Method and system for inducing systemic amyloidosis mouse model

Abstract

The invention relates to an inducing method and system of a systemic amyloid degeneration mouse model, and relates to the technical field of biomedical models, amyloid substances extracted from diseased tissues are injected into a standard strain laboratory mouse as an inducing medium, and the disease progress is controlled by regulating and controlling parameters such as injection dosage and frequency. The method successfully overcomes the defects that an existing model depends on scarce lines or genetic modification, the disease is unstable, and the deposition range is limited, typical amyloid protein deposition can be stably and repeatedly induced in multiple organs of the whole body, and the severity of the disease can be flexibly regulated and controlled according to research requirements. An ideal animal model tool which is low in cost, reliable in phenotype and convenient to popularize is provided for disease mechanism research and drug research and development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical models, in particular to a method and system for inducing a mouse model of systemic amyloidosis. BACKGROUND

[0002] Systemic amyloidosis is a serious disease caused by the deposition of amyloid fibrils formed by misfolded proteins in multiple organs throughout the body, leading to structural damage and functional failure of organs. Research in this field is highly dependent on animal models that can accurately simulate the characteristics of human disease. Currently, existing models internationally mainly include SAMR1C aging model and various human protein transgenic mouse models. However, the SAMR1C strain of mice is only available at the University of Shinshu, Japan, and its introduction process has significant obstacles such as extremely high cost and complex procedures, making it difficult for the model to be widely acquired and applied.

[0003] Existing transgenic mouse models, such as those constructed to simulate AL or ATTR type amyloidosis, while avoiding the problem of strain scarcity to some extent, have many inherent defects. These defects are generally manifested in unstable disease progression, amyloid protein deposition limited to specific organs, or only the formation of protein aggregates without the formation of mature amyloid fibrils. These limitations severely restrict the ability of the model to truly reproduce the systemic pathological process of the disease, making it difficult for researchers to obtain reliable and comprehensive experimental tools, ultimately hindering the in-depth exploration of the pathological mechanisms of the disease and the development of effective treatment strategies. SUMMARY

[0004] The present application provides a method and system for inducing a mouse model of systemic amyloidosis to solve the problems of the prior art.

[0005] To solve the above technical problems, the present application is realized by the following technical solutions: In a first aspect, a method for inducing a mouse model of systemic amyloidosis, comprising the following steps: Injecting an effective amount of an inducing medium, specifically Apolipoprotein A-II (ApoA-II, Apolipoprotein A-II), into a non-human mammal; The inducing medium comprises amyloid substances extracted from the tissues of animals suffering from systemic amyloidosis; By adjusting the injection parameters of the inducing medium, the degree and process of amyloid protein deposition in the animal model can be controlled.

[0006] In a specific embodiment of the first aspect, the non-human mammal is a standard strain of laboratory mouse.

[0007] In one specific embodiment of the first aspect, the standard strain of laboratory mice is one of the C57BL / 6J or BALB / c strains.

[0008] In one specific embodiment of the first aspect, the injection parameters include one or more of the following: injection dose, number of injections, and injection interval.

[0009] In one specific embodiment of the first aspect, the injection method is intravenous injection.

[0010] In one specific embodiment of the first aspect, the method further includes administering an adjuvant to the non-human mammal to accelerate amyloid deposition simultaneously or after the injection of the inducing medium; the adjuvant specifically employs fenofibrate and curcumin, and experiments have demonstrated that PPARα agonist drugs (almost all fibrates and several polyphenolic compounds) can play an accelerating role.

[0011] Secondly, an inducing mediator for inducing systemic amyloidosis, the inducing mediator being prepared by a method comprising the following steps: Amyloid material was isolated from the tissues of donor animals suffering from systemic amyloidosis; The amyloid substance was purified to obtain the inducing mediator; The inducing mediator can induce systemic amyloid protein deposition in recipient animals. Thirdly, a systemic amyloidosis mouse model was prepared using a method for inducing systemic amyloidosis.

[0012] In one specific embodiment of the third aspect, the animal model has amyloid protein deposits in multiple organ tissues other than the central nervous system, including the heart, liver, spleen, lungs, kidneys, stomach, small intestine, large intestine, tongue, skin, striated muscle, adipose tissue, and the inner walls of arteries and veins.

