Construction method and application of peripheral erythrocyte humanized mouse model

Abstract

The invention relates to a construction method of a peripheral red blood cell humanized mouse, which comprises the following steps: transplanting human hematopoietic stem cells into the mouse and then applying a red blood cell inducer to the mouse, or applying a red blood cell inducer to the mouse and then infusing human red blood cells into the mouse. The red blood cell inducer comprises an antibody targeting mouse macrophage, dexamethasone or a derivative thereof, and a human macrophage colony stimulating factor. In a mouse transplanted with human hematopoietic stem cells, one or more of an antibody targeting mouse macrophages, dexamethasone and a human macrophage colony stimulating factor can be used in the red blood cell induced maturation process, and high-level human red blood cell reconstruction can be quickly realized within three weeks; the maturity and the denucleation ratio of peripheral human erythrocytes reconstructed in the mouse are completely consistent with those of normal human blood; in a mouse transplanted with human erythrocytes, the erythrocyte induction process can be single treatment of an antibody or dexamethasone, or combined use of the antibody and dexamethasone. The invention also provides application of the peripheral erythrocyte humanized mouse.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for constructing a peripheral red blood cell humanized mouse model, the constructed peripheral red blood cell humanized mouse model and its application. Background Art

[0002] Immunological research and exploration rely on experimental animal models. Mice, as a classic experimental species, are widely used in a variety of immunological experiments and have driven advancements in the discipline. However, after millions of years of evolutionary divergence, significant differences remain between the mouse immune system and the human immune system, such as differences in the complement system, the composition of peripheral blood immune cell subsets, and the expression of Fc receptors. These differences in the human and mouse immune systems prevent direct clinical application of research results obtained using mouse models, hindering the efficiency of basic research to clinical translation and resulting in a waste of social resources. To address this issue, researchers have developed humanized mouse models incorporating human cells or tissues. These models, which mimic the dynamics of the human immune system, hold promise as a breakthrough. Common mouse models with humanized immune systems are based on immunodeficient mice transplanted with human hematopoietic stem cells to reconstitute the human immune system. These mouse models with humanized immune systems bridge the gap between basic research and clinical translation and are currently being widely used in fields such as oncology and infection. However, red blood cells, the most abundant cell type in the human body, are difficult to efficiently reconstitute in mice with humanized immune systems. Developing humanized immune system mouse models with high levels of human red blood cell reconstitution is a major challenge in this field. Therefore, constructing humanized animal models that reflect the human cell pathology involved in disease progression is urgent.

[0003] Red blood cells (RBCs) play an irreplaceable role in gas exchange and maintaining immune system function. RBC-related diseases, such as anemia and malaria, remain a major global public health threat. Most existing in vivo RBC disease models use mouse RBCs as research subjects. However, because humans and mice are two different species, RBCs differ significantly in size and developmental processes. Therefore, simple mouse models cannot accurately reflect the state of human cells during the disease process, resulting in treatment strategies that cannot be directly extended to clinical applications. Furthermore, many human RBC pathogens, such as Plasmodium falciparum, cannot infect mouse RBCs, leading to a lack of in vivo models in this field. The mouse and human Plasmodium parasites that can infect mouse RBCs are essentially different strains, with significant differences in their targets and mechanisms of infection. Consequently, antimalarial drugs cannot be effectively evaluated and related mechanistic research conducted, severely hindering the global goal of achieving "One Health."

[0004] In the past, there were three main models for humanizing red blood cells (RBCs). The first is the human RBC transfusion model, in which healthy human RBCs are intravenously injected into immunodeficient mice to establish circulating human RBCs in the mouse bloodstream. In this method, mice are injected daily with a human RBC suspension equivalent to half the total mouse blood volume, making the experimental process very cumbersome. More importantly, these transfused human RBCs do not undergo a complete hematopoietic development process in the mouse body, making the transfusion model poorly reflective of the true physiological state of RBCs. The second is the induction model, in which researchers inject liposomes, gadolinium chloride, or other methods into mice with humanized immune systems to deplete mouse macrophages. This reduces the phagocytosis of human RBCs by these mouse macrophages, thereby reestablishing human RBCs. Although the reconstructed human RBCs in this model undergo a complete hematopoietic development process in vivo, the toxicity associated with repeated injections of liposomes or gadolinium chloride results in over half of the mice dying during the injection process, significantly increasing experimental costs. The third type is the spontaneous model. While this model does not require repeated daily injections of red blood cells and drugs, its construction requires the multi-gene (eight-gene) modified mouse model MISTRGF as its foundation. This mouse strain is not available in China, which has seriously hindered the progress of the field of humanized red blood cell in China. In addition, the model construction relies on the co-transplantation of human stem cells and hepatocytes, and the construction time is as long as six months, which severely limits the model's application window.

[0005] The above technology is based on the elimination of mouse macrophages to reduce their phagocytosis of human red blood cells, ignoring the important role of macrophages in supporting the development of red blood cells. In hematopoietic organs, macrophages can form a "hematopoietic island" structure with developing red blood cells, namely erythroblasts. This structure is composed of central macrophages and surrounding erythroblasts. Macrophages can provide anti-apoptotic signals and pro-mitotic signals to erythroblasts, and participate in the denucleation process of erythroblasts, etc., and play an irreplaceable role in maintaining the development and maturation of red blood cells. Previous studies have shown that in mice with humanized immune systems, human erythroblasts and human macrophages, two important components of the "hematopoietic island" structure, have always been difficult to reconstruct efficiently.

[0006] Therefore, there is an urgent need in the art for a humanized mouse model with a large number of mature circulating human red blood cells. The present invention, by replenishing human macrophages in mice with humanized immune systems, provides supportive signals for the development of human erythroblasts, promotes the development of human erythroblasts in hematopoietic organs, and thus reconstitutes circulating mature human red blood cells in vitro. Summary of the Invention

[0007] In response to the problems existing in the above-mentioned prior art, the present invention provides a method for constructing a peripheral red blood cell humanized mouse, which comprises: transplanting human hematopoietic stem cells into the mouse, and then administering a red blood cell inducer to the mouse, or administering a red blood cell inducer to the mouse, and then transfusing human red blood cells into the mouse.

[0008] Humanized immune system (HIS) mice refer to humanized mice with an artificial hematopoietic immune system. The human cells implanted into severely immunodeficient mice are artificial hematopoietic stem cells, which recreate the human immune system in the mice. They are currently widely used in the fields of tumors and infections.

[0009] Hematopoietic stem cells (HSCs) are adult stem cells in the blood system. They are a heterogeneous group with the ability to self-renew over the long term and the potential to differentiate into various mature blood cells. HSCs need to replenish the various mature cell components of the blood system in a timely manner according to the body's physiological needs. At the same time, under stressful conditions such as injury and inflammation, HSCs also play a role in regulating and maintaining the physiological balance of the various cellular components of the blood system in the body. Highly purified HSCs at different stages can be isolated using cell surface markers.

