收集，分离，和人类宫颈样品的流式细胞仪分析
Summary
The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.
Abstract
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Introduction
大多数新感染艾滋病毒引起全世界通过异性性接触传染，女性占新发感染的47％，2011年（联合国艾滋病规划署1）。了解女性生殖道（FGT），主入口门户为HIV和其他性传染病原体之一，是的路径，寻找有效的策略，以防止感染的高度重视。在生殖器粘膜免疫应答是独特的清楚和从那些在外周血2测得不同。然而，在FGT免疫动力学的当前的知识是在最好的限定。迄今为止，黏膜免疫环境的研究主要集中在肠道相关淋巴组织（GALT），它已经很清楚，感染后粘膜组织中的早期事件，对后续的疾病进展3,4的强烈冲击。从生殖器粘膜采集样本代表一个巨大的挑战，并至少部分负责LACk的FGT的免疫学的理解。解决在不同的环境，是FGT就必须有效的方法从该地区采集和分析样品的情况下主机和病原体之间的免疫动态的难题。
在FGT被分成两部分：上部生殖道，包括输卵管，子宫内膜和宫颈管，下部尿路含有宫颈和阴道（由Kaushic评论等 5）。现在还不清楚是什么，这些不同的网站的相对贡献是艾滋病毒感染，但据信，这两个网站可能导致艾滋病毒进入6。 T细胞代表在上下生殖道的白细胞的40-50％，而巨噬细胞含有约10％（以罗德里格斯加西亚评论等 2）。 T细胞可以在阴道，子宫颈，子宫内膜和被检测到。巨噬细胞是更强烈的局部性ZED的子宫内膜和肌层结缔组织比子宫颈，虽然它们可以同时在组织中被检测到。最后，浆细胞样树突细胞（PDC）和朗格汉斯细胞也可以在FGT组织中检测到。表型和免疫人群及其易于感染艾滋病毒的比例可根据荷尔蒙周期变化重要的是，使用荷尔蒙避孕药，细菌性阴道炎，或性活动5,7-9的。
多种方法已经被开发，研究免疫人群的FGT和环境。宫颈活检，宫颈cytobrushes和宫颈阴道灌洗液（CVL）10-12是整个文献中最常用的。 CVL收集由PBS灌洗是最简单的方法，并允许免疫调节蛋白，但结果在极低的细胞产量的研究中，并因此不适合用于研究FGT 13的免疫细胞群。 CVL样品，另一方面，VERY用于通过测量用的方法，如ELISA，细胞因子微球阵列14或质谱15,16的各种细胞因子，趋化因子或抗微生物因子的表达评估FGT的免疫环境是有用的。免疫细胞频率，表型和功能特性可通过宫颈细胞刷或宫颈活检取样采集子宫颈单个核细胞（CMC）来实现。
宫颈活检取样是一种侵入性方法，其增加出血的不适和风险，需要2至11天治愈以下的步骤取决于女人12的免疫状态。另一方面，宫颈cytobrushes，尽管收集的细胞的产量较低，是一种微创的，更方便的方法来收集免疫细胞从FGT。这两种方法都可以达到的CD45 +白细胞中相同的产率，但是两个连续的子cytobrushes是必要的，得到的细胞相同数量的载一n一个活检13。然而，细胞刷取样仍然为进一步体外表型通过流式细胞仪14提供了细胞的可接受的数量（大约5,000的CD45 +细胞/细胞刷）。此外，功能特性，可以进行对这些样品，如刺激和细胞内流式细胞术定量PCR或已使用细胞刷衍生的CMC识别HIV特异性免疫应答17或Th细胞极化18执行。扩张性T细胞的人口也可方便与中医诊所19功能研究。
要注意的是活检和cytobrushes采样FGT不同的部分是很重要的。活检是从宫颈12,13的上皮和基质的优越部导出，而颈cytobrushes采样宫颈内口，收集从宫颈管和推测的转化区的上皮来源的细胞。因此，细胞刷样品品尝重新只园组成的柱状上皮单层的，而活检，包括由鳞状复层上皮衬里5的区域。其结果是，通过宫颈活检和细胞刷收集的白细胞种群的性质不同而不同。活检收集CD3 + T细胞的比例较高，而cytobrushes导致收集的CD14 +单核细胞/巨噬细胞13的比例较高。
研究FGT的免疫学一直是关注了很多年20-22，我们已经积累了大量的专业知识与细胞刷源性间中医诊所的研究。我们的研究重点主要在艾滋病病毒感染，感染和HIV阴性曝光（HESN）女性性工作者来自肯尼亚内罗毕的研究。优先病毒复制在活化的T细胞23和较低的数字活化细胞，可以由艾滋病病毒在FGT有针对性有助于防止感染HIV。根据这一假设，几个STUDI线ES形容HESN性工作者谁是高度暴露于艾滋病毒在较低的免疫激活但仍然未感染的24,25，和这个静态的表型是在FGT 14还观察到。在这里，我们描述的方法进行处理，并通过评估体外流式细胞仪来源于宫颈cytobrushes CMC样品中的T细胞活化。
Protocol
Representative Results
Discussion
面对庞大的知识差距的相对免疫力在女性生殖道（FGT），中医诊所的表型分析可以提供见解的各种各样成多个淋巴细胞群在子宫颈。再加上蛋白质组分析及在宫颈灌洗病毒载量的测量，抗干扰性性传播感染（STI）s和其他病原体可在解剖不同人群。
技术因素-中医诊所：隔离和CMC样品成功染色是具有挑战性的。在CMC收集协议的优化强调第一收集CVL的有效性，随…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| 100uM Cell Strainer for 50 ml Falcon tube | BD | 352360 | CMC processing |
| RPMI 1640 | Hyclone | SH30027.01 | CMC processing |
| Fetal Bovine serum | Life technology | 16000044 | CMC processing |
| Fungizone | Life technology | 15290-018 | CMC processing |
| Penicillin/streptomycin | Sigma | P4333-20ml | CMC processing |
| 50ml Falcon tube | Fisher | 14-959-49A | CMC processing |
| Blood Bank disposable transfer pipette | Fisher | 13-711-6M | CMC processing |
| Cytobrush plus | Cooper surgical | C0121 | CMC sampling |
| Disposable cervical scraper | Quick medical | 2183 | CMC sampling |
| 15 ml Falcon tube | Fisher | 14-959-70c | CVL processsing |
| 1.5ml tube ependroff | Fisher | 05-402-18 | CVL storage |
| LIVE/DEAD Fixable Cell Stain Kit | Invitrogen | Various | Flow cytometry reagent |
| Fixation Buffer (4% PFA) | BD | 554655 | Flow cytometry regeant |
| IgG mouse | Sigma | I8765 | Flow cytometry regeant |
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