Brain glioma animal model construction method based on AAV mediated immune microenvironment

Abstract

The invention discloses a brain glioma animal model construction method based on an AAV mediated immune microenvironment. The method comprises the following steps: selecting an immunodeficient mouse at the right age; human CD34 + hematopoietic stem cells are injected for immune system reconstruction; human CD45 + immune cells in the peripheral blood PBMC are detected; the method comprises the following steps: selecting tumor tissues with high-expression brain glioma marker genes, carrying out rapid qPCR (quantitative polymerase chain reaction) or targeted RNA-seq immunohistochemical multiple verification, and screening consistent tumor tissues; the method comprises the following steps: physically dicing tumor tissues, treating the tumor tissues with pretreatment liquid, digesting the tumor tissues into single-cell suspension, and inoculating the single-cell suspension into a mouse cranium; an AAV vector carrying a specific immune regulation gene is injected into the tail vein of the mouse, and the brain glioma immune microenvironment is regulated; when it is observed that the tumor tissue grows stably and has immune cell infiltration characteristics, construction of the brain glioma animal model is completed. Compared with an existing model, the brain glioma model which is more stable, more scientific and closer to the human immune background is provided, and the brain glioma model is suitable for related immunotherapy drug screening and mechanism research.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor animal models, and particularly to a method for constructing a glioblastoma animal model based on AAV-mediated immune microenvironment. Background Art

[0002] Glioblastoma (GBM) is a highly invasive and malignant brain tumor that usually originates from astrocytes in the brain or spinal cord. GBM is the most common primary brain tumor in adults and one of the most lethal brain tumors. The characteristics of this disease include rapid proliferation, high invasiveness, resistance to conventional treatments (such as radiotherapy and chemotherapy), and a high recurrence rate after treatment. The pathogenesis of GBM is complex, involving multiple molecular pathways and gene mutations, and the tumor microenvironment (such as immune escape mechanisms, blood-brain barrier disorders, etc.) has an important impact on tumor progression. Therefore, it is crucial to study the occurrence and development mechanisms of glioblastoma and optimize existing treatment strategies.

[0003] The research on glioblastoma relies on various experimental models, such as in vitro cell culture, subcutaneous xenograft models, genetically engineered mouse models (GEMM), and orthotopic glioblastoma models. Although these models have played an important role in research, they also have technical limitations. In vitro cell culture models cannot simulate the tumor microenvironment (TME), lack the interaction between immune cells and blood vessels, and thus cannot truly reproduce the immune escape mechanism and growth characteristics of tumors. Subcutaneous xenograft models can be used for drug screening, but due to the microenvironmental differences from the brain tissue, they cannot reflect the growth and drug response of glioblastoma. Genetically engineered mouse models simulate the occurrence of tumors through gene modification. However, their long establishment cycle, the specificity of gene modification, and the limitations of not being able to fully reproduce tumor heterogeneity and microenvironment restrict their application in drug screening. Orthotopic glioblastoma models can better simulate the brain microenvironment, but still face problems such as insufficient accuracy of tumor cell injection, incomplete retention of the tumor microenvironment, insufficient simulation of the immune microenvironment, and lack of the ability of spontaneous metastasis. Therefore, the limitations of these existing models pose challenges in glioblastoma drug R & D and clinical translation, especially in the accuracy of immunotherapy and drug screening.

[0004] Therefore, it is particularly important to develop an animal model that can overcome the limitations of existing glioblastoma experimental models. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing a glioblastoma animal model based on AAV-mediated immune microenvironment to solve one or more technical problems existing in the prior art, and at least provide a beneficial choice or create conditions.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention purpose: The present invention provides a method for constructing a glioma animal model based on AAV-mediated immune microenvironment, including: Selecting immune-deficient mice of appropriate age; Reconstructing the immune system by injecting human CD34+ hematopoietic stem cells via the tail vein; Using flow cytometry to detect human CD45+ immune cells in peripheral blood PBMC, and confirming the successful reconstruction of the immune system; Selecting tumor tissues with high expression of glioma marker genes, performing rapid qPCR or targeted RNA-seq immunohistochemical multiplex verification, and screening tumor tissues that meet the requirements of the glioma animal model; After physically cutting the selected tumor tissues and treating them with a pretreatment solution, digesting them into single-cell suspensions, and inoculating the single-cell suspensions into the mouse intracranial cavity by stereotactic injection; Injecting an AAV vector carrying a specific immune regulatory gene into the mouse tail vein to regulate the glioma immune microenvironment. When the stable expression of the AAV expression product is detected in the mouse body, the immune regulation mediated by the AAV expression product is completed; When it is observed that the tumor tissues grow stably and have the characteristics of immune cell infiltration, it is confirmed that the reconstruction of the immune microenvironment and tumor growth meet the requirements of model construction, and the construction of the glioma animal model is completed.

