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] This invention belongs to the field of biomedical technology, specifically relating to a method for constructing a humanized peripheral erythrocyte mouse model, the constructed humanized peripheral erythrocyte mouse model, and its applications. Background Technology

[0002] Immunological research and exploration rely on experimental animal models. Mice, as classic experimental animals, are widely used in various immunological experiments and have driven the advancement of the discipline. However, after tens of millions of years of divergent evolution, significant differences remain between the mouse and human immune systems. These differences include variations in the complement system, the composition of peripheral blood immune cell subsets, and the expression of Fc receptors. These differences mean that research results obtained using mice as models cannot be directly applied clinically, impacting the efficiency of basic research translation into clinical practice 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, with their ability to simulate the dynamics of the human immune system, hold the promise of being a key breakthrough. Common humanized mouse models of the immune system involve transplanting human hematopoietic stem cells into immunodeficient mice, thereby reconstructing the human immune system within the mice. Humanized mouse models of the immune system have built a "bridge" between basic research and clinical translation and are currently widely used in fields such as oncology and infectious diseases. However, as the most numerous cells in the human body, erythrocytes are difficult to reconstitute efficiently in humanized mice of the immune system. Developing humanized mouse models of the immune system with high-level human erythrocyte reconstitution is one of the challenges in this field. Therefore, constructing humanized animal models that reflect human cellular pathogenesis in disease progression is urgently needed.

[0003] Red blood cells play an irreplaceable role in gas exchange and maintaining the function of the immune system. Red blood cell-related diseases, such as anemia and malaria, remain significant global public health threats. Most existing in vivo red blood cell disease models use mouse red blood cells as the research subject. However, because humans and mice are two different species, red blood cells differ greatly in size and development. Therefore, mouse models alone cannot accurately reflect the state of human cells during disease progression, preventing treatment strategies from being directly applied clinically. Furthermore, many pathogens affecting red blood cells, such as *Plasmodium falciparum*, cannot infect mouse red blood cells, leading to a lack of in vivo models in this field. Moreover, *Plasmodium mucosum*, which can infect mouse red blood cells, and *Plasmodium malariae*, are essentially different strains with significant differences in their infecting targets and mechanisms. This hinders the evaluation of antimalarial drugs and the study of related mechanisms, severely impeding the global goal of achieving "One Health."

[0004] There are three main types of humanized erythrocyte models in the past. The first is the human erythrocyte reinfusion model, in which healthy human erythrocytes are injected intravenously into immunodeficient mice to establish circulating human erythrocytes in the mice's blood. In this method, mice are injected daily with a suspension of human erythrocytes equivalent to half of their total blood volume, making the experimental procedure very cumbersome. More importantly, these reinfused human erythrocytes do not undergo a complete hematopoietic development process in the mice, so the reinfusion model cannot accurately reflect the true physiological state of erythrocytes. The second type is the induction model, in which researchers inject liposomes, gadolinium chloride, or other methods into humanized mice to eliminate mouse macrophages and reconstruct human erythrocytes by reducing the phagocytic activity of mouse macrophages. Although the human erythrocytes reconstructed by this model undergo a complete hematopoietic development process in vivo, the toxicity caused by repeated injections of liposomes or gadolinium chloride leads to the death of more than half of the mice during the injection process, significantly increasing experimental costs. The third type is the spontaneous model. Although this model does not require daily injections of red blood cells and drugs, its construction requires the MISTRGF mouse model, which is modified with eight genes. However, this mouse strain is not available in China, severely hindering the progress of red blood cell humanization in the country. In addition, the construction of this model depends on the co-transplantation of human stem cells and hepatocytes, which takes up to six months, severely limiting the applicability of this model.

[0005] The aforementioned techniques, based on eliminating mouse macrophages to reduce their phagocytosis of human erythrocytes, neglect the crucial role of macrophages in supporting erythrocyte development. In hematopoietic organs, macrophages can form "hematopoietic islands" with developing erythrocytes—erythroblasts. These structures consist of a central macrophage surrounded by erythroblasts. Macrophages provide anti-apoptotic and pro-mitosis signals to erythroblasts and participate in the enucleation process, playing an irreplaceable role in maintaining erythrocyte development and maturation. Previous studies have shown that in humanized mice with a humanized immune system, efficient reconstruction of human erythroblasts and human macrophages—two essential components of the "hematopoietic island" structure—has consistently been difficult.

[0006] Therefore, there is an urgent need in the field for humanized mouse models with a large number of mature circulating human erythrocytes reconstructed. This invention, by reintroducing human macrophages into humanized mice with a human immune system, provides supporting signals for the development of human erythrocytes, promotes the development of human erythrocytes in hematopoietic organs, and thereby reconstructs circulating mature human erythrocytes in the extracellular matrix. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention provides a method for constructing a humanized mouse derived from peripheral erythrocytes. The method includes: transplanting artificial hematopoietic stem cells into a mouse, and then administering an erythrocyte inducer to the mouse, or administering an erythrocyte inducer to the mouse and then infusing human erythrocytes into the mouse.

[0008] Human immune system (HIS) mice are humanized mice with an artificial blood immune system. They are created by implanting human blood stem cells into severely immunodeficient mice, which regenerate the human immune system in the mice. Currently, they are widely used in the fields of oncology and infection.

