Method for constructing animal model of myeloid / lymphoid neoplasm with eosinophilia and tyrosine kinase gene fusion and application thereof

Abstract

The application belongs to the technical field of animal tumor models, and particularly relates to a myeloid / lymphoid tumor animal model with eosinophilia and tyrosine kinase gene fusion and a construction method and application thereof. The application constructs a myeloid / lymphoid tumor animal model with eosinophilia and tyrosine kinase gene fusion, the animal model can stably reproduce the clinical characteristics of peripheral blood eosinophilia, bone marrow hematopoietic system failure, spleen infiltration, decrease of platelets and hemoglobin, and increase of vitamin B12, and the construction method of the application has the advantages of short time consumption, high success rate and good reproducibility. The model can not only be used for evaluating the curative effect of a targeted drug, but also will provide an important in-vivo research platform for exploring the differentiation regulation mechanism of eosinophils, remodeling of bone marrow microenvironment and molecular path of disease progression to acute leukemia, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of animal tumor model technology, specifically relating to the construction method and application of myeloid / lymphoid tumor animal models with eosinophilia and tyrosine kinase gene fusion. Background Technology

[0002] Myeloid / lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK) are a rare and unique disease entity established in the WHO 5th edition of the hematopoietic neoplasm classification. They are characterized by acquired tyrosine kinase gene fusions (such as eosinophilia and tyrosine kinase gene fusions) at the hematopoietic stem cell or progenitor cell level. PDGFRA , PDGFRB , FGFR1 rearrangement or ETV6 :: SYK These tumors are characterized by molecular markers such as eosinophilia, bone marrow proliferative changes, organ infiltration, and a potential risk of blast crisis. Although these tumors show significant therapeutic response to specific tyrosine kinase inhibitors (TKIs), their low incidence, high clinical heterogeneity, and frequent multi-organ damage (heart, lungs, and nervous system) due to eosinophilia make clinical sample acquisition difficult and treatment window assessment limited. Therefore, there is an urgent need to establish experimental models that can simulate the biological characteristics and treatment response of human diseases.

[0003] Current research on MLN-TK mainly relies on in vitro cell line culture or xenograft models. While these methods can reproduce some molecular pathological features, they cannot fully simulate key pathological phenotypes during disease progression, such as eosinophil differentiation and infiltration, the formation of the myelofibrotic microenvironment, and systemic organ involvement. This is especially true for newly discovered [specific types of MLN-TK]. ETV6 :: SYK , ITK :: SYK Rare fusion types, such as MLN-TK, have only been reported in a few transgenic mouse models to induce myeloproliferative neoplasms (MPN) with fibrosis. However, these models still have limitations in terms of the degree of simulation of eosinophilia, the reproducibility of TKI resistance mechanisms, and the reconstruction of the immune microenvironment. They cannot fully meet the needs of drug screening and the development of precision treatment strategies. Moreover, the existing methods are time-consuming and expensive to build models, and cannot obtain a large number of animal models that meet the requirements in a short period of time, which seriously limits the research and development of drugs and precision treatment strategies for MLN-TK.

[0004] Therefore, constructing an efficient, stable, and batch-replicable MLN-TK animal model that exhibits clinical features such as peripheral blood eosinophilia, bone marrow proliferation and fibrosis, and myeloid infiltration of the skin / spleen is an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method and application for constructing animal models of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion.

[0006] This invention provides a method for constructing an animal model of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion, comprising: performing myelodysplasia on the animal, and then injecting bone marrow hematopoietic stem and progenitor cells overexpressing the tyrosine kinase fusion gene into the animal; wherein the tyrosine kinase fusion gene is... PML::SYK .

[0007] Preferably, the nucleotide sequence of the tyrosine kinase fusion gene is shown in SEQ ID NO. 1.

