实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎
Summary
在评价中使用的关键技术<em>念珠菌</em>阴道炎在实验动物模型进行了描述。该方法将允许从阴道标本和淋巴细胞的快速收集，排水腰淋巴结。这些技术可能会引起女性下生殖道的其它疾病的小鼠模型。
Abstract
由念珠菌引起的外阴阴道念珠菌病（VVC），是一个较低的女性生殖道，在他们 18,32-34的生育年龄影响其他健康妇女中约75％的真菌感染。诱发因素包括生殖激素水平的抗生素使用，未经控制的糖尿病和干扰因怀孕，口服避孕药或激素替代疗法 33,34 。复发性VVC（RVVC），定义为三个或三个以上，每年发作，影响一个单独的5至8％的妇女没有诱发因素33。
的VVC的实验小鼠模型已经建立，并用于研究的发病机制和 粘膜念珠菌3,4,11,16,17,19,21,25,37宿主反应。这种模式也得到了测试体内 13,24潜在的抗真菌治疗。该模型要求的动物保持在一个国家选择pseudoestrusimal 念珠菌定植/感染6,14,23。在这种情况下，将已接种过的动物身上检测到的几个星期到几个月的阴道霉菌负担。过去的研究表明一个非常高的动物模型和人类感染的相对免疫和生理特性3,16,21之间的平行。差异，但是，包括念珠菌作为阴道正常菌群的缺乏和中性在小鼠的阴道pH值。
在这里，我们展示了一系列的鼠标阴道炎模型的主要方法，包括阴道接种，迅速收集阴道标本，评估阴道的真菌负担，细胞提取/隔离的组织筹备工作。其次是阴道灌洗液真菌的负担，及引流淋巴结白细胞产量的选民代表结果。随着使用麻醉剂，灌洗样本可以在多个时间点收集纵向评价相同的小鼠感染/定植。此外，这种模式要求没有启动感染的免疫抑制剂，使定义的主机条件下的免疫学研究。最后，模型和每种技术的介绍，这里可能引起的方法来研究降低女性生殖道（细菌，寄生虫，病毒）和各自的局部或全身的宿主防御的其他传染病。
Protocol
1。阴道白色念珠菌接种接种前三天，同时抑制动物暴露腹部，注入100μL芝麻油含有0.1-0.5毫克β-雌二醇皮下注射小腹。前进针约5至10毫米，外侧的皮肤，以尽量减少注射部位的泄漏。 在这个模型中，由于接近生殖道的最佳皮下注射雌激素在小腹。有效剂量可能会有所不同小鼠品系，年龄或雌激素衍生物。在以往的研究中使用CBA – J（H -κ），C3H/HeN（H -κ），C57BL / <…
Discussion
念珠菌性阴道炎的实验小鼠模型已经建立，在过去的几十年历史念珠菌的黏膜宿主反应，以及用于研究测试抗真菌疗法3,4,11,13,16,17,19,21,24 ， 25,37。协议提出，这里纳入效率和劳动密集程度较低的方法，并出现念珠菌性阴道炎的最优化模型系统描述迄今为止。这些技术使真菌负担和收集阴道标本的快速定量分析。此外，以往的研究测试小鼠的几个haplotypic株（N =…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是支持R01 AI32556（NIAID的国立卫生研究院）。这项工作也得到了支持路易斯安那疫苗中部和南部路易斯安那州路易斯安那州摄政理事会主办的传染病研究学会的部分。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Female CBA/J mice | Charles River Laboratories | 01C38 | 5-6 weeks of age |
| Candida albicans (3153A) | National Collection of Pathogenic Fungi, UK | NCPF3153 | |
| Sesame oil | Sigma-Aldrich | S3547 | Does not need to be pre-sterilized before use |
| Β-estradiol 17-valerate | Sigma-Aldrich | E1631 | 0.1-0.5mg in sesame oil |
| Phytone peptone | Becton Dickinson | 211906 | Supplement with 0.1% glucose |
| Trypan blue solution | Sigma-Aldrich | T8154 | |
| Sabouraud dextrose agar | Becton Dickinson | 211584 | |
| Collagenase type IV | Sigma-Aldrich | C5138 | 0.25% |
| Dispase | Invitrogen | 17105-041 | 1.7 U/ml |
| Wire mesh screens | TWP | 060X060S0065W36T | No. 60 mesh, stainless |
| Hanks’ balanced salt solution | Invitrogen | 24020-117 | |
| CytoPrep fixative | Fisher Scientific | 12-570-10 | Preserves smear slides |
| Papanicolaou stain EA-65 | EMD Chemicals | 7054X-85 | |
| Papanicolaou stain OG-6 | EMD Chemicals | 7052X-85 | |
| Harris’ Alum hematoxylin | EMD Chemicals | 638A-85 | |
| Isoflurane | Baxter Healthcare | NDC 10019-773-60 | Used with isoflurane vaporizer or in a drop system closed anesthetic chamber |
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Tags
Vaginal InoculationSample CollectionExperimental Mouse ModelCandida VaginitisVulvovaginal CandidiasisCandida SpeciesFungal InfectionLower Female Genital TractPredisposing FactorsAntibiotic UsageUncontrolled DiabetesReproductive Hormone LevelsPregnancyOral ContraceptivesHormone Replacement TherapiesRecurrent VVCExperimental Mouse Model Of VVCPathogenesisMucosal Host ResponseAntifungal TherapiesPseudoestrusCandida Colonization/infection
Cite This Article
Yano, J., Fidel, Jr., P. L. Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis. J. Vis. Exp. (58), e3382, doi:10.3791/3382 (2011).