原人类单核细胞的隔离、转染和文化
Summary
这里介绍的是一个优化的协议，用于分离、培养、转染和区分人类原发性单核细胞与艾滋病毒感染者和健康对照。
Abstract
尽管1990年代中期采用了联合抗逆转录病毒疗法（cART），但人体免疫机能丧失病毒（艾滋病毒）仍然是一个重大的健康问题。虽然抗逆转录病毒疗法能有效地降低全身病毒载量并恢复正常的CD4+ T细胞计数，但它并不能重建一个完全功能的免疫系统。接受CART的HIV感染者免疫系统功能失调，其特征可能是免疫激活、免疫细胞早衰或持续炎症。这些情况，以及与艾滋病毒感染有关的合并因子，增加了疾病的复杂性，在细胞和动物模型中不容易重现。为了研究这些患者免疫功能障碍背后的分子事件，这里提出了一个在体外培养和操纵人类原发性单核细胞的系统。具体来说，该议定书允许从接受CART的艾滋病毒感染者以及艾滋病毒阴性对照中获得的原发性CD14和单核细胞的培养和转染。该方法涉及单核细胞和单核衍生巨噬细胞的分离、培养和转染。虽然使用了市售试剂盒和试剂，但该协议为使用miRNA模拟和抑制剂以及siRNA成功粘附和转染单核细胞提供了重要提示和优化条件。
Introduction
人体免疫缺陷病毒-1（HIV-1）感染导致严重的免疫功能障碍，可导致机会性感染和后天免疫机能丧失综合症（艾滋病）。虽然接受cART的HIV感染者的特点是病毒载量低和正常的CD4+T细胞计数，免疫系统的功能可能在这些领域受损，导致免疫反应功能失调，这与患癌症的风险增加有关。CART的HIV患者的免疫功能障碍机制在很大程度上仍不为人所知。因此，描述患者衍生的免疫细胞并研究其生物学和功能是当前艾滋病毒研究的重要组成部分。
单核细胞和巨噬细胞是免疫反应的主要调节者，在HIV感染2、3、4、5中起着基础性作用。异构和塑料在自然界中，巨噬细胞可以大致分为经典激活（M1）或替代激活（M2）。虽然这种一般分类在设置实验条件时是必要的，但巨噬细胞的极化状态可能被各种细胞因子6、7、8、9逆转。虽然有多项研究已研究爱滋病毒感染对单核细胞和树突细胞的影响，但单核细胞介导反应的分子细节基本上未知6、7、10、11、12、13、14、15、16、17、18、19。在免疫细胞调节和功能所涉及的因素中，微RNA（miRNA），即转录后调节基因表达的短非编码RNA，已被证明在主要细胞通路（即生长、分化、发育和凋亡）20中发挥重要作用。这些分子被描述为转录因子的重要调节器，对控制巨噬细胞21的功能极化至关重要。miRNA在接受CART的HIV感染者的单核细胞中的潜在作用已经调查，但该领域的进展需要更多的工作22，23，24，25，26。本文讨论了一种从HIV感染者和对照组将miRNA和siRNA转染成原发人类单核细胞的优化方法。
该协议依赖于市售试剂和试剂盒，因为技术程序的连续性有助于消除临床样本工作时不必要的实验变量。尽管如此，该方法提供了重要的提示（即，用无血清培养基培养的细胞数量或短暂孵育，以促进细胞粘附在板中）。此外，该协议中使用的极化条件派生自已发表的作品27、28、29。
Protocol
下面描述的所有方法都已获得路易斯安那州立大学健康科学中心新奥尔良机构审查委员会的批准。所有血液都是在征得知情同意后采集的。 注：整个过程在生物安全2级（BSL2）设施中的无菌条件下进行，以便谨慎处理生物材料。特别是，每个步骤都是在生物安全柜下使用无菌技术执行的。在涉及血液、血液制品、细胞或细胞产品移液的每一步后，在正确处置之前，使用罩内废…
Representative Results
使用所述程序，从艾滋病毒感染者和健康捐赠者中分离出主要的人类单核细胞。这里提供的所有数据均来自艾滋病毒和接受低（<20份/mL）或检测不到的病毒载量和正常CD4+T细胞计数的CART的受试者。分离后，细胞立即被染色，并进行流动细胞测量，以确认细胞群的纯度。结果显示，超过97%的细胞对CD14的染色呈阳性（数据未显示）。对于巨噬细胞的极化，使?…
Discussion
提出的方案演示了使用HIV感染者的初级细胞作为研究单核细胞和巨噬细胞的模型。HIV–接受 CART 的患者感染了多年，并且可能还有其他与免疫系统受损相关的共同感染。为了研究在HIV慢性感染的情况下的免疫调节，细胞直接从患者身上采集。由于miRNA已被证明在细胞发育和分化中起着重要作用，该协议侧重于在这些主细胞中操作miRNA表达的能力（图2，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢HIV临床/肿瘤生物储存核心提供患者样本和细胞免疫学代谢核心提供流式细胞测定分析。该项目由NIH P20GM121288和P30GM114732资助。
Materials
| 0.5M EDTA | Invitrogen | AM9260G | |
| BD Vacutainer Plastic Blood Collection Tubes with K2EDTA | BD Biosciences | 366643 | |
| Brilliant Stain Buffer | BD Horizon | 563794 | Flow cytometry |
