APBA animal model construction method

Abstract

The invention discloses an ABPA animal model building method which comprises the following steps: building an ABPA animal model by injecting a live aspergillus fumigatus spore suspension into an airway, and selecting a mouse for adaptive feeding; a primary sensitization stage; repeating the excitation stage; a live aspergillus fumigatus spore colonization excitation stage; and establishing an evaluation model. The animal model which highly simulates the pathophysiological characteristics of human ABPA is constructed by performing sensitization and excitation in stages and in multiple modes, especially by introducing the key step of lung colonization of live aspergillus fumigatus spores; through increase of serum total IgE and aspergillus fumigatus specific IgE levels, airway eosinophilic granulocyte infiltration and high secretion of mucus, the evaluation model is successfully constructed, and a reliable experimental platform is provided for deep analysis of pathogenesis of diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal models, in particular to an APBA animal model construction method. BACKGROUND

[0002] Allergic Bronchopulmonary Aspergillosis (ABPA) is a complex hypersensitivity disease caused by the colonization of Aspergillus fumigatus in the airway, commonly seen in patients with asthma and cystic fibrosis. The disease has complex pathological features, including significant airway eosinophil and lymphocyte infiltration, excessive mucus secretion, abnormal elevation of serum total immunoglobulin E (IgE) and Aspergillus fumigatus-specific IgE levels, and can progress to central bronchiectasis and other structural lung damage, eventually leading to pulmonary fibrosis and respiratory failure. At present, the pathogenesis of ABPA has not been fully elucidated, and there are challenges in clinical diagnosis and management, and lack of safe and effective treatment.

[0003] One of the important reasons for the limited progress in this field is the lack of an ideal animal model that can highly simulate the core clinical and immunopathological features of human ABPA. Existing mouse models have obvious limitations: first, the immune response is relatively single, most models can only simulate acute allergic inflammation dominated by Th2-type immune response, showing elevated IgE and eosinophil infiltration, but it is difficult to reproduce the mixed immune response mediated by Th1 and Th17 cells and the resulting chronic tissue damage that often accompanies human ABPA; second, the model usually only induces acute and transient inflammatory response, and the inflammation subsides rapidly after stopping the challenge, and cannot stably form chronic persistent airway inflammation and mucus plug formation similar to clinical conditions; finally, the stability and repeatability of the model are poor, and the success rate of modeling is easily affected by animal strain, preparation form of Aspergillus fumigatus antigen (such as the difference between crude protein and spores), sensitization and challenge method (such as nasal inhalation and airway injection), and experimental period, resulting in large differences in model phenotypes between different laboratories and low data comparability.

[0004] Based on the shortcomings of the above prior art, there is an urgent need in the art to develop a new animal model of allergic bronchopulmonary aspergillosis and a construction method. The model should be able to systematically reproduce the core pathophysiological features of human disease, including mixed immune response, chronic airway inflammation, and high mucus secretion, while having good stability and repeatability, providing a key tool for in-depth analysis of the pathogenesis of allergic bronchopulmonary aspergillosis, screening of new therapeutic drugs, and optimization of clinical treatment regimens, and promoting breakthrough progress in the field. SUMMARY

[0005] In order to solve the above-mentioned technical problems, an APBA animal model construction method is provided; the construction method simulates the clinical disease process, sensitizes and excites in stages and multiple ways, especially introduces the key step of lung colonization of live aspergillus fumigatus spores, can construct an animal model highly simulating the pathological and physiological characteristics of human ABPA, the model has the advantages of comprehensive immune response, stable chronic inflammation and strong repeatability, etc., and provides a reliable experimental carrier for related research.

[0006] The technical scheme adopted by the present application is: An APBA animal model construction method, comprising: An ABPA animal model is established by intratracheal injection of live aspergillus fumigatus spore suspension, comprising: S1, selecting mice for adaptive feeding; S2, initial sensitization stage: dissolve aspergillus fumigatus protein in PBS buffer, mix with incomplete Freund's adjuvant to prepare a sensitization working solution; the mice are sensitized by intraperitoneal injection combined with subcutaneous injection at the neck; S3, repeated excitation stage: after a certain period of time after the initial sensitization stage, use aspergillus fumigatus protein dissolved in PBS buffer to repeatedly excite the mice by nose drops; S4, live aspergillus fumigatus spore colonization excitation stage: after the repeated excitation stage ends, a predetermined number of live aspergillus fumigatus spores are suspended in PBS buffer containing a surfactant, i.e. live aspergillus fumigatus spore suspension, which is delivered to the lungs of the mice by intratracheal injection, so that aspergillus fumigatus can colonize in the lungs of the mice; S5, judge model construction: after the live aspergillus fumigatus spore colonization excitation stage ends, the mice are sampled, and blood, alveolar lavage fluid and lung tissue samples are collected; whether the model is successfully constructed is comprehensively judged, wherein the judgment criteria for successful construction are: ① The levels of serum total IgE and aspergillus fumigatus specific IgE are increased; or ② The total number of inflammatory cells and the proportion of eosinophils in the alveolar lavage fluid are significantly increased, and the levels of IFN-γ (Th1 type), IL-17 (Th17 type), IL-5, IL-4, IL-13, IL-33 and TSLP (Th2 type) are increased; or ③ The lung tissue pathological section shows that the airway eosinophil infiltration and mucus secretion are increased.