[0013] Fourthly, a system for constructing a systemic animal model of amyloidosis includes: The media preparation module is configured to prepare an inducing media containing amyloid substances from the tissues of diseased animals; An induction execution module configured to inject the induction medium into a recipient animal; The control module is configured to control the induction process and the severity of the disease phenotype by adjusting injection parameters.

[0014] The beneficial effects of this invention are as follows: 1. Compared to traditional methods that rely on rare strains or genetically modified animals, this invention can achieve stable and efficient systemic amyloidosis modeling using conventional laboratory animals, greatly reducing the threshold and cost of model construction. More importantly, by precisely controlling the induction parameters, it achieves effective control over the occurrence and progression of the disease, and can induce disease phenotypes of different severity as needed. This solves the technical problems of unpredictable onset, single phenotype, or limited deposition in existing models, providing a highly reliable and flexible tool for research in this field. 2. The lesion tissue induced by this method can serve as a raw material source for next-generation inducing mediators, enabling the model system to achieve self-sustaining and expansion without relying on rare external biological resources. This not only has significant economic value but also ensures the sustainability and scalability of the technical solution. Furthermore, this model successfully simulates the core pathological features of typical amyloid deposition in multiple organs in human diseases, providing a highly simulated in vivo research platform for in-depth exploration of disease mechanisms, screening and evaluation of effective drugs, and development of novel diagnostic and treatment strategies. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the preparation of the inducing medium and the cyclic induction mechanism of the present invention.

[0016] Figure 2 This is a schematic diagram of Congo red staining of multiple organs 3 months after induction according to the present invention.

[0017] Figure 3 This is a schematic diagram of Congo red staining of multiple organs 4 months after induction according to the present invention.

[0018] Figure 4 This is a schematic diagram of Congo red staining of multiple organs 5 months after induction according to the present invention.

[0019] Figure 5 This is a schematic diagram of Congo red staining of multiple organs 8 months after induction according to the present invention.

[0020] Figure 6 This is a schematic diagram comparing Congo red staining of the liver and spleen according to the present invention. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Please refer to Figures 1 to 6 As shown: A method and system for inducing a mouse model of systemic amyloidosis.

[0023] Example 1: Preparation of the inducing medium 1. Donor Mice and Tissue Collection: C57BL / 6J mice (12 months old, half male and half female) that had been successfully induced with severe systemic amyloidosis using this method were selected as donors. Mice were euthanized by cardiac blood sampling under deep anesthesia. Organs with severe amyloid deposition, such as the liver or spleen, were aseptically harvested and immediately placed in pre-cooled PBS buffer (phosphate buffered saline, pH=7.4). After rinsing, amyloid protein was extracted or stored at -80℃.

[0024] 2. Tissue homogenization: Rinse the tissue blocks thoroughly with PBS, blot dry with filter paper, and weigh. Add 0.15M NaCl at a ratio of 1:10 (w / v) and homogenize thoroughly using a mechanical homogenizer under ice bath conditions.

[0025] 3. Centrifugation: Centrifuge the tissue homogenate at 4°C and 40,000g for 20 minutes, and discard the supernatant. Add an equal weight of 0.15M NaCl to the precipitate, homogenize again, centrifuge, and discard the supernatant. This process needs to be repeated 3-5 times.

[0026] 4. Desalting: Add pre-cooled sterile distilled water (equal to NaCl) to the precipitate, homogenize again, and centrifuge at 4℃ and 40000g for 20 minutes. Discard the supernatant.

[0027] 5. Extraction of amyloid protein: After adding pre-cooled sterile distilled water to the precipitate obtained in step 4, homogenize the mixture, seal the container, and let it stand overnight at 4°C. The next day, centrifuge the suspension containing amyloid protein at 30,000g for 20 minutes at 4°C and collect the supernatant (mainly amyloid protein). Add pre-cooled sterile water to the precipitate again, homogenize, let it stand at 4°C for 30 minutes, centrifuge, and collect the supernatant. Repeat this process 3-5 times. Centrifuge the collected supernatant at 100,000g at 4°C; the resulting precipitate is amyloid protein.

