Özet

建立与艾滋病毒相关的肝脏发病机制的双人性化TK-NOG小鼠模型

Published: September 11, 2019
doi:

Özet

该协议提供了一种可靠的方法,建立人类免疫系统和肝细胞的人性化小鼠。通过注射人肝细胞和CD34实现的双重组免疫缺陷小鼠造血干细胞易受人体免疫缺陷病毒-1感染和重述肝损伤的影响,如艾滋病毒感染者。

Abstract

尽管感染人体免疫机能丧失病毒-1(HIV-1)的患者预期寿命延长,但肝病已成为其发病的常见原因。HIV-1引起的肝脏免疫病理学仍然难以捉摸。具有人类肝细胞和人类免疫系统的小型异种移植动物模型可以概括人类生物学的发病机制。本文描述了一个方案,通过人类肝细胞和CD34——造血干细胞/祖细胞(HSPCs)移植建立双重人化小鼠模型,以研究HIV感染者观察到的肝脏免疫病理学。为了实现双重重组,男性TK-NOG(NOD)。Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic)小鼠在腹内注射甘西洛韦(GCV)剂量,以消除小鼠转基因肝细胞,并结合三硫丹进行非骨髓性调理,这两种促进人类肝细胞 (HEP) 移植和人类免疫系统 (HIS) 发育。人白蛋白 (ALB) 水平用于肝脏移植,流式细胞测定检测的血液中存在人体免疫细胞,证实了人类免疫系统的建立。使用此处所述的协议开发的模型类似于HIV-1感染造成的肝脏损伤的多个组成部分。其建立对于肝炎病毒合并感染的研究以及抗病毒和抗逆转录病毒药物的评估可能至关重要。

Introduction

自抗逆转录病毒疗法问世以来,与艾滋病毒-1单一感染有关的死亡人数已大大减少。然而,肝病已成为艾滋病病毒感染者发病的常见原因1,2。肝炎病毒感染与HIV-1感染比较常见,占美国艾滋病病毒感染者的10%-30%,3、4、5。

HIV-1和肝炎病毒的宿主特异性限制了小动物模型研究人类特定传染病或研究HIV-1相关肝脏发病机制的多个方面的效用。免疫缺陷小鼠,允许移植人体细胞和/或组织(称为人化小鼠模型)是可以接受的动物模型为临床前研究6,7,8。自21世纪初引入人化小鼠以来,多次对胆囊静物人类肝毒性、人类特异性病原体(包括HIV-1和HIV相关神经认知障碍、爱泼斯坦巴尔病毒、肝炎等)进行临床前研究传染病,已对这些小鼠进行了6、9、10、11的调查。CD34+ HSPCs和/或人类肝细胞移植的多种小鼠模型早已开发出来,并随着时间的推移得到了改进,以研究乙型肝炎病毒(HBV)相关肝病的疾病发病机制12,13,14.HSPC和人肝细胞移植的几个模型基于菌株,称为NOG(NOD)。Cg-Prkdcscid Il2rgtm1Sug/JicTac)813, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ15, 巴尔布/C-拉格2-/- —–(拉格2tm1.1Flv Il2rgtm1.1Flv/J)12,和fah-/- NOD rag1il2ré_null鼠标16。但是,每个模型都有自己的优点和局限性;例如,AFC8 在 Balb/C-Rag2-/- —c -/-背景上为 HEP 和人类干细胞 (HSCs) 提供双人机小鼠,使免疫细胞和 HSC 能够成功移植,但不存在抗原特异性 T 细胞和 B 细胞在此模型12中响应 。在重组双人性小鼠时,主要关切包括次优移植、缺乏支持不同组织的合适模型、不匹配的条件、免疫排斥或移植-宿主疾病(GVHD)和技术困难,如与新生儿的风险操纵和高死亡率由于代谢异常13。