[0007] Furthermore, the AAV vector includes an AAV-TGFβ1-P2A-IL10 expression cassette and an AAV-CSF1 expression cassette for expressing TGF-β1 , IL10 and CSF1 genes; The AAV-TGFβ1-P2A-IL10 expression cassette includes a nucleic acid structure in which the TGF-β1 gene driven by the CAG promoter and the IL10 gene are co-expressed through the P2A self-cleaving peptide sequence, and the total length of the nucleic acid sequence is 3.1±0.2 kb; The AAV-CSF1 expression cassette includes a CSF1 gene expression unit driven by the CAG promoter, and the total length of the nucleic acid sequence is 2.3±0.2 kb.

[0008] Furthermore, AAV9 or AAV-PHP.eB serotype is used for the delivery of TGF-β1 , IL10 and CSF1 genes.

[0009] Furthermore, the injection dose of the AAV vector is 1-5E+11 vg / mouse; The AAV vector is formulated in a buffer solution with a pH of 7.7 to 8.3, and the buffer solution contains 20 mM Tris, 1 mM , 200 mM NaCl and 0.005 mM Tween-20.

[0010] Furthermore, the tumor tissue selected is a tumor tissue pathologically diagnosed as WHO grade III or IV.

[0011] Furthermore, the glioma marker genes include CD133 protein, IDH1 R132H mutation, EGFR amplification, MGMT promoter methylation, and SOX2 protein.

[0012] Furthermore, the pretreatment solution includes Neurobasal medium and Matrigel mixed at a volume ratio of 3:1, and 2× B27, 2 mM glutathione, 50 U / mL penicillin-streptomycin, and 20 ng / mL PDGF-BB are added.

[0013] Furthermore, the growth monitoring of the tumor tissue is obtained by calculating the change in the T2-weighted image of small animal MRI combined with the volume.

[0014] Furthermore, the morphology of the tumor cells is obtained by observing with HE staining.

[0015] Furthermore, the method for detecting the significant expression of the AAV expression product in mice includes: Using qPCR technology to detect the expression level of mRNA; Using Western Blot or ELISA technology to detect the expression level of protein; Using immunohistochemical staining or immunofluorescent staining to further verify the transgenic expression product in the tissue.

[0016] The beneficial effects of the present invention are as follows: The present invention should not only be able to more accurately simulate the tumor microenvironment, immune escape mechanism, and tumor growth characteristics of glioma, but also have the advantages of good reproducibility and rapid construction to improve the success rate of drug screening and clinical transformation.

[0017] The present invention fully considers the defects, deficiencies, and usage limitations of the existing glioma models, uses humanized mice, and takes patient-derived glioma tissues and their appendages as the research objects. By long-term expressing inflammatory factors related to the glioma immune microenvironment through AAV vectors, regulating the immune microenvironment of the mouse glioma model, and optimizing the expression of immune checkpoints, a mouse glioma model closer to the immune characteristics of human glioma is established. Brief Description of the Drawings

[0018] Figure 1 It is the overall flowchart provided according to the embodiments of the present invention; Figure 2 It is the flow cytometry measurement result graph of the mouse immune system reconstruction by injecting human CD34+ hematopoietic stem cells into the mouse tail vein provided according to the embodiments of the present invention; Figure 3 It is the tumor growth curve graph in the mouse intracranial cavity measured by MRI after transplantation of the single-cell suspension digested from glioma tissue provided according to the embodiments of the present invention; Figure 4 It is the graph of the mRNA expression of the vector in peripheral blood detected by qPCR technology after AAV injection provided according to the embodiments of the present invention; Figure 5 It is the graph of the mRNA expression of the vector in tumor tissue detected by qPCR technology after AAV injection provided according to the embodiments of the present invention; Figure 6 It is the protein expression graph of human TGF-β1, IL-10, and CSF1 in tumor tissue verified by IHC method after AAV immune system reconstruction provided according to the embodiments of the present invention; Figure 7 It is the dynamic change graph of mouse glioma under MRI monitoring with different glioma construction factors provided according to the embodiments of the present invention; Figure 8 It is the flow cytometry antibody combination graph provided according to the embodiments of the present invention; Figure 9 It is the quantitative verification result graph of the immune reconstruction of human CD34+ mesenchymal stem cells provided according to the embodiments of the present invention; Figure 10 It is the graph of the influence of different immune reconstruction conditions on the tumor intracranial growth curve after transplantation of mouse glioma tissue cells provided according to the embodiments of the present invention; Figure 11 It is the selection and function graph of the antibody provided according to the embodiments of the present invention; Figure 12 It is the change result graph of the immune microenvironment of tumor tissue measured by flow cytometry under different reconstruction conditions provided according to the embodiments of the present invention; Figure 13 It is the schematic diagram of the AAV-TGFβ1-P2A-IL10 vector design provided according to the embodiments of the present invention; Figure 14 It is the schematic diagram of the AAV-CSF1 vector design provided according to the embodiments of the present invention. Detailed implementation manners

[0019] The present invention will be further described below in combination with specific embodiments. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and cannot be used to limit the protection scope of the present invention.

