用于病毒研究的人源化小鼠研究进展

人源化小鼠从起初的人-鼠嵌合体到目前具有人体免疫活性的模型不断演进,已用于人免疫缺陷病毒、EB病毒、丙型肝炎病毒和登革病毒等病原体的感染、发病机制和防治的研究,取得了很大进展。我们简要介绍几种有代表性的人源化小鼠模型及其在病毒研究中的应用。     

人源化小鼠在呼吸道病原研究中的应用与发展

小鼠模型对于呼吸道病原的研究有着重要意义。然而进化所带来的肺及免疫系统的物种差异,使小鼠模型难以精准模拟人体的呼吸道病原感染。通过构建免疫系统人源化小鼠,可更好地再现人体免疫系统对呼吸道病原感染的应答。构建肺组织人源化小鼠和肺/免疫系统双人源化小鼠可实现人特异性的呼吸道病原感染及其免疫响应。本综述将从免疫系统、肺、肺/免疫系统双人源化3个方面,回顾人源化小鼠在人类呼吸道病原研究方面的进展。     

用于肝脏疾病研究的人源化小鼠模型概述

肝脏疾病是危害人类健康的重要疾病之一,合适的小动物模型的缺乏在很大程度上制约了肝脏疾病的相关研究。人源化小鼠作为重要动物模型之一,在肝脏疾病的研究中有巨大的应用价值。本文对早期的uPA小鼠、FAH小鼠、TK-NOG小鼠和近年来的AFC8小鼠等几种应用较为广泛且有代表性的人源化小鼠模型及其在肝脏疾病研究中的应用进行了对比分析,阐述了它们各自的模型原理和优缺点,以期对人源化小鼠应用于人类肝脏疾病的研究具有较为直观的认识。     

免疫系统人源化小鼠模型构建技术探讨

探讨免疫系统人源化小鼠模型构建技术的方法和技巧,能够提高免疫系统人源化小鼠模型构建的成功率,并为后续的肿瘤免疫治疗药物研发提供临床前动物模型。利用不同移植数量和供体的HuPB-MNC细胞尾静脉注射重度免疫缺陷小鼠NCG,建立免疫系统人源化小鼠模型。每周观察2次,并记录小鼠体重及死亡情况;实验第7、14、21、28和35天眼眶静脉丛试血,流式细胞术监测NCG小鼠外周血中人CD45⁺ T细胞的重建水平。NCG小鼠外周血中人CD45⁺ T细胞水平随注射后时间逐渐升高,约在3~4周达到25%以上。研究结果表明,免疫系统人源化小鼠模型构建应该从构建方法、免疫缺陷小鼠品系、性别和饲养、免疫细胞的供体和数量及细胞状态等各个方面进行考虑,通过采用多种供体克服差异性,优化肿瘤模型和免疫细胞供体联合接种方案最大化利用窗口期等策略提高模型构建成功率及应用转化率,进一步完善实验室免疫系统人源化小鼠模型构建方法,以期为肿瘤免疫疗法提供更有利的药物研发工具。     

肿瘤坏死因子受体超家族成员4(TNFRSF4)敲除和人源化小鼠模型的建立

目的 建立肿瘤坏死因子受体超家族成员4(TNFRSF4)基因人源化小鼠模型,同时建立TNFRSF4基因敲除小鼠模型,为针对TNFRSF4靶点的肿瘤治疗性抗体研发和筛选提供有效的小鼠模型,并为研究TNFRSF4在免疫过程中的作用机制提供小鼠模型.方法 利用CRISPR/Cas9技术,建立TNFRSF4基因人源化和敲除小鼠...     

人造血干细胞人源化小鼠的疝气表型分析

目的研究成年雄性人源化小鼠个体出现疝气症状的原因和对人源化小鼠的影响。方法利用显微注射法构建人造血干细胞人源化小鼠,对疝气表型特征、小鼠行为、生理和病理变化进行了研究。结果 2月龄雄性人源化小鼠出现直接性疝气症状,腹股沟区致密结缔组织结构减少可能是疝气形成的主要原因。疝气小鼠同时伴有耐力和运动协调性下降,但疝气对小鼠的繁殖系统无显著影响。结论雄性人造血干细胞人源化小鼠具有显著的疝气症状,其发生机理有待进一步研究。     

细胞毒性T淋巴细胞相关抗原4(CTLA4)敲除和人源化小鼠模型的建立

目的 建立细胞毒性T淋巴细胞相关抗原4(CTLA4)基因人源化小鼠模型,为靶向CTLA4的肿瘤治疗性抗体研发和筛选提供有效的小鼠模型.方法 利用CRISPR/Cas9技术,建立CTLA4基因人源化和敲除小鼠模型,利用PCR、RT-PCR、Western Blot、免疫组织化学和流式细胞术的方法对其进行鉴定及分析.将小鼠...     