[0010] As certain embodiments of the present application, the erythrocyte inducing agent includes one or more of antibodies targeting mouse macrophages, dexamethasone or its derivatives, and human macrophage colony stimulating factor.

[0011] As certain embodiments of the present application, the dexamethasone derivative includes one or more of pharmaceutical salts, esters, hydrates, and solvates of dexamethasone.

[0012] As certain embodiments of the present application, the dexamethasone derivatives include one or more of dexamethasone sodium phosphate, dexamethasone acetate, dexamethasone palmitate, beclomethasone, beclomethasone dipropionate, hydrocortisone, and prednisone.

[0013] As certain embodiments of the present application, the antibody targeting mouse macrophages includes anti-mouse F4 / 80 antibody and / or anti-mouse CSF1R antibody.

[0014] Anti-mouse F4 / 80 antibodies target the F4 / 80 protein in mice, which is primarily expressed on the surface of macrophages and therefore plays an important role in immunological research. Anti-mouse F4 / 80 antibodies can be used in a variety of experimental methods, including immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry (FC), helping researchers analyze F4 / 80-positive cells in specific cells or tissues. Anti-mouse F4 / 80 antibodies can be used to study various aspects of the immune system, including adaptive immune responses and G protein-coupled receptor signaling pathways. Anti-mouse F4 / 80 antibodies can be polyclonal or monoclonal and can be derived from mice or other animals.

[0015] The anti-murine CSF1R antibody is directed against the mouse colony-stimulating factor 1 receptor (CSF1R), also known as the macrophage colony-stimulating factor receptor (M-CSFR). CSF1R is a single-pass type I membrane protein and a member of the platelet-derived growth factor receptor family. In mice, CSF1R is expressed by monocytes / macrophages, peritoneal exudate cells, plasmacytoid and conventional dendritic cells, and osteoclasts. CSF1R is the receptor for CSF1, and CSF1 signaling through CSF1R regulates the proliferation and differentiation of cells in the monocytic lineage.

[0016] The method for constructing humanized peripheral red blood cell (PRC) mice in this application builds on existing induction models by screening for low-toxic macrophage scavenger Abs (anti-mouse F4 / 80 and CSF1R antibodies). Unlike the highly toxic liposomes and gadolinium chloride used in previous studies, these Abs efficiently eliminate mouse macrophages without significant toxicity. Furthermore, they can also efficiently reconstitute human macrophages, a feat not achieved with previous induction models. The low toxicity of these macrophage scavenger Abs enables humanized PRC mice to rapidly achieve high levels of human erythrocyte reconstitution within three weeks.

[0017] As certain embodiments of the present application, the erythrocyte inducing agent further includes dexamethasone or a derivative thereof.

[0018] Dexamethasone (DXMS) has a chemical structure characterized by the introduction of a fluorine atom at the 9α position of the B ring and a methyl group at the 16α ​​position of the D ring. Both the 9α-fluorine and 16α-methyl groups significantly enhance its anti-inflammatory activity, while the 16α-methyl group significantly reduces the water and sodium retention side effect of dexamethasone. The clinical bioequivalence ratio of dexamethasone to prednisolone is 0.75:5, and its biological half-life is 36-54 hours, making it a long-acting glucocorticoid. Like other glucocorticoids, dexamethasone exhibits pharmacological effects such as anti-inflammatory, anti-endotoxin, immunosuppressive, anti-shock, and stress response enhancement. Therefore, it is widely used in various medical disciplines to treat a variety of conditions, including autoimmune diseases, allergies, inflammation, asthma, and dermatological and ophthalmological conditions. Dexamethasone sodium phosphate injection is an indispensable emergency medicine for critically ill patients.

[0019] Macrophage colony-stimulating factor (M-CSF), also known as colony-stimulating factor-1 (CSF-1), is a lineage-specific cytokine. M-CSF is a dimeric glycoprotein linked by interchain disulfide bonds. It is primarily found in the bone marrow cavity and plays a vital role in the proliferation, differentiation, and maintenance of monocyte activity. It also regulates placental function and promotes bone resorption. M-CSF is synthesized in interstitial cells such as fibroblasts, osteoblasts, and endothelial cells, and is also produced by activated macrophages, B cells, T cells, and various tumor cells.

[0020] This application screened out the small molecule Dex (dexamethasone) that enhances the erythrocyte support ability of human macrophages through in vitro screening of small molecules. This small molecule can enhance the erythrocyte development ability of human macrophages both in vivo and in vitro, and improve the development and maturation of human erythrocytes in mice with humanized immune systems.

[0021] As certain embodiments of the present application, human hematopoietic stem cells are transplanted into mice, and then one or more of antibodies targeting murine macrophages, dexamethasone or its derivatives, and human macrophage colony stimulating factor are administered to the mice.

[0022] As certain embodiments of the present application, human hematopoietic stem cells are transplanted into mice, and antibodies targeting mouse macrophages are then administered to the mice. As certain embodiments of the present application, human hematopoietic stem cells are transplanted into mice, and dexamethasone or a derivative thereof is then administered to the mice. As certain embodiments of the present application, human hematopoietic stem cells are transplanted into mice, and antibodies targeting mouse macrophages and dexamethasone or a derivative thereof are then administered to the mice. As certain embodiments of the present application, human hematopoietic stem cells are transplanted into mice, and antibodies targeting mouse macrophages, dexamethasone or a derivative thereof, and human macrophage colony stimulating factor are then administered to the mice.

[0023] As certain embodiments of the present application, one or more of an antibody targeting murine macrophages, dexamethasone or a derivative thereof is administered to the mouse, and then human red blood cells are transfused into the mouse.

[0024] As certain embodiments of the present application, the mouse is administered with an antibody targeting mouse macrophages, and then human red blood cells are transfused into the mouse. As certain embodiments of the present application, the mouse is administered with dexamethasone or a derivative thereof, and then human red blood cells are transfused into the mouse. As certain embodiments of the present application, the mouse is administered with an antibody targeting mouse macrophages and dexamethasone or a derivative thereof, and then human red blood cells are transfused into the mouse.