虽然人类化小鼠已用于艾滋病研究多年17、18、19,但使用人化小鼠研究HIV-1对肝脏的损害已经有限20。我们之前曾报道建立了双重人性化的TK-NOG小鼠模型,并将其应用于与艾滋病毒相关的肝病8。该模型显示了肝脏和免疫细胞的强健移植,并重述了HIV感染的发病机制。本讨论提出了详细的方案,包括人类肝细胞移植中最关键的步骤。还介绍了成功移植HEP和在TK-NOG小鼠中建立功能免疫系统所需的HSPCs。详细介绍了这些小鼠研究HIV相关肝脏免疫机原机制。使用TK-NOG雄性小鼠携带肝脏特异性疱疹单纯疱疹病毒类型1胸腺激酶(HSV-TK)转基因。表达这种转基因的小鼠肝细胞在短暂接触无毒剂量的GCV后,很容易被消融。移植的人类肝细胞在小鼠肝脏内保持稳定,没有外源性药物21。小鼠还预置了非骨髓剂量的三硫丹,为人类细胞8在小鼠骨髓中创造一个利基。免疫缺陷的TK-NOG小鼠在脾脏注射了HEP和多能的HSPCs。然后,通过血液免疫造影和分别测量血清人-白蛋白水平,定期监测小鼠的血液和肝脏重组。成功重组超过15%的小鼠为人体免疫细胞和HEP注射HIV-1。艾滋病毒对肝脏的影响最早可在感染后4-5周内评估。必须指出,由于使用了HIV-1,在处理病毒并将其注射到小鼠体内时,必须采取一切必要的预防措施。

Protocol

该协议已获得内布拉斯加大学医学中心机构动物护理和使用委员会(IACUC)的批准。 注:在动物实验前,先获得当地IACUC的批准。 1. 脐带血的处理与人体HSPC的分离 在层流柜的无菌条件下执行协议的所有步骤。 取在肝化管中收集的脐带血(CB),通过添加磷酸盐缓冲盐水(PBS)使体积达到35 mL。如图 1所示?…

Representative Results

通过非常简单的ELISA和流式细胞测定,在每一步都可以轻松监测与人类肝脏和免疫细胞的双重人性化小鼠模型。定期进行流式细胞测量,以评估功能免疫系统的发展,并观察艾滋病毒感染对免疫细胞的影响。在双人性小鼠中,功能免疫细胞的发育范围为淋巴细胞门的15%至90%。免疫细胞的代表性子集以点图显示(图3)。为了评估人类肝细胞的移植,ELIS…

Discussion

在HIV感染者24日肝脏受损。实验性小动物模型研究人类肝脏疾病在HIV-1的存在是极其有限的,尽管有一些联合移植的动物模型与CD34+ HSPCs和肝细胞7,12 25.在体外实验中,肝细胞被证明有低水平的HIV-1感染26。携带这两种人类细胞的人类小鼠是理想的模型。在人类调节T细?…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家卫生研究院的拨款R24OD018546(给L.Y.P.和S.G.)的支持。作者要感谢李伟哲博士,在外科手术方面的帮助,阿曼达·科科·伍兹,B.S.,严成免疫性组学,UNMC流式细胞学研究设施成员菲利普·赫克斯利主任,博士,维多利亚B.史密斯,B.S.和萨曼莎沃尔,B.S.,UNMC高级显微镜核心设施成员Janice A. 泰勒,B.S.和詹姆斯R.塔斯卡拉斯,B.S.,技术支持。作者承认CIEA的Mamoru Ito博士和Hiroshi Suemizu博士提供TK-NOG小鼠,约阿希姆·鲍姆加特博士提供硫磺范。作者感谢UNMC的阿德里安·科斯特斯博士对手稿的编辑贡献。