[0008] Preferably, the bone marrow ablation treatment method includes: X-ray radiation, with a total dose of 9±0.5 Gy / animal, performed in 2 sessions with an interval of 3-4 hours between each session.

[0009] Preferably, the method for obtaining bone marrow hematopoietic stem and progenitor cells includes: treating mice with antimetabolite chemotherapy drugs, euthanizing them, and then collecting bone marrow.

[0010] Preferably, the dosage of antimetabolite chemotherapy drugs is 150-200 mg / kg.

[0011] Preferably, the method for constructing bone marrow hematopoietic stem and progenitor cells overexpressing the tyrosine kinase fusion gene includes: infecting bone marrow hematopoietic stem and progenitor cells with a retrovirus containing the tyrosine kinase fusion gene.

[0012] Preferably, the bone marrow hematopoietic stem and progenitor cells overexpressing the tyrosine kinase fusion gene are obtained according to the following steps: Step 1: After stimulating bone marrow hematopoietic stem and progenitor cells, the first round of infection is with a retrovirus containing a tyrosine kinase fusion gene. Step 2: After stimulating bone marrow hematopoietic stem and progenitor cells again, a second round of infection with a retrovirus containing a tyrosine kinase fusion gene is performed.

[0013] Preferably, the retrovirus containing the tyrosine kinase fusion gene is prepared by the following method: the packaging plasmid and the target plasmid are mixed and transfected into the host cell using the calcium phosphate method, and the viral supernatant is collected; the target plasmid contains the tyrosine kinase fusion gene.

[0014] Preferably, the method of the first round of infection includes infection with infection solution 1, the formulation of which includes: a retrovirus containing a tyrosine kinase fusion gene, a stimulating solution, and a positive polymer; And / or, the method of the second round of infection includes infection with infection fluid 2, the formulation of which includes: a retrovirus containing a tyrosine kinase fusion gene and a positive polymer; And / or, in steps 1 and 2, the stimulation method includes stimulation with a stimulating solution; the formulation of the stimulating solution includes: cell stimulating factors and stem cell factors; And / or, the stimulation time is 16-20 hours, and the infection time is 3-4 hours.

[0015] Preferably, the animal is a mouse, and the injection dose of bone marrow hematopoietic stem and progenitor cells overexpressing the tyrosine kinase fusion gene is (0.5-1) × 10⁻⁶. 6 / Only.

[0016] The vector for retroviruses is a viral vector with MSCV as its backbone.

[0017] A "tyrosine kinase fusion gene" refers to a novel gene resulting from the fusion of a tyrosine kinase gene with other genes. The fusion protein produced exhibits sustained kinase activity independent of other signals. This invention requires only one specific tyrosine kinase fusion gene to successfully construct an MLN-TK disease model, without the need for additional synergistic effects from other genes.

[0018] "Bone marrow ablation" refers to the treatment of bone marrow failure caused by radiotherapy or chemotherapy, which kills all bone marrow cells in the bone marrow.

[0019] Numerical designations such as "infectant 1" and "infectant 2" serve only to distinguish different terms and do not indicate any priority or order, nor do they limit the scope of the technical features described.

[0020] This invention successfully constructed an animal model of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion by screening construction methods, conditions, and fusion genes. This animal model comprehensively and stably reproduces clinical features such as peripheral blood eosinophilia, bone marrow hematopoietic system failure and splenic infiltration, decreased platelets and hemoglobin, and increased vitamin B12. Furthermore, the construction method of this invention is time-efficient, has a high success rate, and good reproducibility. This model can not only be used to evaluate the efficacy of targeted drugs but also provide an important in vivo research platform for exploring the regulatory mechanisms of eosinophil differentiation, bone marrow microenvironment remodeling, and the molecular pathways of disease progression to acute leukemia, showing promising application prospects.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the method for constructing a mouse model of bone marrow / lymphoma.

[0024] Figure 2 The image shows the results of detecting the expression of P-SYK, P-STAT5, and P-ERK in 293T cells.