[0007] Further, the selecting mice for adaptive feeding in step S1 is specifically: Select 8-week-old C57BL / 6J female mice without specific pathogen infection; The C57BL / 6J female mice are placed in a standardized feeding environment for adaptive feeding for one week, the feeding environment is controlled to be stable in temperature, suitable in humidity, the light is adopted in day and night alternating mode, and the ventilation is good; During the feeding period, sufficient sterile drinking water and standard feed are provided, the emotional state of the C57BL / 6J female mice is observed every day, and it is ensured that the C57BL / 6J female mice are free of disease infection and have stable emotions.

[0008] Further, in step S2, the amount of the Aspergillus fumigatus protein is 10 μg per mouse, the amount of the PBS buffer is 100 μL, and the amount of the incomplete Freund's adjuvant is 100 μL; the amount of the sensitization working solution for intraperitoneal injection and subcutaneous injection in the neck is 100 μL each; In step S3, the amount of the Aspergillus fumigatus protein used for each challenge is 20 μg per mouse, and the amount of the PBS buffer is 40 μL. In step S4, the PBS buffer containing a surfactant is a PBS buffer containing 0.1% (volume percentage) Tween-80, and the suspension volume is 30 μL.

[0009] Further, in step S4, the number of the live Aspergillus fumigatus spores is 5×10 6 per mouse.

[0010] Further, in step S5, the blood sample is collected, specifically: The mouse blood is collected by enucleation, and the blood is collected in a sterile container. After being placed at room temperature for a period of time, the blood is centrifuged in a centrifuge to obtain serum; The serum is divided into sterile cryogenic tubes, sealed and stored in a low-temperature refrigerator for standby use for subsequent serological index detection.

[0011] Further, in step S4, the collection of the bronchoalveolar lavage fluid and the lung tissue, the mice are divided into two groups, specifically: ① The first group collects the bronchoalveolar lavage fluid by tracheal intubation. A certain amount of PBS buffer is injected, and the lavage fluid is slowly recovered after a short retention. The operation is repeated multiple times, and the recovered lavage fluid is combined. The combined bronchoalveolar lavage fluid is centrifuged in a centrifuge at an appropriate speed to obtain supernatant and bottom cells. The supernatant is divided into cryogenic tubes and stored at low temperature for standby use. The bottom cells are added with a fixing solution, gently blown and mixed, and standby use after a period of fixation. After the collection of the bronchoalveolar lavage fluid, the mouse is dissected to obtain the complete lung tissue, which is placed in a cryogenic tube and stored at low temperature for standby use; ② The second group directly dissects the lung tissue. One side of the lung tissue is completely immersed in a fixing solution for pathological fixation. The other side of the lung tissue is quickly placed in liquid nitrogen for quick freezing, and then transferred to a low-temperature refrigerator for long-term storage for subsequent molecular biology detection.

[0012] Another technical solution adopted by the present application is: The application of the APBA animal model construction method in the preparation of a tool for studying the pathogenesis of ABPA or screening and evaluating therapeutic drugs.