[0025] The present invention relates to a method for constructing mice with humanized peripheral red blood cells, comprising: transplanting human hematopoietic stem cells into mice, and then administering an erythrocyte inducer to the mice (induction model); or first administering an erythrocyte inducer to the mice, and then transplanting human red blood cells into the mice (reinfusion model). The erythrocyte inducer includes: human macrophage colony-stimulating factor, which can reconstitute human macrophages to enhance the in vivo development of human erythroblasts; antibodies such as anti-mouse F4 / 80 antibodies and anti-mouse CSF1R antibodies that target and eliminate mouse macrophages, which can efficiently eliminate mouse macrophages and also efficiently reconstitute human macrophages, improving the development and maturation of human red blood cells in mice with humanized immune systems; and dexamethasone, which can enhance the erythrogenesis-promoting ability of human macrophages and inhibit the phagocytosis of human red blood cells by mouse macrophages. In mice transplanted with human hematopoietic stem cells, the process of inducing red blood cell maturation can be antibody treatment, dexamethasone treatment alone, human macrophage colony-stimulating factor treatment, or a combination of the three. High-level human red blood cell reconstruction can be achieved rapidly within three weeks, and the maturity and denucleation rate of the peripheral human red blood cells reconstructed in mice are completely consistent with normal human blood. In mice transplanted with human red blood cells, the red blood cell induction process can be antibody treatment, dexamethasone treatment alone, or a combination of the two.

[0026] As certain embodiments of the present application, after human hematopoietic stem cells are transplanted into the mouse, the red blood cell inducing agent is administered after the human hematopoietic stem cells have stably formed human immune cells in the mouse.

[0027] As certain embodiments of the present application, human hematopoietic stem cells are transplanted into the mice, and then 8-25 weeks later, an erythrocyte inducing agent is administered to the mice.

[0028] After 8 weeks, or 9 weeks, or 10 weeks, or 11 weeks, or 12 weeks, or 13 weeks, or 14 weeks, or 15 weeks, or 16 weeks, or 17 weeks, or 18 weeks, or 19 weeks, or 20 weeks, or 21 weeks, or 23 weeks, or 24 weeks, or 25 weeks of transplanting human hematopoietic stem cells into the mice, the mice are then administered an erythrocyte inducing agent.

[0029] As certain embodiments of the present application, human hematopoietic stem cells are transplanted into the mice, and 10 to 12 weeks later, an erythrocyte inducing agent is administered to the mice.

[0030] As certain embodiments of the present application, the mice are first administered an erythrocyte inducing agent for 1 to 7 days, and then transplanted with human erythrocytes.

[0031] As certain embodiments of the present application, human hematopoietic stem cells are transplanted into mice, and then antibodies targeting mouse macrophages, dexamethasone or a derivative thereof are administered to the mice. The antibodies targeting mouse macrophages and dexamethasone are administered simultaneously or sequentially.

[0032] As certain embodiments of the present application, the antibody targeting murine macrophages and / or dexamethasone are administered at intervals.

[0033] As certain embodiments of the present application, the antibody targeting murine macrophages and dexamethasone are administered once every three days.

[0034] As certain embodiments of the present application, the mouse is an immunodeficient mouse.

[0035] As certain embodiments of the present application, the immunodeficient mice include one or more of nude mice, NOD scid mice, RAG1 KO mice, RAG2 KO mice, Il2rg KO mice, severely immunodeficient mice, and BLT mice.

[0036] Severely immunodeficient mice are mice whose immune systems are deficient or missing multiple immune components, either due to congenital genetic mutations or artificially induced, typically with B, T, and NK cell deficiencies. They are widely used in research fields such as oncology (e.g., tumor growth, metastasis, and anti-tumor drug screening), immunology (e.g., mechanisms of immune cell development and proliferation, and the pathogenesis of immune diseases), infectious diseases (e.g., mechanisms of viral / bacterial infectious disease pathogenesis), and stem cell biology (e.g., human stem cell transplantation). Due to their lack of T, B, and NK cells, they can efficiently engraft human hematopoietic stem cells (HSCs), human peripheral blood mononuclear cells (PBMCs), xenografts derived from human tumor cell lines (CDXs), xenografts derived from patient tumor tissue (PDXs), or other adult stem cells and tissues, and are widely used in the construction of humanized mice.

[0037] As certain embodiments of the present application, the severely immunodeficient mouse is a mouse comprising at least one of T cell deficiency, B cell deficiency, NK cell deficiency, and hematopoietic stem cell function deficiency.

[0038] As certain embodiments of the present application, the severely immunodeficient mouse is one or more of NOG mouse, NSG mouse, NCG mouse, NPG mouse, NKG mouse, BRG mouse, NRG mouse, and their derivative strains.

[0039] As certain embodiments of the present application, the severely immunodeficient mouse is selected from NBSGW mice, NSGW41 mice, NOG-EXL mice, NCG-M mice, NCG-FLT3-KO mice, NCG-X-TSLP mice, NCG-X-hIL15 mice, NKG mice, NKG-hIL15 mice, NCG-X mice, NCG-MHC-dKO mice, BALB / c Rag2-null IL-2Rγc-null mice, NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ Mouse, NOD-Prkdc em26Cd52 Il2rg em26Cd22 / Nju Mouse, NPG mouse, BRGSF mouse, BRGS mouse, BRGST mouse, NRGS mouse, MISTRG mouse, MISTRGFah - / - mice, MISTRG6 mice, and one or more of their derivative strains.

[0040] The concentration of the anti-mouse F4 / 80 antibody is 0.01-100 mg / ml, and the concentration of the anti-mouse CSF1R antibody is 0.01-100 mg / ml.

[0041] The concentration of the anti-mouse F4 / 80 antibody is selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 , 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3 ,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mg / ml.

[0042] The concentration of the anti-mouse CSF1R antibody is selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3 ,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mg / ml.

[0043] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 0.1-10 mg / ml, and the concentration of the anti-mouse CSF1R antibody is 0.1-10 mg / ml.

[0044] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 1-4 g / ml, and the concentration of the anti-mouse CSF1R antibody is 1-4 mg / ml.

[0045] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 2 mg / ml.

[0046] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 2 mg / ml.

[0047] As certain embodiments of the present application, the concentration of the dexamethasone solution is 0.01-100 mg / ml.

[0048] The concentration of dexamethasone solution is selected from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1. 8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5. 1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mg / ml.

[0049] As certain embodiments of the present application, the concentration of the dexamethasone solution is 0.1-10 mg / ml.

[0050] As certain embodiments of the present application, the concentration of the dexamethasone solution is 1 to 4 mg / ml.

[0051] As certain embodiments of the present application, the concentration of the dexamethasone solution is 2 mg / ml.

[0052] The present application also provides a peripheral red blood cell humanized mouse constructed by the construction method.

[0053] This application establishes a novel humanized erythrocyte mouse model by injecting mice with humanized immune systems with antibodies (anti-mouse F4 / 80 and CSF1R antibodies) and dexamethasone, demonstrating a high proportion of peripheral human erythrocyte reconstitution, high maturity, low toxicity, and a short modeling cycle. This model overcomes the shortcomings of previous models, filling a gap in the field of humanized erythrocyte mouse models in China, and provides a new tool for studying the mechanisms of infectious and inherited diseases of human erythrocytes in vivo, as well as exploring new therapeutic approaches for these diseases.

