Granulomatous mastitis animal model and construction method thereof

Abstract

The application belongs to the field of animal models, and particularly relates to a granulomatous mastitis animal model and a construction method thereof, and aims to solve the problems of low pathological matching degree and poor repeatability of the existing model. The construction method is as follows: SPF level BALB / c female mice (10 weeks old, 20-22g) are selected, fresh lesion tissue homogenate of human breast is prepared, and lesion-derived Staphylococcus capitis bacterial liquid (1.4x10 8 -1.5x10 8 CFU / mL) is mixed and emulsified with Freund's incomplete adjuvant at a ratio of 1:1:1, and then injected into the third pair of mammary glands of the mice, so that the modeling is completed in 2 weeks. The model has high pathological characteristics matching with human diseases, high success rate and strong repeatability, and can be used for GM pathogenesis research and treatment drug screening.

Description

Technical Field

[0001] This invention belongs to the field of animal models, specifically relating to an animal model of granulomatous mastitis and its construction method. Background Technology

[0002] Granulomatous mastitis (GM) is a chronic inflammatory disease characterized by the formation of non-caseating granulomas in breast tissue. Its incidence in women of childbearing age has been increasing year by year in recent years, making it one of the most important diseases affecting women's breast health. Its clinical diagnosis and treatment face multiple challenges: in the early stages, patients often present with painless lumps in one or both breasts, easily confused with breast cancer, leading to a high misdiagnosis rate; as the disease progresses, ulceration of the lumps and sinus tract formation can occur, with the course of the disease lasting from months to years, and a high recurrence rate; current treatment options mainly involve glucocorticoids, immunosuppressants, and surgical resection, but long-term use of hormones can easily cause side effects such as osteoporosis and abnormal blood sugar, while surgical resection may lead to disfigurement of the breast, seriously affecting the patient's quality of life.

[0003] Despite the increasing clinical attention given to mammary gland granuloma (GM), its pathogenesis remains under investigation. Currently, the academic consensus is that GM results from the synergistic effects of multiple factors: autoimmune abnormalities, local infection, and mammary duct dysfunction. On one hand, exposure to autoantigens in breast tissue (such as casein and lipoglobulin released after ductal epithelial damage) may trigger abnormal immune responses, activating macrophages and T lymphocyte aggregation. On the other hand, clinical studies have found pathogens such as Staphylococcus aureus and Corynebacterium in the lesions of GM patients, suggesting that infection may be a key trigger for immune dysregulation. Furthermore, mammary duct obstruction leads to secretion retention, further exacerbating the local inflammatory microenvironment and promoting granuloma formation. However, due to the lack of animal models that closely match human pathological characteristics, the specific regulatory pathways of these mechanisms (such as how pathogens activate immune cells and the specific types of autoantigens) remain unclear, directly hindering the development of targeted therapies.