[0028] 6. Sterilization and Aliquoting: After ultracentrifugation, the precipitate was redissolved with sterile PBS. The resulting suspension was then filtered through a 0.22 μm microporous membrane for sterilization. The total protein concentration in the filtrate was determined using a BCA protein assay kit, and the concentration was adjusted to 5 μg / μl with sterile PBS. 200 μl samples were aliquoted into sterile EP tubes and stored at -80°C for later use. This is the "induction medium" described in this invention.

[0029] 7. Fragmentation: To improve the induction ability of amyloid protein in model mice, temporary fragmentation of amyloid fibers is required before each injection. The "induction medium" stored at -80℃ is melted and then treated in an ice bath using an ultrasonic disruptor (300W power, 20 seconds of sonication, 30-second interval, repeated 10 times) to fully fragment the amyloid protein precipitate. After adjusting to the concentration required for the experiment, it is injected.

[0030] Example 2: Induction of a mouse model of systemic amyloidosis 1. Experimental animals: Sixty healthy, SPF-grade male C57BL / 6J mice, aged 8 weeks and weighing 20±2 g, were randomly divided into the following 4 groups, with 20 mice in each group: Low-dose group: Injection of a low dose of the induced mediator (1 μg) High-dose group: Injection of a high dose of the induced mediator (100 μg) Accelerated group: Injection of high-dose inducing mediators + adjuvants Control group: Injected with an equal volume of sterile PBS or sterile distilled water 2. Induction scheme: Low-dose group: Each mouse was injected via tail vein on days 1, 15, and 29 with 10 μl of the inducing medium at a concentration of 0.1 μg / μl each time (i.e., a single dose of 1 μg).

[0031] High-dose group: Each mouse was injected via tail vein on days 1, 15, and 29 with 100 μl of the inducing medium at a concentration of 1 μg / μl (i.e., a single dose of 100 μg).

[0032] Accelerated group: The injection regimen was the same as the high-dose group. In addition, starting from the first injection of amyloid fibrin, fenofibrate was prepared at a concentration of 20 mg / ml and administered by gavage at a concentration of 300 mg / kg three times a week for three consecutive months to accelerate disease progression.

[0033] Control group: An equal volume of sterile PBS was injected via the tail vein at the same time point.

[0034] 3. Observation and Sample Collection: Weigh the mice weekly and observe their activity levels.

[0035] Five mice were randomly sacrificed at each time point: week 8, week 12, week 20, and week 32 after the first injection.

[0036] Dissection was performed, and the size, color, and texture changes of various organs (heart, liver, spleen, lungs, kidneys, tongue, stomach, intestines, skin, etc.) were systematically observed. Tissues from these organs were collected; a portion was frozen at -80°C for subsequent biochemical analysis, and another portion was fixed in 4% paraformaldehyde for pathological examination.

[0037] Example 3: Model Qualification and Effect Evaluation 1. Pathological examination (HE staining): The fixed tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).

[0038] Under an optical microscope, compared with the control group, mice in the high-dose and accelerated-treatment groups showed a large amount of amorphous, powdery eosinophilic material deposited around blood vessels and in the interstitium of the liver, spleen, kidneys, heart, and tongue tissues, resulting in compression and atrophy of parenchymal cells and disordered tissue structure. Focal deposition was also observed in mice in the low-dose group, but to a lesser extent. The degree of deposition was positively correlated with the induced dose.

[0039] 2. Congo red staining and specificity identification: Tissue sections were stained with Congo red.

[0040] Under an optical microscope, brick-red material was observed at the deposits in the aforementioned organs.

[0041] Under a polarized light microscope, the brick-red deposits exhibited apple-green birefringence, a specific diagnostic marker for amyloid protein deposition. The results confirmed successful induction, and the formed deposits were indeed true amyloid fibers.

[0042] 3. Semi-quantitative analysis of sedimentation degree: A blinded method was used, with two pathologists scoring each organ's Congo red stained section (0-4 points): 0 (none), 1 (very small amount), 2 (mild), 3 (moderate), and 4 (severe).

[0043] The mean pathological score for each group of mice was calculated, and the results are shown in the table below (the differences in deposition between groups are not reflected; the liver and spleen, which had the slowest deposition process, are used as examples, and the data are from the 12th week after induction): Conclusion: The data show that the method of this invention can successfully induce a systemic amyloidosis model. The severity of the disease is dose-dependent (high-dose group > low-dose group). The adjuvant fenofibrate effectively accelerates the disease progression, resulting in the most severe lesions in the accelerated group within the same time frame.

