multi-channel small animal anesthesia machine | RWD | R550 | |
| SEM | Hitachi | TM-1000 | |
| Temperature incubator | Shanghai Zhichu Instrument Co., Ltd | ZQTY-50N | |
| Ultrapure water water generator | Heal Force | NW20VF | |
| Ultrasound machine | Do-Chrom | DS10260D | |
| Xylene | Sinopharm Chemical Reagent Co., Ltd | 10023428 | |
| Yeast extract | Thermo Scientific Oxoid | LP0021B |
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