[0004] Animal models are core tools for elucidating the pathogenesis of GM and validating treatment regimens. Currently, the reported animal models of GM mainly focus on rats, and a standardized mouse model system has not yet been established. The existing rat model construction ideas mostly draw on the "human lesion tissue antigen induction" strategy. For example, the protocol published in Volume 41, Issue 4 of "Experimental Animals and Comparative Medicine" in 2021 uses liquid nitrogen to freeze GM patient lesion tissue, which is then thawed, ground, centrifuged to obtain the supernatant, mixed with complete Freund's adjuvant at a 1:1 ratio, emulsified, and injected into the 3rd and 4th pairs of mammary glands of female rats with a history of pregnancy and childbirth, in an attempt to simulate the pathological process of human disease. However, this type of rat model has significant limitations and is difficult to meet research needs: First, there are significant differences in physiological and immune mechanisms: rat mammary tissue is mainly composed of "compound ducts," with a loose lobular structure and lacking the "terminal duct lobular units" unique to human mammary glands. Its immune cell subsets differ greatly from the immune environment of human female mammary glands, leading to model-induced inflammation often manifesting as diffuse interstitial infiltration rather than the typical "centrilobular granuloma" of human GM, resulting in insufficient pathological matching. Second, the model has low stability and success rate: existing protocols use cryopreserved diseased tissue, and low-temperature storage easily leads to the formation of active antigens (such as protein antigens) within the tissue. The first problem is the lack of a mouse model. Firstly, the use of denaturing and inactivating agents, coupled with the presence of Mycobacterium tuberculosis extract in the complete Freund's adjuvant, can easily induce excessive systemic inflammatory responses in rats (such as rapid weight loss and splenomegaly), interfering with local mammary gland pathological processes and resulting in a low model success rate and significant differences in pathological characteristics among mice from the same batch. Secondly, there is a lack of mouse model research: mice, as model organisms more closely resembling human physiology, especially SPF-grade BALB / c female mice, have mammary gland development cycles (such as the maturation of the lobular ductal system after sexual maturity) and immune response patterns (such as antigen presentation efficiency and antibody production type) that are highly similar to those of human female mammary glands, making them ideal animals for constructing GM models. However, current technologies have not yet combined "fresh lesion tissue antigen + lesion-derived pathogenic bacteria + mild adjuvant," failing to simultaneously simulate the "autoimmune + infection" dual factors of GM pathogenesis. This has resulted in a lack of mouse model construction, severely limiting the depth and translational efficiency of GM mechanism research.

[0005] Therefore, developing a GM animal model based on BALB / c mice with well-defined parameters, high pathological matching, and stable success rate has become a key issue that urgently needs to be addressed in this field. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a method for constructing an animal model of granulomatous mastitis, the method comprising the following steps:

[0007] (I) Selection of experimental animals:

[0008] SPF-grade, specific pathogen-free BALB / c female mice were selected as the model subjects;

[0009] (II) Preparation of reagents:

[0010] (II-1) Select fresh lesion tissue from the patient after surgery, add physiological saline and grind into a homogenate, then centrifuge to separate the supernatant to obtain a homogenate of human breast lesion tissue;

[0011] (II-2) Staphylococcus capillus strains were isolated from human breast lesion tissue culture, and Staphylococcus capillus bacterial suspension was prepared.

[0012] (II-3) Select Freund's incomplete adjuvant;

[0013] (III) Constructing animal models:

[0014] (III-1) The homogenate of the human breast lesion tissue, the staphylococcal bacterial solution and the Freund's incomplete adjuvant are mixed to obtain a mixed reagent;

[0015] (III-2) The mixed reagent is emulsified to obtain an emulsified reagent;

[0016] (III-3) The emulsifying agent was injected into the third pair of mammary glands of mice. After the injection, the mice were returned to their breeding environment. The model was established 2 weeks later.

[0017] Preferably, in step (I), the BALB / c female mice are 10 weeks old and weigh 20-22g.

[0018] Preferably, in step (II-1), the grinding and homogenization process is as follows: grinding and pulverizing at a frequency of 60-70Hz for 3-5 minutes at 0-4℃.

[0019] Preferably, in step (II-1), the centrifugation method is: centrifugation at 3-5℃ and 3500-4500r / min for 10-20min.

[0020] Preferably, in step (II-2), the Staphylococcus aureus strain is inoculated into a solid culture medium for cultivation, and the final bacterial concentration is 1.4 × 10⁻⁶. 8 -1.5×10 8 CFU / mL.

[0021] Preferably, in step (III-1), the volume ratio of the human breast lesion tissue homogenate, the Staphylococcus aureus bacterial solution, and the Freund's incomplete adjuvant is 1:1:1.

[0022] Preferably, in step (III-2), the emulsification method is: oscillating emulsification for 20-30 minutes at 0-4℃ and 2800-3200r / min.

[0023] Preferably, in step (III-3), the injection site is disinfected with 75 vol% ethanol before injection.

[0024] Based on the same technical concept, another aspect of the present invention is to provide an animal model of granulomatous mastitis obtained by the above-described construction method.