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

[0010] As some embodiments of this application, the erythrocyte inducer includes one or more of the following: antibodies targeting mouse macrophages, dexamethasone or derivatives thereof, and human macrophage colony-stimulating factor.

[0011] As some embodiments of this application, dexamethasone derivatives include one or more of the pharmaceutical salts, esters, hydrates, and solvates of dexamethasone.

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

[0013] As some embodiments of this application, the antibodies targeting mouse macrophages include anti-mouse F4 / 80 antibodies and / or anti-mouse CSF1R antibodies.

[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 various experimental methods, including immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry (FC), to help 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] Anti-mouse CSF1R antibodies target mouse colony-stimulating factor 1 receptor (CSF1R) (also known as 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 via CSF1R regulates cell proliferation and differentiation in the monocyte lineage.

[0016] The method for constructing humanized peripheral erythrocyte mice in this application, based on existing induction models, screened for low-toxicity macrophage scavenging agents Ab (anti-mouse F4 / 80 and CSF1R antibodies). Unlike the strong toxicity of previous liposomes and gadolinium chloride, Ab efficiently scavenges mouse macrophages without strong toxicity. Furthermore, it can efficiently reconstitute human macrophages, which is impossible with previous induction models. The low toxicity of the macrophage scavenging agent Ab enables the rapid high-level reconstitution of human erythrocytes in humanized peripheral erythrocyte mice within three weeks.

[0017] As some embodiments of this application, the erythrocyte inducer also includes dexamethasone or a derivative thereof.

[0018] Dexamethasone (DXMS) has the chemical structure of prednisolone with a fluorine atom introduced at the 9α-position of the B ring and a methyl atom introduced 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 effects of dexamethasone. The clinical bioequivalence ratio of dexamethasone to prednisolone is 0.75:5, and its biological half-life is 36-54 hours, classifying it as a long-acting glucocorticoid. Like other glucocorticoids, dexamethasone possesses pharmacological effects such as anti-inflammatory, anti-endotoxin, immunosuppressive, anti-shock, and stress-enhancing effects, thus it is widely used in various departments to treat a variety of diseases, such as autoimmune diseases, allergies, inflammation, asthma, and dermatological and ophthalmic diseases. Dexamethasone sodium phosphate injection is an indispensable emergency medication for rescuing 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, primarily found in the bone marrow cavity, and plays a crucial role in the proliferation, differentiation, and maintenance of monocyte activity. It also regulates placental function and promotes bone resorption. M-CSF can be synthesized in mesenchymal cells such as fibroblasts, osteoblasts, and endothelial cells, and can also be produced in activated macrophages, B cells, T cells, and various tumor cells.

[0020] This application uses in vitro screening of small molecules to screen out Dex (dexamethasone), a small molecule that enhances the erythrocyte support capacity of human macrophages. This small molecule can enhance the erythrocyte development capacity of human macrophages both in vivo and in vitro, and improve the development and maturation of human erythrocytes in humanized mice with immune system.

[0021] As some embodiments of this application, artificial hematopoietic stem cells are transplanted into mice, and then the mice are administered one or more of an antibody targeting mouse macrophages, dexamethasone or a derivative thereof, and human macrophage colony-stimulating factor.

[0022] In some embodiments of this application, artificial hematopoietic stem cells are transplanted into mice, and then the mice are administered antibodies targeting mouse macrophages. In some embodiments of this application, artificial hematopoietic stem cells are transplanted into mice, and then the mice are administered dexamethasone or a derivative thereof. In some embodiments of this application, artificial hematopoietic stem cells are transplanted into mice, and then the mice are administered antibodies targeting mouse macrophages and dexamethasone or a derivative thereof. In some embodiments of this application, artificial hematopoietic stem cells are transplanted into mice, and then the mice are administered antibodies against mouse macrophages, dexamethasone or a derivative thereof, and human macrophage colony-stimulating factor.

[0023] As some embodiments of this application, the mice are administered one or more of an antibody targeting mouse macrophages, dexamethasone, or a derivative thereof, and then human red blood cells are infused into the mice.

[0024] In some embodiments of this application, an antibody targeting mouse macrophages is administered to the mice, followed by the infusion of human red blood cells. In some embodiments of this application, dexamethasone or a derivative thereof is administered to the mice, followed by the infusion of human red blood cells. In some embodiments of this application, an antibody targeting mouse macrophages and dexamethasone or a derivative thereof are administered to the mice, followed by the infusion of human red blood cells.

[0025] This invention relates to a method for constructing humanized mice with peripheral erythrocytes, comprising: transplanting human hematopoietic stem cells into mice, followed by administering an erythrocyte inducer to the mice (induction model); or first administering an erythrocyte inducer to the mice, followed by transplanting human erythrocytes into the mice (reinfusion model). The erythrocyte inducer includes: human macrophage colony-stimulating factor, which can reconstruct human macrophages to enhance the in vivo development of human erythroblasts; antibodies such as anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody that target and clear mouse macrophages, which efficiently clear mouse macrophages and also efficiently reconstruct human macrophages, improving the development and maturation of human erythrocytes in humanized mice with a human immune system; and dexamethasone, which enhances the erythrocyte-stimulating capacity of human macrophages and inhibits the phagocytosis of human erythrocytes by mouse macrophages. In mice transplanted with human hematopoietic stem cells, the process of erythrocyte induction and maturation can be achieved through antibody treatment, dexamethasone treatment alone, human macrophage colony-stimulating factor treatment, or a combination of all three. This can rapidly achieve high-level human erythrocyte reconstitution within three weeks. The maturity and enucleation rate of the reconstituted peripheral human erythrocytes in mice are completely consistent with those of normal human blood. In mice transplanted with human erythrocytes, the erythrocyte induction process can be achieved through antibody treatment, dexamethasone treatment alone, or a combination of both.