[0025] Figure 3 This is a survival curve for mice.

[0026] Figure 4 Image of a mouse spleen.

[0027] Figure 5 Image showing the appearance of the tibia and fibula of a mouse.

[0028] Figure 6 This is a graph showing the results of flow cytometry determination of tumor cell types in a model mouse.

[0029] Figure 7 The graph shows the results of the content of eosinophils (Eso), platelets, hemoglobin and vitamin B12 in the peripheral blood of model mice.

[0030] Figure 8 The images show the flow cytometry results of 3T3 cells infected with MIG-PML::SYK virus; the left image is the flow cytometry plot of control 3T3 cells, and the right image is the flow cytometry plot of 3T3 cells infected with MIG-PML::SYK virus.

[0031] Figure 9 To explore survival curves for recipient mice based on irradiation dose. Detailed Implementation

[0032] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0033] Example 1: Method for constructing a mouse model of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion I. Model Building Methods The method for constructing a mouse model of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion in this embodiment (e.g.) Figure 1 )as follows: 1. Enrichment of mouse bone marrow hematopoietic stem and progenitor cells C57BL / 6J (approximately 8 weeks old) male donor mice were given 200 mg / kg 5-fluorouracil (Sigma, Cat#: F6627) to fully enrich mouse bone marrow stem and progenitor cells (5-fluorouracil needs to be fully dissolved in PBS to 10 mg / mL, filtered for sterilization, and injected via tail vein). (Note that: too low a dose of 5-FU will result in impure enrichment of stem and progenitor cells, while too high a dose will cause excessive damage to tissues and organs, leading to the death of mice).

[0034] 2. Collection of hematopoietic stem and progenitor cells from donor mouse bone marrow (1) Four days later, the donor mice from step 1 were euthanized with carbon dioxide. Dissection tools were sterilized before use. The mice were wiped with 70% ethanol in a sterile laminar flow hood. The femur and tibia of the mice were taken and placed in pre-cooled DMEM high glucose medium (DMEM, Gibco, Cat#: 11965092; containing 10% fetal bovine serum, Gibco, Cat#: A5256701; 1% glutamine, Gibco, Cat#: 25030032; 1% penicillin / streptomycin, Gibco, Cat#: 15140148 and 1% NEAA, Gibco, Cat#: 11140050).

[0035] (2) Using a 10 mL syringe with a 27G 1 / 2 needle, flush the bone marrow cells from the femur and tibia into a 50 mL test tube. Shake well to suspend the bone marrow cells into a single cell state. Take a small amount of cell suspension and remove red blood cells with red blood cell lysis buffer to calculate the total number of cells (Note: It is necessary to prepare a single cell suspension, as cell clumping will affect the efficiency of subsequent virus infection).

[0036] (3) Centrifuge at 300×g for 10 minutes and collect the cells.

[0037] (4) Gently tap the cell pellet at the bottom of the test tube and resuspend the cells in a 10 cm culture dish with stimulation medium (the number of cells in 10 mL of stimulation medium should be less than 3 × 10⁻⁶). 7 Cells were cultured overnight in an incubator (37°C, 5% CO2). The composition of the stimulation medium is shown in Table 1. Table 1 10 mL Stimulation Culture Medium 3. First round of MSCV-PML::SYK-IRES-GFP (MIG-PML::SYK) retroviral infection (1) Collect the cells cultured overnight in a 10cm culture dish obtained in step 2, centrifuge at 300×g for 10 minutes, and collect the cells.