[0020] As Figures 1-14 shown, a method for constructing a glioma animal model based on AAV-mediated immune microenvironment provided by the present invention comprises the following specific steps: S1. Mouse selection and feeding conditions In this study, NOG (NOD.Cg- Prkdc scid Il2rg tm1Sug / JicCrl) mice were used as experimental animals. These mice are a severely immunodeficient strain and are suitable for immune system reconstruction. Three-week-old female mice were used in the experiment. The feeding conditions require a barrier environment at the SPF level, with the environmental temperature controlled at 22-26 °C, the humidity at 40-60%, and a 12-hour light / 12-hour dark light cycle. The mouse feed was a specific pathogen-free (SPF)-grade sterilized feed, and the drinking water was ultrafiltered sterilized water. These feeding conditions ensure that the mice are in a suitable growth and experimental environment.

[0021] S2. Human immune system reconstruction In the experiment, human CD34+ hematopoietic stem cells were used for immune system reconstruction. CD34+ cells can be isolated from human umbilical cord blood or bone marrow, and CD34+ cells with a purity greater than 95% were obtained by FACS sorting. The cell concentration was set at 1-5×10^6 cells / 100 μL PBS. A 30 G fine needle was used for tail vein injection, and the injection process needed to be carried out slowly to ensure the smooth infusion of the cells. The monitoring of immune system reconstruction was usually carried out 4-6 weeks later, and immune cell markers in peripheral blood, such as human CD45+, CD3+, CD19+, etc., were detected by flow cytometry. If the proportion of human CD45+ cells in peripheral blood cells exceeds 25%, it is considered that the immune system reconstruction is successful.

[0022] Here, by tail vein injection of human CD34+ hematopoietic stem cells, the immune system of mice was successfully reconstructed, which can more realistically simulate the human immune microenvironment. Especially in the research on immune escape and immunotherapy of glioma, it provides a more human-like immune response. At the same time, most existing glioma mouse models are immune-deficient mouse models and cannot simulate normal immune responses. However, through the establishment of an immune reconstruction model with a complete immune system in this application, this model can better evaluate the mechanisms of immunotherapy and immune escape, increasing the relevance and translational potential of clinical research.

[0023] S3. Tumor marker gene screening and verification Before establishing the tumor model, select tumor tissues with high expression of glioma marker genes. Human glioblastoma (GBM, WHO grade III or IV) tissues are selected for qPCR or RNA-seq detection. The screened marker genes include CD133 , IDH1 , EGFR , MGMT , SOX2 , SLC1A3 , OLIG2 , etc. The TPM values of these genes need to meet the following requirements (any 3 of the following multiple indicators meet the standards): CD133 > 80 IDH1 > 50 EGFR > 50 MGMT > 30 SOX2 > 100 SLC1A3 > 100 OLIG2 > 25 In addition, the expression level of the protein is further verified by immunohistochemistry (IHC) to ensure that the selected tumor tissues have a high gene expression level.

[0024] S4, Tumor tissue pretreatment After screening out the tumor tissues that meet the standards, cut the tissues into small pieces of 1 - 3 mm³. To ensure the biological activity of the tissue blocks, a specific pretreatment method is adopted. The pretreatment solution is a mixture of Neurobasal medium and Matrigel at a volume ratio of 3:1, supplemented with 2× B27, 2 mM glutathione, 50 U / mL penicillin-streptomycin, and 20 ng / mL PDGF-BB. Subsequently, perform low-speed centrifugation to remove necrotic tissues and digest with a mixed enzyme. After digestion is completed, wash twice with PBS to terminate the digestion process and prepare for the subsequent transplantation.

[0025] Here, an optimized tumor tissue pretreatment method is adopted, including low-speed centrifugation to remove necrotic tissues, moderate digestion, and PBS washing, to ensure the activity of the transplanted tumor tissues and the cell survival rate. Traditional tumor transplantation methods may have problems such as tissue necrosis or low cell survival rate. The finely optimized tissue pretreatment can ensure the activity of tumor cells, improve the tumor growth rate and success rate after transplantation, and enhance the reproducibility and reliability of the model.

[0026] S5, Tissue transplantation After pretreatment, the tumor tissue mass was transplanted into the brains of immunodeficient mice. During the transplantation process, the mice were first anesthetized by inhaling isoflurane, with an induction concentration of 3% and a maintenance concentration of 1.5%. Under aseptic conditions, the mouse's head was fixed, and the scalp was incised to expose the skull. Using stereotactic injection, a dental microdrill was used to drill a hole in the left or right frontal lobe of the mouse (2 mm posterior to the bregma, 2 mm left or right of the midline), and the needle was inserted to a depth of 2.5 - 3 mm. After transplantation, the skull hole was sealed, the scalp was sutured, and an antibiotic (ceftriaxone 25 mg / kg, i.p.) was used to prevent infection.