[0054] Different from previous studies on the phagocytosis of human red blood cells by mouse macrophages, this patent focuses on the research on the support of human red blood cells by human macrophages. It intends to supplement human macrophages to provide support signals for the development of human erythroblasts, thereby efficiently reconstructing circulating mature human red blood cells in mice with humanized immune systems.

[0055] The present application also provides applications of the peripheral red blood cell humanized mice, which include: dynamic analysis and evaluation of human cell subpopulations including human red blood cells, construction of red blood cell infection models, construction of genetic disease models, antibody evaluation, preparation of red blood cell-related drugs, development of red blood cell-related drugs, evaluation of the efficacy and / or toxic side effects of red blood cell-related drugs, development of drug targets for red blood cell-related drugs, antibody drug research and development, vaccine development, efficacy and safety of chimeric antigen receptor T cell (CAR-T) therapy, and one or more of myeloid development research.

[0056] As certain embodiments of the present application, the erythrocyte infection model comprises a Plasmodium falciparum infection model.

[0057] Plasmodium falciparum infection is caused by the parasite Plasmodium falciparum, which uses humans and female Anopheles mosquitoes as its hosts. In humans, schizontogenesis and gametogenesis begin, and in the mosquito, gametogenesis and sporulation are completed. In humans, the parasite develops within hepatocytes and red blood cells. The parasite infects humans through mosquito bites, causing malaria, with typical clinical manifestations of periodic chills, fever, and sweating.

[0058] As certain embodiments of the present application, the genetic disease includes one or more of hereditary megaloblastic anemia, β-thalassemia, and sickle cell anemia.

[0059] Hereditary megaloblastic anemia is a type of anemia caused by an inherited disorder of DNA synthesis, primarily due to a deficiency in vitamin B12 or folic acid. This condition is characterized by macrocytic anemia, with the presence of megaloblasts in the bone marrow. Macromorphological changes are also seen in granulocytes, megakaryocytes, and even certain proliferative somatic cells.

[0060] β-thalassemia is a hereditary hemolytic anemia caused by mutation or deletion of the globin peptide chain gene. It manifests as β-thalassemia and is caused by insufficient synthesis of β-globin chains due to mutation of the β-globin gene.

[0061] Sickle cell anemia, also known as sickle cell anemia or sickle cell anemia, is an inherited hemoglobinopathy caused by the substitution of valine for glutamic acid at the sixth amino acid position of the β-peptide chain, resulting in sickle hemoglobin, which replaces normal hemoglobin. Clinical manifestations include chronic hemolytic anemia, susceptibility to infection, and recurrent painful crises, leading to chronic ischemia and consequent organ and tissue damage.

[0062] As certain embodiments of the present application, the antibody evaluation includes toxicity and efficacy evaluation of the anti-human CD47 antibody.

[0063] As certain embodiments of the present application, the red blood cell-related drugs include one or more drugs for anti-red blood cell infection, drugs for red blood cell-related genetic diseases, and antibody drugs.

[0064] The present application also provides a product combination of erythrocyte inducers for constructing a peripheral erythrocyte humanized mouse model, wherein the product combination of erythrocyte inducers includes one or more of anti-mouse F4 / 80 antibody, anti-mouse CSF1R antibody, dexamethasone or its derivatives, and human macrophage colony stimulating factor.

[0065] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 0.01 to 100 mg / ml.

[0066] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 0.1-10 mg / ml.

[0067] As certain embodiments of the present application, the anti-murine F4 / 80 antibody has a concentration of 1-4 mg / ml.

[0068] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 2 mg / ml.

[0069] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 0.01-100 mg / ml.

[0070] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 0.1-10 mg / ml.

[0071] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 1-4 mg / ml.

[0072] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 2 mg / ml.

[0073] As certain embodiments of the present application, the concentration of the dexamethasone solution is 0.01-100 mg / ml.

[0074] As certain embodiments of the present application, the concentration of the solution of dexamethasone or its derivative is 0.1-10 mg / ml.

[0075] As certain embodiments of the present application, the concentration of the dexamethasone solution is 1-4 mg / ml.

[0076] As certain embodiments of the present application, the concentration of the dexamethasone solution is 2 mg / ml.

[0077] The present application also provides a kit for constructing a peripheral red blood cell humanized mouse model, wherein the kit comprises one or more of an anti-mouse F4 / 80 antibody, an anti-mouse CSF1R antibody, dexamethasone or its derivatives, and a human macrophage colony-stimulating factor.

[0078] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 0.01 to 100 mg / ml.

[0079] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 0.1-10 mg / ml.

[0080] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 1-4 mg / ml.

[0081] As certain embodiments of the present application, the concentration of the anti-mouse F4 / 80 antibody is 2 mg / ml.

[0082] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 0.01-100 mg / ml.

[0083] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 0.1-10 mg / ml.

[0084] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 1-4 mg / ml.

[0085] As certain embodiments of the present application, the concentration of the anti-mouse CSF1R antibody is 2 mg / ml.

[0086] As certain embodiments of the present application, the concentration of the dexamethasone solution is 0.01-100 mg / ml.

[0087] As certain embodiments of the present application, the concentration of the solution of dexamethasone or its derivative is 0.1-10 mg / ml.

[0088] As certain embodiments of the present application, the concentration of the dexamethasone solution is 1-4 mg / ml.

[0089] As certain embodiments of the present application, the concentration of the dexamethasone solution is 2 mg / ml.

[0090] As described above, the method for constructing and using a humanized peripheral red blood cell mouse model of the present invention has the following beneficial effects:

[0091] This application is a new method for inducing reconstitution of peripheral human red blood cells in mice with humanized immune systems. It provides a novel drug combination Ab (anti-mouse F4 / 80 and CSF1R antibodies) + dexamethasone to induce peripheral mature human red blood cells in mice with humanized immune systems. Different from the traditional transfusion model, the present invention adopts an in vivo induction approach to induce circulating peripheral mature human red blood cells in vivo.

[0092] Different from traditional induction models, the present invention has discovered a new drug combination, Ab (anti-mouse F4 / 80 and CSF1R antibody) + dexamethasone, through in vivo drug screening, for low-toxicity depletion of mouse macrophages and reconstruction of human macrophages. This drug combination, Ab (anti-mouse F4 / 80 and CSF1R antibody) + dexamethasone, can induce peripheral mature human red blood cells, while maintaining mouse survival and achieving higher proportions and higher maturity of human red blood cells compared to traditional induction methods.

