GBM animal model and construction method and application thereof

Abstract

The invention discloses a GBM animal model and a construction method and application thereof, and belongs to the technical field of animal model construction. A construction method of a GBM mouse model comprises the following steps that S1, a mouse is anesthetized, the respiratory rate of the mouse is adjusted, and after prepared skin is disinfected, the mouse is cut backwards from the midpoint of a connecting line of inner-eye canthi along a median sagittal line; s2, drilling, injecting a cell suspension, retaining a needle for a certain time, slowly pulling out the needle, and plugging a needle hole; s3, performing disinfection after surgical incision suturing, performing conventional feeding, and checking whether modeling succeeds or not. U87 cells of a female NOD SCID mouse of 8 weeks old are adopted as immune cells, the GBM mouse model is successfully constructed through a specific injection method at a specific position, a specific depth and a specific injection method on the head of the mouse, the GBM of the model mouse is heterogeneous GBM, and the model is stable and reliable and can be used for GBM research and new drug curative effect test; the method provided by the invention is high in success rate of constructing the heterogeneity GBM mouse model, and is simple and easy to operate.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, and in particular to a GBM animal model, its construction method, and its application. Background Technology

[0002] The annual incidence of gliomas in my country is 5–8 cases per 100,000, making it a relatively common type of intracranial tumor, typically accounting for about 40% of all intracranial tumors. Its 5-year mortality rate is second only to pancreatic and lung cancer among all cancers. Glioblastoma (GBM), the most malignant type of glioma, has an incidence of approximately 3.2 cases per 100,000. It is the most common and malignant primary central nervous system tumor, accounting for approximately 35.2%–61.0% of all intracranial tumors. It is the brain tumor with the highest mortality and disability rates, posing the greatest threat to families and society. Even with standard surgery and comprehensive treatment including radiotherapy and chemotherapy, the median overall survival (OS) is only 14.6 months. One of the main reasons for its poor prognosis is that GBM cannot be completely surgically removed. Residual GBM cells continue to infiltrate brain tissue, leading to treatment failure. This process is a result of the interaction between GBM cells and brain cells (neurons, astrocytes, etc.).

[0003] Animal models are experimental animals that are artificially induced to exhibit specific pathological features or pathogenesis of a target disease. Animal models are widely used in medical and life science research to explore the causes and progression of diseases, test potential treatments, and conduct safety and efficacy tests on new drugs.

[0004] Constructing GBM animal models faces numerous challenges, primarily stemming from the complexity of GBM. While replicating this complexity, it's crucial to ensure the model's reliability and reproducibility. The main difficulties in constructing GBM animal models include the following aspects:

[0005] 1) Disease complexity: GBM is a highly heterogeneous tumor, containing multiple cell types and molecular subtypes. The immune systems of humans and animals (such as mice) are significantly different, resulting in a large difference between the immune environment of animals (such as mice) and humans. Therefore, animal models often cannot fully reproduce this complex heterogeneity.

[0006] 2) Tumor microenvironment: GBM invasiveness and angiogenesis depend on a complex tumor microenvironment, including cytokines, growth factors, cell-cell interactions, and matrix components. Animal models struggle to accurately replicate all aspects of this microenvironment.

[0007] 3) Immune rejection: When using human-derived GBM cell lines or tissues to construct models, the host's immune system may recognize and attack these xenografts, leading to unstable or unreliable models, which in turn affects the consistency of experimental or research results.

[0008] 4) Differences in genetic background: Human genetic diversity is far greater than that of laboratory animals. This means that animal models may not fully reflect the genetic variations of human tumors, which in turn affects tumor behavior and response to treatment. Therefore, even if animal models show therapeutic potential in research, it cannot be guaranteed that these findings can be directly translated into clinical applications in humans.

[0009] Therefore, it is essential to develop a stable method for constructing GBM animal models that can reflect the high heterogeneity of GBM as much as possible. Summary of the Invention

[0010] The purpose of this invention is to provide a GBM animal model, its construction method, and its application. The provided method is simple and easy to operate, can reflect the high heterogeneity of GBM, and the constructed GBM animal model has high stability.