[0038] (2) Gently tap the cell pellet at the bottom of the test tube and resuspend the cells in infection medium. Then transfer 4 mL of the cell suspension to a 6-well plate and centrifuge at 1000×g at 37°C for 90 minutes. The composition of the infection medium is shown in Table 2. Table 2 Infection Culture Media The preparation method of MIG-PML::SYK virus is as follows: HEK-293T cells in the exponential growth phase (DMEM high-glucose medium + 10% FBS + 1% penicillin antibody) were cultured at a rate of 2 × 10⁻⁶ cells / year. 6 The cells were seeded into 6-well plates, and when the cells reached 90% confluence, the following steps were taken: One hour before virus inoculation, replace the cell culture medium with fresh medium (containing 25 μM chloroquine) and place the cells in a cell incubator. The calcium phosphate plasmid transfection system is shown in Table 3 below: Table 3. Calcium phosphate plasmid transfection system Mix the mixture in Table 3 with 125 μL of 2×HBS, add to a 6-well plate, and replace with normal culture medium after 10-12 hours. Collect the culture medium containing virus supernatant 36 hours after transfection, filter through a 0.45 μm filter membrane to remove cell debris, and freeze the aliquoted virus solution at -80°C for later use.

[0039] PML::SYK sequence (SEQ ID NO. 1):

[0040] (3) After centrifugation, place the 6-well plate in an incubator and continue culturing for 3-4 hours (37°C, 5% CO2). (Note that after centrifugation, the cells will clump together on one side of the well. Use a 1 mL pipette to disperse them thoroughly to ensure that the virus and cells are in full contact.)

[0041] (4) Transfer the supernatant to a new centrifuge tube using a pipette, centrifuge at 300×g for 10 minutes to remove the infection solution, resuspend the cells at the bottom of the centrifuge tube in 4 mL of fresh stimulation medium, return it to the original well plate, and incubate overnight in an incubator (37°C, 5% CO2). The composition of the stimulation medium is shown in Table 4: Table 4 Stimulation Culture Medium 4. Second round (MIG-PML::SYK) retroviral infection (1) Gently remove 2 mL of supernatant from the overnight cultured cells obtained in step 3 using a pipette, add 2 mL of MIG-PML::SYK virus infection solution, and centrifuge at 1000×g at 37°C for 90 minutes. The composition of the infection solution is shown in Table 5: Table 5 Infection Fluid (2) After centrifugation, place the 6-well plate in an incubator and continue culturing for 3-4 hours (37°C, 5% CO2). (Note that after centrifugation, the cells will clump together on one side of the well. Use a 1 mL pipette to disperse them thoroughly to ensure that the virus and cells are in full contact.)

[0042] (3) Collect all cells in a 6-well plate, centrifuge at 300×g for 10 minutes, remove the supernatant, and wash twice with 5 mL PBS; take a small amount of cell suspension to remove red blood cells with red blood cell lysis buffer and calculate the total number of cells (Note: when taking cells, mix the cell suspension thoroughly to ensure accurate counting).

[0043] 5. Constructing a mouse model (1) Based on the total cell count, adjust the viral-infected bone marrow hematopoietic stem and progenitor cells obtained in step 4 to 5 × 10⁶ cells / year using sterile PBS. 6 Density per mL.

[0044] (2) The specific radiation conditions for C57BL / 6J male recipient mice to receive X-ray radiation were as follows: a total of 9 Gy of lethal X-ray radiation, 4.5 Gy each time, for a total of 2 irradiations, with an interval of 3-4 hours between them. The irradiation instrument model was RS-2000, and the irradiation instrument settings were: voltage 160kV, current 7.6mA (the default conditions are voltage 160kV and current 25mA). Under these conditions, a relatively small irradiation dose rate was ensured while still achieving the purpose of bone marrow cell clearance, so as to ensure the success rate of subsequent model mouse construction. Aseptic conditions were ensured during the operation. If the mice were contaminated during the process, it would lead to bacterial infection and death after transplantation.

[0045] (3) After irradiation, 0.2 mL of virus-infected bone marrow hematopoietic stem cells were injected intravenously into C57BL / 6J receptor mice.