[0025] The beneficial effects of this invention are as follows:

[0026] The granulomatous mastitis animal model and its construction method provided by this invention fill the gap in the field of standardized GM mouse models. By innovatively designing a synergistic induction system of "fresh lesion tissue homogenate + lesion-derived Staphylococcus aureus + Freund's incomplete adjuvant" and clarifying key operating parameters, the model significantly improves the pathological matching degree, construction success rate and reproducibility with human diseases, and provides a reliable experimental tool for the study of GM pathogenesis, targeted drug screening and treatment plan verification.

[0027] 1. Address the issue of low pathological matching in existing rat models and enhance the translational value of research:

[0028] This invention uses SPF-grade BALB / c female mice as the modeling subject (previous technology uses rats with a history of pregnancy), whose mammary tissue is similar to that of humans. The pathological features induced by the model are highly consistent with the pathological manifestations of human GM (e.g., Figure 2 As shown in the figure, it is significantly better than the rat model, and can more accurately simulate the pathological process of human diseases, ensuring that the results of mechanism research can be effectively translated into clinical practice.

[0029] 2. Address the issues of low success rate and poor stability in existing models to meet the needs of large-scale experiments:

[0030] This invention innovatively employs a "three-component synergistic induction" strategy: ① Fresh lesion tissue homogenate retains active autoantigens, providing a material basis for immune activation; ② Staphylococcus aureus bacterial solution derived from lesion tissue simulates clinical infection triggers, synergistically activating local immunity with antigens; ③ Freund's incomplete adjuvant avoids the systemic inflammatory interference of the complete adjuvant, enhancing only the efficiency of local antigen presentation in the mammary gland. The synergistic effect of these three components significantly improves the success rate of model construction, and the coefficient of variation of pathological characteristics (number of granulomas, extent of inflammatory infiltration) between the same and different batches of mice is small, demonstrating significantly better stability than existing rat models.

[0031] 3. Address the issues of ambiguous parameters and poor repeatability in existing schemes to ensure the experiments are scalable:

[0032] This invention sets clear quantitative parameters for key operational steps, such as tissue grinding (0-4℃, 60-70Hz, 3-5min), centrifugation (3-5℃, 3500-4500r / min, 10-20min), and bacterial concentration (1.4×10⁻⁶).8 -1.5×10 8 The parameters include CFU / mL and emulsification conditions (0-4℃, 2800-3200r / min, 20-30min). Experimenters can strictly reproduce the modeling process according to these parameters, ensuring high repeatability and facilitating widespread use in different laboratories. Furthermore, the injection dose is only 50μL, far lower than the 0.2-0.3mL required for rat models.

[0033] 4. Address the issue of numerous interfering factors in existing models and improve research accuracy:

[0034] This invention uses Freund's incomplete adjuvant instead of the complete adjuvant, eliminating systemic inflammation (such as weight loss and increased spleen index in mice) induced by Mycobacterium tuberculosis extract, ensuring that the inflammatory response is limited to the mammary gland. At the same time, standardized BALB / c mice weighing 20-22g at 10 weeks of age are used to avoid interference from physiological developmental differences in mice (such as immature mammary glands in juvenile mice) on the model, making the experimental results more accurate and reducing the impact of non-specific factors on mechanism research and drug screening. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0036] Figure 1 These are photographs of the mammary gland appearance of female BALB / c mice taken on days 1-13 after modeling.

[0037] Figure 2 This is an H&E staining image of mammary gland tissue taken from female BALB / c mice two weeks after modeling. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] Example

[0040] This embodiment provides a method for constructing an animal model of granulomatous mastitis, the method comprising the following steps:

[0041] (I) Selection of experimental animals:

[0042] The experimental animals used in this invention were SPF-grade (specific pathogen-free) female BALB / c mice, all purchased from Beijing Spaford Co., Ltd. The selection criteria were as follows: Mice must be at least 10 weeks old, at which stage their mammary glands are mature and their immune function is stable, reducing interference from physiological developmental differences in the model construction effect; their weight must be controlled within the range of 20-22g, and the weight difference between mice in the same batch should not exceed 2g to avoid uneven injection dosage due to weight deviation, which would affect experimental repeatability; regarding health status, mice were observed for 3 days before the experiment to ensure they had no mammary gland developmental abnormalities (such as mammary nodules, nipple deformities), no infectious diseases (such as skin ulcers, lethargy), and normal activity, with regular eating and drinking habits, meeting the ethical requirements for animal experiments.

