Mouse PDX cell and construction method of humanized systemic lupus erythematosus mouse model

Abstract

The invention provides a mouse PDX cell and a construction method of a humanized systemic lupus erythematosus (SLE) mouse model, and belongs to the technical field of animal model construction. The invention provides a mouse PDX cell L0407-6-P3 which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2024181. After the mouse PDX cell L0407-6-P3 is intravenously injected into an NSG mouse, the mouse disease is stable, the human SLE disease phenotype can be generated, the multi-system and multi-organ inflammatory response and injury of human SLE can be better simulated, the mouse PDX cell L0407-6-P3 can be used for constructing a humanized systemic lupus erythematosus mouse model, the blank of the current humanized SLE animal model is filled, and the mouse PDX cell L0407-6-P3 has better popularization and application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and in particular relates to a method for constructing a mouse PDX cell and a humanized systemic lupus erythematosus mouse model. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by inflammation and immune-mediated damage to multiple organ systems, including the skin, mucosa, musculoskeletal system, blood, renal system, and nervous system.

[0003] The ideal mouse disease model is the main tool for studying the pathogenesis of SLE and drug development. At present, SLE mouse models mainly include three types: gene knockout spontaneous model, induced model and humanized model. Compared with gene knockout spontaneous model and induced model, humanized disease model is closer to the pathogenesis of human SLE and can more comprehensively reproduce the complexity of human SLE. It is the most ideal tool for studying the pathogenesis of SLE and drug development. However, the current humanized model has the defect of not being able to stably develop the disease or not being able to fully reflect the pathogenesis of SLE with systemic organ involvement. Summary of the invention

[0004] The purpose of the present invention is to provide a mouse PDX cell line and a method for constructing a humanized systemic lupus erythematosus mouse model. The humanized systemic lupus erythematosus mouse model of the present invention can stably develop the disease and can fully reflect the pathogenesis characteristics of systemic organ involvement of SLE.

[0005] The invention provides a mouse PDX cell strain L0407-6-P3, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024181.

[0006] The present invention also provides the use of the mouse PDX cell L0407-6-P3 described in the above scheme in constructing a humanized systemic lupus erythematosus animal model.

[0007] The present invention also provides a method for constructing a humanized systemic lupus erythematosus mouse model, comprising the following steps:

[0008] The peripheral blood mononuclear cells of patients with active systemic lupus erythematosus were intravenously injected into P1 generation NSG mice, and the spleens of the P1 generation NSG mice were taken out 1 to 2 months later to prepare a single cell suspension; the single cell suspension was intravenously injected into P2 generation NSG mice, and a humanized systemic lupus erythematosus mouse model was obtained 3 to 4 weeks later;

[0009] Alternatively, peripheral blood mononuclear cells from patients with active systemic lupus erythematosus are intravenously injected into P1 generation NSG mice; mouse PDX cells L0407-6-P3 described in the above protocol are intravenously injected into P2 generation NSG mice, and a humanized systemic lupus erythematosus mouse model is obtained after 3 to 4 weeks;

[0010] Alternatively, the mouse PDX cells L0407-6-P3 described in the above scheme were intravenously injected into NSG mice, and a humanized systemic lupus erythematosus mouse model was obtained after 3 to 4 weeks.

[0011] Preferably, the number of cells injected intravenously in the single cell suspension is 1×10 6 / P2 generation NSG mice.

[0012] Preferably, the spleen of the P1 mouse contains mouse PDX cells.

[0013] Preferably, the marker of mouse PDX cells is hCD45 + hCD3 + hCD8 + .

[0014] Preferably, the number of mouse PDX cells L0407-6-P3 injected intravenously is 1×10 6 / P2 generation NSG mice.

[0015] Preferably, the intravenous injection comprises tail vein injection.

[0016] The present invention also provides the use of a humanized systemic lupus erythematosus mouse model constructed by the construction method described in the above scheme in studying the pathogenesis of systemic lupus erythematosus disease progression.

[0017] The present invention also provides the use of a humanized systemic lupus erythematosus mouse model constructed by the construction method described in the above scheme in the development of drugs for systemic lupus erythematosus.

