WO2024088412A1 - 一种小鼠细胞、人源化小鼠及其构建方法和应用 - Google Patents

一种小鼠细胞、人源化小鼠及其构建方法和应用 Download PDF

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WO2024088412A1
WO2024088412A1 PCT/CN2023/127313 CN2023127313W WO2024088412A1 WO 2024088412 A1 WO2024088412 A1 WO 2024088412A1 CN 2023127313 W CN2023127313 W CN 2023127313W WO 2024088412 A1 WO2024088412 A1 WO 2024088412A1
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mouse
mice
humanized
cells
lupus erythematosus
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李颜
朱茹洁
任德善
丁帅
刘爽
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南京大学
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Definitions

  • the present invention belongs to the technical field of animal experimental models and their preparation, and specifically relates to a type of mouse cell with an activated TLR7-IFN I signaling pathway, a humanized systemic lupus erythematosus mouse having the cell, a method for constructing the humanized systemic lupus erythematosus mouse, and its application in the field of medical research.
  • the present invention uses humanized mice to construct a systemic lupus erythematosus (SLE) model.
  • SLE systemic lupus erythematosus
  • Humanized mice stably reconstruct the human immune system based on severely immunodeficient mice, which is of great significance for the study of human immunology, especially for the study of human autoimmune diseases.
  • FIG. 1 Chinese patent document CN111149767B can construct a simple humanized systemic lupus erythematosus model by transplanting peripheral blood mononuclear cells (PBMC) from systemic lupus erythematosus patients into severely immunodeficient mice, but it has very obvious limitations.
  • peripheral blood mononuclear cells are terminally differentiated cells and cannot simulate the various stages of differentiation of hematopoietic stem cells (HSC) in vivo. After the infused peripheral blood mononuclear cells enter the body of severely immunodeficient mice, they will also cause severe graft-versus-host disease, leading to T cell activation and secretion of a large number of inflammatory factors.
  • HSC hematopoietic stem cells
  • hematopoietic stem cells Compared with peripheral blood mononuclear cells, hematopoietic stem cells have become the first choice for rebuilding the human immune system due to their self-renewal and multidirectional differentiation potential. After being transplanted into immunodeficient recipients, they can differentiate and develop a relatively complete set of human immune systems, including T, B, monocytes, NK, DC, etc. at various stages, and can maintain long-term hematopoiesis. Recently, a research team injected pristane intraperitoneally into hematopoietic stem cell humanized mice.
  • the present invention provides a cell in a mouse body with an activated TLR7-IFN I signaling pathway, a humanized systemic lupus erythematosus mouse having the cell, a method for constructing the mouse, and its application in the field of medical research.
  • TLR7-IFN I signaling pathway By inducing and reconstructing the TLR7-IFN I signaling pathway in a mouse with a humanized immune system, T cell abnormalities, upregulation of inflammatory factors, and lupus nephritis manifestations similar to those in patients with systemic lupus erythematosus are induced.
  • mice described in the present invention refer to mice after the interferon signaling pathway is activated
  • the cells in the mouse body described in the present invention include the mouse's own cells and/or human immune cells in the mouse body.
  • the present invention provides the following:
  • TLR7-IFN I signaling pathway TLR7 signaling pathway is activated and triggers downstream IFN I response
  • TLRs are genetically encoded pattern recognition receptors found on the cell surface. TLRs recognize pathogen-associated molecular proteins (PAMPs), which include TLR7 agonists or TLR8 agonists. TLR7, as a transmembrane receptor in the endosomal compartment of the cell, is located on the lysosomal membrane and can recognize and bind to viral single-stranded (ss) RNA and activate the interferon response through the MyD88 signaling pathway, that is, triggering downstream signaling pathways to induce type I interferon response, namely IFN-I response.
  • PAMPs pathogen-associated molecular proteins
  • type I IFN depends on a complex containing MyD88 and IRF7. After TLR7 is activated, it can recruit MyD88 and further recruit IRAK4. The complex also contains TRAF3, TRAF6, IRAK1, IKK ⁇ , etc. MyD88 directly or indirectly recruits IRF7, causing it to be phosphorylated by IRAK1 and/or IKK ⁇ and translocated to the nucleus to regulate the expression of type I IFN. In addition, the MyD88-IRAK4-TRAF6 complex drives the induction of NF- ⁇ b-dependent inflammatory factors.
  • RNA and DNA released by dead or damaged cells can be recognized by the B cell receptor (BCR) on autoreactive B cells.
  • BCR transports RNA and DNA to intracellular lysosomes, where they bind to TLR7 and TLR9, respectively.
  • Activation of TLR7 and TLR9 in B cells can induce their proliferation, cytokine production (IFN-I), and differentiation into plasma cells.
  • IFN-I cytokine production
  • plasma cells secrete autoantibodies that can bind to RNA, DNA, or RNA/DNA-related proteins to form immune complexes.
  • Fc ⁇ RIIa (CD32) expressed by plasmacytoid dendritic cells (pDCs) binds to immune complexes containing nucleic acids and transports them to and binds to TLR7/TLR9 on the lysosomal membrane.
  • Activation of TLR7 and TLR9 in pDCs can induce the production of proinflammatory cytokines and chemokines, leading to a malignant cytokine storm and inflammatory cell infiltration of target organs, further causing tissue lesions.
  • IFN- ⁇ secreted by pDCs further enhances B cell proliferation and autoantibody production by upregulating TLR7 expression on B cells, thus forming a positive feedback loop that leads to the exacerbation of autoimmunity.
  • the present invention provides cells in a mouse body.
  • the mouse is a mouse with an activated interferon signaling pathway, and the cells in the mouse body include the mouse's own cells and/or human immune cells in the mouse body.
  • the TLR7-IFN I signaling pathway in the cell is activated by administering a TLR7 agonist.
  • the TLR7 agonist is resiquimod and imiquimod.
  • the dose range of resiquimod administration is 30-300 ⁇ g/week/mouse.
  • the dose of resiquimod administered each time is 10-100 ⁇ g/mouse.
  • the administration frequency of resiquimod is twice a week or three times a week.
  • the administration frequency of imiquimod is 0.5-3 mg/mouse/time, twice a week.
  • the present invention also provides a humanized systemic lupus erythematosus mouse, wherein the mouse has the above-mentioned cell.
  • the mouse has abnormal T cell content, upregulated levels of inflammatory factors, manifestations of lupus nephritis, dysregulation of B cell subsets and autoantibody production.
  • the mouse is used for a period of 8-30 weeks, preferably 8-16 weeks.
  • the present invention also provides a method for constructing a humanized systemic lupus erythematosus mouse, the method comprising:
  • the process of injecting a plasmid expressing human Flt3L into a mouse comprises:
  • the process of injecting autologous activated lymphocyte-derived DNA into mice comprises:
  • the present invention provides the use of the humanized systemic lupus erythematosus mice obtained by the above construction method in medical research.
  • the present invention successfully constructed a humanized systemic lupus erythematosus mouse by transplanting human hematopoietic stem cells into severely immunodeficient mice.
  • the TLR7 agonist resiquimod was used to activate the key mechanism of the TLR7-IFN I signaling pathway, and induced T cell abnormalities, upregulation of inflammatory factors and lupus nephritis similar to those in patients with systemic lupus erythematosus in mice.
  • mice expressing human Flt3L plasmids were expressing human Flt3L plasmids and intraperitoneal injection of immune ALD-DNA in mice. Furthermore, through the process of high-pressure tail vein injection of mice expressing human Flt3L plasmids and intraperitoneal injection of immune ALD-DNA in mice, the humoral immune response of mouse B cells was promoted, inducing B cell subset disorders and autoantibody production.
  • the induction period is significantly shortened. Compared with the six-month induction period of the humanized Pristane model, the induction time of this disease model is shortened from six months to two months.
  • mice of the present invention are more obvious than those of other existing humanized lupus erythematosus mice.