[0043] Throughout the study, the mice were housed in a specific pathogen-free environment with strictly controlled conditions: the temperature was maintained at 22-25℃, the humidity at 50%-60%, and the light cycle was 12 hours of light and 12 hours of darkness. The mice had free access to standard mouse feed and sterile drinking water. The housing environment was regularly cleaned and disinfected to ensure no external pathogen contamination, thus guaranteeing the health of the experimental animals and the reliability of the experimental results.

[0044] (II) Preparation of reagents:

[0045] (II-1) Human breast lesion tissue homogenate (liquid fraction): Derived from fresh lesion tissue after surgery in patients with granulomatous mastitis. For preparation, approximately 1g of lesion tissue was first taken and placed in a sterile grinding tube at 4°C. Sterile physiological saline was added at a mass-to-volume ratio of 3mL physiological saline per 1g of tissue. The homogenate was then homogenized at 64Hz for 3 minutes using a tissue homogenizer, maintaining an ice bath (temperature controlled at 0-4°C) throughout the process to prevent tissue denaturation. After homogenization, the mixture was transferred to a 10mL sterile centrifuge tube and centrifuged at 4000r / min and 4°C for 15 minutes. The clear supernatant (i.e., the homogenate liquid fraction) was collected and stored at 4°C for no more than 2 hours. This reagent provides human disease-related antigens, providing material support for simulating the pathological basis of autoimmune induction.

[0046] (II-2) Staphylococcus capitella bacterial suspension: The strain was isolated from human breast lesion tissue. Strictly controlled conditions were required during the culture process to ensure stable bacterial activity and concentration. First, the frozen strain was inoculated onto LB solid medium and incubated at 37°C for 12 hours. After single colonies formed, a single colony was picked and inoculated onto LB liquid medium and incubated at 37°C and 220 rpm for 12 hours. At this point, the strain entered the logarithmic growth phase (vigorous growth phase). Subsequently, the OD of the bacterial suspension was measured using a UV spectrophotometer. 600Value, if OD 600 If the value is less than 1.5, the concentration needs to be adjusted using LB medium until the bacterial concentration is stabilized at 1.5 × 10⁻⁶. 8 After adjustment, the reagent is stored at 4°C for no more than 2 hours. This reagent can simulate clinical infection-related factors, synergistically activate the local immune response in mouse mammary glands with antigens, and promote granuloma formation.

[0047] (II-3) Freund's incomplete adjuvant: purchased from Sigma (catalog number: F5506), without Mycobacterium tuberculosis extract. The core function of this reagent is to enhance the local antigen presentation efficiency, prolong the retention time of antigen in breast tissue, and at the same time avoid the systemic excessive inflammation caused by the presence of Mycobacterium tuberculosis components in the complete Freund's adjuvant, ensuring that the inflammatory response is limited to the breast area.

[0048] (III) Constructing animal models:

[0049] (III-1) Preliminary Preparation (Reagent Pretreatment and Environmental Control):

[0050] Environmental control: All experimental operations must be carried out in a Class II biosafety cabinet. Before operation, wipe the surface of the biosafety cabinet and all equipment (including centrifuge tubes, pipette tips, syringes, etc.) with 75% ethanol. All equipment must be sterile disposable products to avoid cross-contamination. Experimenters must wear sterile gloves, masks and protective clothing and strictly follow aseptic operation procedures.