| CD14 PerCP | Invitrogen | 46-0149-42 | Flow cytometry- conjugated antibody |
| CD163 BV711 | BD Horizon | 563889 | Flow cytometry- conjugated antibody |
| CD209 BV421 | BD Horizon | 564127 | Flow cytometry- conjugated antibody |
| CD80 FITC | BD Horizon | 557226 | Flow cytometry- conjugated antibody |
| CD83 APC | BD Horizon | 551073 | Flow cytometry- conjugated antibody |
| Easy 50 EasySep Magnet | StemCell Technologies | 18002 | |
| Easy Sep Direct Human Monocyte Isolation Kit | StemCell Technologies | 19669 | |
| EIF4EBP1 mAb | Cell Signaling | 9644 | Monoclonal antibody for Western blot |
| EIF4EBP1 siRNA | Santa Cruz | sc-29594 | |
| Fetal Bovin Serum Defined Heat Inactivated | Hyclone | SH30070.03HI | |
| Gallios Flow Cytometer | Beckman Coulter | B43618 | |
| GAPDH mAb | Santa Cruz | SC-47724 | Monoclonal antibody for Western blot |
| HuFcR Binding Inhibitor | eBiosciences | 14-9161-73 | Flow cytometry- blocking buffer |
| Kaluza Analysis Software | Beckman Coulter | B16406 | Software to analyze flow cytometry data |
| Lipopolysaccharides from Escherichia coli O55:B5 | Sigma | L4524 | |
| miRCURY LNA microRNA Mimic hsa-miR-146a-5p | Qiagen | YM00472124 | |
| MISSION miRNA Negative Control | Sigma | HMC0002 | Scrambled miRNA conjugated with a near infrared dye |
| Nunc 35mm Cell Culture Dish | Thermo Scientific | 150318 | |
| PBS | Gibco | 20012027 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Recombinant Human GM-CSF | R&D Systems | 215-GM-050 | |
| Recombinant Human IFN-γ | R&D Systems | 285-IF-100 | |
| Recombinant Human IL-4 | R&D Systems | 204-IL-010 | |
| Recombinant Human M-CSF | R&D Systems | 216-MC-025 | |
| RPMI 1640 with L-Glutamine | Corning | 10040CVMP | |
| Scrambled Control siRNA | Santa Cruz | sc-37007 | |
| Viromer Blue Transfection Reagent Kit | Lipocalyx | VB-01LB-01 | |
| WST-1 Cell Proliferation Reagent | Roche | 5015944001 | Colorimetric assay to assess cell viability |
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Cite This Article
Plaisance-Bonstaff, K., Faia, C., Wyczechowska, D., Jeansonne, D., Vittori, C., Peruzzi, F. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (154), e59967, doi:10.3791/59967 (2019).