  • the construction method disclosed in the present invention and the systemic lupus erythematosus mice obtained after the construction may become the humanized systemic lupus erythematosus disease model that best reflects the pathological conditions of systemic lupus erythematosus patients.
  • the construction method disclosed in the present invention is also stable and reliable, which facilitates the long-term evaluation of the efficacy and mechanism of action of candidate drugs, and is expected to provide a reliable drug screening platform for screening new drugs for systemic lupus erythematosus.
  • Figure 1 T lymphocyte activation in mice in the TLR7 agonist administration group.
  • Figure 2 Changes in the proportion of Th17 and Tfh cells in mice in the TLR7 agonist administration group.
  • FIG. 3 Plasma IL-6, IL-17, IFN- ⁇ and IL-22 inflammatory factors were upregulated in mice in the TLR7 agonist administration group.
  • Figure 4 Proliferation of glomerular endothelial and mesangial cells was observed in mice in the TLR7 agonist administration group.
  • FIG. 5 Immune complexes (IgG+IgM) were deposited in the kidneys of mice in the TLR7 agonist administration group (the picture shows a single glomerulus).
  • FIG. 6 Immune complexes (IgG+IgM) were deposited in the kidneys of mice in the TLR7 agonist administration group (the picture shows a cross-section of the entire kidney).
  • Figure 7 The total plasma IgG of mice in the TLR7 agonist administration group increased.
  • Figure 8 Mice in the TLR7 agonist-administered group showed facial erythema similar to that of lupus erythematosus patients.
  • FIG. 10 The urine protein content of mice in the TLR7 agonist administration group increased, and the spleen enlarged, resulting in weight gain.
  • Figure 11 After intraperitoneal injection of DNA derived from autologous activated lymphocytes, the proportion of plasmablasts and IgG + B cells in total B cells in mice increased.
  • Figure 12 After intraperitoneal injection of DNA derived from autologous activated lymphocytes, a large number of follicular helper T cells appeared in the spleen and lymph nodes of mice.
  • FIG. 13 After intraperitoneal injection of DNA derived from autologous activated lymphocytes, mice produced certain titers of anti-nuclear antibodies and anti-dsDNA antibodies.
  • Figure 14 After intraperitoneal injection of DNA derived from autologous activated lymphocytes into mice, massive activation and proliferation of T cells were detected in the spleen and bone marrow 3-5 days later.
  • FIG. 15 The titer of antinuclear antibodies in the serum of humanized SLE mice after TLR7 agonist induction.
  • Figure 16 Flow cytometry detection results and data statistics of kidney B cells in humanized SLE mice;
  • Figure 16A is the gating result of kidney B cells in the flow cytometry detection data;
  • Figure 16B is the statistical data comparison result of the proportion of plasma cells in kidney B cells.
  • Figure 17 Survival curve of humanized SLE mice.
  • Figure 18 The therapeutic effects on total serum IgG and anti-double-stranded DNA autoantibodies after administration of two monoclonal antibody preparations;
  • Figure 18A shows the results of total serum IgG antibody levels detected after administration of the vehicle group (control G1’), belimumab preparation (G2’) and rituximab preparation (G3’);
  • Figure 18B shows the results of anti-double-stranded DNA autoantibody levels detected after administration of the vehicle group (control G1’), belimumab preparation (G2’) and rituximab preparation (G3’).
  • Pristane-induced lupus mouse model In 1994, Satoh M used pristane to induce BALB/c mice to develop symptoms similar to human systemic lupus erythematosus, and established the pristane-induced lupus mouse model for the first time. This method has become the most commonly used method for inducing lupus mouse models.
  • Pristane also known as 2,6,10,14-tetramethylpentadecane, TMPD, is an isoprenoid alkane compound that can be found in plants, marine organisms and byproducts of petroleum distillation.
  • pristane is also a membrane-activating compound that interacts with the phospholipid bilayer.
  • Pristane can induce programmed cell death in mouse lymphocyte lines and peritoneal exudate cells both in vitro and in vivo, suggesting that pristane-induced apoptosis provides sufficient autoantigen substrates, leading to immune disorders associated with excessive production of interferon ⁇ and ⁇ (IFN- ⁇ and IFN- ⁇ ).
  • TLR7-IFN I signaling pathway The TLR7 signaling pathway activates and triggers downstream IFN I responses.
  • TLRs are genetically encoded pattern recognition receptors found on the cell surface.
  • TLRs recognize pathogen-associated molecular proteins (PAMPs), which are compounds such as TLR7 agonists or TLR8 agonists.
  • PAMPs pathogen-associated molecular proteins
  • TLR7 as a transmembrane receptor in the endosomal compartment of the cell, can recognize viral single-stranded RNA and then trigger downstream signaling pathways to induce a type I interferon response, namely an IFN I response.
  • Flt3L (Flt3Ligand) is a membrane protein with homology to SCF and M-CSF, and can stimulate cytokines of early hematopoiesis. In vivo and in vitro experiments have confirmed that Flt3-L can regulate the proliferation and differentiation of non-erythroid hematopoietic stem/progenitor cells.
  • Flt3-L can promote the proliferation, differentiation and maturation of pre-B lymphocytes, dendritic cells (DC), natural killer (NK) cells, and cytotoxic T lymphocytes (CTL), thus playing an important immunomodulatory role.
  • DC dendritic cells
  • NK natural killer
  • CTL cytotoxic T lymphocytes
  • mice with imperfect innate immunity and completely lacking functional T, B and NK cells can be used for human tumor cell transplantation (CDX) and human tumor tissue transplantation (PDX), as well as human peripheral blood mononuclear cells (PBMC) and human hematopoietic stem cells (CD34+HSC) transplantation for immune reconstruction.
  • CDX human tumor cell transplantation
  • PDX human tumor tissue transplantation
  • PBMC peripheral blood mononuclear cells
  • CD34+HSC human hematopoietic stem cells
  • TLR7 agonists are drugs that have both affinity and intrinsic activity. They can bind to receptors and stimulate receptors to produce effects. TLR activation can initiate innate and adaptive immune responses. TLR7 is one of the members of TLRs, responsible for recognizing single-stranded RNA of pathogens. It is mainly distributed in plasma dendritic cells (pDC) and B cells, and plays an important role in the human body's recognition and elimination of pathogenic microorganisms. TLR7 agonists can stimulate pDC to secrete interferon- ⁇ and act on other immune cells (such as NK cells and macrophages) to enhance immunity.
  • pDC plasma dendritic cells
  • B cells plays an important role in the human body's recognition and elimination of pathogenic microorganisms.
  • TLR7 agonists can stimulate pDC to secrete interferon- ⁇ and act on other immune cells (such as NK cells and macrophages) to enhance immunity.
  • TLR7 agonists are also expected to be developed for the treatment of viral diseases such as HIV-1 infection and hepatitis B.
  • Resiquimod also known as R848, is an immune response modifier that acts as a potent TLR 7/TLR8 agonist and can induce upregulation of cytokines such as TNF- ⁇ , IL-6 and IFN- ⁇ , acting as a potent TLR 7 agonist.
  • Imiquimod is an imidazoquinolineamine interleukin agonist, a drug for the treatment of genital warts. It is used in mouse skin to induce cytoplasmic division and can produce ⁇ -interferon, tumor necrosis factor and a variety of interleukins. It is also a common TLR 7 agonist.
  • the Mn adjuvant used in the present invention was purchased from Qimange Biotechnology, other reagents were purchased from Biolegend/Thermo Fisher and BD Biosciences, and severely immunodeficient NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the method for constructing a humanized immune system mouse model is as follows:
  • mice constructed in the same batch, kill the mice, obtain spleen and bone marrow, and detect the reconstruction of the human immune system in the tissues.
  • Drug dosage The experimental group was divided into three groups, and the doses of TLR7 agonist resiquimod applied weekly were 10 ⁇ g*3 times, 60 ⁇ g*3 times and 100 ⁇ g*3 times, that is, the dose range of resiquimod used per week was 30-300 ⁇ g/mouse.