[0093] Low-toxicity macrophage scavenger Abs (anti-mouse F4 / 80 and CSF1R antibodies) efficiently eliminate mouse macrophages without significant toxicity. Furthermore, they are also highly effective in reconstituting human macrophages. Their low toxicity enables humanized peripheral red blood cell (PRC) mice to rapidly achieve high levels of human erythrocyte reconstitution within three weeks. Dexamethasone (Dex), a small molecule that enhances the erythrocyte-supporting capacity of human macrophages, can enhance the erythropoiesis-stimulating ability of human macrophages and improve the development and maturation of human erythrocytes in mice with humanized immune systems. The maturity and denucleation rates of these reconstructed peripheral human erythrocytes are completely consistent with those of normal human blood.

[0094] Different from the complex mouse strains and construction strategies of spontaneous models, the present invention uses the most basic immune system humanized mouse strain NCG-X and a simple construction strategy;

[0095] This application uses the red blood cell induction drug combination of Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone to produce a new peripheral red blood cell humanized mouse strain, and uses this as a basis to study the dynamics of human cell subpopulations, including human red blood cells in the new strain, and protects the research of the new peripheral red blood cell humanized mouse strain in red blood cell infection and genetic diseases, and the evaluation of new antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 The main technical roadmap for the construction of humanized peripheral red blood cell mice of this application is shown;

[0097] Figure 2 It showed that compared with traditional liposomes, Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) treatment can prolong the survival time of transfused human red blood cells in vivo;

[0098] Figure 3 showed that compared with traditional liposomes, Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) treatment significantly depleted mouse macrophages, including the liver and spleen, and efficiently reconstituted human macrophages in these tissues;

[0099] Figure 4 Dexamethasone was shown to promote the expression of erythroid support-related genes in human macrophages in vitro, enhance the binding ability of human macrophages to human erythroid precursors, and promote the maturation and denucleation of human erythroid precursors.

[0100] Figure 5 The results showed that Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) plus dexamethasone injection into mice significantly induced human erythrocytes in peripheral blood, with a ratio of up to 15%. Compared with traditional liposome methods, this method had a higher induction ratio and lower lethal toxicity to mice. The human erythrocyte precursors in the bone marrow and spleen also had a higher degree of maturity.

[0101] Figure 6 The results showed that the maturation and denucleation of human erythrocytes reconstituted in the peripheral blood of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone were completely consistent with those of normal human blood, while traditional liposome injection could not achieve the mature phenotype of normal human blood;

[0102] Figure 7 The results showed that the peripheral red blood cell humanized mouse model induced by Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone injection supports the infection experiment of Plasmodium falciparum;

[0103] Figure 8 The humanized red blood cell mouse model of this application supports the evaluation of the toxicity and efficacy of anti-human CD47 antibodies;

[0104] Figure 9 It was shown that red blood cells from patients with hereditary megaloblastic anemia can be reconstructed in this model and exhibit pathological characteristics; mice transplanted with stem cells from patients with red blood cell anemia exhibited larger peripheral red blood cell size, lower denucleation rate and abnormal red blood cell morphology. In tissues, immunohistochemical staining showed accumulation of human hemoglobin in multiple tissues, such as the liver, spleen, kidney and liver, and tissue damage caused by abnormal red blood cells was detected. DETAILED DESCRIPTION

[0105] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0106] [Experimental Materials]

[0107] NCG-X newborn mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0108] Anti-mouse F4 / 80 antibody manufactured by BioXcell, catalog number BE0206

[0109] Anti-mouse CSF1R antibody manufactured by BioXcell, catalog number BE0213

[0110] Dexamethasone manufacturer MCE, product number HY-14648

[0111] PEG300 manufacturer MCE, product number HY-Y0873

[0112] DMSO manufacturer Sigma, product number D8418

[0113] Flow cytometer manufacturer Aglient, model Novocyte Penteon

[0114] Disodium clodophosphate liposome manufacturer Liposoma, product number CP-005-005

[0115] CFSE manufacturer: Thermo Fisher, catalog number 65-0850-84

[0116] Nuclear dye Hoechst 33342 (manufactured by Yeasen, catalog number 40731ES10)

[0117] The human tumor cell line Raji was purchased from ATCC Cell Bank, catalog number CCL-86.

[0118] Anti-human CD47 antibody clone IBI188 was donated by Innovent Biologics

[0119] Anti-human CD47 antibody clone Hu5F9-G4 (manufactured by Absin, catalog number abs141277)

[0120] Human Plasmodium falciparum Pfs16-GFP reporter gene strain, a gift from Zhang Qingfeng's laboratory at Tongji University

[0121] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0122] Example 1

[0123] Figure 1 The main technical route of the present invention for constructing a novel humanized red blood cell mouse model is presented, and a novel humanized red blood cell mouse model is generated:

[0124] CD34 purified from aborted fetal liver by magnetic bead separation + Human hematopoietic stem cells (ethical review number: 2021-488-01), the purified CD34 + Human hematopoietic stem cells are injected into NCG-X newborn mice 3 to 7 days old. 10 to 12 weeks after the injection, human leukocytes and various immune cell subsets are analyzed by flow cytometry to determine the efficiency of hematopoietic stem cell engraftment and the proportion of human immune cell subsets reconstituted. A human leukocyte proportion greater than 10% is considered successful reconstitution. Thus, NCG-X mice with a humanized immune system are obtained.

[0125] Subsequently, erythrocyte inducer was injected into NCG-X immune system humanized mice: the formula of erythrocyte inducer Ab+dexamethasone of the present application is: 200 μg anti-mouse F4 / 80 antibody, 200 μg anti-mouse CSF1R antibody, 100 μl 100 μg / ml dexamethasone.

[0126] The erythrocyte induction agent was prepared by dissolving dexamethasone at a stock concentration of 67 mg / ml in DMSO and adjusting the concentration to 2 mg / ml by adding equal proportions of PEG300 and 0.9% saline. Subsequently, 200 μg each of anti-mouse F4 / 80 and anti-mouse CSF1R antibodies were dissolved in 100 μl of 0.9% saline and an equal volume of 2 mg / ml dexamethasone solution was added.

[0127] The injection method is: 100 μl of the prepared antibody solution and 2 mg / ml dexamethasone solution were mixed and then injected intraperitoneally. The mice were given the drug once every three days. After each injection, the peripheral human CD235 was detected by flow cytometry. + After 3 to 7 injections, the induction process takes effect, with human red blood cells clearly visible. To maintain human red blood cells in the model mice, continue injecting the antibody solution and dexamethasone solution at the above frequency.