从人体尿液中分离肾源细胞构建拟肾血管单位小鼠皮下嵌合模型及其初步评价

目的 将健康人体尿液中分离培养的肾源细胞与人源间充质干细胞(mesenchymal stem cells, MSCs)、人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)以3∶3∶2的比例混合,并移植到NOD-SCID免疫缺陷小鼠皮下而构建人源化嵌合小鼠模型。方法 (1)体外培养MSCs、HUVECs,同时从健康人体尿液中分离培养肾源细胞,并对该细胞进行免疫荧光鉴定。(2)将上述3种细胞以3∶3∶2的比例混合后与Matrigel基质胶一起注射到NOD-SCID小鼠皮下形成人源组织,通过HE染色、免疫组织化学对移植模型进行鉴定。结果 (1)成功地从尿液中分离培养出肾源细胞,并检测鉴定了肾小管上皮细胞和集合管细胞;(2)将3种细胞混合后注射到NOD-SCID免疫缺陷小鼠皮下可初步形成小管结构以及与小鼠血管相连通的人源血管结构。结论 具有肾小管上皮细胞和集合管细胞的肾源细胞与HUVECs和MSCs皮下移植入NOD-SCID免疫缺陷小鼠体内可形成嵌合的人肾血管单位小鼠模型。该模型可为肾病个体化治疗和相关病毒感染研究提供一个人源化动物...     

人成体肝细胞在Fah-/-Nod/Scid小鼠肝脏中的显著增殖

利用人肝细胞异种移植到受体鼠肝内建立人源化肝脏的嵌合体小鼠(人鼠嵌合肝)对药物代谢、乙型肝炎病毒等嗜肝性病毒及其疫苗的研究具有重要意义. 研究表明, 利用Fah^-/-Rag2^-/-Il2rg^-/-三基因剔除小鼠可获得人肝细胞在小鼠肝脏中的显著再殖, 但较高的死亡率及纯合子不能用于繁殖, 制约着该小鼠模型的规模化应用. 本研究结合延胡索酰乙酰乙酸水解酶基因剔除(Fah^-/-)小鼠的肝脏再殖优势和Nod/Scid小鼠异种移植的特点, 将这两种小鼠进行杂交繁育建立Fah^-/-Nod/Scid小鼠品系, Fah^-/-Nod/Scid小鼠可以纯合保种并能正常繁殖. 采用提前停药的预处理方案, 结合FK506处理, 移植的人成体肝细胞能够实现在Fah^-/-Nod/Scid小鼠肝脏中的显著增殖, 肝脏再殖程度达到30%以上. 采用体重曲线、肝功能和人肝细胞功能蛋白表达等三方面指标评价嵌合肝脏中人肝细胞的功能, 结果表明再殖的肝细胞具有正常的人肝细胞功能. 这些结果表明, Fah^-/-Nod/Scid小鼠可以作为理想的可规模应用的人鼠嵌合肝模型, 该技术体系的改进简化了Fah^-/-小鼠作为人源化肝脏小鼠模型的实用性问题.     

胰腺癌免疫系统人源化小鼠模型的构建及评估

目的 构建并评估胰腺癌免疫系统人源化小鼠模型,以期为胰腺癌的免疫治疗研究提供理想的临床前模型。方法 应用Ficoll密度梯度离心法,从健康人外周血中分离出新鲜的单个核细胞(peripheral blood mononucear cells, PBMC),经尾静脉注射植入重度联合免疫缺陷小鼠NCG体内,以构建免疫系统人源化小鼠模型,随后在小鼠皮下植入人胰腺癌细胞系Aspc1,并定期监测肿瘤生长情况,在PBMC植入后第3周,应用断尾法采集小鼠外周血进行流式分析,检测人CD45⁺细胞的水平,当肿瘤生长至100～200 mm³时开始给予抗PD-1单抗治疗,持续治疗3周后,对小鼠施行安乐死并取材,应用流式细胞术、免疫组化等方法分析胰腺癌免疫系统人源化小鼠外周血、脾、骨髓及肿瘤组织中人免疫细胞的浸润及活化情况。结果 植入人PBMC 3周后,在小鼠外周血、脾及骨髓中可检测到较高水平的人CD45⁺细胞;重建的人源免疫系统能够显著抑制人胰腺癌肿瘤的生长(P<0.01,P<0.001),并被人抗PD-1单抗活化,促进肿瘤组织中细...     