  • mice were injected intraperitoneally with 5 ⁇ g Flt3L protein three times a week for two weeks.
  • the injection dose for each mouse is 10 ⁇ g, and the concentration of plasmid stock solution is 500 ⁇ g/ml
  • the injection solution was prepared as shown in Table 2:
  • Plasmids were injected into the tail vein of mice at high pressure to induce human Flt3L expression in the liver.
  • mice were intraperitoneally injected with DNA (20 ⁇ g) + Mn adjuvant + R848 (20 ⁇ g).
  • DNA (20 ⁇ g) was dissolved in 200 ⁇ l ddH2O.
  • R848 (60 ⁇ g) was dissolved in 30 ⁇ l acetone.
  • Mn adjuvant 200 ⁇ l, purchased from Qimeng Biotechnology.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • a mouse model with a humanized immune system was constructed by the above method to verify the construction effect of the mouse with a humanized immune system.
  • mice severely immunodeficient mice were injected with human hematopoietic stem cells after irradiation.
  • the level of human immune cell reconstruction was tested 8-16 weeks after the injection of human hematopoietic stem cells.
  • the results showed that the human immune system in the peripheral blood of mice was well reconstructed, with an average of more than 20%, and all major immune cell subsets such as B cells, T cells, monocytes, and dendritic cells (DC) were stably reconstructed.
  • B cells B cells, T cells, monocytes, and dendritic cells (DC) were stably reconstructed.
  • TLR7 agonist resiquimod was administered to the ears of humanized mice obtained by the above operation.
  • Control group no TLR7 agonist resiquimod was administered, only solvent acetone was administered
  • Dosage groups 30 ⁇ g group, 180 ⁇ g group and 300 ⁇ g group
  • the three groups were administered via ear administration of TLR7 agonist resiquimod at doses of 10 ⁇ g*3 times, 60 ⁇ g*3 times and 100 ⁇ g*3 times per week, that is, the weekly dose of resiquimod was 30-300 ⁇ g/mouse.
  • mice developed partial systemic lupus erythematosus-like phenotypes, while the level of peripheral blood humanization was relatively stable.
  • T lymphocyte abnormalities similar to those of systemic lupus erythematosus patients appeared, including T cell activation, dysregulation of helper T cell (Th) subsets, and increase in follicular helper T cells (Tfh).
  • Th helper T cell
  • Tfh follicular helper T cells
  • plasma IL-6, IFN- ⁇ , IL-17A, IL-22 inflammatory factors and total immunoglobulin G (IgG) increased significantly.
  • HE staining of the kidneys in the induction group showed proliferation of endothelial and mesangial cells in the glomeruli, and immunofluorescence staining showed a large amount of human IgG and IgM deposits in the glomeruli, which are typical manifestations of lupus nephritis.
  • human Flt3L is supplemented to promote the development of dendritic cells to induce a more obvious systemic lupus erythematosus phenotype and B cell stress response.
  • a humanized mouse model of the immune system was constructed by the above method, and a plasmid expressing human Flt3L was injected into the humanized mouse. Then, the TLR7 agonist resiquimod was administered to the mouse ears.
  • Control group no TLR7 agonist resiquimod
  • Flt3L group only the plasmid expressing human Flt3L was injected
  • TLR7 group only TLR7 agonist 60 ⁇ g*3 times
  • Flt3L+TLR7 group first inject the plasmid expressing human Flt3L, then apply TLR7 agonist group 60 ⁇ g*3 times
  • Flt3L can expand human conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC), and induce T cells, especially the upregulation of CD4+T cells, which can better help B cells obtain antigens and activate proliferation.
  • cDC dendritic cells
  • pDC plasmacytoid dendritic cells
  • T cells especially the upregulation of CD4+T cells, which can better help B cells obtain antigens and activate proliferation.
  • the phenotype of the model mice was more obvious, with splenomegaly and facial erythema similar to that of patients with systemic lupus erythematosus.
  • the urine protein level of the induced group also gradually increased over time, and was significantly different from that of the control group at the end point.
  • the test results of each group are shown in Table 4.
  • the results of the control group without the TLR7 agonist resiquimod were normal, and no obvious changes were observed in the Flt3L group injected with the human Flt3L plasmid (indicating that the single administration of the Flt3L plasmid would not produce a significant induction effect); however, the TLR7 group only with the TLR7 agonist had symptoms such as splenomegaly, increased urine protein, and facial erythema similar to systemic lupus erythematosus, proving that the addition of the TLR7 agonist has activated the TLR7-IFN I signaling pathway in the constructed model to a certain extent, thereby producing symptoms similar to lupus erythematosus; To further prove the correlation between the two, a Flt3L+TLR7 combination group was set up in which the Flt3L plasmid was injected first and then the TLR7 agonist was applied.
  • This example compares the effects of two TLR7 agonists (resiquimod and imiquimod) on mouse ears.
  • the immune system humanized mouse model was constructed by the above method, and two TLR7 agonists, resiquimod and imiquimod, were administered to the ears of the humanized mice, respectively.
  • the dosages of the two agonists are shown in Table 5 below:
  • the specific operation is to squeeze 5% imiquimod ointment into a 1ml insulin needle, and squeeze out a certain volume of ointment each time to apply to the mouse ear for easy quantitative measurement.
  • Resiquimod is dissolved in acetone and slowly dripped into the mouse ear with a 100 ⁇ l pipette.
  • IMQ group Imiquimod ointment group
  • R848 group resiquimod group
  • the humanized mouse model of immune system was constructed by the above method, and the humanized mouse was injected with a plasmid expressing human Flt3L. Then, two TLR7 agonists, resiquimod and imiquimod, were administered to the mouse ears respectively.
  • the specific operation is to squeeze out a certain volume of 5% imiquimod ointment each time and apply it to the mouse ear for easy quantitative measurement.
  • Resiquimod is dissolved in acetone and then slowly dripped into the mouse ear with a 100 ⁇ l pipette.
  • Control group injection of plasmid expressing human Flt3L, without administration of TLR7 agonist resiquimod (Ctrl group)
  • IMQ group Imiquimod ointment group
  • R848 group resiquimod group
  • mice in the drug administration group had facial erythema (Figure 8), which was similar to that of lupus erythematosus patients.
  • the urine protein content of mice in the drug administration group increased, and the spleen was enlarged, resulting in weight increase ( Figures 9 and 10).
  • a humanized mouse model of the immune system was constructed by the above method, and activated lymphocyte-derived DNA (ALD-DNA) was intraperitoneally infused into the humanized mice to induce T cell and B cell subset disorders and the production of autoantibodies in humanized systemic lupus erythematosus mice.
  • ALD-DNA activated lymphocyte-derived DNA
  • ALD-DNA Activated lymphocyte-derived DNA
  • Anti-double-stranded DNA antibodies which are the serological characteristics of patients with systemic lupus erythematosus, have been proven to be pathogenic, can cause immune complex deposition and tissue damage, and are closely related to the severity of systemic lupus erythematosus. Genetic studies of patients with systemic lupus erythematosus have found that most anti-dsDNA autoantibodies belong to the IgG subclass with high affinity for dsDNA, which is different from antibodies produced by somatic mutations. Studies have shown that self-DNA can induce the production of anti-dsDNA antibodies. In general, the immunogenicity of mammalian DNA is weak and will not cause an immune response. Finding the driving components that cause autoimmune reactions and the production of anti-dsDNA antibodies is a hot topic for immunologists.
  • the present invention found that DNA from activated lymphocytes was used in the process of searching for the driver of systemic lupus erythematosus.
  • mice immunized with ALD-DNA can develop a series of systemic lupus erythematosus symptoms, including high levels of anti-dsDNA autoantibodies, proteinuria, immune complex deposition and glomerulonephritis. These symptoms simulate the systemic lupus erythematosus symptoms caused by a large amount of self-DNA derived from apoptotic cells in patients. Therefore, ALD-DNA-immunized mice can be used as an ideal mouse lupus model to explore the possible cellular and molecular immunological mechanisms of systemic lupus erythematosus.