[0128] Example 2

[0129] Disodium clodronate liposomes (100 μl) and Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) were intraperitoneally injected into NCG-X humanized mice whose immune systems had been reconstituted for 10 to 12 weeks in Example 1. After the three injections were completed, CFSE-labeled peripheral blood red blood cells from healthy humans were injected into the mice, and tail vein blood was collected from the mice at specific time points (1 h, 2 h, 4 h, 7 h, 10 h, 24 h, 30 h). Figure 2 A), the proportion of CFSE-positive human erythrocytes was then detected by flow cytometry, and the changing trend of CFSE-positive human erythrocytes over time was statistically analyzed. Figure 2 Figure B shows that in the Ab (anti-mouse F4 / 80 and anti-mouse CSF1R)-injected mice, human erythrocyte survival was significantly improved in vivo. Thirty hours after transfusion, the erythrocyte survival rate in the liposome-injected group was approximately 50%, while that in the Ab (anti-mouse F4 / 80 and anti-mouse CSF1R)-treated group was approximately 80%, an approximately 1.6-fold increase compared to clodronate liposome injection. These results demonstrate that, compared to traditional liposomes, Ab (anti-mouse F4 / 80 and anti-mouse CSF1R) treatment can prolong the survival of transfused human erythrocytes.

[0130] Disodium clodronate liposomes (100 μl) and Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) were respectively injected intraperitoneally into the NCG-X humanized mice whose immune system had been reconstructed for 10 to 12 weeks in Example 1. After the three injections were completed, the mice were anesthetized and liver and spleen tissue samples were removed. Immunofluorescence staining was then used to identify the distribution of human macrophages (hCD68) and mouse macrophages (mF4 / 80) in the tissues. The specific number of human and mouse macrophages in one field of view was counted and statistically analyzed. The immunofluorescence results showed that in the livers of mice in the Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) treatment group, the number of mouse F4 / 80-positive macrophages was significantly reduced, while the number of human CD68-positive macrophages was significantly increased ( Figure 3 A); the same phenomenon was also observed in spleen tissue ( Figure 3 B). The statistical results of the data showed that compared with the traditional clodronate disodium liposome, Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) treatment can significantly clear the liver ( Figure 3 C) and spleen ( Figure 3 D) and efficiently reconstituted human macrophages in these tissues.

[0131] Figure 4Dexamethasone can promote the expression of erythrocyte support-related genes in human macrophages under in vitro conditions, enhance the binding ability of human macrophages to human erythrocyte precursors, and promote the maturation and denucleation of human erythrocyte precursors.

[0132] Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) was intraperitoneally injected into NCG-X humanized mice whose immune systems had been reconstituted for 10 to 12 weeks in Example 1. After the three injections, the mice were anesthetized and femur and tibia samples were obtained. Human macrophages in the bone marrow were then purified by flow cytometry. The macrophages in the experimental group were pretreated with dexamethasone in vitro, while those in the control group were treated with physiological saline. The treated human macrophages were then cultured with human erythroblasts, and the support capacity of the macrophages for erythroblasts was tested ( Figure 4 A). Real-time quantitative PCR results showed that human macrophages treated with dexamethasone upregulated genes related to erythrocyte development, such as the denuclearization genes AXL and MERTK; the adhesion genes CD169 and CD163; and the iron transport-related gene SLC40A1 ( Figure 4 B) Imaging flow cytometry analysis of co-cultured human macrophages and erythroblasts showed that dexamethasone-treated macrophages were able to form larger hematopoietic island structures with erythroblasts. Counting the number of erythroblasts adhered to their surfaces showed that more human erythroblasts adhered to their surfaces ( Figure 4 C), indicating that their ability to adhere to erythrocytes and form hematopoietic islands is enhanced; statistical analysis of the maturation of human erythroblasts showed that after co-culture of dexamethasone-treated macrophages with erythroblasts, the maturity of erythroblasts (proportion of late erythrocytes) and the rate of nuclear-free erythrocytes were significantly improved compared with the control group ( Figure 4 D) The above data indicate that dexamethasone treatment can enhance the ability of macrophages to support erythrocyte development.

[0133] Example 3

[0134] Clodronate disodium liposomes (100 μl), Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone were intraperitoneally injected into NCG-X humanized mice whose immune systems had been reconstituted for 10 to 12 weeks as described in Example 1. Blood was collected from the tail vein of the mice after each injection, and human CD235 expression was analyzed by flow cytometry. +The proportion of positive cells to total cells was plotted and statistically analyzed. In addition, the mortality rate of mice was counted daily and statistically analyzed. Human erythrocyte development was also examined in tissues. After three injections of liposomes or Ab, the mice were anesthetized and their bone marrow and spleen were removed. Immune cells were extracted and analyzed by flow cytometry staining for human CD71 and human CD235a. The denucleation rate of human erythrocytes was analyzed by staining with the nuclear dye Hoechst 33342.

[0135] Figure 5 It was shown that Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone injection into mice can significantly induce human erythrocytes in peripheral blood, with a ratio of up to 15% ( Figure 5 A), compared with the traditional clodronate liposome injection method, it has a higher induction ratio and shows an overall upward trend, while the proportion of human red blood cells in the peripheral blood of liposome-injected mice does not exceed 5% ( Figure 5 B); Survival statistical analysis results showed that mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone had lower lethal toxicity, with a survival rate of more than 80% at 21 days after injection, while the survival rate of traditional clodronate disodium liposomes was only 10% ( Figure 5 C). In addition, flow cytometry showed that the proportion of late red blood cells in the bone marrow was significantly increased, the proportion of early red blood cells was significantly decreased, and the proportion of nuclear-free red blood cells was also significantly increased ( Figure 5 D), indicating that human erythroblasts have a higher degree of maturity in the bone marrow of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone; in addition, we also observed a similar phenomenon in the spleen ( Figure 5 E) It shows that Ab+dexamethasone promotes the development of human erythroblasts in tissues.

[0136] Clodronate disodium liposomes (100 μl), Abs (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone were intraperitoneally injected into NCG-X humanized mice whose immune systems had been reconstituted for 10 to 12 weeks as described in Example 1. After three injections of liposomes or Abs, blood was collected from the mouse tail vein. The blood was then analyzed for human erythrocyte developmental stages by flow cytometry staining for human CD71 and human CD235a. The rate of human erythrocyte denucleation was analyzed by staining with the nuclear dye Hoechst 33342. Control human blood samples were obtained from healthy individuals and were stained and analyzed using the same staining protocol and analysis methods as the mouse tail vein blood samples.