[0013] Compared with the prior art, the present application has the following beneficial effects: 1) By optimizing the sensitization method and the challenge strategy, the sensitization method combining intraperitoneal injection with subcutaneous injection can effectively induce comprehensive immune response in mice, overcoming the defect of the existing model that can only simulate single Th2 type immune response; by multi-stage nasal challenge and subsequent Aspergillus fumigatus spore lung colonization, the immune system is gradually induced to produce sustained mixed immune response, and Th1 type, Th2 type and Th17 type immune cells are activated, so that the model can reproduce the complex immune imbalance state of human ABPA, providing an ideal carrier for studying the interaction of immune cells in the disease; 2) By reasonably designing the experimental period and the challenge method, the model animal is successfully induced to produce sustained chronic airway inflammation, avoiding the problem of short-term inflammation reaction in the existing model; the stable colonization of Aspergillus fumigatus spores in the lung can continuously stimulate the airway mucosa, trigger long-term inflammation, and further promote chronic pathological changes such as mucus hypersecretion, so that the model can reflect the long-term development process of the disease, and is closer to the clinical characteristics of human ABPA, providing a reliable tool for studying the intervention strategies of chronic inflammation regulation mechanism; 3) By standardizing the experimental process, including preparation of sensitization working solution, standardization of challenge operation, unification of sample collection and processing, etc., the human error and environmental interference in the experimental process are effectively reduced, and the stability and repeatability of the model are improved; when different laboratories use the method of the present application to construct the model, consistent and good pathological phenotypes and experimental results can be obtained, solving the problems of large phenotype difference and poor repeatability of the existing model, which is conducive to the horizontal comparison of research data and academic exchanges, and promotes the standardized development of the field research; 4) The operation is simple, and does not require complex experimental equipment and technology, and the experimental process is easy to popularize and apply; at the same time, the model has various sampling methods, and various samples such as serum, lung lavage fluid, lung tissue, etc. can be obtained, which can support immune index detection, cell analysis, pathological observation, molecular biology detection, etc. and has wide applicability; 5) The animal model constructed by the application can comprehensively reproduce the core pathophysiological characteristics of human ABPA. The success of the model construction is judged by the increase of serum total IgE and Aspergillus fumigatus specific IgE levels, airway eosinophil infiltration and high mucus secretion, which provides a reliable experimental platform for in-depth analysis of the pathogenesis of the disease. Through the model, researchers can carry out research from multiple angles such as immune cell interaction, inflammatory factor regulation, airway eosinophil damage repair, etc., to reveal the key molecular targets of disease occurrence and development, and provide a theoretical basis for the research and development of new treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0014] The application will be further described below in conjunction with the drawings and specific embodiments: Figure 1 is a flowchart of the APBA animal model construction method of the application; Figure 2 is a flowchart comparison of the APBA model in the application and the Aspergillus fumigatus sensitized asthma (ASA) model; Figure 3 is the difference in immunological indicators of the APBA model in the application and the Aspergillus fumigatus sensitized asthma (ASA) model on the 3rd and 7th day; Figure 4 is the HE and PAS staining chart of the APBA model in the application and the Aspergillus fumigatus sensitized asthma (ASA) model on the 3rd and 7th day; Figure 5 is a comparison chart of the serum total IgE level and the Aspergillus fumigatus specific IgE level of the APBA model in the application and the control group. DETAILED DESCRIPTION EMBODIMENT