[0027] S6, AAV Vector Injection and Immune Microenvironment Regulation To regulate the immune microenvironment of glioma, an AAV vector carrying a specific gene was injected into the tail vein of the model animals. The packaging capacity limit of AAV is 4.7 kb, so the length of the gene elements of the AAV vector needs to be strictly calculated. The specific AAV vectors were designed as: AAV-TGFβ1-P2A-IL10 (about 3.1 kb) or AAV-CSF1 (about 2.3 kb). Both vectors used the CAG promoter to improve the gene expression efficiency. The CAG promoter has high expression stability in the nervous system, avoiding common epigenetic silencing problems, ensuring long-term expression and the effectiveness of immune regulation, and facilitating the study of the effects of long-term immune microenvironment regulation. In terms of serotype selection, AAV9 or AAV-PHP.eB is recommended to penetrate the blood-brain barrier and achieve a high glioma cell transduction efficiency. The injection dose was 1 - 5E+11 vg / mouse, diluted with 100 μL of formulation buffer.

[0028] Here, the AAV vector has a higher blood-brain barrier penetration ability (especially the AAV9 and AAV-PHP.eB serotypes), can continuously and stably express gene products, and has low immunogenicity in small animals. This technology improves gene delivery efficiency, reduces off-target effects, and can more effectively regulate the tumor immune microenvironment.

[0029] S7, AAV Transgene Expression Monitoring To monitor the expression of the AAV transgene, it can be detected by various methods. The qPCR technique was used to detect the mRNA expression level, and Western Blot or ELISA techniques were used to detect the protein expression level. In addition, immunohistochemistry (IHC) or immunofluorescence (IF) staining was used to further verify the transgene expression products in tissues.

[0030] S8, Immune Microenvironment and Tumor Growth Monitoring After establishing the immune microenvironment, the immune cells in the tumor were analyzed by flow cytometry, especially the infiltration of immune cells such as CD8+ T cells, Tregs, MDSCs, macrophages, etc. Compared with the existing glioma mouse models that usually can only evaluate tumor growth, this application can monitor the immune cell composition and its changes in the tumor microenvironment in real time, helping to deeply understand the immune escape mechanism and the dynamic changes of the immune microenvironment, and providing a more refined research tool for the evaluation and optimization of immunotherapy.

[0031] Meanwhile, the expression of immune co-stimulatory / co-inhibitory molecules (such as PD-L1, CD80 / CD86, etc.) was detected by immunofluorescence staining. In addition, the levels of cytokines (such as IL-6, IFN-γ, etc.) can also be detected by ELISA or Luminex. The growth of the tumor was monitored by small animal MRI or CT scan, the change of tumor volume was evaluated, and the morphology of tumor cells was observed by HE staining. Compared with the traditional tumor models, the use of non-invasive imaging technology can monitor the growth process of the tumor in real time and continuously, reduce the harm to experimental animals, improve the experimental efficiency, and increase the repeatability of the data.

[0032] S9. Validation and advantages of the model The repeatability of the model was verified by statistically analyzing the tumor incidence rate and growth rate among different experimental batches. In addition, this model was compared with other existing glioma models to evaluate its response in immunotherapy or targeted therapy. Finally, the scientific nature and clinical translation potential of this model were evaluated through indicators such as survival time, immune infiltration, and gene expression profile.

[0033] The above steps will be further introduced in detail in combination with multiple groups of examples as follows: Example 1: Verification of immune reconstruction of human CD34+ mesenchymal stem cells based on NOG mice Experimental animals and grouping In this experiment, 3-week-old female NOG mice (NOD.Cg- Prkdc scid Il2rg tm1Sug / JicCrl) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0034] The irradiated NOG mice were randomly divided into 2 groups according to the experimental requirements, with 5 mice in each group. Among them, the mice in the blank control group were injected with 100 μL of PBS into the tail vein of each mouse; Stem cell immune reconstruction group: Each mouse was injected with human CD34+ mesenchymal stem cells (suspended in 100 μL of PBS) into the tail vein.

[0035] Immune reconstruction monitoring Peripheral blood was collected from mice about 4 weeks after transplantation of human CD34+ mesenchymal stem cells (50 - 100 μL each time, anticoagulated with EDTA); the results of immune reconstruction were verified by flow cytometry, and the antibody combinations for flow cytometry (all purchased from BioLegend), with specific information as Figure 8 shown.

[0036] Flow cytometry operation Red blood cell lysis: Incubate with ACK lysis buffer (Thermo Fisher) at room temperature for 3 - 5 minutes; centrifuge at 300 × g for 5 minutes at room temperature to collect white blood cells; aspirate the supernatant, leaving about 50 µL of liquid to avoid disturbing the precipitate; gently mix the cells and the remaining liquid, then add 5 mL of pre-cooled PBS buffer; mix the cells and the buffer, and then collect the cells by centrifuging at 300 × g for 5 minutes at 2 - 8 °C; aspirate the supernatant, and then resuspend the cells in PBS buffer added as needed at 2 - 8 °C.

[0037] Antibody staining: Add antibodies as needed and according to the antibody instruction manual, incubate in the dark for 20 minutes (4 °C); add about 1 mL of PBS to resuspend and dilute, centrifuge at 300 × g for 5 minutes to collect the cells; resuspend in 100 µL of FACS buffer and load onto the machine.

[0038] Data analysis: Collected using BD FACSDiva software and analyzed with FlowJo v10.8.