[0018] The present invention provides a mouse PDX cell L0407-6-P3, which is deposited in the China Center for Type Culture Collection and has a deposit number of CCTCC NO: C2024181. After the mouse PDX cell L0407-6-P3 of the present invention is intravenously injected into NSG mice, the mice develop stable disease, can produce human SLE disease phenotypes, and can better simulate the multi-system and multi-organ inflammatory response and damage of human SLE, can be used for the construction of a humanized systemic lupus erythematosus mouse model, fill the gap in the current humanized SLE animal model, and have good promotion and application value.

[0019] Biological Deposit Description

[0020] Mouse PDX cells L0407-6-P3 were deposited in the China Center for Type Culture Collection on June 12, 2024. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is: CCTCC NO: C2024181. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 The spleen of the P2 generation of diseased mice of the humanized SLE mouse model constructed in the present invention and its pathological staining results; wherein, A. Gross photo of spleen; B. HE staining result of spleen;

[0023] Figure 2 The liver of the P2 generation of diseased mice in the humanized SLE mouse model constructed in the present invention and its pathological staining results; wherein, A. a gross photo of the liver; B. HE staining results of the liver;

[0024] Figure 3 The diagrams show the kidney damage and urine evaluation results of the P2 generation mice of the humanized SLE mouse model constructed in the present invention; wherein, A. gross kidney photos; B-D. HE staining results of the kidneys of the SLE-P2 generation mice; E. urine protein detection results of the SLE-P2 generation mice;

[0025] Figure 4 The flow cytometry test results of human inflammatory cells in the peripheral blood of P1-generation diseased mice of the humanized SLE mouse model constructed in the present invention; wherein A. flow cytometry display and isotype negative control comparison results; B. further typing results of immune cells in A;

[0026] Figure 5 The immunohistochemistry (IHC) detection results of human inflammatory cells in the P1 generation of diseased mice of the humanized SLE mouse model constructed by the present invention; wherein, A. The results of staining with the human-specific antibody hCD45 in the spleen and liver of the P1 generation of diseased mice of the SLE mouse model; B. The results of staining with the human-specific antibody hCD3 for T cells in the kidneys of the P1 generation of diseased mice of the SLE mouse model;

[0027] Figure 6 The immunofluorescence detection results of human IgG deposition in the kidneys of P1 generation diseased mice of the humanized SLE mouse model constructed in the present invention;

[0028] Figure 7This is a graph showing the results of blood autoantibody detection in the P1 generation of diseased mice of the humanized SLE mouse model constructed in the present invention; wherein, A. anti-dsDNA; B. anti-Smith (SM) antibody;

[0029] Figure 8 This is a diagram showing the results of TUNEL staining of the spleen of P2-generation diseased mice in the humanized SLE mouse model constructed in the present invention;

[0030] Fig. 9 The humanized SLE mouse model constructed in the present invention is the spleen autoreactive T cells (hCD45 + hCD3 + ) flow cytometry results. DETAILED DESCRIPTION

[0031] The invention provides a mouse PDX cell strain L0407-6-P3, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024181.

[0032] The mouse PDX cell L0407-6-P3 of the present invention can produce human SLE disease phenotype after being injected into NSG mice, and is a seed cell for constructing a humanized systemic lupus erythematosus animal model.

[0033] In the present invention, the mouse PDX cells L0407-6-P3 are derived from the spleen of NSG mice.

[0034] In the present invention, the NSG mouse is a mouse lacking T, B and NK lymphocytes. In the present invention, the NSG mouse is purchased from Jicui Yaokang Biotechnology Co., Ltd. In the present invention, the NSG mouse is preferably an 8-week-old female NSG mouse.

[0035] The present invention also provides the use of the mouse PDX cell L0407-6-P3 described in the above scheme in constructing a humanized systemic lupus erythematosus animal model.

[0036] In the present invention, the animal model includes a mouse model.