[0026] As some embodiments of this application, after transplanting artificial hematopoietic stem cells into the mice, the erythrocyte inducer is administered after the artificial hematopoietic stem cells have stabilized and formed human immune cells in the mice.

[0027] As some embodiments of this application, artificial hematopoietic stem cells are transplanted into the mice, and erythrocyte inducers are administered to the mice 8-25 weeks later.

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

[0029] As some embodiments of this application, artificial hematopoietic stem cells are transplanted into the mice, and erythrocyte inducers are administered to the mice 10-12 weeks later.

[0030] As some embodiments of this application, the mice are first treated with an erythrocyte inducer for 1 to 7 days, followed by transplantation of human erythrocytes.

[0031] As some embodiments of this application, artificial hematopoietic stem cells are transplanted into mice, and then the mice are administered an antibody targeting mouse macrophages, dexamethasone or a derivative thereof, wherein the antibody targeting mouse macrophages and dexamethasone are administered simultaneously or sequentially.

[0032] As some embodiments of this application, the antibody targeting mouse macrophages and / or dexamethasone are administered at intervals.

[0033] As some embodiments of this application, the antibody targeting mouse macrophages is administered with dexamethasone once every three days.

[0034] In some embodiments of this application, the mouse is an immunodeficient mouse.

[0035] As some embodiments of this 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] Severe immunodeficient mice are mice whose immune systems are deficient or lacking in multiple immune components due to congenital genetic mutations or artificial methods. They typically lack B cells, T cells, and NK cells. These mice are widely used in oncology (e.g., tumor growth, metastasis, anti-tumor drug screening), immunology (e.g., immune cell development and proliferation mechanisms, immune disease pathogenesis), infectious diseases (e.g., viral / bacterial infectious disease pathogenesis), and stem cell biology (e.g., human stem cell transplantation). Due to the lack of T, B, and NK immune cells, they can be efficiently implanted with human hematopoietic stem cells (HSCs), human peripheral blood mononuclear cells (PBMCs), xenografts derived from human tumor cell lines (CDX), xenografts derived from patient tumor tissue (PDX), or other adult stem cells and tissues, and are widely used in the construction of humanized mice.

[0037] As some embodiments of this application, the severely immunodeficient mouse is a mouse containing at least one of the following: T cell deficiency, B cell deficiency, NK cell deficiency, and hematopoietic stem cell dysfunction.

[0038] As some embodiments of this application, the severely immunodeficient mice are one or more of NOG mice, NSG mice, NCG mice, NPG mice, NKG mice, BRG mice, NRG mice, and their derivative strains.

[0039] As some embodiments of this application, the severely immunodeficient mice are 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, and NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ Mice, NOD-Prkdc em26Cd52 Il2rg em26Cd22 / Nju Mice, NPG mice, BRGSF mice, BRGS mice, BRGST mice, NRGS mice, MISTRG mice, MISTRGFah - / - One or more of the following: mice, MISTRG6 mice, and 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 was 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, and 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 some embodiments of this 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 some embodiments of this 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 some embodiments of this application, the concentration of the anti-mouse F4 / 80 antibody is 2 mg / ml.

[0046] In some embodiments of this application, the concentration of the anti-mouse CSF1R antibody is 2 mg / ml.

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

[0048] The solution concentrations of dexamethasone were 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, and 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 some embodiments of this application, the concentration of dexamethasone in the solution is 0.1-10 mg / ml.

[0050] In some embodiments of this application, the concentration of dexamethasone solution is 1–4 mg / ml.

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

[0052] This application also provides a humanized mouse derived from peripheral erythrocytes constructed using the described method.

[0053] This application describes a novel humanized erythrocyte mouse model characterized by high reconstitution rate, high maturity, low toxicity, and short modeling period. This model overcomes the shortcomings of previous models, fills a gap in the field of humanized erythrocyte mouse models in China, and provides a novel tool for studying the mechanisms of human erythrocyte infectious and genetic diseases in vivo, as well as exploring new treatment methods.

[0054] Unlike previous studies on mouse macrophages engulfing human erythrocytes, this patent focuses on research on human macrophages supporting human erythrocytes. It aims to provide support signals for the development of human erythrocytes by supplementing human macrophages, thereby efficiently rebuilding circulating mature human erythrocytes in humanized mice of the immune system.

[0055] This application also provides one or more applications of the aforementioned humanized peripheral erythrocyte mice, including: dynamic analysis and evaluation of human cell subsets, including human erythrocytes; construction of erythrocyte infection models; construction of models of hereditary diseases; antibody evaluation; preparation of erythrocyte-related drugs; development of erythrocyte-related drugs; evaluation of the efficacy and / or toxicity of erythrocyte-related drugs; development of drug targets for erythrocyte-related drugs; antibody drug development; vaccine development; efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy; and myeloid development research.