[0137] Figure 6The figures show the ratio of mature and denucleated human erythrocytes reconstructed in the peripheral blood of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone. The representative flow cytometric graphs of erythrocyte maturation detection based on CD71 and CD235a show that in the peripheral blood of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone, the ratio of human CD235a single positive erythrocytes is about 96.8%, while the ratio of human CD235a single positive erythrocytes in normal human blood samples is about 98.3%. In mice induced by traditional clodronate disodium injection, the ratio of human erythrocytes CD235a single positive is only 63.8% ( Figure 6 A), the statistical results show the same trend ( Figure 6 B), indicating that the mature phenotype of human erythrocytes in the peripheral blood of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone is very similar to that of normal human blood samples, while clodronate disodium liposomes cannot achieve a similar proportion of human CD235a single-positive erythrocytes; in addition, the denucleation detection of human erythrocytes based on the nuclear dye Hoechst33342 showed that in the human erythrocytes in the peripheral blood of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone, the proportion of Hoechst33342-negative anucleated human erythrocytes was approximately 97.7%, while the proportion in normal human blood samples was approximately 98.9%, while in mice induced by traditional clodronate disodium injection, the proportion of anucleated Hoechst33342-negative human erythrocytes was only 61.7%, ( Figure 6 C), the statistical results show the same trend ( Figure 6 D), showing that the maturation of peripheral human erythrocytes induced by Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone injection has almost the same phenotype as that of normal human blood samples.

[0138] Example 4

[0139] The humanized red blood cell mouse model constructed in Example 2 was used for application research:

[0140] Ab + dexamethasone was intraperitoneally injected into NCG-X humanized mice whose immune system had been reconstituted for 10 to 12 weeks. After 3 injections of Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone, 1×10 6 The Pfs16-GFP reporter gene strain of Plasmodium falciparum was used to analyze GFP-positive Plasmodium falciparum by flow cytometry 7 days after injection of Plasmodium falciparum. The proportion of Plasmodium falciparum-positive cells in human red blood cells was counted. Figure 7 A).

[0141] Figure 7The results showed that the humanized mouse model of peripheral red blood cells induced by Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone injection can support the infection experiment of human Plasmodium falciparum: that is, one week after Plasmodium falciparum infection, flow cytometry showed that the infection ratio of human red blood cells in peripheral blood was about 10% ( Figure 7 B), and we performed imaging flow cytometry analysis on human erythrocytes and found that GFP and Hoechst33342-labeled Plasmodium falciparum parasites existed inside human erythrocytes ( Figure 7 C), demonstrating that infection with human Plasmodium falciparum was successfully achieved in this erythrocyte humanized mouse.

[0142] Take the NCG-X humanized mice whose immune system has been reconstructed for 10 to 12 weeks in Example 1 and place 5×10 6 Raji, a human B-cell lymphoma cell line, was inoculated subcutaneously and the tumors were grown to approximately 300 mm. 3 Afterwards, IBI188 and Hu5F9-G4, two anti-human CD47 antibodies, were injected into the tail vein at a dose of 1 mg / kg. The tumors of the mice in the control group injected with IgG grew to 1000 mm. 3 Afterwards, the mice were euthanized. During the whole process, the mice were injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone every three days by intraperitoneal injection. After each injection, blood was collected from the tail vein of the mice and human CD235 was analyzed by flow cytometry. + The proportion of positive cells to total cells was plotted and statistically analyzed ( Figure 8 A).

[0143] Figure 8 This study demonstrates that the humanized erythrocyte mouse model can also support the evaluation of the toxicity and efficacy of anti-human CD47 antibodies. As shown in the figure below, after transplanting the human tumor cell line Raji into this humanized mouse model, anti-human CD47 antibody treatment was used. It was observed that in mice treated with anti-human CD47 antibodies, tumor growth was significantly slowed compared to the IgG control group. Furthermore, injections of both IBI188 and Hu5F9-G4 anti-human CD47 antibodies achieved similar tumor inhibitory effects. Figure 8 B) In mice injected with anti-human CD47 antibodies, the growth rate of human red blood cell count was significantly slowed compared to the control group mice injected with IgG, achieving a 3-5-fold increase after nine days of injection. In the control group mice injected with IgG, the proportion of human red blood cells increased by 1000-fold, indicating that anti-human CD47 antibodies have a certain degree of human red blood cell toxicity in vivo ( Figure 8 C).

[0144] Example 5

[0145] In order to explore the application potential of the present invention in hereditary red blood cell diseases, human CD34 was enriched by magnetic bead sorting in bone marrow biopsy samples of patients with hereditary megaloblastic anemia. + Hematopoietic stem cells (ethics review number: 2022-176-02) were injected into newborn NCG-X mice. 20 weeks after the injection, the mice were intraperitoneally injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone ( Figure 9 A), with the frequency and dosage consistent with the construction method in Example 1. After seven injections, mice were euthanized and blood and tissue samples were collected. Peripheral blood samples were stained for CD235a and nuclei, and human red blood cell maturation and morphology were analyzed by flow cytometry and imaging flow cytometry. After tissue fixation, human hemoglobin α subunit was detected by immunohistochemistry, and the tissues were scanned using a whole-slide scanner.

[0146] like Figure 9 As shown, compared with mice transplanted with normal human stem cells (the group marked with Normal in the figure), mice transplanted with patients with red blood cell anemia (the group marked with MgA in the figure) showed larger peripheral red blood cell size ( Figure 9 B), lower denucleation rate ( Figure 9 C), Imaging flow cytometry showed that human erythrocytes in the peripheral blood of mice transplanted with hematopoietic stem cells from patients with megaloblastic anemia exhibited abnormal erythrocyte morphology, such as an abnormal round shape with nuclei ( Figure 9 D and E). In tissues, immunohistochemical staining of the α-subunit of human hemoglobin showed that human hemoglobin accumulated in multiple tissues in mice transplanted with megaloblastic stem cells. Pulmonary capillary hemorrhage and pulmonary thrombosis were observed in the lungs. Figure 9 F); formation of erythrocyte microthrombi was observed in liver samples, and abnormal accumulation of erythrocytes was observed in the sinusoids ( Figure 9 G); pathological features such as renal capillary hemorrhage and glomerular congestion were observed in the kidneys ( Figure 9 H), severe fibrosis and extramedullary hematopoiesis were observed in the spleen ( Figure 9 I), the above results show that the red blood cells of patients with hereditary megaloblastic anemia can be reconstructed in this model and show certain pathological characteristics.

[0147] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for constructing a mouse with humanized peripheral red blood cells, characterized in that: The construction method comprises: transplanting human hematopoietic stem cells into mice and then administering a red blood cell inducing agent to the mice, or administering a red blood cell inducing agent to the mice and then transfusing human red blood cells into the mice.