[0015] As Figure 1 shown, an APBA animal model construction method comprises: The ABPA animal model is established by intratracheal injection of live Aspergillus fumigatus spore suspension, comprising: S1, selecting mice for adaptive feeding; In this embodiment, 8-week-old C57BL / 6J female mice are selected, which are free of specific pathogen infection. The C57BL / 6J female mice are placed in a standardized feeding environment for adaptive feeding for one week. The feeding environment is controlled to have stable temperature, suitable humidity, and alternating day and night light mode, and good ventilation. During the feeding period, sufficient sterile drinking water and standard feed are provided. The emotional state of the C57BL / 6J female mice is observed daily to ensure that the C57BL / 6J female mice are free of disease infection and have stable emotions. After the adaptive feeding is completed, the subsequent process is entered; S2, initial sensitization stage: dissolve the A. fumigatus protein in PBS buffer, mix with incomplete Freund's adjuvant to prepare a sensitization working solution; through a combination of intraperitoneal injection and subcutaneous injection at the neck, the mouse is initially sensitized to simulate the repeated onset of allergic airway inflammation; In this embodiment, 10 μg of A. fumigatus protein is added to 100 μL of PBS buffer, fully stirred and dissolved to obtain an A. fumigatus protein solution; then, 100 μL of incomplete Freund's adjuvant is added to the A. fumigatus protein solution, which is fully mixed by vortexing for a sufficient time to ensure that a uniform and stable sensitization working solution is formed, which is used immediately after preparation; The C57BL / 6J female mice are sensitized by intraperitoneal injection and subcutaneous injection at the neck, i.e. each C57BL / 6J female mouse is injected intraperitoneally with 100 μL of the sensitization working solution, and at the same time, the same amount of the sensitization working solution is injected subcutaneously at the neck, the injection process is standardized to avoid injuring the internal organs or blood vessels of the C57BL / 6J female mice and to ensure that the sensitization working solution is accurately injected into the body; S3, repeated challenge stage: at a specific period after the initial sensitization stage, the A. fumigatus protein dissolved in PBS buffer is used to repeatedly challenge the mouse by nasal drops to simulate the pathological state of the continuous presence of A. fumigatus allergens in the lungs of a clinical patient; In this embodiment, after the sensitization process is completed, nasal drop challenge is started after a certain period of time, specifically: 20 μg of A. fumigatus protein is dissolved in 40 μL of PBS buffer to prepare a nasal drop challenge solution; during the challenge, the C57BL / 6J female mouse is fixed, the head is slightly tilted backward, the nasal cavity is upward, a certain amount of the nasal drop challenge solution is taken with a micropipette, and is slowly dropped into the nasal cavity of the C57BL / 6J female mouse to ensure that the challenge solution can fully contact the nasal mucosa and avoid coughing or liquid leakage; the nasal drop challenge operation is repeated according to the set time interval to complete the multi-stage repeated challenge; S4, live A. fumigatus spore colonization challenge stage: after the repeated challenge stage is completed, a predetermined number of live A. fumigatus spores are suspended in a PBS buffer containing a surfactant, i.e. a live A. fumigatus spore suspension, which is delivered to the lungs of the C57BL / 6J female mouse by intratracheal injection to allow A. fumigatus to colonize in the lungs of the C57BL / 6J female mouse; In this embodiment, 5 x 10 6A live Aspergillus fumigatus spore is added into a PBS buffer containing a surfactant, mixed uniformly by gently blowing, to obtain a live Aspergillus fumigatus spore suspension; the spore concentration is determined by using a blood cell counting board, and the spore viability is detected by a plate culture method, to ensure that the proportion of live spores in the spore suspension meets the experimental requirements; wherein the PBS buffer containing the surfactant is a PBS buffer containing 0.1% (by volume) Tween-80, and the suspension volume is 30 μL; After the nasal challenge is completed, the C57BL / 6J female mice are anesthetized, the anesthetic is a commonly used animal anesthetic, and the standard dose is injected to ensure that the C57BL / 6J female mice are moderately anesthetized and have stable vital signs; the anesthetized C57BL / 6J female mice are fixed on an operating table with the head fixed, and the prepared live Aspergillus fumigatus spore suspension is slowly injected into the lungs of the C57BL / 6J female mice by intratracheal injection, and the C57BL / 6J female mice are kept in the same position for a period of time after the injection is completed to ensure that the spore suspension is uniformly distributed in the lung tissue, and the stable colonization of Aspergillus fumigatus in the lung is achieved. S5, judgment model construction: after the live Aspergillus fumigatus spore colonization challenge stage is completed, the mice are sampled, and blood, lung lavage fluid and lung tissue samples are collected; the inflammatory cells in the lung lavage fluid are classified, counted and identified, the total IgE and Aspergillus fumigatus specific IgE levels in the serum are detected, and the pathological staining analysis of the lung tissue is combined to comprehensively judge whether the model is successfully constructed; the inflammatory cells include eosinophils, neutrophils, macrophages and lymphocytes; the pathological staining includes HE staining or PAS staining; Specifically, S51, the C57BL / 6J female mice are subjected to fasting treatment, sufficient water is provided during the fasting period, and then the samples are collected: 1) Blood sample collection: the blood of the C57BL / 6J female mice is collected by enucleation, and the blood is collected in a sterile container, centrifuged after being placed at room temperature for a period of time, and the serum is separated; the serum is aliquoted into sterile cryogenic tubes, sealed and stored in a low-temperature refrigerator for standby use for subsequent serological index detection; 2) Lung lavage fluid and lung tissue collection: the C57BL / 6J female mice are divided into two groups: ① The first group adopts the tracheal intubation method to collect the alveolar lavage fluid, first injects a certain amount of PBS buffer, and then slowly recovers the lavage fluid after a short stay. Repeat the operation several times, combine the recovered lavage fluid, and then put it into the centrifuge for centrifugation at an appropriate speed to separate the supernatant and the bottom cells. The supernatant is subpackaged into a cryogenic tube for low-temperature storage. The bottom cells are added to the fixing solution, gently blown and mixed, and then stored for a period of time. After collecting the alveolar lavage fluid, the C57BL / 6J female mice are dissected to remove the complete lung tissue, which is then placed in a cryogenic tube and stored at low temperature for future use; ② The second group directly dissects the lung tissue, one side of which is immersed in the fixing solution for pathological fixation, and the other side is quickly frozen in liquid nitrogen and then stored in a low-temperature refrigerator for long-term storage for subsequent molecular biology detection; S52, cell staining and observation: The bottom cell suspension after centrifugation of the alveolar lavage fluid is evenly spread on a glass slide to make a cell smear, which is naturally dried and then stained by HE and PAS staining methods. The staining steps follow the conventional HE and PAS staining process, that is, first fixation, then addition of HE and PAS staining solution, and finally washing with PBS buffer and air-drying; The stained smear is placed in a full-automatic cell scanning analyzer for whole-slide scanning to observe and count the number of eosinophils, neutrophils, macrophages, and lymphocytes, and analyze the airway inflammatory cell infiltration; S53, the evaluation model is successfully constructed, and the criteria include: ① The levels of serum total IgE and Aspergillus fumigatus-specific IgE are elevated; or ② The total number of inflammatory cells and the proportion of eosinophils in the alveolar lavage fluid are significantly increased, and the levels of IFN-γ (Th1 type), IL-17 (Th17 type), IL-5, IL-4, IL-13, IL-33, and TSLP (Th2 type) are elevated; or ③ The lung tissue pathological section shows increased airway eosinophil infiltration and mucus secretion; The operation is as follows: Immune index detection: The levels of serum total IgE and Aspergillus fumigatus-specific IgE are detected by enzyme-linked immunosorbent assay; the total number of inflammatory cells and the proportion of eosinophils in the alveolar lavage fluid, as well as the levels of IFN-γ (Th1 type), IL-17 (Th17 type), IL-5, IL-4, IL-13, IL-33, and TSLP (Th2 type), are detected by flow cytometry; Pathological observation: The fixed lung tissue is dehydrated, embedded, and sectioned, and then stained by HE and PAS staining methods to observe the airway mucosal eosinophil infiltration and mucus secretion; The above verification confirmed that the model animals exhibited pathophysiological characteristics similar to human ABPA, including elevated serum total IgE and Aspergillus fumigatus-specific IgE levels, or significantly elevated total number of inflammatory cells and proportion of eosinophils in bronchoalveolar lavage fluid, elevated levels of IFN-γ (Th1 type), IL-17 (Th17 type), IL-5, IL-4, IL-13, IL-33, and TSLP (Th2 type), or increased airway eosinophil infiltration and mucus secretion, indicating that the model was successfully constructed.