[0046] II. Characterization of Animal Models 1. Characterization methods The MIG-PML::SYK virus in the construction method was replaced with a blank MIG vector as a control. The survival rate of mice 40 days after model construction was counted. 21 days after model construction, control mice and model mice were dissected to measure spleen size, examine the appearance of the tibia and fibula, determine the tumor cell type of the model mice (identifying whether the tumor cells are B cells, T cells, progenitor cells, or granulocytes), peripheral blood platelet and hemoglobin levels, eosinophil count, and vitamin B12 concentration.

[0047] The MIG-PML::SYK plasmid vector was transfected into 293T cells, and the expression levels of P-SYK, P-STAT5, and P-ERK were measured. The specific methods are as follows: HEK-293T cells in the exponential growth phase (DMEM high-glucose medium + 10% FBS + 1% penicillin antibody) were inoculated at 1×10⁻⁶ cells / year. 6 The cells were seeded into 6-well plates. When the cells reached a density of approximately 40%, the following transfection procedure was performed: One hour before transfection, replace the cell culture medium with fresh medium (containing 25 μM chloroquine) and place the cells in a cell incubator. The calcium phosphate plasmid transfection system is shown in Table 6 below: Table 6. Calcium phosphate plasmid transfection system Mix the mixture in Table 6 with 125 μL of 2×HBS, add to 6-well plates, and replace with normal culture medium after 10-12 hours. Collect 293T cells 24 hours after transfection and lyse them with RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime: RIPA, P0013B; protease phosphatase inhibitor, P1045) to obtain protein lysis buffer. Analyze the activation of STAT5 and ERK, downstream targets of PML::SYK, by Western blotting to reflect the kinase activity of PML::SYK. The Western blotting method is as follows: Determine the concentration of the extracted protein sample using a quinoline carboxylic acid (BCA) protein quantification kit. Mix an equal volume of protein (usually 15-20 μg) with 5×sodium dodecyl sulfate (SDS) loading buffer and denature in a 100°C metal bath for 10 minutes. Denatured protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then the proteins in the gel were transferred to a polyvinylidene fluoride (PVDF) membrane using a wet transfer method. After transfer, the PVDF membrane was placed in Tris-buffered saline (TBST) containing 5% skim milk powder and blocked at room temperature for 1 hour to block non-specific binding sites on the membrane. After blocking, the membrane was incubated overnight at 4°C with working solutions of primary antibodies that specifically recognize phosphorylated SYK (p-SYK), total SYK, PML, phosphorylated STAT5 (p-STAT5), total STAT5, phosphorylated ERK (p-ERK), total ERK, and the internal control protein ACTIN. The next day, the membrane was washed three times with TBST buffer for 10 minutes each time to remove unbound primary antibodies. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. The membrane was then washed three more times with TBST buffer for 10 minutes each time. Finally, an enhanced chemiluminescence (ECL) substrate was added, and the image was developed and acquired using a chemiluminescence imaging system.

[0048] 2. Characterization Results (1) Expression of P-SYK, P-STAT5, and P-ERK The expression results of P-SYK, P-STAT5, and P-ERK in 293T cells are as follows: Figure 2 As shown in the figure. The results indicate that MIG-PML::SYK can autophosphorylate and activate STAT and ERK, thus proving that the MIG-PML::SYK gene can lead to autophosphorylation and continuous activation of the STAT and ERK signaling pathways without additional stimulation.

[0049] (2) Model survival rate results Survival curves Figure 3As shown, there were 7 mice in the control group and 7 mice in the model group. The results showed that, compared with the control group mice, all the model mice died within 30 days, indicating that the model mice constructed in this invention were severely diseased and the model was successfully constructed.

[0050] (3) Pathological phenotype results Spleen results as follows Figure 4 As shown, the results indicate that, compared with the blank control group, the spleen of the model mice was infiltrated by a large number of tumor cells and the spleen was enlarged.