[0051] Pretreatment of core reagents: In addition to the separate preparation and treatment of human breast lesion tissue homogenate, Staphylococcus aureus bacterial suspension, and incomplete Freund's adjuvant mentioned above, a final check is required before mixing the three: confirming that the human breast lesion tissue homogenate is free of turbidity and precipitation, and that the OD of the Staphylococcus aureus bacterial suspension is [not specified]. 600 The value remained stable at 1.5, and the incomplete Freund's adjuvant showed no stratification, ensuring that the activity and purity of each reagent met the requirements.

[0052] (III-2) Preparation of composite induction emulsion (including reagent ratios for each group):

[0053] This invention sets up one experimental group and three control groups. The emulsion in each group is prepared according to the "50 μL injection volume per mouse". The specific ratio and preparation process are as follows:

[0054] (i) Experimental group (homogeneous + bacteria + adjuvant group): The human breast lesion tissue homogenate liquid fraction: Staphylococcus aureus bacterial solution: Freund's incomplete adjuvant = 1:1:1 volume ratio was mixed. During preparation, the above three reagents were added to a 5 mL sterile centrifuge tube in sequence. The centrifuge tube was fixed on a vortex mixer and vortexed at 3000 r / min for 25 minutes. The centrifuge tube was kept in an ice bath (0-4℃) throughout the process. The vortexing was paused once every 5 minutes. One drop of emulsion was dropped into cold water. If the emulsion drop was spherical and did not spread, it indicated that the emulsification was complete (if it spread, the vortexing should be continued for 5-10 minutes). After the emulsification was completed, a small amount of emulsion was drawn with a sterile syringe for observation. If it was confirmed that there was no layering, no precipitation and uniform viscosity, it was a qualified emulsion. It was stored at 4℃ and used within 4 hours. Repeated freeze-thaw cycles should be avoided.

[0055] (ii) Control group 1 (adjuvant-only group): Incomplete Freund's adjuvant was used as the induction agent, with a single mouse injection of 50 μL of incomplete Freund's adjuvant. During preparation, 50 μL of incomplete Freund's adjuvant was directly placed into a sterile centrifuge tube without adding any other reagents. Since the adjuvant itself is an oil-phase liquid, emulsification is unnecessary, but it must be ensured that the adjuvant is free of stratification and impurities. It should be stored at 4°C and used within 4 hours.

[0056] (iii) Control Group 2 (Adjuvant plus bacteria group): Staphylococcus aureus bacterial solution and incomplete Freund's adjuvant were mixed at a volume ratio of 1:1, with a total injection volume of 50 μL. The preparation process was similar to that of the experimental group: the two reagents were added to a sterile centrifuge tube and vortexed at 3000 rpm under ice bath conditions for 20 minutes. The emulsification status was checked every 5 minutes (the emulsion droplets should not spread when placed in cold water to indicate that it is qualified). After passing the test, the emulsion was stored at 4°C and used within 24 hours.

[0057] (iv) Control group 3 (adjuvant plus homogenate group): The liquid portion of human breast lesion tissue homogenate and incomplete Freund's adjuvant were mixed at a volume ratio of 1:1, with a total injection volume of 50 μL. During preparation, both reagents were first added to a sterile centrifuge tube and vortexed at 3000 rpm under ice bath conditions for 20 minutes. The emulsification status was checked every 5 minutes. After passing the emulsification test, the emulsifier was stored at 4°C and used within 24 hours.

[0058] (III-3) Animal injection and model observation:

[0059] Injection procedure: Fix the mouse with its abdomen facing upward. Disinfect the skin of the third pair of mammary glands (1 mm around the nipple from the head to the tail, in the mammary fat pad area) with 75% ethanol. Using a sterile insulin syringe, insert the needle subcutaneously and slowly inject the emulsion over a period of ≥10 seconds to avoid leakage. After injection, observe the injection site for swelling, oozing, etc., mark the injection site, and return the mouse to its rearing environment.

[0060] It should be noted that the injection method is as follows: Using ophthalmic forceps, lift the nipple; using an insulin syringe, insert the different inoculum for each group obliquely along the base of the nipple to a depth of 4-6 mm, injecting it into the third pair of mammary glands of the rat. This completes the artificial implantation and embedding process. Care must be taken not to insert the needle too deeply, which could cause the inoculum to be injected into the muscle layer.