[0039] The experimental results and schematic diagrams are as Figure 2 and Figure 9 shown. Among them, Figure A: Total cell FSC / SSC population plot, showing granulocytes (high FSC / SSC), monocytes (medium FSC / SSC), and lymphocytes (low FSC / SSC); Figure B: CD45+ cell sorting (abscissa CD45 PerCP-Cy5.5, ordinate FSC); Figure C: CD3+ and CD3- cell population (abscissa CD3 APC, ordinate CD45PerCP-Cy5.5); Figure D: CD4+ Th and CD8+ Tc subset analysis (abscissa CD4 BV421, ordinate CD8a PE-Cy7); Figure E: CD19+ B cells and CD56+ NK cell population (abscissa CD19 FITC, ordinate CD56 PE).

[0040] Experimental conclusion In this example, it was verified by flow cytometry that transplantation of CD34+ MSCs could significantly reconstruct human T cells (CD3+) and B cells (CD19+). Figures A - E clearly show the cell populations and subset ratios, proving that the method described in the present invention can effectively construct the immune basis of a humanized glioma PDX model.

[0041] Example 2: Effects of Different Immunological Reconstitution Conditions on the Intracranial Tumor Growth Curve of Mouse Glioma Tissue Cells after Transplantation Experimental Animals and Grouping In this experiment, 3-week-old female NOG mice (NOD.Cg- Prkdc scid Il2rg tm1Sug / JicCrl) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0042] The irradiated NOG mice were randomly divided into 3 groups according to the experimental requirements, with 5 mice in each group. Among them, each mouse in the blank control group was injected with 100 μL of PBS via the tail vein; each mouse in the stem cell immunological reconstitution group was injected with human CD34+ mesenchymal stem cells (suspended in 100 μL of PBS) via the tail vein, and each mouse in the non-immunological intervention group was injected with mouse embryonic fibroblast NIH3T3 (suspended in 100 μL of PBS) via the tail vein.

[0043] Monitoring of Immunological Reconstitution About 4 weeks after transplantation of human CD34+ mesenchymal stem cells, peripheral blood was collected from the mice (50 - 100 μL each time, anticoagulated with EDTA); the results of immunological reconstitution were verified by flow cytometry. The antibody combinations for flow cytometry (all purchased from BioLegend), and the specific antibody information and relevant information on the flow cytometry detection process were the same as in Example 1.

[0044] Tumor Transplantation after Immunological Reconstitution The tumor tissue (pathologically diagnosed as IDH wild-type glioblastoma) from patients that met the screening criteria was digested into a cell suspension and implanted into the left or right striatal region of the mice (coordinates: 2.0 mm posterior to the bregma, 2.0 mm left or right of the midline, depth 2.5 - 3.0 mm). The specific digestion process is as follows: Preparation of Single-Cell Suspension - Mixed Enzyme Digestion Method a) After all the tumors were dissected, the tumors were transferred to the pretreatment solution. Using a curved scissors by hand, the tumors were fully cut into pieces of 1 - 3 mm 3 in size, left standing for a few seconds, and the upper smaller particles were aspirated using a 1 mL pipette. The tissue was further cut into pieces, and normal saline was repeatedly added until the size of all the tissues met the requirements. This process was carried out on ice and should not exceed 30 minutes as much as possible.

[0045] b) Transfer the tumor tissue suspension into a 50 mL centrifuge tube, add Neurobasal medium, centrifuge at 250 × g for 5 minutes, discard the supernatant, and add 20 mL of tumor tissue digestive solution (Neurobasal medium with a final concentration of 0.1% collagenase IV, 0.125% trypsin, and 0.002% DNase).

[0046] c) Gently pipette to mix well, transfer to a culture flask, and incubate in a 37 °C shaker for 1 - 2 hours. Observe the digestion status under the microscope every 30 minutes to determine whether to terminate the digestion.

[0047] d) After digestion, dilute with PBS, then use a 200 - mesh sieve to remove the remaining tissue blocks, and wash twice with 5 - 10 volumes of PBS buffer until there are no tissue blocks left, obtaining a single - cell suspension.

[0048] e) Collect the single - cell suspension, centrifuge at 300 × g for 5 minutes, and discard the supernatant.

[0049] f) Resuspend the cells with PBS (added with one - tenth volume of Matrigel), and adjust the cell concentration to 5×10 8 / mL.

[0050] Tumor growth monitoring After the establishment of the glioma animal model in this invention, the dynamic growth of the tumor was monitored by a small - animal MRI imaging system as follows: Using a Bruker BioSpec 70 / 30 type 7T small - animal magnetic resonance imaging system, perform T2 - weighted fast spin - echo (T2WI - TSE) scanning. The scanning parameters are set as: TR / TE = 3000 / 50 ms, slice thickness 0.5 mm, field of view 20×20 mm, matrix 256×256, NEX = 2. Scan regularly once after the tumor is implanted in situ.