[0056] As some embodiments of this application, the red blood cell infection model includes a Plasmodium falciparum infection model.

[0057] Plasmodium falciparum infection is a form of malaria caused by the parasite in humans and female Anopheles mosquitoes. In humans, the parasite begins schizogony and gametogenesis, while in mosquitoes, gametogenesis and spore proliferation are completed. In humans, the parasite develops within liver cells and red blood cells. Plasmodium infects humans through mosquito bites, causing malaria. Typical clinical manifestations include periodic chills, fever, and sweating.

[0058] As some embodiments of this application, the hereditary 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 a hereditary DNA synthesis disorder, primarily due to a deficiency of vitamin B12 or folic acid. This condition is characterized by macrocytic anemia, with the presence of megaloblasts in the bone marrow, and giant cell morphological changes also observed in granulocytes, megakaryocytes, and even some proliferating somatic cells.

[0060] β-thalassemia is a hereditary hemolytic anemia caused by mutations or deletions in the globin peptide chain gene. It manifests as β-globin production disorder anemia, which is caused by insufficient synthesis of β-globin chains due to mutations in the β-globin gene.

[0061] Sickle cell anemia, also known as sickle cell type anemia, is a hereditary hemoglobinopathic disorder. It occurs when valine replaces glutamic acid at the 6th amino acid position of the β-peptide chain, forming sickle-shaped hemoglobin that replaces normal hemoglobin. Clinical manifestations include chronic hemolytic anemia, susceptibility to infection, and recurrent painful crises leading to chronic local ischemia and subsequent organ and tissue damage.

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

[0063] As some embodiments of this application, the erythrocyte-related drugs include one or more of the following: drugs for treating erythrocyte infections, drugs for treating erythrocyte-related genetic diseases, and antibody drugs.

[0064] This application also provides a commercially available combination of erythrocyte inducers for constructing a humanized mouse model of peripheral erythrocytes, the commercially available combination of erythrocyte inducers comprising one or more of the following: anti-mouse F4 / 80 antibody, anti-mouse CSF1R antibody, dexamethasone or a derivative thereof, and human macrophage colony-stimulating factor.

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

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

[0067] As some embodiments of this application, the concentration of the anti-mouse F4 / 80 antibody is 1-4 mg / ml.

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

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

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

[0071] In some embodiments of this application, the concentration of the anti-mouse CSF1R antibody is 1-4 mg / ml.

[0072] In some embodiments of this application, the concentration of the anti-mouse CSF1R antibody is 2 mg / ml.

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

[0074] As some embodiments of this application, the solution concentration of dexamethasone or its derivatives is 0.1-10 mg / ml.

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

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

[0077] This application also provides a kit for constructing a humanized mouse model of peripheral erythrocytes, the kit comprising one or more of the following: anti-mouse F4 / 80 antibody, anti-mouse CSF1R antibody, dexamethasone or a derivative thereof, and human macrophage colony-stimulating factor.

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

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

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

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

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

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

[0084] In some embodiments of this application, the concentration of the anti-mouse CSF1R antibody is 1-4 mg / ml.

[0085] In some embodiments of this application, the concentration of the anti-mouse CSF1R antibody is 2 mg / ml.

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

[0087] As some embodiments of this application, the solution concentration of dexamethasone or its derivatives is 0.1-10 mg / ml.

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

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

[0090] As described above, the method for constructing and applying a humanized mouse model of peripheral erythrocytes according to the present invention has the following beneficial effects:

[0091] This application provides a novel method for inducing the reconstitution of peripheral human erythrocytes in humanized mice with the immune system. It provides the mechanism and method for inducing peripheral mature human erythrocytes in humanized mice with the immune system by combining a novel drug combination Ab (anti-mouse F4 / 80 and CSF1R antibody) + dexamethasone. Unlike the traditional reinfusion model, this invention uses an in vivo induction method to induce circulating peripheral mature human erythrocytes in vivo.

[0092] Unlike traditional induction models, this invention has identified a novel drug combination, Ab (anti-mouse F4 / 80 and CSF1R antibodies) + dexamethasone, through in vivo drug screening. By inducing peripheral mature human erythrocytes using this drug combination, a higher proportion and higher maturity of human erythrocytes can be reconstituted while maintaining mouse survival.

[0093] The low-toxicity macrophage scavenger Ab (anti-mouse F4 / 80 and CSF1R antibodies) effectively scavenges mouse macrophages without strong toxicity. Furthermore, it efficiently reconstitutes human macrophages. Its low toxicity allows peripheral erythrocyte humanized mice to rapidly achieve high-level human erythrocyte reconstitution within three weeks. The small molecule dexamethasone (Dex), which enhances the erythrocyte support capacity of human macrophages, can enhance the erythrocyte-stimulating development capacity of human macrophages and improve the development and maturation of human erythrocytes in immune system humanized mice. The maturity and enucleation rate of these reconstituted peripheral human erythrocytes in mice are completely consistent with normal human blood.

[0094] Unlike spontaneous models which involve complex mouse strains and construction strategies, this invention uses the most basic humanized immune system mouse strain NCG-X and a simple construction strategy.