[0051] Examination results of the tibia and fibula are as follows Figure 5 As shown, the results indicate that, compared with the blank control group, the tibia / fibula of the model mice was observed to be white in color and the translucency of the bone cortex was increased (no red blood cells), suggesting that bone marrow tumor infiltration and bone marrow hematopoietic system failure occurred in the tibia / fibula of the model mice.

[0052] (4) Tumor cell types in model mice The results are as follows Figure 6 As shown, all cells expressing PML-SYK in the peripheral blood of diseased mice were granulocytes, indicating that the tumor cell type in the model mice was granulocytes.

[0053] (5) Determination of important phenotypes in peripheral blood of model mice The results are as follows Figure 7 As shown in the figure. The results indicate a significant increase in eosinophil (Eso) count, suggesting that the mice constructed using the method described in this embodiment exhibit eosinophilia. Furthermore, the peripheral blood of the model mice showed decreased platelets and hemoglobin, and increased vitamin B12, indicating severe suppression of normal hematopoiesis and severe anemia. This further demonstrates that this embodiment successfully constructed a mouse model of the disease and comprehensively simulated all phenotypes of the disease. The phenotype of this model mouse closely matches the characteristics of clinically diagnosed myeloid / lymphoid tumor patients with eosinophilia and tyrosine kinase gene fusion.

[0054] Moreover, the construction method described in this embodiment was repeated multiple times, and myeloid / lymphoid tumor mouse models with eosinophilia and tyrosine kinase gene fusion were successfully constructed each time.

[0055] The above results demonstrate that this embodiment successfully constructed a mouse model of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion, which can comprehensively and stably reproduce clinical features such as peripheral blood eosinophilia, bone marrow hematopoietic system failure, and spleen infiltration.

[0056] The following experiments further illustrate the effectiveness of the technical solution of the present invention.

[0057] Experiment 1: MIG-PML::SYK Virus Infectivity Test I. Experimental Methods To ensure sufficient viral titer, the infectivity of the MIG-PML::SYK virus was tested before model construction. The specific method is as follows: 5.0×10 4 3T3 cells were placed in 6-well plates containing 2 mL of culture medium. The next day, the cell culture medium was removed, and 1 mL of virus solution and 1 mL of culture medium (DMEM medium + 10% FBS) mixture (with 20 μL HEPES and 2 μL Polybrene infection aid) were added to simulate virus infection of primary mouse bone marrow hematopoietic stem and progenitor cells. After 3 hours, the normal culture medium was replaced, and after 48 hours, the percentage of GFP was tested by flow cytometry.

[0058] II. Experimental Results The results are as follows Figure 8 As shown in the figure. The results indicate that the GFP infection efficiency is above 95%, and the MIG-PML::SYK virus can be used to infect host cells.

[0059] Experiment Example 2: Screening of Irradiation Conditions I. Experimental Methods Male C57BL / 6J recipient mice were irradiated with total doses of 7.5 Gy, 8.0 Gy, 8.5 Gy, 9.0 Gy, and 9.5 Gy, respectively, using an RS-2000 irradiation instrument set at 160 kV and 7.6 mA. Each dose was administered in two separate irradiations with a 3-hour interval between doses. Six mice were used in each group, and the survival rate was calculated. Aseptic conditions were maintained throughout the procedure.

[0060] II. Experimental Results Results of radiation dose exploration in C57BL / 6J male receptor mice are as follows: Figure 9 As shown, within the irradiation dose range of 7.5-9.5 Gy, some mice did not die after irradiation with normal bone marrow cells at 7.5 Gy, 8.0 Gy, and 8.5 Gy, indicating incomplete myelination and failure of the model. However, mice died after irradiation with normal bone marrow cells at 9.0 Gy and 9.5 Gy without reinfusion. This result indicates that the irradiation condition with a total dose of 9.0 Gy is the most suitable. Under this optimized irradiation condition, a relatively low irradiation dose rate is ensured while achieving the goal of bone marrow cell clearance, thus guaranteeing the success rate of subsequent mouse model construction.