[0051] Tumor volume analysis was performed using Paravision 6.0 software. The specific operation is as follows: Import the acquired images into the software, identify the high - signal tumor area, depict the ROI layer by layer, and use the built - in volume measurement module of the system to automatically calculate the tumor volume based on the area of each layer of ROI and the slice thickness, and export the data changing with time to judge the growth of the tumor in the model and the establishment effect of the immune regulation effect. The specific results are as follows.

[0052] The experimental results are as Figure 3 and Figure 10 , in which the tumor mass volumes of each model construction group can increase with time, indicating that the model construction meets the expectations. The tumor volumes of the PBS control group and the non - immune intervention group (NIH3T3) increased significantly faster than those of the stem cell injection group ( P<0.01), but there was no significant difference between the PBS control group and the non-immune intervention group (NIH3T3), indicating that non-human cells could not effectively regulate the tumor microenvironment; after the reconstruction of the human immune system, the tumor growth itself was inhibited to a certain extent, reflecting the importance of immune microenvironment construction.

[0053] Example 3: Target protein after AAV injection TGF-β1 , IL10 , CSF1 mRNA expression in peripheral blood and tumor tissues This example was used to verify the expression effect of the constructed AAV-mediated expression system in mice with glioma model, and to detect TGF-β1 , IL10 , CSF1 mRNA levels of three immune regulatory factors.

[0054] NOG mice reconstructed with human CD34+ hematopoietic stem cells were selected as experimental animals, and orthotopic inoculation of donor tumor cells of glioma was completed. After successful modeling, the animals were randomly divided into two groups, with 8 model mice in each group.

[0055] Control group (blank group): The same volume of preparation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl and 0.005 mM Tween-20) was injected through the tail vein; Experimental group (AAV immune reconstruction group): The mixed AAV vector was injected through the tail vein. The mixed AAV vector included: AAV-TGFβ1-P2A-IL10 vector (co-expression of TGF-β1 and IL10 driven by the CAG promoter); AAV-CSF1 vector (CSF1 expression driven by the CAG promoter), and the structure is as Figure 13 and Figure 14 shown.

[0056] The AAV injection dose was 1E+11 vg per mouse and was dissolved in 100 μL of preparation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl and 0.005 mM Tween-20) for injection.

[0057] At 3-4 weeks after injection, peripheral blood of mice was collected through the submandibular vein, PBMC cells were separated, and at the same time, some animals were sacrificed and tumor tissues were separated.

[0058] Total RNA in peripheral blood PBMC and tumor tissues was extracted using an RNA extraction kit, cDNA was synthesized using a reverse transcription kit, and then qPCR was used to detect TGF-β1 ,IL10 and CSF1 mRNA expression levels GAPDH as internal reference genes

[0059] The experimental results showed that, compared with the control group, in the peripheral blood PBMC and tumor tissues of the AAV injection group TGF-β1 、 IL10 and CSF1 the mRNA expression levels of the genes were all significantly up-regulated ( P <0.01), indicating that the constructed AAV vector could effectively express the target immune regulatory factors in vivo and had the functional basis for mediating the regulation of the immune microenvironment of glioma. The specific results were as shown in Figure 4 and Figure 5 shown

[0060] Example 4: Immunohistochemistry after AAV injection to determine the expression of target proteins TGF-β1, IL10, and CSF1 in tumor tissues To further verify whether the constructed AAV vector could successfully express the target proteins related to immune regulation in vivo, immunohistochemical staining was used to detect the expression of TGF-β1, IL10, and CSF1 in glioma tissues

[0061] NOG mice with a humanized CD34+ hematopoietic stem cell-reconstituted immune system were selected as experimental animals, and orthotopic inoculation of donor tumor cells of glioma was completed. After successful modeling, the animals were randomly divided into 2 groups, with 8 model mice in each group

[0062] Control group (blank group): An equal volume of formulation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl, and 0.005 mM Tween-20.) was injected via the tail vein Experimental group (AAV immune reconstitution group): A mixed AAV vector was injected via the tail vein. The mixed AAV vector contained AAV-TGFβ1-P2A-IL10 vector (co-expression of TGFβ1 and IL10 driven by the CAG promoter); AAV-CSF1 vector (expression of CSF1 driven by the CAG promoter), and the structure was as shown in Figure 13 and Figure 14 shown

[0063] The AAV injection dose was 1 × 10¹¹ vg per mouse and was dissolved in 100 μL of formulation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl, and 0.005 mM Tween-20) for injection

[0064] At 3 - 4 weeks after injection, some animals were sacrificed and glioma tissues were isolated, fixed in 4% paraformaldehyde for 24 hours, and paraffin sections (section thickness 4 μm) were prepared after conventional dehydration and embedding for subsequent immunohistochemical staining.

[0065] The staining procedure is as follows: After the sections were dewaxed with xylene and rehydrated with gradient ethanol, antigen retrieval was performed using citrate buffer; Use 3% Block the activity of endogenous peroxidase; Use normal goat serum to block non - specific binding sites; According to the experimental design, primary antibodies were separately dropped onto the section tissues: rabbit anti - mouse TGF - β1, IL10, CSF1 antibodies, and incubated overnight at 4°C; The next day, add HRP - labeled goat anti - rabbit secondary antibody and incubate at room temperature for 1 hour; Develop color with DAB, counterstain with hematoxylin, dehydrate and mount the slides; Observe and photograph under a light microscope.