[0037] The present invention also provides a method for constructing a humanized systemic lupus erythematosus mouse model, comprising the following steps: intravenously injecting peripheral blood mononuclear cells of systemic lupus erythematosus patients in the active stage of the disease into P1 (Passage 1) generation NSG mice, and taking the spleen of the P1 generation NSG mice 1 to 2 months later to prepare a single cell suspension; intravenously injecting the single cell suspension into P2 generation NSG mice, and obtaining a humanized systemic lupus erythematosus mouse model 3 to 4 weeks later; or, intravenously injecting peripheral blood mononuclear cells of systemic lupus erythematosus patients in the active stage of the disease into P1 generation NSG mice; intravenously injecting the mouse PDX cells L0407-6-P3 described in the above scheme into P2 generation NSG mice, and obtaining a humanized systemic lupus erythematosus mouse model 3 to 4 weeks later; or, intravenously injecting the mouse PDX cells L0407-6-P3 described in the above scheme into NSG mice, and obtaining a humanized systemic lupus erythematosus mouse model 3 to 4 weeks later.

[0038] The present invention first intravenously injects peripheral blood mononuclear cells of systemic lupus erythematosus patients in the active stage of the disease into P1 generation NSG mice, and takes the spleen of the P1 generation NSG mice 1 to 2 months later to prepare a single cell suspension. After obtaining the single cell suspension, the present invention intravenously injects the single cell suspension into P2 generation NSG mice, and obtains a humanized systemic lupus erythematosus mouse model 3 to 4 weeks later.

[0039] In the present invention, the active stage of the disease refers to a disease activity score (SLEDAI score) of ≥ 8 under regular treatment with hormones and other immunosuppressants. At the same time, the patient is diagnosed with SLE without other chronic underlying diseases, including diabetes, hypertension, hyperlipidemia, etc.

[0040] In the present invention, the mononuclear cell collection method preferably includes: collecting peripheral blood from patients with systemic lupus erythematosus in the active stage of the disease and separating the blood with Ficoll to obtain a mononuclear cell suspension.

[0041] After intravenous injection of P1 generation NSG mice, the present invention preferably further comprises collecting blood at 2, 4, 8, 12, and 16 weeks to detect hCD45 by flow cytometry. + The proportion is used to initially determine whether the model is successfully constructed.

[0042] After the single cell suspension is intravenously injected into P2 generation NSG mice or after the mouse PDX cells L0407-6-P3 described in the above scheme are intravenously injected into P2 generation NSG mice, the present invention preferably further comprises collecting blood at 2 and 4 weeks to detect hCD45 by flow cytometry + The proportion is used to initially determine whether the model is successfully constructed.

[0043] In one embodiment of the present invention, the active activated immune cells from SLE patients were used for intravenous injection into immunodeficient NSG mice to construct a SLE disease model, and human autoimmune T cells (hCD45 + hCD3 + hCD8 + The T cells were injected again into immunodeficient NSG mice to obtain the second generation of mice (Passage 2, P2), that is, mice with stable disease of SLE. P2 mice showed that human autoimmune T cells (hCD45 + hCD3 + hCD8 + ), and showed typical pathological changes of SLE patients, including glomerular damage, proteinuria, liver / spleen enlargement accompanied by inflammatory cell infiltration, etc. It is currently the best animal model to simulate the multi-system and multi-organ involvement of human SLE.

[0044] In another embodiment of the present invention, the present invention first intravenously injects peripheral blood mononuclear cells of patients with active systemic lupus erythematosus into P1 generation NSG mice; then intravenously injects the mouse PDX cells L0407-6-P3 described in the above scheme into P2 generation NSG mice, and obtains a humanized systemic lupus erythematosus mouse model after 3 to 4 weeks;

[0045] In another embodiment of the present invention, the mouse PDX cells L0407-6-P3 described in the above scheme were intravenously injected into NSG mice, and a humanized systemic lupus erythematosus mouse model was obtained after 3 to 4 weeks.

[0046] In the present invention, the number of cells injected intravenously in the single cell suspension is preferably 1×10 6 / P2 generation NSG mouse; the intravenous injection volume of the single cell suspension is preferably 200 μl / P2 generation NSG mouse; the number of cells of the mouse PDX cell L0407-6-P3 intravenous injection is preferably 1×10 6 / P2 generation NSG mouse; the intravenous injection volume of the mouse PDX cells L0407-6-P3 is preferably 200 μl / P2 generation NSG mouse.