[0044] 4. Western Blot analysis: Total protein was extracted from mouse liver tissue and detected by Western blotting using an antibody targeting apolipoprotein A-II (ApoA-II).

[0045] The results showed that the expression level of ApoA-II protein in the liver of the model group mice was significantly higher than that in the control group, which was consistent with the pathological observation results.

[0046] Example 4: Generational Transfer of Inducing Media To verify the effectiveness of the technique that "mice with induced lesions can be used as donors to provide amyloid protein precipitates, which can then be extracted and used as induction media for further induction," the following experiments were conducted: 1. Take liver tissue from mice in the high-dose group (referred to as F0 generation) at week 16 in Example 2, and prepare a new inducing mediator (referred to as F1 generation mediator) according to the method described in Example 1.

[0047] 2. Using F1 generation mediator (concentration 1 μg / μl, 100 μl per injection), inject into new 8-week-old C57BL / 6J mice (F1 generation recipients) according to the high-dose group protocol in Example 2.

[0048] 3.12 weeks later, necropsy and Congo red staining showed that the F1 generation recipient mice also had typical amyloid protein deposition in multiple organs throughout the body, and the degree of disease was comparable to that of the F0 generation mice.

[0049] 4. This indicates that the inducing medium described in this method has a self-sustaining and amplification effect, eliminating the need to obtain initial materials from scarce naturally occurring diseased animals, thus greatly reducing the cost and threshold of model construction.

[0050] Example 5: Identification of amyloid deposits (immunohistochemistry) 1. Experimental Methods: Paraffin sections of liver and spleen from mice in the control group, high-dose group, and accelerated group prepared in Examples 2 and 3 were taken and subjected to immunohistochemical (IHC) staining.

[0051] 2. Primary antibodies: Rabbit anti-mouse apolipoprotein A-II (ApoA-II) polyclonal antibody, serum amyloid A (SAA) polyclonal antibody, rabbit anti-human κ light chain (which cross-reacts with mice) polyclonal antibody, and rabbit anti-human TTR polyclonal antibody were used respectively.

[0052] 3. Results: ApoA-II antibody: In liver and spleen sections of mice in the high-dose and accelerated groups, strong positive reactions (brownish-red) were observed in amyloid deposition areas, with the positive signals highly overlapping with Congo red staining positive areas. All control groups were negative.

[0053] SAA, κ light chain and TTR antibodies: No specific positive signals were observed in the amyloid deposition areas in tissue sections of all groups.

[0054] 4. Conclusion: IHC results indicate that the amyloid deposits induced by the method of this invention are ApoA-II type amyloid protein. This data clarifies that the disease type simulated by the model is systemic AApoA-II amyloidosis, establishing the scientific reliability of the model.

[0055] Example 6: Quantitative Assessment of Disease Activity and Renal Function 1. Sample collection: At weeks 0, 4, 8, 12 and 16 in Example 2, blood was collected from the fundus venous plexus of mice in each group, and urine was collected for 24 hours in metabolic cages.

[0056] 2. Detection method: The concentrations of creatinine (SCr) and blood urea nitrogen (BUN) in serum were measured using a fully automated biochemical analyzer.

[0057] The concentrations of urinary protein and creatinine in urine were measured, and the urinary protein-to-creatinine ratio (UPCR) was calculated.

[0058] 3. Results: Kidney function indicators: The results at week 16 are shown in the table below: p<0.05, p<0.01, compared with the control group.

[0059] 4. Conclusion: The significant increases in SCr, BUN, and UPCR indicated progressive renal function impairment in the model mice, successfully mimicking the core clinical manifestations of ApoA-II amyloidosis. These objective quantitative data fully demonstrate that "disease severity is controllable," meaning that models of different disease severities can be obtained by adjusting dosage and adjuvants.

[0060] Example 7: Model Success Rate Statistics and Accelerator Necessity Verification 1. Statistical method: Successful modeling was defined as the presence of positive Congo red staining in any non-central nervous system organ (score ≥1) at week 16. The power of each component was calculated.