[0066] As Figure 6 The results showed that the protein expressions of TGF - β1, IL10, and CSF1 in the tumor tissues of the AAV - injected group of mice were significantly enhanced. The brown - yellow granules were mainly distributed in the tumor cells and the surrounding infiltrating immune cell regions. Compared with the control group, the expression levels were significantly increased ( P <0.01).

[0067] This result was consistent with the mRNA detection result, further verifying that the AAV - TGFβ1 - P2A - IL10 vector and the AAV - CSF1 vector could synergistically drive the expression of the target immune regulatory factors in vivo, thus laying a foundation for regulating the tumor immune microenvironment.

[0068] Example 5: Determination of the changes in the tumor tissue immune microenvironment after immune system reconstruction by flow cytometry The purpose of this example was to compare the compositional differences in the immune microenvironment in glioma tissues before and after stem cell immune reconstruction and AAV - mediated immune regulation by flow cytometry, so as to verify the effect of the AAV - mediated immune microenvironment construction method of the present invention on the tumor immune state.

[0069] Three - week - old female NOG mice were selected for the experiment. The mice were randomly divided into 3 groups, with 6 mice in each group. According to the experimental design, the mice in each group were operated according to the following plan.

[0070] Single glioma model control group: No immune reconstruction was performed, and only a glioma model was established; Stem cell reconstruction + glioma model group: Received CD34+ stem cell reconstruction and established a glioma model after reconstruction; Stem cell + AAV + glioma model group: Received CD34+ stem cell reconstruction. After reconstruction, a glioma model was established and an AAV vector mixture was injected via the tail vein to further construct an immune regulatory microenvironment.

[0071] The mixed AAV vector contains: AAV-TGFβ1-P2A-IL10 vector (co-expression of TGF-β1 and IL10 driven by the CAG promoter); AAV-CSF1 vector (CSF1 expression driven by the CAG promoter), with the structure as Figure 13 and Figure 14 shown.

[0072] The AAV injection dose was 1E+11 vg per mouse and was dissolved in 100 μL of formulation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl and 0.005 mM Tween-20) for injection.

[0073] Four weeks after AAV injection (i.e., the 5th week after tumor inoculation), the mice were sacrificed, the tumor tissues were isolated, and single-cell suspensions were prepared by enzymatic digestion. The enzymatic digestion solution was RPMI-1640 medium containing 1 mg / mL collagenase IV and 0.1 mg / mL DNase I. After incubation in a 37°C water bath for 30 minutes, it was filtered through a 70 μm cell sieve. ACK lysate was used to remove red blood cells, and after resuspension with PBS, it was used for subsequent staining. Human-specific antibodies were used for surface staining, including: T cell subsets: ; Myeloid cells: ; Immunosuppressive cells: type macrophages, ; Cytokine-related markers: ,, cells; Surface and intracellular staining was performed using human antibodies as described in Figure 11 . All antibodies were purchased from Biolegend or BD.

[0074] After staining, the cells were resuspended in FACS buffer for detection.

[0075] Data were acquired using BD FACSDiva software and analyzed using FlowJo v10.8.

[0076] The main results are as shown in Figure 12 : The proportion of Treg cells in the tumor tissues in the combined reconstruction group was significantly increased; simultaneously, it was accompanied by M2-type macrophages Increased; Compared with the overall proportion of cells, it decreased slightly compared with the stem cell reconstruction group; Compared with cells were significantly increased in the combined reconstruction group; the above factors showed an increasing trend in the single AAV intervention group, but were lower than those in the combined group; the levels of the above immunosuppressive cells / factors were the lowest in the single stem cell reconstruction group.

[0077] Conclusion: The results of this example show that although single stem cell reconstruction can partially restore the infiltration of human immune cells, it has not formed a tumor-related immune microenvironment; while further using the AAV-mediated method of the present invention on the basis of stem cell reconstruction can effectively induce the expression of immune regulatory factors such as TGF-β1, IL10, and CSF1, significantly change the types and proportions of immune cells in the tumor microenvironment, tend to be enriched in M2 macrophages and upregulate regulatory T cells, presenting the characteristics of an "immunosuppressive microenvironment", verifying the effectiveness and feasibility of constructing an immune microenvironment by the present invention.

[0078] In summary, the present invention has achieved significant improvements in the following aspects: Precisely simulate the immune microenvironment of human GBM.

[0079] Traditional models have failed to effectively reproduce the immunosuppressive characteristics of GBM, while this scheme enhances Treg activity and increases M2 macrophage infiltration through AAV-mediated immune regulation (such as key immune factors such as PD-L1, CSF1, and TGF-β1), making the immune environment closer to that of human GBM patients.

[0080] This strategy is more in line with the tumor immune characteristics of GBM, making it more suitable for studying the immune escape mechanism and developing immune therapy strategies.

[0081] By single injection of AAV, long-term regulation of the immune microenvironment is achieved.