[0095] This application protects the use of a combination of erythrocyte-inducing drugs, namely, Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) and dexamethasone, to generate a novel humanized peripheral erythrocyte mouse strain, and to study the dynamics of human cell subsets, including human erythrocytes in the new strain, based on this strain, and to protect the research on the novel humanized peripheral erythrocyte mouse strain in erythrocyte infection and genetic diseases, and the evaluation of novel antibodies. Attached Figure Description

[0096] Figure 1 This diagram illustrates the main technical roadmap for constructing humanized peripheral erythrocyte mice according to this application.

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

[0098] Figure 3 Compared to traditional liposomes, treatment with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) can significantly clear mouse macrophages, including those in the liver and spleen, and efficiently reconstitute human macrophages in these tissues;

[0099] Figure 4 The results showed that dexamethasone can promote the expression of human macrophage erythrocyte support-related genes, enhance the binding ability of human macrophages to human erythrocyte precursors, and promote the maturation and enucleation of human erythrocyte precursors under in vitro conditions.

[0100] Figure 5 The results showed that injecting mice with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone could significantly induce human erythrocytes in peripheral blood, with a proportion of up to 15%. Compared with the traditional liposome method, it has a higher induction ratio and lower lethal toxicity in mice. The human erythrocyte precursors in bone marrow and spleen also have higher maturity.

[0101] Figure 6 The results showed that mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone had peripherally reconstituted human erythrocytes that matured and denucleated in a manner completely consistent with normal human blood, while traditional liposome injection could not achieve the mature phenotype of normal human blood.

[0102] Figure 7 The humanized mouse model of peripheral erythrocytes induced by Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone injection supports the infection experiments of Plasmodium falciparum;

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

[0104] Figure 9 The red blood cells of patients with hereditary megaloblastic anemia can be reconstructed in this model and exhibit pathological features. Mice transplanted with stem cells from patients with megaloblastic anemia showed larger peripheral red blood cell size, lower enucleation 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 Implementation

[0105] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0106] [Experimental Materials]

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

[0108] BioXcell, manufacturer of anti-mouse F4 / 80 antibody, catalog number BE0206

[0109] BioXcell, manufacturer of the anti-mouse CSF1R antibody, catalog number BE0213

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

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

[0112] DMSO manufacturer Sigma, part number D8418

[0113] The flow cytometer manufacturer is Aglient, and the model is Novocyte Penteon.

[0114] Liposoma, manufacturer of disodium chlorophosphate liposomes, catalog number CP-005-005

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

[0116] The manufacturer of the nuclear dye Hoechst 33342 is Yeasen, catalog number 40731ES10.

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

[0118] The anti-human CD47 antibody clone IBI188 was donated by Innovent Biologics.

[0119] Anti-human CD47 antibody clone Hu5F9-G4, manufacturer: Absin, catalog number abs141277

[0120] The human Plasmodium falciparum Pfs16-GFP reporter gene strain was donated by Zhang Qingfeng's laboratory at Tongji University.

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

[0122] Example 1

[0123] Figure 1 This invention demonstrates the main technical route for constructing a novel humanized erythrocyte mouse model, resulting in a novel humanized erythrocyte mouse model:

[0124] CD34 purified from the liver of aborted fetuses via magnetic bead sorting + Artificial hematopoietic stem cells (ethics review number: 2021-488-01), using purified CD34 + Human hematopoietic stem cells were injected into NCG-X neonatal mice aged 3-7 days. Ten to twelve weeks after injection, flow cytometry was used to detect human leukocytes and various human immune cell subsets to determine the engraftment efficiency of the human hematopoietic stem cells and the proportion of reconstituted human immune cell subsets. A proportion of human leukocytes greater than 10% was considered successful reconstitution. This resulted in NCG-X-humanized immune system mice.

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

[0126] The erythrocyte inducer was prepared as follows: Dexamethasone was dissolved in DMSO at a storage concentration of 67 mg / ml, and the concentration was adjusted to 2 mg / ml by adding an equal proportion of PEG300 and 0.9% physiological saline solution. Subsequently, 200 μg each of anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody were dissolved in 100 μl of 0.9% physiological saline, and an equal volume of 2 mg / ml dexamethasone solution was added.

[0127] The injection method was as follows: 100 μl of the prepared antibody solution and 2 mg / ml of dexamethasone solution were mixed and injected intraperitoneally. Mice were administered the drug every three days. Peripheral human CD235 levels were measured by flow cytometry after each injection. + The chimerism rate of positive erythrocytes was determined after 3-7 injections, during which the induction process took effect, and human erythrocytes were clearly visible. To maintain human erythrocytes in the mouse model, antibody and dexamethasone solutions should be continuously injected at the above-mentioned frequency.

[0128] Example 2

[0129] Disodium chlorophosphate liposomes (100 μl) and antibodies (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) were administered intraperitoneally to NCG-X humanized mice that had undergone immune system reconstitution for 10–12 weeks as described in Example 1. After three injections, CFSE-labeled peripheral blood erythrocytes from healthy individuals were injected into the mice. Tail vein blood samples were 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) Subsequently, the proportion of CFSE-positive human erythrocytes was detected by flow cytometry, and the trend of CFSE-positive human erythrocytes over time was statistically analyzed. Figure 2 B showed that in mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody), the survival of human erythrocytes in vivo was significantly improved. After 30 hours of reinfusion, the erythrocyte survival rate in the liposome-injected group was approximately 50%, while the erythrocyte survival rate in the Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody)-treated group was approximately 80%, representing an increase of approximately 1.6 times compared to disodium chlorophosphate liposome injection. These results indicate that, compared to conventional liposomes, Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) treatment can prolong the survival time of reinfused human erythrocytes.