  • This comparative example uses the mice to explore the therapeutic effect of anti-aging drugs (Senolytics) on lupus erythematosus.
  • mice Humanized systemic lupus erythematosus mice were constructed by the above method, and two anti-aging drugs were used in combination: ABT263 (BCL2 inhibitor) and S63845 (MCL-1 inhibitor).
  • the experimental groups were as follows:
  • ABT263 group mice were given 10 mg/kg twice a week by oral administration;
  • mice The mice were given 25 mg/kg twice a week by intraperitoneal injection
  • ABT263+S63845 group The mice were given ABT263 10 mg/kg, twice a week, by oral gavage; S63845 25 mg/kg, twice a week, by intraperitoneal injection.
  • the present invention can induce lupus phenotype in humanized mice through the above method, and administering anti-aging drugs can treat lupus. Therefore, the humanized systemic lupus erythematosus mice can be used for drug efficacy evaluation.
  • the present invention induces T cell abnormalities, upregulation of inflammatory factors and lupus nephritis similar to those in patients with systemic lupus erythematosus by activating the key mechanism of TLR7-IFN I signaling pathway in humanized immune system mice. And by injecting plasmids expressing human Flt3L and DNA derived from autologous activated lymphocytes into mice, the humoral immune response of mouse B cells is promoted, and the disorder of B cell subpopulations and the production of autoantibodies are induced, thereby constructing the humanized systemic lupus erythematosus mouse.
  • the SLE phenotype was induced in the third-generation humanized mice by TLR7 agonist mouse ear wipe.
  • the induced group was divided into 3 groups according to the disease situation.
  • One group was not given any treatment (G1'), one group was treated with belimumab (G2'), and the other group was treated with rituximab (G3').
  • the grouping and dosing strategy are shown in Table 8. During the period, the survival rate of mice, the phenotype of disease, etc. were monitored. The endpoint was reached at 4 weeks of treatment, and the immunological and pathological related indicators were evaluated.
  • FIG. 17 shows the survival curves of the above three groups of humanized SLE mice after administration.
  • FIG. 18 shows the therapeutic effects on serum total IgG and anti-double-stranded DNA autoantibodies after administration of two monoclonal antibody preparations, respectively.
  • the application scope of the humanized SLE mouse model constructed by this patent includes, but is not limited to: drug evaluation and pharmacological mechanism exploration of various cell therapies, biological targeted preparations, small molecule drugs, and compound drugs (such as traditional Chinese medicine, etc.).
  • drug evaluation and pharmacological mechanism exploration of various cell therapies, biological targeted preparations, small molecule drugs, and compound drugs such as traditional Chinese medicine, etc.
  • compound drugs such as traditional Chinese medicine, etc.

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Abstract

公开了一种TLR7-IFN I信号通路活化的小鼠体内细胞，具有该细胞的人源化系统性红斑狼疮小鼠，该小鼠的构建方法及其在医学研究中的应用。具体地，通过施用TLR7激动剂瑞喹莫德活化细胞的TLR7-IFN I信号通路，使该小鼠表现出系统性红斑狼疮患者类似的表型，并通过向小鼠注射表达人Flt3L的质粒及自体活化淋巴细胞来源的DNA，促进了小鼠B细胞的体液免疫反应，诱导其B细胞亚群失调和自身抗体产生，从而构建该人源化系统性红斑狼疮小鼠。该人源化系统性红斑狼疮小鼠，是在人的免疫系统中诱导出了免疫失调和相应的表型，因此其更有利于筛选新型靶向药物。