[0082] Traditional GBM models usually rely on local injection or gene editing to change the immune environment, while this scheme can stably express specific immune regulatory factors in the central nervous system (CNS) for a long time through single tail vein injection of AAV.

[0083] This method not only reduces the stress response of experimental animals and surgery-related injuries, but also can dynamically regulate the immune microenvironment throughout the disease process, making the model more physiologically relevant.

[0084] The model establishment is stable and rapid, improving the experimental efficiency.

[0085] By optimizing the model establishment process, the repeatability and reliability of the model are improved, accelerating the research process and improving the experimental efficiency.

[0086] More suitable for immunotherapy research and improve the clinical translation value.

[0087] Traditional GBM mouse models have poor predictability for immunotherapy (such as anti-PD-1 antibody and CAR-T therapy). However, this protocol improves the research reliability of strategies such as immune checkpoint inhibitors (ICIs), CAR-T cell therapy, and tumor vaccines by optimizing the immune microenvironment.

[0088] This model better simulates the immune response characteristics of clinical GBM and can be used as an important tool for screening and optimizing immunotherapy strategies.

[0089] This protocol breaks through the limitations of existing GBM animal models and provides a more accurate and efficient model for studying the occurrence, development, and immune microenvironment of GBM. This model not only improves the research accuracy of immunotherapy but also accelerates drug screening and clinical translation, providing more powerful support for the basic research and clinical treatment of GBM.

[0090] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing a glioma animal model based on AAV-mediated immune microenvironment, characterized in that It includes the following steps: Select immunodeficient mice of appropriate age; Reconstruct the immune system by injecting human CD34+ hematopoietic stem cells via the tail vein; Use flow cytometry to detect human CD45+ immune cells in peripheral blood PBMCs to confirm successful reconstruction of the immune system; Select tumor tissues with high expression of glioma marker genes, perform rapid qPCR or targeted RNA-seq immunohistochemical multiplex verification, and screen tumor tissues that meet the requirements of the glioma animal model; Physically cut the selected tumor tissues, treat them with a pretreatment solution, digest them into single-cell suspensions, and inoculate the single-cell suspensions into the mouse intracranium using stereotactic injection; Inject an AAV vector carrying a specific immune regulatory gene into the mouse tail vein to regulate the glioma immune microenvironment. When stable expression of the AAV expression product in the mouse body is detected, complete the immune regulation mediated by the AAV expression product; When it is observed that the tumor tissue grows stably and has the characteristics of immune cell infiltration, confirm that the reconstruction of the immune microenvironment and tumor growth meet the requirements of model construction, and complete the construction of the glioma animal model.

2. The method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, characterized in that: The AAV vector includes an AAV-TGFβ1-P2A-IL10 expression cassette and an AAV-CSF1 expression cassette for expressing TGF-β1 , IL10 and CSF1 genes; The AAV-TGFβ1-P2A-IL10 expression cassette comprises a nucleic acid structure in which the TGF-β1 gene and the IL10 gene are co-expressed via a P2A self-cleaving peptide sequence and are driven by a CAG promoter, and the total length of the nucleic acid sequence is 3.1±0.2 kb; TGF-β1 gene and IL10 gene, with a total nucleic acid sequence length of 3.1±0.2 kb; The AAV-CSF1 expression cassette includes a CSF1 gene expression unit driven by the CAG promoter, and the total length of the nucleic acid sequence is 2.3 ± 0.2 kb.

3. The method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 2, wherein Performed using AAV9 or AAV-PHP.eB serotype for TGF-β1 , IL10 and CSF1 gene delivery.

4. A method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, characterized in that, The injection dose of the AAV vector is 1 - 5E+11 vg / mouse; The AAV vector is formulated in a buffer with a pH of 7.7 to 8.3, and the buffer contains 20 mM Tris, 1 mM , 200 mM NaCl, and 0.005 mM Tween-20.

5. A method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, characterized in that The glioma marker genes include CD133 protein, IDH1 R132H mutation, EGFR amplification, MGMT promoter methylation, and SOX2 protein.

6. The method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, wherein The pretreatment solution includes Neurobasal medium and Matrigel mixed at a volume ratio of 3:1, and supplemented with 2×B27, 2 mM glutathione, 50 U / mL penicillin-streptomycin, and 20 ng / mL PDGF-BB.

7. A method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, characterized in that, The growth monitoring of the tumor tissue is obtained by calculating the change in the T2-weighted image of small animal MRI combined with the volume.

8. A method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, characterized in that, The morphology of the tumor cells is obtained by observing HE staining.

9. The method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, wherein, The method for detecting significant expression of the AAV expression product in the mouse body includes: Using qPCR technology to detect the expression level of mRNA; Using Western Blot or ELISA technology to detect the expression level of protein; Using immunohistochemical staining or immunofluorescent staining to further verify the transgenic expression product in the tissue.

10. A method for constructing a glioma animal model based on AAV-mediated immune microenvironment according to claim 1, characterized in that, The tumor tissue selected is a tumor tissue pathologically diagnosed as WHO grade III or IV.

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