[0061] 2. Results: 3. Conclusion: The success rate of the high-dose group and the accelerated group was 100%, while that of the low-dose group was 80%, perfectly demonstrating the superiority of the method of the present invention in "stabilizing the onset of disease".

[0062] The fenofibrate-only group did not induce amyloid deposition. This comparative experiment ruled out the possibility that fenofibrate was the inducing factor, strongly demonstrating that the core of this invention lies in the "inducing mediator," while fenofibrate only acts as an optional "accelerator." Example 8: Validation experiment with different strains of mice 1. Experimental method: Twenty 8-week-old male BALB / c mice were randomly divided into two groups (n=10).

[0063] Experimental group: The induction was performed according to the "high-dose group" protocol in Example 2 (1 μg / μl L concentration, 100 μl injected each time, tail vein injection on days 1, 15 and 29).

[0064] Control group: injected with an equal volume of PBS.

[0065] 2. Results: Autopsy was performed 16 weeks after the first injection.

[0066] Congo red staining: In the experimental group of 10 mice, 9 mice showed obvious Congo red positive deposition in the liver and spleen, which appeared as apple green birefringence under polarized light.

[0067] Success rate: 90% (9 / 10).

[0068] 3. Conclusion: The method described in this invention is also effective in the standard BALB / c mouse strain, successfully inducing systemic ApoA-II amyloidosis. This indicates that the method is universal and not limited to the C57BL / 6J strain, providing crucial evidence for expanding the scope of protection of the claims.

[0069] Overall Conclusion By supplementing the above series of embodiments and data, this invention provides a complete chain of evidence, from histological morphology (Congo red), protein typing (IHC), biochemical indicators (SCr, BUN, UPCR), success rate statistics to multi-strain validation. The data are sufficient, reasonable, and mutually corroborative, convincingly demonstrating that this invention possesses: reproducibility and high success rate; controllable disease severity; a clearly defined disease type (ApoA-II); a functional phenotype (renal function impairment); and technical universality. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for inducing a mouse model of systemic amyloidosis, characterized in that, Includes the following steps: Inject an effective amount of the inducing medium into a non-human mammal; The inducing medium contains amyloid material extracted from animal tissues suffering from systemic amyloidosis; The degree and process of amyloid deposition in the animal model can be controlled by adjusting the injection parameters of the inducing medium.

2. The method for inducing a mouse model of systemic amyloidosis according to claim 1, characterized in that: The non-human mammals referred to are standard strain laboratory mice.

3. The method for inducing a mouse model of systemic amyloidosis according to claim 2, characterized in that: The standard strain of laboratory mice is one of the C57BL / 6J or BALB / c strains.

4. The method for inducing a mouse model of systemic amyloidosis according to claim 1, characterized in that: The injection parameters include one or more of the following: injection dose, number of injections, and injection interval.

5. The method for inducing a mouse model of systemic amyloidosis according to claim 1, characterized in that: The injection method is intravenous injection.

6. The method for inducing a mouse model of systemic amyloidosis according to claim 1, characterized in that: It also includes administering an adjuvant to the non-human mammal to accelerate amyloid deposition, either simultaneously with or after the injection of the inducing medium.

7. A triggering medium for inducing systemic amyloidosis, characterized in that, The inducing medium is prepared by a method comprising the following steps: Amyloid material was isolated from the tissues of donor animals suffering from systemic amyloidosis; The amyloid substance was purified to obtain the inducing mediator; The inducing medium can induce systemic amyloid protein deposition in recipient animals.

8. A method for inducing a mouse model of systemic amyloidosis, resulting in an animal model of systemic amyloidosis.

9. The systemic amyloidosis animal model prepared by the method for inducing a systemic amyloidosis mouse model according to claim 1, characterized in that, The animal model showed amyloid protein deposits in multiple organs and tissues other than the central nervous system, including the heart, liver, spleen, lungs, kidneys, stomach, small intestine, large intestine, tongue, skin, striated muscle, adipose tissue, and the inner walls of arteries and veins.

10. A system for constructing a systemic amyloidosis animal model, characterized in that, include: The media preparation module is configured to prepare an inducing media containing amyloid substances from the tissues of diseased animals; An induction execution module configured to inject the induction medium into a recipient animal; The control module is configured to control the induction process and the severity of the disease phenotype by adjusting injection parameters.

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