2. The construction method according to claim 1, characterized in that The erythrocyte inducing agent includes one or more of an antibody targeting mouse macrophages, dexamethasone or its derivatives, and human macrophage colony stimulating factor; Preferably, the dexamethasone derivative includes one or more of the pharmaceutical salts, esters, hydrates and solvates of dexamethasone; Preferably, the dexamethasone derivatives include one or more of dexamethasone sodium phosphate, dexamethasone acetate, dexamethasone palmitate, beclomethasone, beclomethasone dipropionate, hydrocortisone, and prednisone; The antibodies targeting mouse macrophages include anti-mouse F4 / 80 antibodies and / or anti-mouse CSF1R antibodies; Preferably, human hematopoietic stem cells are transplanted into mice, and then one or more of antibodies targeting murine macrophages, dexamethasone or its derivatives, and human macrophage colony stimulating factor are administered to the mice; Preferably, one or more of an antibody targeting murine macrophages, dexamethasone or a derivative thereof is administered to the mouse, and then human red blood cells are transfused into the mouse.

3. The construction method according to any one of claims 1 to 2, characterized in that: After transplanting human hematopoietic stem cells into the mice, the red blood cell inducing agent is administered after the human hematopoietic stem cells stably form human immune cells in the mice; Alternatively, the mice are first treated with an erythroid inducer for 1 to 7 days, and then transplanted with human erythrocytes; Preferably, human hematopoietic stem cells are transplanted into the mice, and then an erythrocyte inducing agent is administered to the mice 8-25 weeks later; More preferably, human hematopoietic stem cells are transplanted into the mice, and then 10-12 weeks later, an erythrocyte inducing agent is administered to the mice.

4. The construction method according to claim 2, characterized in that Transplanting human hematopoietic stem cells into mice, and then administering an antibody targeting mouse macrophages, dexamethasone or a derivative thereof to the mice, wherein the antibody targeting mouse macrophages and dexamethasone are administered simultaneously or sequentially; Alternatively, the antibody targeting murine macrophages and / or dexamethasone are administered at intervals; Preferably, the antibody targeting murine macrophages and dexamethasone are administered once every three days.

5. The construction method according to claim 1, characterized in that The mice are immunodeficient mice; The immunodeficient mice include one or more of nude mice, NOD scid mice, RAG1 KO mice, RAG2 KO mice, Il2rg KO mice, severe immunodeficient mice, and BLT mice; Preferably, the severely immunodeficient mouse is a mouse comprising at least one of T cell deficiency, B cell deficiency, NK cell deficiency, and hematopoietic stem cell function deficiency; Preferably, the severely immunodeficient mouse is one or more of NOG mouse, NSG mouse, NCG mouse, NPG mouse, NKG mouse, BRG mouse, NRG mouse, and their derivative strains; More preferably, the severely immunodeficient mouse is selected from the group consisting of NBSGW mouse, NSGW41 mouse, NOG-EXL mouse, NCG-M mouse, NCG-FLT3-KO mouse, NCG-X-TSLP mouse, NCG-X-hIL15 mouse, NKG mouse, NKG-hIL15 mouse, NCG-X mouse, NCG-MHC-dKO mouse, BALB / c Rag2-null IL-2Rγc-null mouse, NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ Mouse, NOD-Prkdc em26Cd52 Il2rg em26Cd22 / Nju Mouse, NPG mouse, BRGSF mouse, BRGS mouse, BRGST mouse, NRGS mouse, MISTRG mouse, MISTRGFah - / - mice, MISTRG6 mice, and one or more of their derivative strains.

6. The construction method according to claim 1, characterized in that The concentration of the anti-mouse F4 / 80 antibody is 0.01-100 mg / ml, and the concentration of the anti-mouse CSF1R antibody is 0.01-100 mg / ml; Preferably, the concentration of the anti-mouse F4 / 80 antibody is 0.1 to 10 mg / ml, and the concentration of the anti-mouse CSF1R antibody is 0.1 to 10 mg / ml; More preferably, the concentration of the anti-mouse F4 / 80 antibody is 1 to 4 mg / ml, and the concentration of the anti-mouse CSF1R antibody is 1 to 4 mg / ml; Further preferably, the concentration of the anti-mouse F4 / 80 antibody is 2 mg / ml; Further preferably, the concentration of the anti-mouse CSF1R antibody is 2 mg / ml.

7. The construction method according to claim 2, characterized in that: The concentration of dexamethasone solution is 0.01-100 mg / ml; Preferably, the concentration of dexamethasone solution is 0.1-10 mg / ml; More preferably, the concentration of dexamethasone solution is 1-4 mg / ml; More preferably, the concentration of the dexamethasone solution is 2 mg / ml.

8. Use of the peripheral red blood cell humanized mouse constructed according to the construction method according to any one of claims 1 to 7, characterized in that: The applications include: dynamic analysis of human cell subpopulations including human red blood cells, construction of red blood cell infection models, construction of genetic disease models, antibody evaluation, preparation of red blood cell-related drugs, development of red blood cell-related drugs, evaluation of the efficacy and / or toxic side effects of red blood cell-related drugs, development of drug targets for red blood cell-related drugs, antibody drug development, vaccine development, efficacy and safety of chimeric antigen receptor T cell (CAR-T) therapy, and myeloid development research; Preferably, the red blood cell infection model comprises a Plasmodium falciparum infection model; Preferably, the genetic disease includes one or more of hereditary megaloblastic anemia, β-thalassemia and sickle cell anemia; Preferably, the antibody evaluation includes toxicity and efficacy evaluation of the anti-human CD47 antibody; Preferably, the red blood cell-related drugs include one or more drugs for anti-red blood cell infection, drugs for red blood cell-related genetic diseases, and antibody drugs.

9. A product or kit of erythrocyte inducer combination for constructing a peripheral erythrocyte humanized mouse model, characterized in that: The red blood cell inducing agent combination product or kit includes one or more of anti-mouse F4 / 80 antibody, anti-mouse CSF1R antibody, dexamethasone, and human macrophage colony stimulating factor; Preferably, the concentration of the anti-mouse F4 / 80 antibody is 0.01 to 100 mg / ml; Preferably, the concentration of the anti-mouse CSF1R antibody is 0.01 to 100 mg / ml; Preferably, the concentration of dexamethasone solution is 0.01-100 mg / ml.

10. The red blood cell inducing agent combination product or kit according to claim 9, characterized in that: The concentration of the anti-mouse F4 / 80 antibody is 0.1 to 10 mg / ml; or, the anti-mouse CSF1R antibody concentration is 0.1 to 10 mg / ml; Alternatively, the concentration of dexamethasone solution is 0.1-10 mg / ml; Preferably, the concentration of the anti-mouse F4 / 80 antibody is 1 to 4 mg / ml; Preferably, the anti-mouse CSF1R antibody concentration is 1 to 4 mg / ml; Preferably, the concentration of dexamethasone solution is 1-4 mg / ml; More preferably, the anti-mouse F4 / 80 antibody concentration is 2 mg / ml; More preferably, the anti-mouse CSF1R antibody concentration is 2 mg / ml; More preferably, the concentration of dexamethasone solution is 2 mg / ml.

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