[0047] In the present invention, the spleen of the P1 generation mouse contains human autoreactive T cells (human autoimmune T cells, hCD45 + hCD3 + hCD8 + ), which is the core cell line for constructing stable SLE mice. In the present invention, the marker of mouse PDX cells is hCD45 + hCD3+ hCD8 + .

[0048] In the present invention, the mouse PDX cells L0407-6-P3 are isolated from the human autoreactive T cells described in the above scheme.

[0049] In the present invention, human autoreactive T cells still survive and proliferate in the P2 generation NSG mice, and have typical characteristics of active SLE patients, as shown below: decreased peripheral blood cell count and positive antibodies; enlarged spleen with inflammatory cell infiltration and significantly reduced apoptotic cells; liver damage with lymphocyte infiltration; kidney damage (glomerular damage with proteinuria), kidney IgG deposition, etc.

[0050] In the present invention, the intravenous injection preferably includes tail vein injection.

[0051] The present invention also provides the use of a humanized systemic lupus erythematosus mouse model constructed by the construction method described in the above scheme in studying the pathogenesis of systemic lupus erythematosus disease progression.

[0052] The present invention also provides the use of a humanized systemic lupus erythematosus mouse model constructed by the construction method described in the above scheme in the development of drugs for systemic lupus erythematosus.

[0053] The humanized systemic lupus erythematosus mouse model provided by the present invention is an animal model of multi-system and multi-organ involvement, which better simulates the multi-system and multi-organ inflammatory response produced by abnormally activated autoimmune cells (T cells) of patients, and is currently the animal model of multi-system and multi-organ damage closest to human SLE. This model can be used to explore the pathogenesis of SLE and drug development, and accelerate the advancement of basic scientific research.

[0054] To further illustrate the present invention, a mouse PDX cell strain and a method for constructing a humanized systemic lupus erythematosus mouse model provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1: Preparation of peripheral blood mononuclear cell suspension from active SLE patients:

[0056] a) Screening of active SLE patients: Information on SLE patients was collected in the outpatient clinic and wards of the hospital, the medical history of SLE patients was tracked in detail, and the laboratory test data of patients were collected, such as routine blood test, routine urine test and urine sediment, anti-dsDNA antibodies, anti-SM antibodies, complement C3 and C4, etc. At the same time, SLEDAI score was calculated based on the clinical symptoms of patients. The inclusion criteria for SLE patients included three items: 1) meeting the SLE diagnostic criteria established by the American College of Rheumatology; 2) being in the active stage of the disease, with a SLEDAI score ≥ 8 points; 3) not having any other autoimmune diseases.

[0057] b) Collect 5-10 ml of peripheral blood from the patient, add an equal volume of 1× PBS buffer and dilute it 1 / 2 times. Use a pipette to slowly transfer the diluted peripheral blood along the tube wall to the upper layer of the Ficoll separation solution (lymphocyte separation solution).

[0058] c) Centrifuge at 2,000 rpm for 30 min in a low-temperature horizontal centrifuge, and then aspirate the white cloudy cell layer at the interface between the upper and middle liquid layers, i.e., mononuclear cells. Wash twice with saline at low speed, and then resuspend in 0.5 mL of saline or 1× PBS buffer, and count the cells.

[0059] Example 2: NSG mouse breeding, inoculation and modeling success evaluation indicators

[0060] NSG mouse breeding and inoculation:

[0061] a) Mouse breeding conditions: 8-week-old female NSG mice were raised under SPF conditions. The room temperature was 18-25°C, the relative humidity was 40%-60%, and the feed and drinking water were sterilized by high pressure. The bedding was changed at least twice a week.

[0062] b) NSG mice inoculation: 1×10 L0407-6-P3 PDX cells were injected into the tail vein. 6 Before injection, the cells were filtered with a 0.2 μm filter membrane and resuspended in PBS. The injection volume was 200 μL.