[0061] As demonstrated by the above embodiments and experimental examples, this invention successfully constructed an animal model of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion. This animal model can comprehensively and stably reproduce clinical features such as peripheral blood eosinophilia, bone marrow hematopoietic system failure and splenic infiltration, decreased platelets and hemoglobin, and increased vitamin B12. Moreover, the construction method of this invention is time-efficient, has a high success rate, and good reproducibility. This model can not only be used to evaluate the efficacy of targeted drugs but will also provide an important in vivo research platform for exploring the regulatory mechanisms of eosinophil differentiation, bone marrow microenvironment remodeling, and the molecular pathways of disease progression to acute leukemia, showing promising application prospects.

Claims

1. A method for constructing an animal model of myeloid / lymphoid tumors with eosinophilia and tyrosine kinase gene fusion, characterized in that, It includes: performing myeloablation on animals, then injecting bone marrow hematopoietic stem and progenitor cells overexpressing a tyrosine kinase fusion gene into the animals; the tyrosine kinase fusion gene is... PML::SYK .

2. The construction method according to claim 1, characterized in that: The nucleotide sequence of the tyrosine kinase fusion gene is shown in SEQ ID NO.

1.

3. The construction method according to claim 1, characterized in that: The bone marrow ablation treatment method includes: X-ray radiation, with a total dose of 9±0.5 Gy / animal, performed in 2 sessions with an interval of 3-4 hours between each session.

4. The construction method according to any one of claims 1-3, characterized in that: The method for obtaining bone marrow hematopoietic stem and progenitor cells includes: treating mice with antimetabolite chemotherapy drugs, sacrificing them and then collecting bone marrow.

5. The construction method according to claim 4, characterized in that: The dosage of antimetabolite chemotherapy drugs is 150-200 mg / kg.

6. The construction method according to any one of claims 1-3, characterized in that, The method for constructing bone marrow hematopoietic stem and progenitor cells overexpressing the tyrosine kinase fusion gene includes: infecting bone marrow hematopoietic stem and progenitor cells with a retrovirus containing the tyrosine kinase fusion gene.

7. The construction method according to claim 6, characterized in that, The bone marrow hematopoietic stem and progenitor cells overexpressing the tyrosine kinase fusion gene were obtained according to the following steps: Step 1: After stimulating bone marrow hematopoietic stem and progenitor cells, the first round of infection is with a retrovirus containing a tyrosine kinase fusion gene. Step 2: After stimulating bone marrow hematopoietic stem and progenitor cells again, a second round of infection with a retrovirus containing a tyrosine kinase fusion gene is performed.

8. The construction method according to claim 7, characterized in that, Retroviruses containing a tyrosine kinase fusion gene are prepared by the following method: the packaging plasmid and the target plasmid are mixed and transfected into host cells using the calcium phosphate method, and the viral supernatant is collected; the target plasmid contains a tyrosine kinase fusion gene.

9. The construction method according to claim 7, characterized in that: The method of the first round of infection includes infection with infection solution 1, the formulation of which includes: a retrovirus containing a tyrosine kinase fusion gene, a stimulating solution, and a positive polymer; And / or, the method of the second round of infection includes infection with infection fluid 2, the formulation of which includes: a retrovirus containing a tyrosine kinase fusion gene and a positive polymer; And / or, in steps 1 and 2, the stimulation method includes stimulation with a stimulating solution; the formulation of the stimulating solution includes: cell stimulating factors and stem cell factors; And / or, the stimulation time is 16-20 hours, and the infection time is 3-4 hours.

10. The construction method according to claim 1, characterized in that: The animals were mice, and the injection dose of bone marrow hematopoietic stem and progenitor cells overexpressing the tyrosine kinase fusion gene was (0.5-1) × 10⁻¹⁰. 6 / Only.

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