Description

一种小鼠细胞、人源化小鼠及其构建方法和应用 技术领域
本发明属于动物实验模型及其制备技术领域，具体涉及一种TLR7-IFN I信号通路活化的小鼠体内细胞，具有该细胞的人源化系统性红斑狼疮小鼠，该人源化系统性红斑狼疮小鼠的构建方法及其在医学研究领域的应用。
背景技术
本发明选用了人源化小鼠来构建系统性红斑狼疮(SLE)模型。人源化小鼠在重度免疫缺陷的小鼠的基础上稳定地重建了人体免疫系统，对研究人体免疫学，尤其是针对人类自身免疫性疾病的研究有重大意义。
中国专利文件CN111149767B通过移植系统性红斑狼疮病人外周血单个核细胞(PBMC)到重度免疫缺陷小鼠可以构建简单的人源化系统性红斑狼疮模型，但其有非常明显的限制性。首先，外周血单个核细胞是终末分化的细胞，无法模拟造血干细胞(HSC)在体内分化的各个阶段，输注的外周血单个核细胞进入重度免疫缺陷鼠体内后也会引起严重的移植物抗宿主病，导致T细胞被活化，分泌大量炎症因子等。其次，该模型中，只有T细胞重建良好，其他细胞如B细胞、NK细胞、单核巨噬细胞等均重建极低甚至没有，这也将该模型的应用限制于研究T细胞相关作用，使其应用价值大大被削弱。此外，该小鼠模型寿命只有4周左右，其使用时间窗较短，无法进行长时程持续性的研究。
相较于外周血单个核细胞，造血干细胞因其自我更新和多向分化潜能而成为重建人免疫系统的首选。移植入免疫缺陷受体体内后，其能分化发育出相对完整的一套人免疫系统，包括各阶段的T、B、单核、NK、DC等，且能维持长期造血。近来，有研究团队给造血干细胞人源化小鼠腹腔注射了降植烷，6个月后可出现人抗核自身抗体、TB细胞过度活化、炎症因子分泌增加、狼疮性肾炎和肺浆膜炎等免疫学和临床特征。这一模型的出现将人源化系统性红斑狼疮模型带入了一个更广阔的领域，相较于外周血单个核细胞人源化系统性红斑狼疮小鼠模型而言，其不仅有T细胞重建，还有各发育阶段和类型的其他免疫细胞，更接近于生理状态下的人免疫系统，且拥有更长的寿命和使用时间窗。然而，在此模型中，尽管出现了多种自身抗体谱，其滴度都极其低，血浆总IgG和IgM的浓度也远小于在人体内的浓度。疾病最经典的，也是Pristane诱导狼疮小鼠模型中较为明确的IFN-I信号通路也仅显示出了转录水平的上调，并未有蛋白水平的变化。此外，同普通Pristane诱导狼疮小鼠模型类似，该模型诱导时间长达6个月，可能会错过人源化小鼠最佳使用时期或为后期的进一步药物验证带来时间上的限制。
发明内容
本发明提供了一种TLR7-IFN I信号通路活化的小鼠体内细胞，具有该细胞的人源化系统性红斑狼疮小鼠，该小鼠的构建方法及其在医学研究领域的应用。通过在免疫系统人源化小鼠体内诱导重建TLR7-IFN I信号通路，诱导出与系统性红斑狼疮患者类似的T细胞异常、炎症因子上调和狼疮肾炎表现。进一步，通过向小鼠注射表达人Flt3L的质粒及自体活化淋巴细胞来源的DNA，促进了小鼠的B细胞体液免疫反应，诱导B细胞亚群失调和自身抗体产生，来构建人源化系统性红斑狼疮小鼠模型，以解决现有技术中存在的技术缺陷。
本发明中所述的小鼠，是指干扰素信号通路活化后的小鼠；
本发明中所述的小鼠体内细胞，包括了小鼠自身细胞和/或小鼠体内的人源免疫细胞。
具体地，本发明提供了如下内容：
1.TLR7-IFN I信号通路：TLR7信号通路激活并触发下游IFN I反应
TLR是在细胞表面发现的基因编码模式识别受体。TLR识别病原体相关分子蛋白(PAMP)，这类化合物包括TLR7激动剂或TLR8激动剂。TLR7作为细胞内体区室的一种跨膜受体，位于溶酶体膜，能够识别并结合病毒单链(ss)RNA并通过MyD88信号通路激活干扰素反应，即触发下游信号通路来诱导I型干扰素反应即IFN-I反应。
I型IFN(即IFN-I)的分泌依赖于包含MyD88和IRF7的复合物。TLR7活化后，能够招募MyD88，进一步招募IRAK4。该复合体还包含TRAF3、TRAF6、IRAK1、IKKα等。MyD88直接或间接招募IRF7，使其被IRAK1和/或IKKα磷酸化，并易位到细胞核中调节I型IFN的表达。此外，MyD88-IRAK4-TRAF6复合体驱动NF-κb依赖性炎症因子的诱导。
死亡或受损细胞释放的RNA和DNA可被自身反应性B细胞上的B细胞受体(BCR)识别。BCR将RNA和DNA运输到细胞内溶酶体，分别与TLR7和TLR9结合。B细胞中TLR7和TLR9的激活可诱导其增殖、细胞因子的产生(IFN-I)和向浆细胞分化。随后，浆细胞分泌自身抗体，可结合RNA、DNA或RNA/DNA相关蛋白形成免疫复合物。由浆细胞样树突状细胞(pDC)表达的FcγRIIa(CD32)结合含有核酸的免疫复合物，并将其运输到溶酶体膜上的TLR7/TLR9且与之结合。pDCs中TLR7和TLR9的激活可诱导促炎细胞因子和趋化因子的产生，从而导致恶性细胞因子风暴和靶器官炎症细胞浸润，进一步引起组织病变。此外，pDCs分泌的IFN-α通过上调B细胞TLR7表达进一步增强B细胞增殖和自身抗体的产生，从而形成一个正反馈循环，导致自身免疫的恶化。
2.小鼠体内细胞
本发明提供一种小鼠体内细胞，小鼠为干扰素信号通路活化后的小鼠，其体内细胞包括了小鼠自身细胞和/或小鼠体内的人源免疫细胞。
可选地，通过施用TLR7激动剂活化该细胞内的TLR7-IFN I信号通路。
可选地，所述TLR7激动剂是瑞喹莫德和咪喹莫特。
可选地，所述瑞喹莫德施用的剂量范围为30-300μg/周/鼠。
可选地，所述瑞喹莫德每次施用的剂量为10-100μg/鼠。
可选地，所述瑞喹莫德的施用频率为每周两次或每周三次。
可选地，所述咪喹莫特的施用频率0.5-3mg/只/次，一周两次。
3.人源化系统性红斑狼疮小鼠
本发明还提供一种人源化系统性红斑狼疮小鼠，其中该小鼠具有上述细胞。
可选地，该小鼠具有T细胞含量异常、炎症因子水平上调、狼疮肾炎表现、B细胞亚群失调和自身抗体产生。
可选地，该小鼠的使用周期为8-30周，优选为8-16周。
4.人源化系统性红斑狼疮小鼠的构建方法
进一步地，本发明还提供一种人源化系统性红斑狼疮小鼠的构建方法，该方法包括：
1)对出生一周内的免疫缺陷鼠进行辐照清髓；
2)将人胎肝来源造血干细胞注射入小鼠肝脏部位，剂量为0.5-10×104个细胞/鼠；
3)注射造血干细胞8-16周后检测小鼠外周血人免疫细胞重建情况；
4)向小鼠注射表达人Flt3L的质粒。
5)向小鼠注射自体活化淋巴细胞来源的DNA。
在某些实施例中，向小鼠注射表达人Flt3L质粒的过程包括：
1)构建表达人Flt3L的pLIVE质粒；
2)提取质粒并配成储备液；
3)配置新鲜的注射液；
4)通过小鼠高压尾静脉注射该注射液。
在某些实施例中，向小鼠注射自体活化淋巴细胞来源DNA的过程包括：
1)通过小鼠高压尾静脉注射表达人IL-2的质粒，活化及扩增免疫系统人源化小鼠体内人免疫细胞；
2)提取步骤(1)中小鼠的脾脏及骨髓免疫细胞；
3)提取活化淋巴细胞来源的基因组DNA。
4)小鼠腹腔注射该自体活化淋巴细胞来源的DNA。
5.人源化系统性红斑狼疮小鼠的应用
进一步地，本发明提供上述构建方法得到的人源化系统性红斑狼疮小鼠在医学研究中的应用。
本发明通过移植人造血干细胞至重度免疫缺陷鼠，成功构建免疫系统人源化系统性红斑狼疮小鼠。并在此基础上，应用TLR7激动剂瑞喹莫德活化TLR7-IFN I信号通路这一关键机制，在小鼠体内诱导出了系统性红斑狼疮患者类似的T细胞异常、炎症因子上调和狼疮肾炎表现。进一步，通过小鼠高压尾静脉注射表达人Flt3L质粒的过程及小鼠腹腔注射免疫ALD-DNA，促进了小鼠B细胞的体液免疫反应，诱导B细胞亚群失调和自身抗体产生。
