人类免疫缺陷小鼠模型中的慢性、急性和再激活HIV感染
Summary
本文介绍了三种研究人类小鼠HIV感染动态的实验方法。前者允许研究慢性感染事件，而后者允许研究原发感染或病毒重新激活后的急性事件。
Abstract
人性化NOD/SCID/IL-2受体+链空小鼠重述人体免疫的一些特征，在传染病的基础和临床前研究中可以加以利用。本文介绍了三种用于研究HIV感染动态的人性化免疫缺陷小鼠模型。第一种是基于CD34的肝内注射-在新生小鼠的造血干细胞，这允许重组几个血液和淋巴组织封闭细胞，其次是感染与参考HIV菌株。此模型允许在感染后长达 36 周的监测，因此称为慢性模型。第二和第三个模型称为急性和再激活模型，其中外周血单核细胞在成年小鼠中腹中注射。在急性模型中，来自健康供体的细胞通过腹管内途径移植，然后感染参考HIV菌株。最后，在复活模型中，来自抗逆转录病毒疗法的受艾滋病毒感染捐赠者的细胞通过腹内途径进行移植。在这种情况下，鼠标中的无药物环境允许病毒重新激活和增加病毒载量。此处提供的方案描述了对HIV感染的人性化免疫缺陷小鼠模型的传统实验方法。
Introduction
人性化的NOD/SCID/白细胞素（IL）-2受体+链空（以下简称huNS+链空）小鼠模型已被广泛用于研究感染、自身免疫和癌症的发病机制，以及药物和基于人体细胞的疗法1、2的临床前研究。这些小鼠基于非肥胖糖尿病（NOD）背景，在IL-2受体β链位点（IL-2、IL-4、IL-7、IL-9、IL-15和IL-21）的共发β链上具有杀伤性突变和靶向突变，这在小鼠T-、B-和自然杀伤细胞（NK）的发育中引起严重损害。因此，它们支持人体组织、人类CD34+造血干细胞（HSCs）和人类外周血单核细胞（PBMC）3、4、5的移植。 此外，人类造血因子的转基因表达，如干细胞因子（SCF）、粒细胞因子/巨噬细胞菌群刺激因子（GM-CSF）和IL-3促进人类骨髓群6、7、8的移植。
在HIV研究中，描述了几种HuNS-链空小鼠模型，这些模型在小鼠菌株、使用的人类细胞类型、用于移植的组织类型以及细胞的来源（即健康与） 之间有所不同。HIV感染者）9日，10日。原始菌株，然而，被广泛使用，由于高水平的人体细胞移植和病毒复制后感染与参考HIV菌株11，12，13。具有人类造血因子（如NOG-EXL或NSG-SGM3）或植入人类肝脏和胸腺组织（骨髓-肝-胸腺[BLT]小鼠）的类似免疫缺陷小鼠菌株，对于评估骨髓群在抗HIV免疫反应中的作用、HIV对这些组织的影响及其作为病毒库的参与非常有用。此外，一些具有转基因表达的人类白细胞抗原（HLA）分子，以及BLT小鼠，可用于研究T细胞对HIV感染的反应16，17。
一般来说，在这些小鼠中，人性化取决于细胞来源、分娩途径（腹内、内肝、静脉内、心脏内）和小鼠年龄，在移植时为18、19、20。关于细胞来源，从脐带血、胎儿肝脏或动员的外周血中提取的人类CD34+HSC可注射在新生儿或幼鼠3、21。此外，成人β链空小鼠可以通过注射PBMC（这里称为hu-PBL-NS+链空小鼠）进行人体化，允许这些细胞在血液、继发性淋巴器官和发炎组织22、23、24中进行时间循环。
本文介绍了建立HUNS+链空鼠模型以研究HIV感染的详细方案。第一种是慢性模型，其中从健康供体脐带血中提取的人类CD34+HC被注射到新生小鼠中，随后在人类免疫系统重组14周后感染参考HIV菌株。此模型允许在感染后长达 36 周对小鼠进行监测。第二个模型是急性模型，其中从健康捐赠者中提取的PBMC注射到成年NS+链空小鼠中，随后在小鼠中人类T细胞扩张3周后感染参考HIV菌株。最后，第三个模型是重新激活模型，其中从受艾滋病毒感染的捐赠者在抑制性抗逆转录病毒疗法（ART）下获得的PBMC注射到成人NS+链空小鼠中。在这种情况下，无药物环境允许病毒重新激活和增加病毒载量。后两种模型允许在移植后长达 9 周的监测。
总体而言，这三种模型对于病毒学研究、新药临床前研究以及HIV感染对全球免疫反应的影响评估都很有用。还必须考虑，使用感染艾滋病毒的人性化小鼠需要机构生物安全委员会（IBC）以及机构动物护理和使用委员会（IACUC）在进行任何实验之前进行审查和批准。这确保了该研究遵循所有内部和外部机构条例，规定使用危险生物材料和人道处理实验动物。
Protocol
在这项工作中，所有动物护理和程序都按照马里兰大学医学院机构动物护理和使用委员会（IACUC）审查和批准的协议进行（协议号1018017、1018018和0318009）。 1. 人类CD34=新生小鼠的HSC移植 始终使用一次性个人防护设备 （PPE），包括无菌磨砂、手套、专用鞋、鞋套、面罩、护目镜、头发/胡须发动机罩和无菌实验室外套。 在 10 mL 的 RPMI 1640 介质 10% FBS 中，?…
Representative Results
如上所述，在HSC注射后14周（慢性模型）或PBMC注射后3周（急性和再活化模型）时，小鼠通过流动细胞测定筛选人体细胞移植水平。图1A显示了用于评估1人CD45+细胞重组和2）CD4+和CD8+T细胞百分比的代表性门控策略。通常，移植水平（人类CD45+细胞的百分比）在CD34+HSC注射后为10%-80%，取决于注射和小鼠菌株的路线，除上述其他因素外…
Discussion
在开发用于人性化的免疫缺陷小鼠菌株方面取得了重要进展，根据研究兴趣1，可以使用许多不同的选择。这里提供了一个一般方案，用于NS +链空小鼠和基因相似的菌株的人性化，用于三种不同的模型，用于研究艾滋病毒感染。在第一种实验方法中，辐照的新生小鼠被注射人类CD34+HSC，可以从脐带血、胎儿肝脏或动员的外周血3，21。<sup class="xref"…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了IHV临床部门内部资金对JCZ的支持。
Materials
| 0. 5 ml Microcentrifuge tubes | Neptune | 3735.S.X | |
| 1. 5 ml Microcentrifuge tubes | Neptune | 3745.S.X | |
| 10 ml Serologial pipetes | stellar sceintific | VL-4090-0010 | |
| 15 ml conical tubes | Stellar scientific | T15-600 | |
| 25 ml Serologial pipetes | stellar sceintific | VL-4090-0025 | |
| 5 ml Serologial pipetes | stellar sceintific | VL-4090-0005 | |
| 50 ml conical tubes | Stellar scientific | T50-600 | |
| ACK lysis buffer | Quality biological | 118-156-101 | |
| Alcohol prep pads | Fisher scientific | 06-669-62 | Sterile |
| Anti-Human CD3 clone UCHT1 | Biolegend | 300439 | APC conjugated |
| Anti-Human CD4 clone OKT4 | Biolegend | 317420 | AF488 conjugated |