[0061] Experimental period and testing: Daily testing was performed after injection. Two weeks later, mice were euthanized by cervical dislocation, and the third pair of mammary gland tissue (injection site) was quickly removed for pathological examination (H&E staining).

[0062] Pathological section observation (HE staining): Breast tissue was fixed in 4% paraformaldehyde solution for 24 hours, followed by gradient dehydration (70% ethanol → 80% ethanol → 95% ethanol → anhydrous ethanol, 30 minutes each), xylene clearing (twice, 15 minutes each), paraffin embedding, and preparation of 4μm thick sections; after dewaxing to water (xylene → anhydrous ethanol → 95% ethanol → 80% ethanol → 70% ethanol, 5 minutes each), the sections were stained with an HE staining kit: hematoxylin staining for 5 minutes, hydrochloric acid ethanol differentiation for 30 seconds, eosin staining for 3 minutes, followed by gradient dehydration and clearing, and then mounting with neutral resin; the sections were observed, and the judgment criteria were: whether there were non-caseating granulomas composed of macrophages and multinucleated giant cells.

[0063] Results and Analysis Examples

[0064] like Figure 1 As shown, these are photographs of the mammary gland appearance of female BALB / c mice from day 1 to day 13 after modeling. The images are arranged in a column-row format: columns represent different experimental groups (4 columns in total, corresponding to 4 experimental groups), and rows represent specific days after modeling. Each row in each column is a frontal view of the third pair of mammary glands (injection site) of mice in the same experimental group on the corresponding day. The shooting conditions are uniform (same light intensity, same shooting distance, same angle, all taken with the mice fixed with their abdomens facing upwards, clearly showing the skin texture, color, and shape of the mammary gland area), allowing for a direct comparison of the dynamic changes in mammary gland appearance over time among different groups.

[0065] (1) First column: group with Freund's incomplete adjuvant alone (control group 1)

[0066] Day 1 after modeling: The skin of the third pair of mammary glands in mice is normal light pink, consistent with the skin color of the surrounding non-injection areas. The mammary glands are flat, without protrusion, swelling or tightness. The injection needle holes have closed and there is no residual exudate.

[0067] Days 2-10 after modeling: There were no changes in the appearance of the mammary glands, the skin color remained light pink, and the mammary gland contour was completely consistent with that of normal unmodeled mice. There were no local tissue bulges or skin abnormalities, proving that injection of incomplete Freund's adjuvant alone could not induce pathological appearance changes in the mammary glands during days 1-10.

[0068] (2) Second column: Freund's incomplete adjuvant + Staphylococcus aureus group (control group 2)

[0069] Day 1 after modeling: The right side of the third pair of mammary glands in mice showed a slight bulge, but no exudate.

[0070] Days 2-13 after modeling: The right breast begins to protrude more, the borders of the protruding area become blurred, and the skin color deepens to a light red, while the left breast remains relatively flat;

[0071] (3) Third column: Freund's incomplete adjuvant + homogenate group (control group 3)

[0072] Day 1 after modeling: The skin on both sides of the third pair of mammary glands of mice was normal light pink, the mammary glands were flat, the injection sites had healed, and there was no exudation or local swelling.

[0073] Days 2-13 after modeling: There were no changes in the appearance of the breasts at any time, the skin color remained light pink, and there were no protrusions, redness or tissue hardening of the breasts, which was completely consistent with the appearance of the group with single incomplete adjuvant (first column);

[0074] Key findings: Injection of human breast lesion tissue homogenate alone failed to activate the local immune response in mouse mammary glands within 1-13 days and did not induce any pathological changes in appearance. This indicates that human lesion tissue antigens alone, without adjuvant assistance, cannot form visible breast pathological appearances.

[0075] (4) Fourth column: Freund's incomplete adjuvant + Staphylococcus aureus + homogenate group (experimental group)

[0076] Day 1 after modeling: The third pair of mammary glands in mice showed slight bilateral bulging with symmetrical color.