[0011] To achieve the above objectives, this invention provides a method for constructing a GBM mouse model, comprising the following steps:

[0012] S1. NOD SCID mice were used. They were fasted for 12 hours and deprived of water for 4 hours before the operation. The mice were anesthetized and their respiratory rate was controlled at 60-90 breaths / min. After the head was prepared and disinfected, the skull was fully exposed by making an incision along the midline of the line connecting the inner canthi of the two eyes, starting from the midpoint of the line connecting the inner canthi. The external auditory canal of the mice was then symmetrically fixed at the two needles of the stereotactic instrument, and the incisors were fixed in front.

[0013] S2. The intersection of the mouse's anterior fontanelle and the midline is set as the origin. After determining the target location, a hole is drilled, and the cell suspension is injected. After the injection is completed, the needle is left in place for a certain period of time, then slowly pulled out and the needle hole is sealed.

[0014] S3. The wound made in S1 was sutured on a sterile operating table, disinfected again, placed in a cage, and kept warm with clean bedding for routine feeding; then an ultrasound examination was performed to examine the tumor-forming mice and identify the mice that had successfully modeled the tumor.

[0015] Preferably, the NOD SCID mice in S1 are 6-8 week old female mice, and anesthesia is performed using 1-1.5% isoflurane in an anesthesia induction box.

[0016] Preferably, after successful anesthesia induction, the mouse's head is fixed on a stereotaxic apparatus, and the respiratory rate of the mouse is adjusted to 60-90 breaths / min by adjusting the isoflurane concentration.

[0017] Preferably, iodine tincture is used for disinfection in both S1 and S2.

[0018] Preferably, the target position in S2 is 3mm from the origin and 2mm behind it, and the drilling is performed using a dental drill.

[0019] Preferably, the cell suspension in S2 is a suspension of U78 with a concentration of 4 × 10⁻⁶. 4 / mL.

[0020] Preferably, the injection in S2 is performed using a micro-syringe with an injection volume of 5 μL. During injection, the needle is slowly and vertically inserted along the drilled bone hole to 3 mm below the dura mater, and then withdrawn by 1 mm. The injection is performed slowly at a rate of 1 μL / min.

[0021] Preferably, the needle is left in S2 for 1 minute, and the needle hole is sealed with bone wax; the day of modeling is recorded as day 1, and ultrasound examination is performed starting on day 5; if any functional impairment occurs during the modeling process, the mouse is euthanized by cervical dislocation.

[0022] A GBM mouse model constructed using the method described above.

[0023] Application of a GBM mouse model as described above in the testing of new GBM drugs.

[0024] Therefore, the GBM animal model, its construction method, and its application provided by this invention have the following specific technical effects:

[0025] (1) The GBM mouse model was successfully constructed using the method provided in this invention. The GBM in the model mice is heterogeneous GBM. The model is stable and reliable and can be used for GBM research and new drug efficacy testing.

[0026] (2) The method provided by this invention has a high success rate in constructing heterogeneous GBM mouse models and is simple and easy to operate.

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a flowchart of the GBM mouse model construction in Embodiment 2 of the present invention;

[0030] Figure 2This is an MRI image of the GBM mouse model in Embodiment 3 of the present invention; the arrow points to the tumor. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] The instruments, equipment, reagents and materials used in the examples were all obtained commercially; the methods and steps not described in detail are all conventional techniques in the field; the animal experiments were approved by the Animal Welfare and Ethics Committee of the Beijing Neurosurgical Institute.

[0034] Example 1

[0035] The specific steps for preparing a U78 cell suspension are as follows:

[0036] (1) Culture medium: DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin was used.