与现有红斑狼疮小鼠构建方法相比，如注射Pristane诱导人源化红斑狼疮小鼠模型相比，
本发明具有以下技术优势：
①诱导周期显著缩短，相比于Pristane模型人源化六个月的诱导周期，本疾病模型的诱导时间从六个月缩短至两个月。
②本发明小鼠的系统性红斑狼疮相关表型相比于现有其他人源化红斑狼疮小鼠更加明显。
③模型的免疫学应答机制更明确，即TLR7-IFN I信号通路的活化过程。
因此本发明公开的构建方法以及构建后获得的系统性红斑狼疮小鼠可能成为最能反映系统性红斑狼疮患者病理情况的人源化系统性红斑狼疮疾病模型。同时本发明公开的构建方法还具有稳定可靠的特点，为长期评价候选药物的疗效及作用机制带来了便利，有望为筛选系统性红斑狼疮新型药物提供可靠的药筛平台。
附图说明
图1：TLR7激动剂给药组小鼠T淋巴细胞活化情况。
图2：TLR7激动剂给药组小鼠Th17及Tfh细胞比例变化情况。
图3：TLR7激动剂给药组小鼠血浆IL-6、IL-17、IFN-γ及IL-22炎症因子上调。
图4：TLR7激动剂给药组小鼠肾小球内皮、系膜细胞出现增生。
图5：TLR7激动剂给药组小鼠肾脏免疫复合物(IgG+IgM)出现沉积(图为单个肾小球)。
图6：TLR7激动剂给药组小鼠肾脏免疫复合物(IgG+IgM)出现沉积(图为整个肾脏横截面)。
图7：TLR7激动剂给药组小鼠血浆总IgG升高。
图8：TLR7激动剂给药组小鼠呈现与红斑狼疮患者类似的面部红斑。
图9：TLR7激动剂给药组小鼠尿蛋白含量上升。
图10：TLR7激动剂给药组小鼠尿蛋白含量上升，脾脏肿大导致重量增加。
图11：腹腔注射自体活化淋巴细胞来源的DNA后，小鼠浆母细胞及IgG+B细胞在总B细胞中的比例增加。
图12：腹腔注射自体活化淋巴细胞来源的DNA后，小鼠脾脏及淋巴结中出现大量滤泡辅助性T细胞。
图13：腹腔注射自体活化淋巴细胞来源的DNA后，小鼠体内产生一定滴度的抗核抗体及抗dsDNA抗体。
图14：小鼠腹腔注射自体活化淋巴细胞来源的DNA，3-5天后检测到脾脏和骨髓中T细胞大量活化增殖。
图15：TLR7激动剂诱导后，人源化SLE小鼠血清中抗核抗体的滴度检测结果。
图16：人源化SLE小鼠肾脏B细胞的流式细胞检测结果及数据统计结果；其中，图16A为流式细胞检测数据中肾脏B细胞的门控结果；图16B为肾脏B细胞中浆细胞含量占比的统计数据对比结果。
图17：人源化SLE小鼠的生存曲线。
图18：分别给予两种单抗制剂后，所呈现的针对血清总IgG和抗双链DNA自身抗体的治疗效果；其中，图18A为溶媒组(对照G1’)、贝利尤单抗制剂(G2’)以及利妥昔单抗制剂(G3’)分别给药后检测的血清总IgG抗体水平结果；图18B为溶媒组(对照G1’)、贝利尤单抗制剂(G2’)以及利妥昔单抗制剂(G3’)分别给药后检测的抗双链DNA自身抗体水平结果。
具体实施方式
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。
同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
Pristane诱导狼疮小鼠模型：1994年Satoh M应用Pristane诱导BALB/c小鼠出现类似于人类系统性红斑狼疮的症状，首次建立了Pristane诱导的狼疮小鼠模型，该方法已成为诱导狼疮小鼠模型最常用方法。Pristane即姥鲛烷，又称2，6，10，14-四甲基十五烷、TMPD，是一种类异戊二烯烷烃类化合物，可以在植物、海洋生物和石油蒸馏的副产品中找到。同时，Pristane也是一种与磷脂双分子层相互作用的膜激活化合物。在体外和活体条件下，Pristane均能诱导小鼠淋巴细胞系和腹腔渗出液细胞程序性死亡，这提示Pristane诱导的细胞凋亡提供了充足的自身抗原底物，从而导致与干扰素α和β(IFN-α和IFN-β)的过度产生有关的免疫紊乱。
TLR7-IFN I信号通路：即TLR7信号通路激活并触发下游IFN I反应。TLR是在细胞表面发现的基因编码模式识别受体。TLR识别病原体相关分子蛋白(PAMP)，这类化合物包括TLR7激动剂或TLR8激动剂。TLR7作为细胞内体区室的一种跨膜受体，可识别病毒单链RNA，然后触发下游信号通路来诱导I型干扰素反应即IFN I反应。
表达人Flt3L的pLIVE质粒：即表达人Flt3L基因的pLIVE质粒载体。Flt3L(Flt3Ligand)是一种膜蛋白，与SCF和M-CSF有同源性，能够刺激早期造血的细胞因子。在体内和离体试验均证实，Flt3-L可调节非红系造血干/祖细胞的增殖和分化，与多种细胞因子协同，Flt3-L可促进前B淋巴细胞、树突状细胞(DC)、自然杀伤(NK)细胞、细胞毒T淋巴细胞(CTL)的增殖、分化和成熟，从而具有重要的免疫调节作用。
重度免疫缺陷鼠：即拥有不健全的先天免疫，完全缺乏有功能T、B和NK细胞的小鼠，该类小鼠可以用于人源肿瘤细胞移植(CDX)和人源肿瘤组织移植(PDX)，以及人外周血单个核细胞(PBMC)及人源造血干细胞(CD34+HSC)移植进行免疫重建。具有完整免疫系统的小鼠通过强大的先天性和适应性免疫反应迅速清除外来移植的人源细胞，只有免疫缺陷小鼠可以作为异种移植的受体。这种通过重度免疫缺陷小鼠移植人类的细胞或者组织的即为人源化小鼠。
TLR7激动剂：激动剂为既有亲和力又有内在活性的药物，它们能与受体结合并激动受体而产生效应。TLR的激活可以启动先天性和适应性免疫反应。TLR7是TLRs成员之一，负责识别病原体单链RNA，主要分布在浆树突状细胞(pDC)和B细胞的胞内，在人体识别和清除病原微生物过程中发挥重要作用。TLR7激动剂可通过刺激pDC分泌干扰素-α，并作用于其它免疫细胞(如NK细胞和巨噬细胞)发挥免疫增强的作用。同时，它还可激活pDC，提高pDC的抗原提呈能力，促进CD4+T细胞的增殖，并进一步激活CD8+T细胞杀伤肿瘤细胞，发挥抗肿瘤作用。除了用于治疗癌症，TLR7激动剂还有望开发用于治疗病毒性疾病，如HIV-1感染和乙肝等。
瑞喹莫德：即Resiquimod，简称R848，是一种免疫反应调节剂，用作有效的TLR 7/TLR8激动剂并可诱导如TNF-α、IL-6和IFN-α等细胞因子的上调，用作有效的TLR 7激动剂。
咪喹莫特：简称IMQ。咪喹莫特是一种咪唑喹啉胺类白细胞介素激动剂，属生殖疣治疗药物，用于小鼠皮肤致细胞质分裂，可产生α干扰素、肿瘤坏死因子以及多种白细胞介素，也是常见的TLR 7激动剂。
本发明所用Mn佐剂购买自启锰生物，其他试剂采购于Biolegend/Thermo Fisher以及BD Biosciences，重度免疫缺陷NCG小鼠购自江苏集萃药康生物科技有限公司。
以下为本发明实施例中采取的实验方法：
1.免疫系统人源化小鼠模型的构建方法如下：
1)对重度免疫缺陷鼠辐照清髓，辐照强度为70cGy，63s；
2)对小鼠肝脏注射人胎肝来源造血干细胞，剂量为5*104/鼠；
3)注射造血干细胞十到十二周后脸颊采血，检测外周血人免疫细胞重建情况；
4)选取同批次构建的人源化小鼠，杀鼠取脾脏骨髓，检测组织中人免疫系统重建情况。
2.TLR7激动剂瑞喹莫德的鼠耳给药过程：
1)药物剂量：实验组分为三组，每周施加TLR7激动剂瑞喹莫德的剂量分别为10μg*3次，60μg*3次和100μg*3次，即每周使用瑞喹莫德的剂量范围为30-300μg/鼠。
2)将三种剂量的瑞喹莫德分别溶于丙酮后，用移液枪缓慢滴加至鼠耳。
3.腹腔注射Flt3L蛋白促进DC的发育和成熟