[0130] Disodium chlorophosphate liposomes (100 μl) and Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) were administered intraperitoneally to NCG-X humanized mice that had undergone immune system reconstitution for 10–12 weeks, as described in Example 1. After three injections, the mice were anesthetized, and liver and spleen tissue samples were harvested. 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 a single field of view was counted and statistically analyzed. Immunofluorescence results showed that in the livers of mice treated with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody), 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). Statistical analysis of the data showed that, compared to traditional disodium chlorophosphate liposomes, treatment with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) significantly cleared [the virus], including in the liver (…). Figure 3 C) and spleen ( Figure 3 Mouse macrophages in D) and efficiently reconstructed human macrophages in these tissues.

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

[0132] Anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody were administered intraperitoneally to NCG-X humanized mice that had undergone immune system reconstitution for 10–12 weeks as described in Example 1. After three injections, the mice were anesthetized, and femoral and tibial bone samples were obtained. Human macrophages from the bone marrow were then purified by flow cytometry. The experimental group macrophages were pretreated with dexamethasone in vitro, while the control group was treated with physiological saline. The treated human macrophages were then cultured together with human erythroblasts, and the macrophages' ability to support erythroblasts was tested. Figure 4 A). Real-time quantitative PCR results showed that dexamethasone-treated human macrophages upregulated genes related to erythrocyte development, such as the enucleation genes AXL and MERTK; adhesion genes CD169 and CD163; and iron transport-related genes SLC40A1, etc. 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, and the number of erythroblasts adhering to their surface showed that more human erythroblasts adhered to their surface. Figure 4 C) indicates that its ability to adhere to erythrocytes and form hematopoietic islands is enhanced; statistical analysis of human erythroblast maturation shows that after co-culturing macrophages with dexamethasone-treated macrophages, the maturity (proportion of late erythrocytes) and enucleation rate (nuclear-free erythrocytes) of erythroblasts 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] 100 μl of disodium chlorophosphate liposomes, Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone were administered intraperitoneally to NCG-X humanized mice that had undergone immune system reconstitution for 10–12 weeks, as described in Example 1. Blood was collected from the tail vein of the mice after each injection, and human CD235 was analyzed by flow cytometry. +The proportion of positive cells to total cells was plotted and statistically analyzed. In addition, the daily mortality rate of mice was recorded and statistically analyzed, and the development of human erythrocytes in tissues was detected. After three injections of liposomes or Ab, mice were anesthetized, and bone marrow and spleen were harvested. Immune cells were extracted, and then human CD71 and human CD235a were stained by flow cytometry to analyze the developmental stages of human erythrocytes. The enucleation rate of human erythrocytes was analyzed by staining with the nuclear dye Hoechst 33342.

[0135] Figure 5 The study showed that injection of Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone into mice significantly induced human erythrocytes in peripheral blood, reaching a proportion of up to 15%. Figure 5 A) Compared with the traditional method of injecting mice with disodium chlorophosphate liposomes, it has a higher induction rate and an overall upward trend, while the proportion of human erythrocytes in the peripheral blood of liposome-injected mice does not exceed 5%. Figure 5 B); Survival statistical analysis 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 over 80% at 21 days post-injection, while the survival rate of conventional disodium chlorophosphate liposomes was only 10%. Figure 5 C). Furthermore, flow cytometry showed a significant increase in the proportion of late erythrocytes and a significant decrease in the proportion of early erythrocytes in the bone marrow, while the proportion of nuclear-free erythrocytes also significantly increased. Figure 5 D) indicates that human erythroblasts have higher 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) indicates that Ab+dexamethasone promotes the development of human erythroblasts in tissues.

[0136] Disodium chlorophosphate liposomes (100 μl), Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone were administered intraperitoneally to NCG-X humanized mice that had undergone immune system reconstitution for 10–12 weeks, as described in Example 1. After three injections of the liposomes or Ab, blood was collected from the mouse tail vein. Human erythrocyte developmental stages were analyzed by flow cytometry staining for human CD71 and human CD235a; the enucleation rate of human erythrocytes was analyzed by staining with the nuclear dye Hoechst 33342. Human blood samples used as controls were derived from normal human examination samples and were stained and analyzed using the same methods as the mouse tail vein blood samples.

[0137] Figure 6The diagram shows the proportion of maturing and enucleated human erythrocytes in peripheral blood of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone. Flow cytometry representations based on CD71 and CD235a erythrocyte maturation assays show that in the peripheral blood of mice injected with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone, the proportion of human CD235a single-positive erythrocytes was approximately 96.8%, compared to approximately 98.3% in normal human blood samples. In contrast, the proportion of human CD235a single-positive erythrocytes in mice induced by conventional disodium chlorophosphate injection was only 63.8%. Figure 6 A), the statistical results show the same trend ( Figure 6 B) indicates 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 liposome chlorophosphate 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 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%, and in mice induced by conventional disodium chlorophosphate 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) indicates that the maturity of peripheral human erythrocytes induced by injection of Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone has an almost identical phenotype to that of normal human blood samples.