[0037] (2) Resuscitation: The live cells from the received T25 flasks are first disinfected with 75% alcohol. Any T25 flasks with broken culture flasks, liquid spillage, or cell contamination are removed. Then, the intact and uncontaminated T25 flasks are placed in an incubator at 37°C and 5% CO2 for about 2 to 3 hours.

[0038] (3) Passage: Passage is performed when the cell density reaches 80-90%.

[0039] For adherent cells, follow these steps: ① Discard the culture supernatant, rinse the cells twice with calcium- and magnesium-free PBS, then add 2 mL of 0.25% trypsin solution and digest at 37°C for 3 min. Observe whether the cells begin to detach, and gently tap the culture flask to promote cell detachment. ② Add culture medium containing FBS serum to stop the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells, and seed them into new culture flasks at a ratio of 1:2 to 1:4. ③ Incubate the cells in a 37°C, 5% CO2 incubator, changing the culture medium every 2–3 days.

[0040] (4) Observe the state of rat U78 glioma cells under a microscope, prepare a single-cell suspension and place it in a 2mL centrifuge tube (steps as above), stain with trypan blue and count the cells using a cell counter, add DMEM culture medium and adjust the concentration to 4×10⁻⁶. 4 Cells / mL, take 2mL and place it in a centrifuge tube and keep it in an incubator for later use.

[0041] Example 2

[0042] The specific steps for constructing a GBM mouse model are as follows:

[0043] S1. Eight-week-old female NOD SCID mice were selected. After fasting for 12 hours and withholding water for 4 hours, the mice were placed in an anesthesia induction box and anesthetized with 1.5% isoflurane. After successful anesthesia induction, the mouse head was fixed to a stereotaxic apparatus. The isoflurane concentration was adjusted to maintain the mouse's respiratory rate at 60–90 breaths / min. After shaving the head and disinfecting with iodine, an incision was made along the midline of the line connecting the inner canthi of both eyes, extending posteriorly to fully expose the skull. The mouse's external auditory canal was then symmetrically fixed to the two pin points of the stereotaxic apparatus, with the incisors fixed anteriorly.

[0044] S2. Based on the mouse head anatomy atlas, the intersection of the anterior fontanelle and the midline was designated as the origin. A hole was drilled 3 mm from the origin and 2 mm posterior to it using a dental drill. Then, 5 μL of the U78 cell suspension prepared in Example 1 (4 × 10⁻⁶ cells) was aspirated using a microsyringe. 4 (1 cell / mL) The needle is slowly and vertically inserted 3 mm below the dura mater through the drilled bone hole, then withdrawn 1 mm to allow space for the injected cells and prevent leakage. The cell suspension is slowly injected at a rate of 1 μL / min. After injection, the needle is left in place for 1 minute, then slowly withdrawn and sealed with bone wax. The surgical incision made in step S1 is sutured, and the wound is disinfected again with povidone-iodine. The mouse is then placed in a cage lined with clean bedding to keep it warm.

[0045] S3. Starting from day 1 of modeling, an ultrasound examination is performed on day 5. The mice with tumors are used as experimental subjects. If any functional impairment occurs in the mice during the modeling process, they are euthanized by cervical dislocation.

[0046] Example 3

[0047] To investigate whether the tumor-bearing mice obtained in Example 2 were successfully modeled GBM mice, the following steps were taken 5 days post-surgery using MRI:

[0048] Mice were anesthetized by intraperitoneal injection of 0.8% sodium pentobarbital and placed in a mouse coil. T1-weighted fast spin echo (TSE) enhanced scans of the mouse head were performed in the transverse, coronal, and sagittal planes using a 7.0T MRI system. Specific parameters were: transverse plane slice thickness 0.8 mm, interval 0.2 mm, FOV 39 mm × 50 mm, matrix 296 × 384, TR / TE = 1130 ms / 15 ms; coronal plane slice thickness 1.0 mm, interval 0.2 mm, FOV 39 mm × 50 mm, matrix 296 × 384, TR / TE = 1130 ms / 15 ms; sagittal plane slice thickness 1.0 mm, interval 0.2 mm, FOV 31 mm × 40 mm, matrix 296 × 384, TR / TE = 1000 ms / 17 ms. The imaging and location of xenografts in the brains of nude mice were observed, and the maximum anteroposterior, lateral, and superior-inferior diameters of the tumors were measured using the built-in software. The tumor volume (mm) at different ages was calculated. 3 = Front-to-back diameter × Left-to-right diameter × Top-to-bottom diameter. The result is as follows: Figure 2 As shown, after intracranial glioma implantation, a round or oval high-signal mass with localized heterogeneous mixed signal was observed in the caudate nucleus region on plain MRI scan.