方法：对小鼠腹腔注射5μg Flt3L蛋白，一周三次，持续两周。
结果：促进小鼠DC的扩增和成熟。
4.注射表达人Flt3L的质粒过程如下：
1)构建表达人Flt3L的pLIVE质粒；
2)提取质粒并配成储备液储存于冰箱-20℃；
3)配置新鲜的注射液；
根据质粒浓度和每只鼠需要的量，配成质粒溶液：
其中小鼠数量为n时，每只鼠注射剂量为10μg，质粒储存液浓度:500μg/ml
注射液组分如下表1所示：
表1
将上述成分混匀后用0.22um滤膜过滤后备用。
根据小鼠体重，配成注射液，如表2所示：
表2
3)小鼠高压尾静脉注射质粒以诱导肝脏中人Flt3L表达。
5.小鼠腹腔注射自体活化淋巴细胞来源的DNA(ALD-DNA)
1)小鼠高压尾静脉注射表达人IL-2的CMV质粒50μg，以活化及扩增免疫系统人源化小鼠体内人免疫细胞，3-5天后检测脾脏和骨髓中T细胞大量活化增殖。
2)杀鼠提取脾脏及骨髓免疫细胞；
3)提取活化淋巴细胞来源的基因组DNA；
4)小鼠腹腔注射DNA(20μg)+Mn佐剂+R848(20μg)。
具体为：DNA(20μg)溶于200μl ddH2O。
R848(60μg)溶于30μl丙酮。
Mn佐剂(200μl，购买自启锰生物)。
混匀后腹腔注射。
实验结果参见附图14。
实施例1：
1)实验目的：
本实施例通过上述方法构建免疫系统人源化小鼠模型，以验证免疫系统人源化鼠的构建效果。
2)实验操作：
本实施例对重度免疫缺陷鼠，辐照后通过注射人造血干细胞。
3)实验结果：
注射人造血干细胞8-16周后检测人免疫细胞重建水平，结果表示，小鼠外周血人免疫系统重建良好，平均可达20％以上，各主要免疫细胞亚群如B细胞、T细胞、单核细胞、树突状细胞(DC)等均得到稳定重建。进一步深入分析B细胞在脾脏和骨髓中的重建情况示，人源B细胞重建尚可，各个分化阶段及状态的B细胞如初始B细胞、过渡期B细胞、浆细胞样B细胞以及记忆B细胞均得到重建。
实施例2
1)实验目的：
不同剂量TLR7激动剂瑞喹莫德对小鼠系统性红斑狼疮表型指征的影响。
2)实验操作：
本实施例采用TLR7激动剂瑞喹莫德，对通过上述操作获得的人源化小鼠进行鼠耳给药。
对照组：不施加TLR7激动剂瑞喹莫德，只给溶剂丙酮
给药组：30μg组，180μg和300μg组
三组通过鼠耳给药，每周施加TLR7激动剂瑞喹莫德的剂量分别为10μg*3次，60μg*3次和100μg*3次，即每周使用瑞喹莫德的剂量为30-300μg/鼠。
3)实验结果，如表3所示：
表3

结果表明，TLR7激动剂组鼠耳给药两周后，小鼠出现部分系统性红斑狼疮样表型，同时外周血人源化水平相对较为稳定，且在处理8周之后出现了类似系统性红斑狼疮患者的T淋巴细胞异常，包括T细胞活化，辅助性T细胞(Th)亚群失调，滤泡状辅助性T细胞(Tfh)增加等。诱导组血浆IL-6、IFN-γ、IL-17A、IL-22炎症因子及总免疫球蛋白G(IgG)有较明显的升高。诱导组肾脏HE染色示肾小球有内皮和系膜细胞的增生，免疫荧光染色示肾小球存在大量人IgG、IgM沉积，是狼疮肾炎的典型表现。
实施例3
1)实验目的：
本实施例通过补充人Flt3L，促进树突状细胞的发育以诱导更明显的系统性红斑狼疮表型和B细胞应激反应。
2)实验操作：
本实施例通过上述方法构建免疫系统人源化小鼠模型，对该人源化小鼠注射表达人Flt3L的质粒，之后采用TLR7激动剂瑞喹莫德，进行鼠耳给药。
对照组：不施加TLR7激动剂瑞喹莫德
Flt3L组：只注入表达人Flt3L的质粒
TLR7组：只施加TLR7激动剂60μg*3次
Flt3L+TLR7组：先注入表达人Flt3L的质粒，再施加TLR7激动剂组60μg*3次
3)实验结果：结果如下表4所示：
表4
Flt3L能够扩增人常规树突状细胞(cDC)及浆细胞样树突状细胞(pDC)，并诱导T细胞，特别是CD4+T细胞的上调，可以更好地帮助B细胞获得抗原及活化增殖等。在8周药物诱导后，模型小鼠的表型更为明显，出现了脾脏肿大及与系统性红斑狼疮患者类似的面部红斑。诱导组的尿蛋白水平也随着时间逐渐上升，并在终点时与对照组有显著性差异。各组检测结果如表4所示，其中，不施加TLR7激动剂瑞喹莫德的对照组各项结果均显示正常，注入表达人Flt3L质粒的Flt3L组也未见明显变化(表明单独施用Flt3L质粒不会产生明显诱导作用)；但只施加TLR7激动剂的TLR7组已经出现了脾脏肿大、尿蛋白升高以及类似于系统性红斑狼疮的面部红斑等表征，证明加入TLR7激动剂已经一定程度上活化了所构建模型体内的TLR7-IFN I信号通路，进而产生了类似红斑狼疮的症状；为进一步证明二者的关联性，所设置的先注入Flt3L质粒再施加TLR7激动剂的Flt3L+TLR7联合组，其施药后的小鼠模型，已经出现了明显的症状，即脾脏肿大加剧、尿蛋白大幅升高以及面部呈现明显的掉毛和发红等，此联合用药组的结果，充分表明：补充人Flt3L(并通过激动剂TLR7充分活化)，相较于不补充人Flt3L或只单独施加激动剂，确实能够促进树突状细胞的发育，进而诱导更明显的系统性红斑狼疮表型和B细胞应激反应。
实施例4
1)实验目的：
本实施例对两种TLR7激动剂(瑞喹莫德和咪喹莫特)的鼠耳给药效果进行了比较。
2)实验操作：
通过上述方法构建免疫系统人源化小鼠模型，对该人源化小鼠采用两种TLR7激动剂瑞喹莫德和咪喹莫特，分别进行鼠耳给药。两种激动剂的使用剂量参见下表5所示：
表5
具体操作为，将5％的咪喹莫特软膏挤入1ml胰岛素针中，每次挤出一定体积的药膏涂抹鼠耳，方便定量。瑞喹莫德溶于丙酮后用100μl移液枪缓慢滴加至鼠耳。
对照组：不施加TLR7激动剂，只滴加溶剂丙酮组(Ctrl组)
给药组：咪喹莫特软膏组(IMQ组)，瑞喹莫德组(R848组)
3)实验结果：
结果表明，TLR7激动剂诱导后，小鼠的T淋巴细胞活化程度增加(附图1)。同时，低剂量TLR7激动剂诱导出现辅助性T细胞亚群Th17及Tfh细胞比例失调(附图2)，小鼠血浆炎症因子的比例发生变化，血浆IL-6、IL-17、IFN-γ及IL-22炎症因子上调(附图3)，肾小球内皮、系膜细胞增生明显(附图4)，出现狼疮肾炎表现，肾脏免疫复合物(IgG+IgM)出现沉积情况(附图5和6)，血浆总IgG升高(附图7)。且诱导结果如图15、图16所示，从图15可以看出，人源化SLE小鼠血清呈现出较高滴度的抗核抗体，从图16的流式细胞检测结果及统计数据可以看出，人源化SLE小鼠肾脏中出现了大量的浆细胞浸润。
实施例5
1)实验目的：
本实施例在对上述人源化小鼠注射表达人Flt3L质粒的基础上，对瑞喹莫德和咪喹莫特两种TLR7激动剂的鼠耳给药效果进行了比较。
2)实验操作：
通过上述方法构建免疫系统人源化小鼠模型，对该人源化小鼠注射表达人Flt3L的质粒，之后采用两种TLR7激动剂瑞喹莫德和咪喹莫特，分别进行鼠耳给药。
两种激动剂的施用剂量参见下表6所示：
表6
具体操作为，5％的咪喹莫特软膏每次挤出一定体积的药膏涂抹鼠耳，方便定量。瑞喹莫德溶于丙酮后用100μl移液枪缓慢滴加至鼠耳。
对照组：注射表达人Flt3L的质粒，不施加TLR7激动剂瑞喹莫德(Ctrl组)
给药组：咪喹莫特软膏组(IMQ组)，瑞喹莫德组(R848组)
3)实验结果：
结果表明，给药组小鼠出现面部红斑表现(附图8)，呈现与红斑狼疮患者类似的面部红斑。同时，给药组小鼠尿蛋白含量上升，脾脏肿大导致重量增加(附图9和10)。
咪喹莫特和瑞喹莫德给药后，小鼠T细胞活化水平和亚群变化情况相似。
实施例6
1)实验目的：
通过上述方法构建免疫系统人源化小鼠模型，对该人源化小鼠腹腔输注活化淋巴细胞来源的DNA(ALD-DNA)，诱导人源化系统性红斑狼疮小鼠体内T细胞及B细胞亚群失调及自身抗体的产生过程。