| Anti-Human CD45 clone 2D1 | Biolegend | 368522 | BV421 conjugated |
| Anti-Human CD8 clone SK1 | Biolegend | 344710 | PerCP-Cy5.5 conjugated |
| Biosafaty cabinet level 2 | If posible connected to an exauste chimeny when handling Isoflurane | ||
| Bonnet | Fisher scientific | 17-100-900 | Single use cap for basic protection |
| Cavicide | Metrex | 13-1000 | Surface desinfectant |
| CD34+ cells | Lonza | 2C-101 | As many vials available from a single donor |
| Centrifuge | Beckman | 65-6KR | |
| Clear jar | Amazon | 77977 | |
| Cotton gauze pad | Fisher scientific | 22-415-468 | Sterile |
| Disposable lab coats | Fisher scientific | 19-472-422 | |
| EDTA micro tubes | Greiner bio-one | 450480 | |
| Face Mask | Fisher scientific | 17-100-897 | |
| FACS lysing solution | BD | 340202 | |
| FBS premium HI | Atlanta biologicals | S1115OH | |
| Ficoll | GE health one | 17-1440-02 | |
| Flow cytometer | We used FACS Aria II | ||
| Flow cytometry tubes | Falcon | 352054 | 5 ml polystyrene and round bottom |
| HIV BaL | Prepared in our uQUANT core facility | ||
| Human PBMCs | HIV positive and negative volunteers | ||
| Infrared warming pad | Venet scientific | DCT-25 | Temporary therapeutic warming pad for small animals |
| Isentress (Raltegravir) | Merck | NSC 0006-0227061 | Antiretroviral medication to treat human immunodeficiency virus (HIV)-Integrase inhibitor |
| Isoflurane | Henry Schein | NDC 11695-6776-2 | |
| Mark I irradiator | Equipment belonging to university of Maryland | ||
| Micro pipettes | |||
| Microcentrifuge | Eppendorf | ||
| Mouse ear tags | National Band & Tag company | 1005-1L1 | |
| Natelson blood collection tubes | Fisher scientific | 02-668-10 | |
| NOG-EXL | Taconic | HSCFTL-13395-F | |
| NSG mice | Jackson | 5557 | Time pregnant females for CD34 engraftment and Juveniles for PBMCs engraftment |
| NSG-SGM3 | Jackson | 13062 | |
| Paraformaldehyde 16% | Electron microscopy sciences | 15710 | |
| PBS 1X pH 7.4 | Gibco | 100-10-023 | |
| Petri dishes | Fisher scientific | 08-757-28 | |
| Quantistudio qPCR machine | Thermo | QS3 | |
| Reagent reservoirs | Costar | 4870 | |
| RPMI media 1640 1X | Gibco | 11875-093 | |
| Shoe covers | Fisher scientific | 17-100-911 | |
| Sterile disposable Gloves | Microflex | SUF-524 | |
| SuperScript II First-Strand Synthesis SuperMix | Invitrogen | 10080-400 | cDNA synthesis |
| Syringes 28-G x 1/2 | BD | 329-461 | |
| Syringes 29-G x 1/2 | BD | 324-702 | |
| Truvada (Emtricitabine and Tenofovir | Gilead | NDC 61958-0701-1 | Antiretroviral medication to treat human immunodeficiency virus (HIV)-Nicleoside analog-transcriptase inhibitor |
| Trypan blue | Sigma | T8154 | Cell count and viability |
| Vick Vaporub | School health | 43214 | Ointment based on menthol and eucalyptus |
| Water molecular biology grade | Quality biological | 351-029-131 |
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Diesen Artikel zitieren
Perdomo-Celis, F., Medina-Moreno, S., Heredia, A., Davis, H., Bryant, J., Zapata, J. C. Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models. J. Vis. Exp. (154), e60315, doi:10.3791/60315 (2019).