[0063] Modeling success criteria:

[0064] a) General indicators: After inoculation, observe whether the mice have decreased activity, weight loss, hunched back, diarrhea, hair loss, hemiplegia, etc. Weigh the mice and record the weight every week.

[0065] b) Blood collection from tail vein: 300-500 μL of blood was collected from the tail vein of mice every 2 weeks after injection, lysed with red blood cell lysis buffer for 20 minutes, washed once with PBS, and resuspended in 500 μL PBS to make a nucleated cell suspension. Labeled with mCD45, hCD45, or combined with hCD3, hCD33, hCD19 and other antibody combinations, flow cytometry detection of cell ratio to determine whether the transplantation was successful and the type of implanted human immune cells (T lymphocytes, B lymphocytes, myeloid leukocytes).

[0066] c) Examination of various tissues and organs of diseased mice: One month after modeling, mice with significant weight loss were immediately killed or dissected immediately after death, and peripheral blood, spleen, liver, and kidneys of the mice were collected, and the organ sizes were recorded by taking photos. Peripheral blood was subjected to complete blood cell count (lymphocyte counts in SLE patients were reduced) and autoantibody detection (anti-dsDNA or anti-SMITH positive). Peripheral blood, spleen, and bone marrow cell suspensions were labeled with mCD45, hCD45, or combined with hCD3, hCD33, hCD19 and other antibody combinations, and immunophenotypes were analyzed by flow cytometry to clarify the proportion of implanted human cells. Spleen, liver, and kidney tissues were routinely fixed, paraffin-embedded, and sectioned, and then stained with hematoxylin-eosin staining (HE staining) to observe inflammatory cell infiltration and tissue cell destruction. In addition, tissue sections were stained with IHC to clarify the infiltration of human cells in mice, and TUNEL staining was performed to determine whether the proportion of apoptotic cells was reduced.

[0067] d) Collecting urine from mice for urine protein detection.

[0068] Example 3: Validation experiment of multi-system and multi-organ damage in the humanized SLE mouse model constructed in Example 2

[0069] 1) Preparation of paraffin sections of spleen, liver and kidney tissues of humanized SLE mice

[0070] a) Humanized experimental mice were euthanized, and the spleen, liver, and left and right kidneys were completely separated. After rinsing with pre-cooled saline, the organs were quickly placed in 4% paraformaldehyde and fixed at room temperature for 24 to 72 hours;

[0071] b) After washing, the specimens were immersed in 50% alcohol → 75% alcohol → 95% alcohol (I) → 95% alcohol (II) → anhydrous alcohol (I) → anhydrous alcohol (II) for 2 hours in each concentration of alcohol;

[0072] c) Place three wax cups filled with 58°C paraffin in a wax melting box to melt, and keep the temperature of the wax melting box constant (about 58°C), then place the transparent specimens in wax cup (Ⅰ) → wax cup (II) → wax cup (Ⅲ), and soak them in sequence for 1 hour; add melted paraffin to the holes of a fixed plate with a size of 1×1×1cm per hole, and then put the wax-soaked material in the third cup into it, with the cut surface facing down and the position correct, then quickly immerse half of the fixed plate in cold water, and after the surface of the paraffin solidifies, immerse the entire fixed plate in water to cool. After the paraffin is completely solidified, take out the wax block, fix the wax block on the container of the microtome base, and install it on the microtome fixture to wait for slicing;

[0073] d) Cut the wax block into 5 μm thick slices on a rotary microtome. Use a brush to gently remove the slices and wax tape and place them in a wax tape box for later use;

[0074] e) Cut the wax strip into wax slices of appropriate length as required, use tweezers to gently place the wax slices on the water surface of the slide, and then place the slide on the slide table (temperature is about 45°C), the wax slices will slowly flatten out after being heated;

[0075] f) After the wax slide is completely flattened, use a dissecting needle to adjust the slice position and pour off the remaining water on the slide;

[0076] Place it in a baking box and bake it in a 50℃ constant temperature oven to dry.