[0138] Example 4

[0139] Application studies were conducted on the humanized erythrocyte mouse model constructed in Example 2:

[0140] NCG-X humanized mice that had undergone immune system reconstruction for 10–12 weeks were injected intraperitoneally with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone. Following three injections of Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone, 1 × 10⁻⁶ oz. of dexamethasone was injected intraperitoneally. 6 A Pfs16-GFP reporter gene strain of Plasmodium falciparum was used to analyze GFP-positive Plasmodium falciparum by flow cytometry 7 days after injection. The proportion of Plasmodium falciparum-positive cells in human erythrocytes was also counted. Figure 7 A).

[0141] Figure 7The study demonstrated that a humanized mouse model of peripheral erythrocytes induced by injection of Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone can support human Plasmodium falciparum infection experiments: specifically, flow cytometry showed that approximately 10% of human erythrocytes in peripheral blood were infected with Plasmodium falciparum one week after infection. Figure 7 B), and our imaging flow cytometry analysis of human erythrocytes revealed the presence of Plasmodium falciparum labeled with GFP and Hoechst33342 within the human erythrocytes. Figure 7 C) indicates that human Plasmodium falciparum infection was successfully achieved in this humanized erythrocyte mouse.

[0142] Take NCG-X humanized mice that underwent immune system reconstruction for 10-12 weeks as described in Example 1, and then take 5×10 6 The individual's B-cell lymphoma cell line, Raji, was subcutaneously inoculated until the tumor grew to approximately 300 mm. 3 Subsequently, two anti-human CD47 antibodies, IBI188 and Hu5F9-G4, were injected via tail vein at a dose of 1 mg / kg. In the control group mice that were to be injected with IgG, tumors grew to 1000 mm. 3 Subsequently, the mice were euthanized. Throughout the process, the mice were injected intraperitoneally every three days with Ab (anti-mouse F4 / 80 antibody and anti-mouse CSF1R antibody) + dexamethasone. Blood was collected from the tail vein of the mice after each injection, 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 demonstrates that humanized erythrocyte mouse models can also support the evaluation of the toxicity and efficacy of anti-human CD47 antibodies. For example... Figure 8 As shown, after transplanting the human tumor cell line Raji into this humanized mouse model, treatment with anti-human CD47 antibodies resulted in a significant slowdown in tumor growth compared to the IgG-treated control group. Furthermore, injections of both IBI188 and Hu5F9-G4 anti-human CD47 antibodies achieved similar tumor-suppressive effects. Figure 8 B). In mice injected with anti-human CD47 antibodies, compared with the control group injected with IgG, the rate of increase in human erythrocyte count was significantly slowed, achieving an increase of approximately 3-5 times by the ninth day after injection. In contrast, in the control group injected with IgG, the proportion of human erythrocytes increased by 1000 times, indicating that anti-human CD47 antibodies have a certain degree of erythrocyte toxicity in vivo. Figure 8 C).

[0144] Example 5

[0145] To explore the potential application of this invention in erythrocyte-related genetic diseases, human CD34 was enriched in bone marrow biopsy samples from patients with hereditary megaloblastic anemia using magnetic bead sorting. + Hematopoietic stem cells (ethics review number: 2022-176-02) were injected into newborn NCG-X mice. Twenty 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) The frequency and dosage were consistent with the construction method in Example 1. After seven injections, mice were euthanized and blood and tissue samples were collected. CD235a and cell nuclei in the peripheral blood samples of mice were stained, and the maturity and morphology of human erythrocytes were analyzed by flow cytometry and imaging flow cytometry. After tissue fixation, the human hemoglobin α subunit was detected by immunohistochemistry, and the tissue was scanned under a whole-slide scanner.

[0146] like Figure 9 As shown, compared with mice transplanted with stem cells from normal human donors (the group labeled Normal in the figure), mice transplanted with stem cells from patients with erythrocytic anemia (the group labeled MgA in the figure) exhibited larger peripheral red blood cell size. Figure 9 B), lower nucleus exfoliation rate ( Figure 9 C), Imaging flow cytometry showed that human erythrocytes exhibited abnormal morphology in the peripheral blood of mice that had undergone hematopoietic stem cell transplantation from patients with megaloblastic anemia, such as abnormally nucleated disc-shaped cells. Figure 9 (D and E). Immunohistochemical staining of the hemoglobin α subunit in tissues showed accumulation of human hemoglobin in multiple tissues in mice with megaloblastic anemia who had undergone stem cell transplantation. Pulmonary capillary hemorrhage and pulmonary thrombosis were observed in the lungs. Figure 9 F); erythrocyte microthrombi were observed in liver samples, and abnormal accumulation of erythrocytes was observed in blood sinuses. Figure 9 G); pathological features such as renal capillary hemorrhage and glomerular congestion were observed in the kidneys. Figure 9 Severe fibrosis and extramedullary hematopoiesis were observed in the spleen (H), 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 exhibit certain pathological characteristics.