[0049] Therefore, this invention uses 8-week-old female NOD SCID mice and U87 cells as immune cells to successfully construct a GBM mouse model by injecting the cells at specific locations and depths in the mouse head using a specific method. The GBM in the model mice is heterogeneous, and the model is stable and reliable, and can be used for GBM research and new drug efficacy testing. The method provided by this invention has a high success rate in constructing heterogeneous GBM mouse models and is simple and easy to operate.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a GBM mouse model, characterized in that, The steps are as follows: S1. NOD SCID mice were used. They were fasted for 12 hours and deprived of water for 4 hours before the operation. The mice were anesthetized and their respiratory rate was controlled at 60-90 breaths / min. After the head was prepared and disinfected, the skull was fully exposed by making an incision along the midline of the line connecting the inner canthi of the two eyes, starting from the midpoint of the line connecting the inner canthi. The external auditory canal of the mice was then symmetrically fixed at the two needles of the stereotactic instrument, and the incisors were fixed in front. S2. The intersection of the mouse's anterior fontanelle and the midline is set as the origin. After determining the target location, a hole is drilled, and the cell suspension is injected. After the injection is completed, the needle is left in place for a certain period of time, then slowly pulled out and the needle hole is sealed. S3. The wound made in S1 was sutured on a sterile operating table, disinfected again, placed in a cage, and kept warm with clean bedding for routine feeding; then an ultrasound examination was performed to examine the tumor-forming mice and identify the mice that had successfully modeled the tumor.

2. The method for constructing a GBM mouse model according to claim 1, characterized in that: In S1, NOD SCID mice were 6-8 week old females, and anesthesia was performed using 1-1.5% isoflurane in an anesthesia induction box.

3. The method for constructing a GBM mouse model according to claim 1, characterized in that: After successful anesthesia induction, the mouse's head was fixed on a stereotaxic apparatus, and the respiratory rate of the mouse was adjusted to 60-90 breaths / min by adjusting the isoflurane concentration.

4. The method for constructing a GBM mouse model according to claim 1, characterized in that: Iodine tincture is used for disinfection in both S1 and S2.

5. The method for constructing a GBM mouse model according to claim 1, characterized in that: The target position in S2 is 3mm from the origin and 2mm behind it. The drilling is done using a dental drill.

6. The method for constructing a GBM mouse model according to claim 1, characterized in that: The cell suspension in S2 was a suspension of U78 cells with a concentration of 4 × 10⁻⁶. 4 / mL.

7. The method for constructing a GBM mouse model according to claim 1, characterized in that: In S2, a micro-syringe is used for injection, with an injection volume of 5 μL. During injection, the needle is slowly and vertically inserted along the drilled bone hole to 3 mm below the dura mater, and then withdrawn 1 mm. The injection is slow and is carried out at a rate of 1 μL / min.

8. The method for constructing a GBM mouse model according to claim 1, characterized in that: The needle was left in the S2 for 1 minute, and the puncture hole was sealed with bone wax. The day of modeling was recorded as day 1, and ultrasound examination was performed starting on day 5. If any functional impairment occurred during the modeling process, the mouse was euthanized by cervical dislocation.

9. A GBM mouse model constructed by the construction method according to any one of claims 1 to 8.

10. The application of the GBM mouse model as described in claim 9 in the testing of new GBM drugs.

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