[0077] 2) HE staining of humanized experimental mouse organs

[0078] a) Select humanized experimental mouse organ (spleen or kidney) tissue paraffin sections with a thickness of 5 μm and bake at 60°C for 1 h;

[0079] b) xylene I for 20 min, xylene II for 20 min, 95% ethanol I for 5 min, 95% ethanol II for 5 min, 75% ethanol I for 5 min, 75% ethanol II for 5 min, and ddH2O for 5 min;

[0080] c) Hematoxylin for 3 min, differentiate for 8 s, and then add 0.5% eosin for counterstaining for 2 min;

[0081] d) Gradient dehydration, 95% ethanol III for 1 min, 95% ethanol IV for 1 min, anhydrous ethanol I for 1 min, anhydrous ethanol II for 1 min, xylene I for 1 min, xylene II for 1 min;

[0082] e) Seal the slide with colorless nail polish and observe and take photos under a microscope;

[0083] 3) IHC detection of autoreactive T cells in the kidneys of humanized SLE mice;

[0084] a) Select paraffin sections of mouse kidney tissue, bake at 70°C for 2 h, and then hydrate in a gradient manner;

[0085] b) Place in sodium citrate buffer for antigen retrieval, microwave the sections at high temperature for 2 min, and cool to room temperature;

[0086] c) Prepare BSA (2.5%) blocking solution and incubate with the slices at 4°C for 30 minutes. Add primary anti-human CD3 antibody (Abcam, Ab5690) at a dilution ratio of 1:75 and incubate at 4°C overnight.

[0087] d) Add secondary antibody (Abcam, ab205722) at a dilution ratio of 1:500 and incubate at room temperature for 1 h;

[0088] e) Add DAB (3,3′-Diaminobenzidine) colorimetric solution and incubate for 3.5 min;

[0089] f) staining in hematoxylin solution for 45 seconds;

[0090] g) Seal the slide with colorless nail polish and observe and take photos under a microscope.

[0091] 4) Detection of organ apoptosis in humanized SLE mice

[0092] a) TUNEL staining relies on terminal deoxynucleotidyl transferase (TdT), an enzyme that ligates deoxynucleotides to the 3'-hydroxyl ends of DNA breaks. In TUNEL staining, the TdT-ligated nucleotides are directly labeled with a fluorescent marker or a chemical label that can be indirectly linked to a fluorescent marker or enzyme;

[0093] b) Humanized SLE mouse spleen sections were fixed with formaldehyde for 15 min on ice and washed three times with PBS buffer;

[0094] c) add ice-cold 70% ethanol and incubate for 30 min;

[0095] d) Incubate in TUNEL staining solution (Abcam, TUNEL detection kit-FITC (ab66108) at 37°C for 1 h, then add buffer for washing;

[0096] e) Incubate in propidium iodide / RNase A solution at room temperature for 30 min;

[0097] f) Add the prepared DAPI working solution (Vector Laboratories) dropwise and incubate at room temperature in the dark for 1 hour.

[0098] g) Observe the green fluorescence under a fluorescence microscope and take photos.

[0099] 5) Urine protein determination in humanized SLE mice

[0100] a) Use the Urit 1VP urine protein test strips (model: URIT 1VP);

[0101] b) Take a humanized SLE mouse and gently massage the bladder along the urethra and collect at least 50 μL of mouse urine using a sterile Ep tube;

[0102] c) Hold the end of the test strip, immerse the test strip into the urine sample for 1 to 2 seconds, take it out, wipe off the excess urine on the side on a paper towel, compare the color comparison time indicated on the label with the color standard, and find the color standard with the closest color according to the depth of the color reaction of the test strip. The value marked on the color standard is the test result.

[0103] 6) Detection of serum autoantibodies in humanized SLE mice

[0104] a) Collect tail vein blood from mice and centrifuge at 4°C;

[0105] b) 100 μL of concentration standard, sample and control group were added to the well plate embedded with Anti-Mouse dsDNAIg antibody (Cat No.: 31A040, InnoBio) in sequence; incubated at room temperature for 1 hour;

[0106] c) Add PBS solution to wash 3-4 times, and then dry the solution in the wells;

[0107] d) Add 100uL Anti-Mouse Ig HRP diluent to each well and incubate at room temperature for 30min;

[0108] e) Add PBS solution to wash the plate 3 to 4 times, and then dry the solution in the wells;

[0109] f) TMB reactant was added to each well, and the solution in each well began to turn blue, and incubated at room temperature in a dark room for 15 minutes;

[0110] g) Add the stop reaction solution to each well, and the solution turns yellow;

[0111] h) Use an ELISA reader to measure the absorbance at 450 nm, draw a standard curve, and calculate the autoantibody concentration in the sample serum.