[0077] Days 2-13 after modeling: Both breasts showed obvious bulges with clear borders. The skin color deepened to light red. The height of the bulges on both sides was consistent with that of the breasts, with no obvious difference. The borders of the bulges were clear and there was no spread or signs of regression. The appearance characteristics were consistent with the early clinical manifestation of "bilateral breast lumps" in granulomatous mastitis.

[0078] like Figure 2The image shows H&E staining of mammary gland tissue samples from female BALB / c mice two weeks after modeling. The images are arranged in a column-row format: columns represent different experimental groups (4 columns in total, corresponding to 4 experimental groups), the first row is the overall image, and the second and third rows are localized or magnified images. Columns 1 (control group 1: injected with Freund's incomplete adjuvant) and 2 (control group 2: Freund's incomplete adjuvant + Staphylococcus aureus) both showed tissue necrosis, inflammatory cell infiltration, and fibrous tissue hyperplasia, but no granulomas formed. Column 3 (control group 3: Freund's incomplete adjuvant + homogenate) showed a small amount of inflammatory cell infiltration accompanied by fibrous tissue hyperplasia. Column 4, the experimental group, showed typical granulomatous structures with epithelioid cells and neutrophils. These results indicate successful modeling.

[0079] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for constructing an animal model of granulomatous mastitis, characterized in that, The construction method includes the following steps: (I) Selection of experimental animals: SPF-grade, specific pathogen-free BALB / c female mice were selected as the model subjects; (II) Preparation of reagents: (II-1) Select fresh lesion tissue from the patient after surgery, add physiological saline and grind into a homogenate, then centrifuge to separate the supernatant to obtain a homogenate of human breast lesion tissue; (II-2) Staphylococcus capillus strains were isolated from human breast lesion tissue culture, and Staphylococcus capillus bacterial suspension was prepared. (II-3) Select Freund's incomplete adjuvant; (III) Constructing animal models: (III-1) The homogenate of the human breast lesion tissue, the staphylococcal bacterial solution and the Freund's incomplete adjuvant are mixed to obtain a mixed reagent; (III-2) The mixed reagent is emulsified to obtain an emulsified reagent; (III-3) The emulsifying agent was injected into the third pair of mammary glands of mice. After the injection, the mice were returned to their breeding environment. The model was established 2 weeks later.

2. The method for constructing an animal model of granulomatous mastitis according to claim 1, characterized in that, In step (I), the BALB / c female mice are 10 weeks old and weigh 20-22g.

3. The method for constructing an animal model of granulomatous mastitis according to claim 1, characterized in that, In step (II-1), the grinding and homogenization method is as follows: grinding and pulverizing at a frequency of 60-70Hz for 3-5 minutes at 0-4℃.

4. The method for constructing an animal model of granulomatous mastitis according to claim 1, characterized in that, In step (II-1), the centrifugation method is as follows: centrifuge for 10-20 minutes at 3-5℃ and 3500-4500r / min.

5. The method for constructing an animal model of granulomatous mastitis according to claim 1, characterized in that, In step (II-2), the Staphylococcus aureus strain was inoculated into a solid culture medium for cultivation, and the final bacterial concentration was 1.4 × 10⁻⁶. 8 -1.5×10 8 CFU / mL.

6. The method for constructing an animal model of granulomatous mastitis according to claim 1, characterized in that, In step (III-1), the volume ratio of the human breast lesion tissue homogenate, the Staphylococcus aureus bacterial solution, and the Freund's incomplete adjuvant is 1:1:

1.

7. The method for constructing an animal model of granulomatous mastitis according to claim 1, characterized in that, In step (III-2), the emulsification method is: oscillating emulsification for 20-30 minutes at 0-4℃ and 2800-3200r / min.

8. The method for constructing an animal model of granulomatous mastitis according to claim 1, characterized in that, In step (III-3), the injection site is disinfected with 75 vol% ethanol before injection.

9. The animal model of granulomatous mastitis obtained by the construction method according to any one of claims 1-8.

Images