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing humanized mice from peripheral erythrocytes, characterized in that, The construction method includes: transplanting artificial hematopoietic stem cells into mice and then administering an erythrocyte inducer to the mice, or administering an erythrocyte inducer to the mice and then infusing human erythrocytes into the mice.

2. The construction method according to claim 1, characterized in that, The erythrocyte inducer includes one or more of the following: antibodies targeting mouse macrophages, dexamethasone or its derivatives, and human macrophage colony-stimulating factor; Preferably, the dexamethasone derivative includes one or more of the pharmaceutically acceptable salts, esters, hydrates, and solvates of dexamethasone; Preferably, the dexamethasone derivative includes 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 antibody and / or anti-mouse CSF1R antibody; Preferably, artificial hematopoietic stem cells are transplanted into mice, and then the mice are administered one or more of the following: an antibody targeting mouse macrophages, dexamethasone or a derivative thereof, and human macrophage colony-stimulating factor. Preferably, the mice are administered one or more of an antibody targeting mouse macrophages, dexamethasone, or a derivative thereof, and then human red blood cells are infused into the mice.

3. The construction method according to any one of claims 1 to 2, characterized in that, After transplanting artificial hematopoietic stem cells into the mice, and after the artificial hematopoietic stem cells have stabilized and formed human immune cells in the mice, the erythrocyte inducer is then administered. Alternatively, the mice were first treated with an erythrocyte inducer for 1–7 days, followed by transplantation of human erythrocytes; Preferably, artificial hematopoietic stem cells are transplanted into the mice, and erythrocyte inducers are administered to the mice 8-25 weeks later. More preferably, artificial hematopoietic stem cells are transplanted into the mice, and erythrocyte inducers are administered to the mice 10-12 weeks later.

4. The construction method according to claim 2, characterized in that, Human hematopoietic stem cells were transplanted into mice, and then the mice were administered an antibody targeting mouse macrophages, dexamethasone or a derivative thereof, wherein the antibody targeting mouse macrophages and dexamethasone were administered simultaneously or sequentially. Alternatively, the antibody targeting mouse macrophages and / or dexamethasone can be administered at intervals. Preferably, the antibody targeting mouse macrophages is administered with dexamethasone once every three days.

5. The construction method according to claim 1, characterized in that, The mice were immunodeficient mice; The immunodeficient mice include one or more of the following: nude mice, NOD scid mice, RAG1 KO mice, RAG2 KO mice, Il2rg KO mice, severely immunodeficient mice, and BLT mice. Preferably, the severely immunodeficient mouse is a mouse containing at least one of the following: T cell deficiency, B cell deficiency, NK cell deficiency, and hematopoietic stem cell dysfunction. Preferably, the severely immunodeficient mice are one or more of NOG mice, NSG mice, NCG mice, NPG mice, NKG mice, BRG mice, NRG mice, and their derivative strains; More preferably, the severely immunodeficient mice are 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, and NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ Mice, NOD-Prkdc em26Cd52 Il2rg em26Cd22 / Nju Mice, NPG mice, BRGSF mice, BRGS mice, BRGST mice, NRGS mice, MISTRG mice, MISTRGFah - / - One or more of the following: mice, MISTRG6 mice, and 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 0.1–10 mg / ml, and the concentration of the anti-mouse CSF1R antibody is 0.1–10 mg / ml; More preferably, the concentration of the anti-mouse F4 / 80 antibody is 1-4 mg / ml, and the concentration of the anti-mouse CSF1R antibody is 1-4 mg / ml; More preferably, the concentration of the anti-mouse F4 / 80 antibody is 2 mg / ml; More 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 in a solution ranges from 0.01 to 100 mg / ml; Preferably, the concentration of dexamethasone in the solution is 0.1-10 mg / ml; More preferably, the concentration of dexamethasone in the solution is 1-4 mg / ml; More preferably, the concentration of dexamethasone in the solution is 2 mg / ml.

8. The application of the peripheral erythrocyte humanized mouse constructed according to any one of claims 1 to 7, characterized in that, The applications include: dynamic analysis of human cell subsets, including human erythrocytes; construction of erythrocyte infection models; construction of models of hereditary diseases; antibody evaluation; preparation of erythrocyte-related drugs; development of erythrocyte-related drugs; evaluation of the efficacy and / or toxicity of erythrocyte-related drugs; development of drug targets for erythrocyte-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 the following in myeloid development research: Preferably, the red blood cell infection model includes a Plasmodium falciparum infection model; Preferably, the hereditary disease includes one or more of hereditary megaloblastic anemia, β-thalassemia, and sickle cell anemia; Preferably, the antibody evaluation includes an evaluation of the toxicity and efficacy of the anti-human CD47 antibody; Preferably, the erythrocyte-related drugs include one or more of the following: drugs for treating erythrocyte infections, drugs for treating erythrocyte-related genetic diseases, and antibody drugs.

9. A commercially available combination or kit of erythrocyte inducers for constructing a humanized mouse model of peripheral erythrocytes, characterized in that, The erythrocyte inducer combination product or kit includes one or more of the following: 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 0.01–100 mg / ml; Preferably, the concentration of the anti-mouse CSF1R antibody is 0.01–100 mg / ml; Preferably, the concentration of dexamethasone in the solution is 0.01-100 mg / ml.

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

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