[0112] 7) Conventional methods were used to perform TUNEL staining of the spleen of P2-generation diseased mice in the humanized SLE mouse model and flow cytometry detection of autoreactive T cells (hCD45+hCD3+) in the spleen of P2-generation diseased mice in the humanized SLE mouse model.

[0113] 8) Evaluation criteria and results of humanized cutaneous lupus erythematosus mouse model

[0114] a) Humanized SLE mice showed continuous weight loss and decreased activity; peripheral blood tests showed decreased white blood cell counts, with or without thrombocytopenia and decreased hemoglobin.

[0115] b) Spleen and liver were enlarged, infiltrated with human immune cells, and histiocytes were degenerated and necrotic; kidney tissue structure was destroyed, glomeruli were enlarged, cells increased, cysts became smaller, and basement membranes were discontinuous; the specific results are as follows:

[0116] The spleen and pathological staining results of the P2 generation of diseased mice in the humanized SLE mouse model constructed by the present invention are shown in Figure 1 Among them, A is a macroscopic photo of the spleen. Compared with the healthy control mice (not modeled), the spleen of SLE-P2 mice was significantly enlarged; B is the result of HE staining of the spleen. The spleen of SLE-P2 mice showed extensive inflammatory cell infiltration and subcapsular hyalinization.

[0117] The liver and pathological staining results of the P2 generation of diseased mice in the humanized SLE mouse model constructed in the present invention are shown in Figure 2 Among them, A is a macroscopic photo of the liver, and the liver of the SLE-P2 generation mouse is significantly enlarged; B is the result of liver HE staining, and the spleen of the SLE-P2 generation mouse shows extensive inflammatory cell infiltration and the appearance of inflammatory cell groups. The liver cells are enlarged and arranged in a disordered manner.

[0118] The kidney damage of P2-generation mice in the humanized SLE mouse model constructed in the present invention can be found in Figure 3 Among them, A is a gross photo of the kidney, and subcapsular hemorrhage appeared in the kidney of SLE-P2 mice; B is the HE staining result of the kidney of SLE-P2 mice, and the enlarged image shows subcapsular hemorrhage; C is the HE staining result of the kidney of SLE-P2 mice, and the glomerulus volume, cell number, and basement membrane thickening are increased; D is the HE staining result of the kidney of SLE-P2 mice, and the basement membrane of the glomerular blood vessels is thickened.

[0119] c) Proteinuria determination: Compared with the blank control, the urine protein of SLE-P2 mice was judged as ++ (positive), see Figure 3 E in.

[0120] d) Human immune cells in tissues show scattered hCD3 + hCD8 + Cells. The specific results are as follows:

[0121] The results of flow cytometry detection of human inflammatory cells in the peripheral blood of P1-generation diseased mice in the humanized SLE mouse model constructed in the present invention are shown in Figure 4; A is the flow cytometry result compared with the isotype negative control, and the results show that a significant proportion of human (h) immune cells (hCD45 + ); B is a further classification of the immune cells in A, confirming that the human immune cells are autoreactive T cells (hCD45 + hCD3 + hCD8 + ), but not B cells (hCD19 - ), nor myeloid inflammatory cells (hCD33 - ). mCD45 indicates mouse-derived cells, and m stands for mouse.

[0122] The immunohistochemistry (IHC) test results of human inflammatory cells in P1 generation diseased mice of the humanized SLE mouse model constructed by the present invention are shown in Figure 5 ; Among them, A is the spleen and liver of the P1 generation of SLE mouse model mice stained with human-specific antibody hCD45, showing positive cells, namely human immune cells; B is the kidneys of the P1 generation of SLE mouse model mice stained with human-specific antibody hCD3, showing positive cells, namely human T cells.