Mouse models with human immunity and their application in biomedical research

Biomedical research in human beings is largely restricted to in vitro studies that lack complexity of a living organism. To overcome this limitation, humanized mouse models are developed based on immunodeficient characteristics of severe combined immunodeficiency (SCID) or recombination activating gene (Rag)^null mice, which can accept xenografts. Peripheral constitution of human immunity in SCID or Rag^null mice has been achieved by transplantation of mature human immune cells, foetal human thymus, bone marrow, liver tissues, lymph nodes or a combination of these, although efficiency needs to be improved. These mouse models with constituted human immunity (defined as humanized mice in the present text) have been widely used to investigate the basic principles of human immunobiology as well as complex pathomechanisms and potential therapies of human diseases. Here, elements of an ideal humanized mouse model are highlighted including genetic and non-genetic modification of recipient mice, transplantation strategies and proposals to improve engraftments. The applications of the humanized mice to study the development and response of human immune cells, human autoimmune diseases, virus infections, transplantation biology and tumour biology are reviewed as well.     

The Establishment of an In Vivo HIV-1 Infection Model in Humanized B-NSG Mice

Suitable animal models for human immunodeficiency virus type 1(HIV-1) infection are important for elucidating viral pathogenesis and evaluating antiviral strategies in vivo. The B-NSG(NOD-PrkdcscidIl2rgtm1/Bcge) mice that have severe immune defect phenotype are examined for the suitability of such a model in this study. Human peripheral blood mononuclear cells(PBMCs) were engrafted into B-NSG mice via mouse tail vein injection, and the repopulated human T-lymphocytes were observed at as early as 3-weeks post-transplantation in mouse peripheral blood and several tissues.The humanized mice could be infected by HIV-1, and the infection recapitulated features of T-lymphocyte dynamic observed in HIV-1 infected humans, meanwhile the administration of combination antiretroviral therapy(cART) suppressed viral replication and restored T lymphocyte abnormalities. The establishment of HIV-1 infected humanized B-NSG mice not only provides a model to study virus and T cell interplays, but also can be a useful tool to evaluate antiviral strategies.     

Evolution of SIVsm in humanized mice towards HIV-2

Through the accumulation of adaptive mutations, HIV-2 originated from SIVsm. To identify these evolutionary changes, a humanized mouse model recapitulated the process that likely enabled this cross-species transmission event. Various adaptive mutations arose, as well as increased virulence and CD4⁺ T-cell decline as the virus was passaged in humanized mice.     

Inhibition of Megakaryocyte Development in the Bone Marrow Underlies Dengue Virus-Induced Thrombocytopenia in Humanized Mice

A characteristic clinical feature of dengue virus infection is thrombocytopenia, though its underlying mechanism is not definitively determined. By adoptive transfer of human CD34⁺ fetal liver cells into immunodeficient mice, we have constructed humanized mice with significant levels of human platelets, monocytes/macrophages, and hepatocytes. Infection of these mice with both lab-adapted and clinical strains of dengue virus induces characteristic human hematological changes, including transient leukopenia and thrombocytopenia. We show that the specific depletion of human platelets is not mediated by antibodies in the periphery or reduced production of human thrombopoietin in the liver but reduction of human megakaryocytes and megakaryocyte progenitors in the bone marrow of the infected mice. These findings identify inhibition of platelet production in the bone marrow as a key mechanism underlying dengue-induced thrombocytopenia and suggest the utility of the improved humanized mouse model in studying dengue virus infection and pathogenesis in a human cell context.     

Dengue virus tropism in humanized mice recapitulates human dengue fever

Animal models of dengue virus disease have been very difficult to develop because of the virus' specificity for infection and replication in certain human cells. We developed a model of dengue fever in immunodeficient mice transplanted with human stem cells from umbilical cord blood. These mice show measurable signs of dengue disease as in humans (fever, viremia, erythema and thrombocytopenia), and after infection with the most virulent strain of dengue serotype 2, humanized mice showed infection in human cells in bone marrow, spleen and blood. Cytokines and chemokines were secreted by these human cells into the mouse bloodstream. We demonstrated that the pathology of dengue virus infection in these mice follows that reported in human patients, making this the first valid and relevant model for studying dengue fever pathogenesis in humans.     

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

5S is a mouse monoclonal IgG1 that binds to the ‘a’ epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K_D = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.     

Applicability of bioengineered human skin: from preclinical skin humanized mouse models to clinical regenerative therapies

Ongoing progress in the field of regenerative medicine, in combination with the development of tissue-engineered skin products, has opened new possibilities for the treatment of certain diseases in which current treatments are aimed at alleviating symptoms but are not able to get a permanent cure. Our laboratory has developed a fibrin-based bioengineered human skin that has been successfully used for permanent regenerative therapies in different situations in the clinic. Moreover, we have been able to stably regenerate human skin by orthotopic grafting of this skin equivalent onto the back of immunodeficient mice. The so-called skin-humanized mouse model system has permitted us to model several monogenic skin diseases, when keratinocytes and fibroblasts harboring the genetic defect were used. In most cases different gene therapy approaches for ex vivo correction of cells have proved effective in reverting the phenotype using this model. More importantly, the feasibility of the system has allowed us to generate a skin humanized mouse model for psoriasis, a common chronic inflammatory disease where the immune component has a pivotal role in the pathogenesis. Establishing reliable humanized animal models for skin diseases is necessary to gain a deeper knowledge of the pathogenesis and to develop novel therapeutic strategies. In this sense, the skin humanized mouse model developed in our laboratory meets the needs of this field of research.     