2)实验操作：
给小鼠腹腔输注活化淋巴细胞来源的DNA(ALD-DNA)，具体操作如上所述。
3)实验结果：
在Flt3L和TLR7激动剂的基础上，增加自身活化淋巴细胞来源的DNA免疫小鼠后，脾脏和淋巴结中出现大量滤泡辅助性T细胞(附图12)，抗体分泌的浆母细胞显著增加，也诱导出了较高比例的发生类别转换的IgG+B细胞(附图11)。血浆中检测到较低滴度的抗核抗体，抗dsDNA IgM型自身抗体较单纯TLR7激动剂诱导有1.5倍左右的上升(附图13)。
作为系统性红斑狼疮病人血清学特征的抗双链DNA抗体，已经被证明是致病性的，可以引起免疫复合物沉积和组织损伤，与系统性红斑狼疮疾病的严重程度密切相关。系统性红斑狼疮病人遗传学的研究发现抗dsDNA自身抗体大多属于对dsDNA具有高亲和力的IgG亚类，不同于体细胞突变产生的抗体。研究显示自身DNA可以诱导抗dsDNA抗体产生。一般情况下，哺乳动物的DNA免疫原性较弱，不会引起免疫应答。寻找引起自身免疫反应和抗dsDNA抗体产生的驱动成份是免疫学家关注的热点。
本发明在寻找系统性红斑狼疮驱动原的过程中发现用活化淋巴细胞来源的DNA
(ALD-DNA)免疫同系的雌性小鼠，可以产生一系列的系统性红斑狼疮症状，包括高水平的抗dsDNA自身抗体、蛋白尿、免疫复合物沉积和肾小球肾炎，这些症状模拟病人体内由大量凋亡细胞来源的自身DNA引起的系统性红斑狼疮症状，因此ALD-DNA免疫的小鼠可以被作为理想的小鼠狼疮模型进行探讨系统性红斑狼疮疾病可能的细胞与分子免疫学机制。
应用例1：
1)实验目的：
本对比例应用该小鼠探究抗衰老药(Senolytics)对红斑狼疮的治疗作用。
2)实验操作：
通过上述方法构建人源化系统性红斑狼疮小鼠，联合使用两种抗衰老药：ABT263(BCL2抑制剂)和S63845(MCL-1抑制剂)，实验分组如下：
ABT263组：对小鼠给药剂量为10mg/kg，一周两次，给药方式为灌胃给药；
S63845组：对小鼠给药剂量为25mg/kg，一周两次，给药方式为腹腔注射
ABT263+S63845组：对小鼠给药剂量为ABT263 10mg/kg，一周两次，给药方式为灌胃给药；S63845 25mg/kg，一周两次，给药方式为腹腔注射。
3)实验结果，如表7所示：
表7
本发明通过上述方法能够在人源化小鼠体内诱导出狼疮表型，而给予抗衰老药能够治疗狼疮。因此该人源化系统性红斑狼疮小鼠能够进行药物疗效评估。
本发明通过在免疫系统人源化小鼠中活化TLR7-IFN I信号通路这一关键机制，诱导出了系统性红斑狼疮患者类似的T细胞异常、炎症因子上调和狼疮肾炎表现。并通过向小鼠注射表达人Flt3L的质粒及自体活化淋巴细胞来源的DNA，促进了小鼠B细胞的体液免疫反应，诱导其B细胞亚群失调和自身抗体产生，从而构建该人源化系统性红斑狼疮小鼠。
与现有的注射Pristane诱导人源化系统性红斑狼疮模型相比，其诱导周期显著缩短，表型更明显，TLR7-IFN I信号通路的活化机制也相对更明确。
应用例3：
人源化SLE小鼠应用于生物制剂的疗效评估：
同前所述，在第三代人源化小鼠中通过TLR7激动剂鼠耳抹药诱导SLE表型。诱导4周时，将诱导组根据发病情况平均分为3组，一组未给予任何治疗(G1’)，一组给予贝利尤单抗治疗(G2’)，另一组给予利妥昔单抗治疗(G3’)，分组及给药策略如表8所示。期间监测小鼠生存率、发病表型等，治疗4周时到达终点，进行免疫学和病理学相关指标的评估。
图17显示了上述三组给药后的人源化SLE小鼠的生存曲线。
图18显示了分别给予两种单抗制剂后，所呈现的针对血清总IgG和抗双链DNA自身抗体的治疗效果。
结果表明，在人源化SLE小鼠中，利妥昔单抗和贝利尤单抗治疗均能降低自身抗体水平(图18，贝利尤单抗和利妥昔单抗两种生物制剂治疗后，能够降低血清总IgG和抗双链DNA自身抗体)，提高生存率(图17)，贝利尤单抗的效果略好于利妥昔单抗。这一结论很好地复现了临床结果，证明了本专利中构建的人源化SLE模型在临床前药筛等方面具有极大的应用前景。
表8
除以上所记载的应用例2和应用例3之外，本专利所构建的人源化SLE小鼠模型的应用范围包括但不限于：各种细胞疗法、生物靶向制剂、小分子药物以及复方药物(如中药等)的药物评价与药理机制探究。以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. 一种小鼠体内细胞，其特征在于，所述小鼠为干扰素信号通路活化后的小鼠，其体内细胞包括了小鼠自身细胞和/或小鼠体内的人源免疫细胞。
  2. 根据权利要求1所述的小鼠体内细胞，其特征在于，所述活化的干扰素信号通路为TLR7-IFN I信号通路。
  3. 根据权利要求1所述的小鼠体内细胞，其特征在于，通过施用TLR7激动剂活化该TLR7-IFN I信号通路。
  4. 根据权利要求1所述的小鼠体内细胞，其特征在于，所述TLR7激动剂是咪喹莫特或瑞喹莫德中的一种或两种联合使用；
    优选地，所述瑞喹莫德施用的剂量范围为30-300μg/周/鼠；
    优选地，所述瑞喹莫德每次施用的剂量为10-100μg/鼠；
    优选地，所述瑞喹莫德的施用频率为每周两次或每周三次。
  5. 一种人源化系统性红斑狼疮小鼠，其特征在于，该小鼠具有如权利要求1至4中任一项所述的小鼠体内细胞。
  6. 根据权利要求5所述的人源化系统性红斑狼疮小鼠，其特征在于，该小鼠呈现T细胞水平异常、炎症因子水平异常、狼疮肾炎表型、B细胞亚群失调和自身抗体产生；
    优选地，该小鼠的使用周期为8-30周，更优选为8-16周。
  7. 一种人源化系统性红斑狼疮小鼠的构建方法，其特征在于，所述方法为对含有权利要求1至4任一项所述的小鼠体内细胞的小鼠进行模型构建或构建权利要求5至6中任一项所述的人源化系统性红斑狼疮小鼠模型；
    具体地，所述构建方法包括：A、构建人源化小鼠；B、将所述的人源化小鼠构建为人源化系统性红斑狼疮小鼠模型；
    优选地，该步骤B包括B’、向所构建的人源化小鼠注射表达人Flt3L质粒或Flt3L蛋白，或转基因表达Flt3L蛋白过程；
    优选地，该步骤B包括B”、向小鼠注射自体活化淋巴细胞来源DNA的过程；
    若该方法中同时含有步骤B’和步骤B”，步骤B’位于步骤B”前。
  8. 根据权利要求7所述的构建方法，其特征在于，所述步骤B’中的注射表达人Flt3L质粒的过程包括：
    1)构建表达人Flt3L的pLIVE质粒；
    2)提取质粒并配成储备液；
    3)配置新鲜的注射液；
    4)向小鼠注射该注射液；
    所述步骤B”中的自体活化淋巴细胞来源的DNA按照以下方法制备：
    1)向小鼠注射表达人IL-2的质粒，以活化及扩增免疫系统人源化小鼠体内人免疫细胞；
    2)提取步骤1)中小鼠的脾脏及骨髓免疫细胞；
    3)提取活化淋巴细胞来源的基因组DNA；
    4)向小鼠注射该DNA。
  9. 根据权利要求7所述的构建方法，其特征在于，所述步骤A中构建人源化小鼠的方法包括：
    1)对免疫缺陷小鼠进行辐照清髓；
    2)将造血干细胞注入小鼠，剂量为(0.5-10)×104个细胞/鼠。
  10. 一种权利要求1至4任一项所述的小鼠体内细胞在构建人源化系统性红斑狼疮小鼠或在医学研究/医药制备/商业销售中的应用，或
    权利要求5-6中任一项所述的小鼠，或
    权利要求7-9中任一项所述的构建方法得到的小鼠在医学研究/医药制备/商业销售中的应用。
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