[0123] The results of immunofluorescence detection of human IgG deposition in the kidneys of P1-generation diseased mice in the humanized SLE mouse model constructed in the present invention are shown in Figure 6 The kidney sections of the P1-generation SLE mouse model were immunostained with human-specific antibody hIgG, and the secondary antibody was coupled with green fluorescence and observed under a laser confocal microscope. The widely distributed green fluorescence signal in the kidneys represented the deposition of human IgG.

[0124] e) Serum autoantibody test: Anti-dsDNA and / or anti-SM antibodies are positive. The specific results are as follows:

[0125] The results of blood autoantibody detection of P1-generation diseased mice in the humanized SLE mouse model constructed by the present invention are shown in Figure 7 ELISA kits were used to detect autoantibodies in the plasma of SLE mouse models, including anti-dsDNA and anti-smith (SM) antibodies.

[0126] The results of TUNEL staining of spleen of P2-generation diseased mice in the humanized SLE mouse model constructed by the present invention are shown in Figure 8 The reduction of apoptosis is one of the important signs of SLE. TUNEL (TdT-mediated dUTPNick-End Labeling) staining was used to detect the apoptosis of cells in the spleen. The green signal is apoptotic cells. The results showed that the apoptotic cells in the spleen of P2 mice were significantly reduced.

[0127] The flow cytometry results of spleen autoreactive T cells (hCD45+hCD3+) in the humanized SLE mouse model P2 mice constructed by the present invention are shown in Fig. 9 Flow cytometry showed that compared with the isotype negative control, the spleen of the P2 generation diseased mice contained human immune cells that were self-reactive T cells, indicating that the spleen of the P2 generation diseased mice still contained abnormal SLE T cells.

[0128] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A mouse PDX cell line L0407-6-P3, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: C2024181.

2. Use of the mouse PDX cell L0407-6-P3 described in claim 1 in constructing a humanized systemic lupus erythematosus animal model.

3. A method for constructing a humanized systemic lupus erythematosus mouse model, characterized in that: The following steps are involved: The peripheral blood mononuclear cells of patients with active systemic lupus erythematosus were intravenously injected into P1 generation NSG mice, and the spleens of the P1 generation NSG mice were taken out 1 to 2 months later to prepare a single cell suspension; the single cell suspension was intravenously injected into P2 generation NSG mice, and a humanized systemic lupus erythematosus mouse model was obtained 3 to 4 weeks later; Alternatively, peripheral blood mononuclear cells from patients with active systemic lupus erythematosus are intravenously injected into P1 generation NSG mice; the mouse PDX cells L0407-6-P3 described in claim 1 are intravenously injected into P2 generation NSG mice, and a humanized systemic lupus erythematosus mouse model is obtained after 3 to 4 weeks; Alternatively, the mouse PDX cells L0407-6-P3 described in claim 1 are intravenously injected into NSG mice, and a humanized systemic lupus erythematosus mouse model is obtained after 3 to 4 weeks.

4. The construction method according to claim 3, characterized in that: The number of cells injected intravenously in the single cell suspension was 1×10 6 / P2 generation NSG mice.

5. The construction method according to claim 3, characterized in that: The spleen of the P1 mice contains mouse PDX cells.

6. The construction method according to claim 5, characterized in that: The marker of the mouse PDX cells is hCD45 + hCD3 + hCD8 + .

7. The construction method according to claim 3, characterized in that: The number of mouse PDX cells L0407-6-P3 injected intravenously was 1×10 6 / P2 generation NSG mice.

8. The construction method according to claim 3, characterized in that: The intravenous injection includes tail vein injection.

9. Use of the humanized systemic lupus erythematosus mouse model constructed by the construction method according to any one of claims 3 to 8 in studying the pathogenesis of systemic lupus erythematosus disease progression.

10. Use of the humanized systemic lupus erythematosus mouse model constructed by the construction method according to any one of claims 3 to 8 in drug development for systemic lupus erythematosus.

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