Materials

27G1/2" needles BD biosciences 305109
30G1/2" needles BD biosciences 305106
5 mL polystyrene  round-bottom tube 12 x 75 mm style Corning 352054
BD 1 mL Tuberculin Syringe Without Needle BD biosciences 309659
BD FACS array bioanalyzer  BD Biosciences For purity check of eluted CD34+ cells 
BD FACS array software BD Biosciences Software to analysis acquired CD34+ cell on FACS array
BD FACS lysing solution BD Biosciences 349202 To lyse red blood cells
BD LSR II BD Biosciences Instrument for acquisiton of flow cytometry samples
BD Vacutainer Plastic Blood Collection Tube BD biosciences BD 367874 To collect Cord blood
Bovine Serum Albumin  Sigma-aldrich A9576
Buprenorphine Controlled substance and pain-killer
CD14-PE BD Biosciences 555398 Specific to human
CD19-BV605 BD Biosciences 562653 Specific to human
CD34 MicroBead Kit, human Miltenyi Biotec 130-046-702 For isoation of   CD34+ HSPC
CD34-PE, human Miltenyi Biotec 130-081-002 Antibody used for purity check of eluted CD34+ cells 
CD3-AF700 BD Biosciences 557943 Specific to human
CD45-PerCPCy5.5 BD Biosciences 564105 Specific to human
CD4-APC BD Biosciences 555349 Specific to human
CD8-BV421 BD Biosciences 562428 Specific to human
Cell counting slides Bio-rad 1450015
ChargeSwitch gDNA Mini Tissue Kit Thermofisher scientific CS11204 for extraction of genomic DNA from ear piece
Cobas Amplicor system v1.5  Roche Molecular Diagnostics bioanalyzer to measure viral load
Cotton-tipped applicators   McKesson 24-106-2S
Cytokeratin-18 (CK18) DAKO M7010 Specific to human
DMSO (Dimethyl sulfoxide) Sigma-aldrich D2650-5X5ML
Extension set Microbore Slide Clamp(s) Fixed Male Luer Lock. L: 60 in L: 152 cm PV: 0.55 mL Fluid Path Sterile BD biosciences 30914 Attached to dispensing pippet and to load with HSPC and HEP suspesion
FACS Diva version 6 BD Biosciences flow cytometer software required for  acqusition of sample
Fetal Bovine Serum (FBS) Gibco 10438026
FLOWJO analysis software
v10.2
FLOWJO, LLC flow cytometry analysis software
Ganciclovir APP Pharmaceuticals, Inc. 315110 Prescripition drug
Greiner MiniCollect EDTA Tubes Greiner bio-one 450475
Hepatocytes thawing medium  Triangle Research Labs  MCHT50
Horizon Open Ligating Clip Appliers Teleflex 537061 To hold the ligating clips
Hospira Sterile Water for Injection ACE surgical supply co. Inc. 001-1187 For dilution of Buprenorphine (pain-killer)
Human Albumin ELISA Quantitation Set Bethyl laboratories E80-129 For assesing human albumin levels in mouse serum
Human hepatocyte Triangle Research Labs  HUCP1  Cryopreserved human hepatocytes, induction qualified 
Iris Scissors, Straight Ted Pella, Inc. 13295
Lancet MEDIpoint Goldenrod 5 mm
LS columns  Miltenyi Biotec 130-042-401 Used to entrap CD34+ microbeads (positive selection)
Lymphocyte Separation Medium (LSM) MP Biomedicals 50494 For isoation of   lymphocytes from peripheral blood
MACS MultiStand Miltenyi Biotec 130-042-303 holds Qudro MACS seperator and LS columns
McPherson-Vannas Micro Dissecting Spring Scissors Roboz Surgical Instrument Co. RS-5605 Used to make an incision on skin to expose spleen
Micro Dissecting Forceps Roboz Surgical Instrument Co. RS-5157  to hold and pull out spleen from peritoneal cavity
mouse CD45-FITC BD Biosciences 553080 mouse-specific
PBS (Phosphate Buffered Saline) Hyclone SH30256.02
Qudro MACS separator  Miltenyi Biotec 130-090-976 holds four LS columns
RPMI 1640 medium Gibco 11875093
StepOne Plus Real Time PCR  Applied Biosystems Instrument used  to  genotype
Stepper Series Repetitive Dispensing Pipette 1ml DYMAX CORP T15469 Used to  dispense  HSPC and HEP supension in controlled manner
Suturevet PGA synthetic absorbale suture Henry Schein Animal Health 41178 Suturing of skin and peritoneum
TaqMan Gene Expression Master Mix Thermofisher scientific 4369016
TC20 automated cell counter Bio-rad 1450102
TK-NOG mice  Provided by the Central Institute for Experimental Animals (CIEA, Japan; Drs. Mamoru Ito and Hiroshi Suemizu)
Treosulfan Medac GmbH Provided by  Dr. Joachim Baumgart (medac GmbH) 
Trypan Blue Bio-rad 1450022
Vannas-type Micro Scissors, Straight, 80mm L Ted Pella, Inc. 1346 Used to make an incision on skin to expose spleen
Weck hemoclip traditional titanium ligating clips Esutures 523700 To ligate the spleen post-injection

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Bu Makaleden Alıntı Yapın
Dagur, R. S., Wang, W., Makarov, E., Sun, Y., Poluektova, L. Y. Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis. J. Vis. Exp. (151), e58645, doi:10.3791/58645 (2019).

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