Generation of a functional humanized Delta-like ligand 4 transgenic mouse model

Humanized mouse models are important tools in many areas of biological drug development including, within oncology research, the development of antagonistic antibodies that have the potential to block tumor growth by controlling vascularization and are key to the generation of in vivo proof-of-concept efficacy data. However, due to cross reactivity between human antibodies and mouse target such studies regularly require mouse models expressing only the human version of the target molecule. Such humanized knock-in/knock-out, KIKO, models are dependent upon the generation of homozygous mice expressing only the human molecule, compensating for loss of the mouse form. However, KIKO strategies can fail to generate homozygous mice, even though the human form is expressed and the endogenous mouse locus is correctly targeted. A typical strategy for generating KIKO mice is by ATG fusion where the human cDNA is inserted downstream of the endogenous mouse promoter elements. However, when adopting this strategy it is possible that the mouse promoter fails to express the human form in a manner compensating for loss of the mouse form or alternatively the human protein is incompatible in the context of the mouse pathway being investigated. So to understand more around the biology of KIKO models, and to overcome our failure with a number of ATG fusion strategies, we developed a range of humanized models focused on Delta-like 4 (Dll4), a target where we initially failed to generate a humanized model. By adopting a broader biologic strategy, we successfully generated a humanized DLL4 KIKO which led to a greater understanding of critical biological aspects for consideration when developing humanized models.     

PD-1 Blockade in Chronically HIV-1-Infected Humanized Mice Suppresses Viral Loads

An estimated 34 million people are living with HIV worldwide (UNAIDS, 2012), with the number of infected persons rising every year. Increases in HIV prevalence have resulted not only from new infections, but also from increases in the survival of HIV-infected persons produced by effective anti-retroviral therapies. Augmentation of anti-viral immune responses may be able to further increase the survival of HIV-infected persons. One strategy to augment these responses is to reinvigorate exhausted anti-HIV immune cells present in chronically infected persons. The PD-1-PD-L1 pathway has been implicated in the exhaustion of virus-specific T cells during chronic HIV infection. Inhibition of PD-1 signaling using blocking anti-PD-1 antibodies has been shown to reduce simian immunodeficiency virus (SIV) loads in monkeys. We now show that PD-1 blockade can improve control of HIV replication in vivo in an animal model. BLT (Bone marrow-Liver-Thymus) humanized mice chronically infected with HIV-1 were treated with an anti-PD-1 antibody over a 10-day period. The PD-1 blockade resulted in a very significant 45-fold reduction in HIV viral loads in humanized mice with high CD8⁺ T cell expression of PD-1, compared to controls at 4 weeks post-treatment. The anti-PD-1 antibody treatment also resulted in a significant increase in CD8⁺ T cells. PD-1 blockade did not affect T cell expression of other inhibitory receptors co-expressed with PD-1, including CD244, CD160 and LAG-3, and did not appear to affect virus-specific humoral immune responses. These data demonstrate that inhibiting PD-1 signaling can reduce HIV viral loads in vivo in the humanized BLT mouse model, suggesting that blockade of the PD-1-PD-L1 pathway may have therapeutic potential in the treatment of patients already infected with the AIDS virus.     

Dimeric 2G12 as a potent protection against HIV-1

We previously showed that broadly neutralizing anti-HIV-1 antibody 2G12 (human IgG1) naturally forms dimers that are more potent than monomeric 2G12 in in vitro neutralization of various strains of HIV-1. In this study, we have investigated the protective effects of monomeric versus dimeric 2G12 against HIV-1 infection in vivo using a humanized mouse model. Our results showed that passively transferred, purified 2G12 dimer is more potent than 2G12 monomer at preventing CD4 T cell loss and suppressing the increase of viral load following HIV-1 infection of humanized mice. Using humanized mice bearing IgG "backpack" tumors that provided 2G12 antibodies continuously, we found that a sustained dimer concentration of 5-25 μg/ml during the course of infection provides effective protection against HIV-1. Importantly, 2G12 dimer at this concentration does not favor mutations of the HIV-1 envelope that would cause the virus to completely escape 2G12 neutralization. We have therefore identified dimeric 2G12 as a potent prophylactic reagent against HIV-1 in vivo, which could be used as part of an antibody cocktail to prevent HIV-1 infection.