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
Summary
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）8，13， NSG （NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ）15， 巴尔布/C-拉格2-/- —–（拉格2tm1.1Flv Il2rgtm1.1Flv/J）12，和fah-/- NOD rag1—il2ré_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?…
Divulgations
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 |
References
- Smith, C., et al. Factors associated with specific causes of death amongst HIV-positive individuals in the D:A:D Study. AIDS. 24 (10), 1537-1548 (2010).
- Puoti, M., et al. Mortality for liver disease in patients with HIV infection: a cohort study. Journal of Acquired Immune Deficiency Syndromes. 24 (3), 211-217 (2000).
- Rodriguez-Mendez, M. L., Gonzalez-Quintela, A., Aguilera, A., Barrio, E. Prevalence, patterns, and course of past hepatitis B virus infection in intravenous drug users with HIV-1 infection. The American Journal of Gastroenterology. 95 (5), 1316-1322 (2000).
- Scharschmidt, B. F., et al. Hepatitis B in patients with HIV infection: relationship to AIDS and patient survival. Annals of Internal Medicine. 117 (10), 837-838 (1992).
- Lacombe, K., Rockstroh, J. HIV and viral hepatitis coinfections: advances and challenges. Gut. 61, 47-58 (2012).
- Brehm, M. A., Jouvet, N., Greiner, D. L., Shultz, L. D. Humanized mice for the study of infectious diseases. Current Opinion in Immunology. 25 (4), 428-435 (2013).
- Billerbeck, E., et al. Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells. The Journal of Hepatology. 65 (2), 334-343 (2016).
- Dagur, R. S., et al. Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice. Biology Open. 7 (2), (2018).
- Gaska, J. M., Ploss, A. Study of viral pathogenesis in humanized mice. Current Opinion in Virology. 11, 14-20 (2015).
- Gorantla, S., Poluektova, L., Gendelman, H. E. Rodent models for HIV-associated neurocognitive disorders. Trends in Neurosciences. 35 (3), 197-208 (2012).
- Xu, D., et al. Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity. The Journal of Pharmacology and Experimental Therapeutics. 352 (2), 274-280 (2015).
- Washburn, M. L., et al. A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease. Gastroenterology. 140 (4), 1334-1344 (2011).
- Gutti, T. L., et al. Human hepatocytes and hematolymphoid dual reconstitution in treosulfan-conditioned uPA-NOG mice. The American Journal of Pathology. 184 (1), 101-109 (2014).
- Strick-Marchand, H., et al. A novel mouse model for stable engraftment of a human immune system and human hepatocytes. PLoS One. 10 (3), 0119820 (2015).
- Keng, C. T., et al. Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection. Gut. 65 (10), 1744-1753 (2016).
- Li, F., Nio, K., Yasui, F., Murphy, C. M., Su, L. Studying HBV Infection and Therapy in Immune-Deficient NOD-Rag1-/-IL2RgammaC-null (NRG) Fumarylacetoacetate Hydrolase (Fah) Knockout Mice Transplanted with Human Hepatocytes. Methods in Molecular Biology. 1540, 267-276 (2017).
- Poluektova, L. Y., Garcia, J. V., Koyanagi, Y., Manz, M. G., Tager, A. M. . Humanized Mice for HIV Research. , (2014).
- Cheng, L., Ma, J., Li, G., Su, L. Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy. Frontiers in Immunology. 9, 817 (2018).
- Zhang, L., Su, L. HIV-1 immunopathogenesis in humanized mouse models. Cellular & Molecular Immunology. 9 (3), 237-244 (2012).
- Nunoya, J., Washburn, M. L., Kovalev, G. I., Su, L. Regulatory T cells prevent liver fibrosis during HIV type 1 infection in a humanized mouse model. The Journal of Infectious Diseases. 209 (7), 1039-1044 (2014).
- Hasegawa, M., et al. The reconstituted ‘humanized liver’ in TK-NOG mice is mature and functional. Biochemical and Biophysical Research Communications. 405 (3), 405-410 (2011).
- Higuchi, Y., et al. The human hepatic cell line HepaRG as a possible cell source for the generation of humanized liver TK-NOG mice. Xenobiotica. 44 (2), 146-153 (2014).
- Kosaka, K., et al. A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections. Biochemical and Biophysical Research Communications. 441 (1), 230-235 (2013).
- Crane, M., Iser, D., Lewin, S. R. Human immunodeficiency virus infection and the liver. World Journal of Hepatology. 4 (3), 91-98 (2012).
- Bility, M. T., Li, F., Cheng, L., Su, L. Liver immune-pathogenesis and therapy of human liver tropic virus infection in humanized mouse models. Journal of Gastroenterology and Hepatology. 28, 120-124 (2013).
- Kong, L., et al. Low-level HIV infection of hepatocytes. Virology Journal. 9, 1-7 (2012).
- Dash, P. K., et al. Long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in HIV-1-infected humanized mice. AIDS. 26 (17), 2135-2144 (2012).
- Sun, S., Li, J. Humanized chimeric mouse models of hepatitis B virus infection. International Journal of Infectious Diseases. 59, 131-136 (2017).
- Shafritz, D. A., Oertel, M. Model systems and experimental conditions that lead to effective repopulation of the liver by transplanted cells. The International Journal of Biochemistry & Cell Biology. 43 (2), 198-213 (2011).
- Almeida-Porada, G., Porada, C. D., Chamberlain, J., Torabi, A., Zanjani, E. D. Formation of human hepatocytes by human hematopoietic stem cells in sheep. Blood. 104 (8), 2582-2590 (2004).
- Streetz, K. L., et al. Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells. Hepatology. 47 (2), 706-718 (2008).
Citer Cet Article
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).