[0016] To highlight the advantages of the animal model of this invention, it was constructed in comparison with a conventional allergic asthma model, the ASA model; the ASA model construction steps are as follows: Week 1: Sensitization method is the same as the ABPA model of this invention; Weeks 3, 4, and 5: The protein nasal drop stimulation method is the same as the ABPA model of this invention; Week 6: 20 μg of Aspergillus fumigatus protein was dissolved in 30 μL of PBS containing 0.1% Tween-80 and administered via intratracheal injection for the final challenge; Week 7: Sample collection, testing, and observation.

[0017] The comparison revealed that the two models were completely identical in the initial sensitization and protein challenge phases. The only difference was the form of the antigen used for the final challenge: the ASA model administered Aspergillus fumigatus protein dissolved in PBS buffer via intratracheal injection in week 6, while the APBA model administered a suspension of live Aspergillus fumigatus spores via intratracheal injection in week 6.

[0018] The types and numbers of inflammatory cells were observed on days 3 and 7, and the results were as follows: Figure 3 As shown, the levels of IgE, IFN-γ, IL-17, IL-5, IL-4, IL-13, IL-33, and TSLP in the ABPA group were higher than those in the ASA model and the control group; this trend was more pronounced on day 7. HE and PAS staining were performed on days 3 and 7, respectively, to observe eosinophil infiltration and mucus secretion. The results are as follows... Figure 4 As shown, the ABPA model exhibited higher levels of eosinophil infiltration and mucus secretion than the ASA model and the control group; this trend was more pronounced on day 7. The difference between the control group and the ASA and ABPA models was that all steps involved treatment with physiological saline.

[0019] like Figure 5 The figure shows a comparison between the serum total IgE level and the Aspergillus fumigatus-specific IgE level detected by the APBA model and the control group. The serum total IgE level and the Aspergillus fumigatus-specific IgE level detected by the APBA model are significantly higher than those of the control group. Therefore, the APBA model in this invention has been successfully constructed.

[0020] In summary, the ABPA model of the present application, due to the introduction of live Aspergillus fumigatus spores airway colonization, provides a sustained endogenous antigen source, which is expected to produce a more intense and persistent inflammatory response, more complex immune cell infiltration, higher and more persistent serum total IgE levels, and a greater likelihood of observing more significant signs of mucus hypersecretion and even early airway structural changes pathologically, which is necessary to simulate the unique clinical pathology of ABPA.

[0021] The present application successfully constructs an animal model that can more comprehensively and stably simulate the key characteristics of human allergic bronchopulmonary aspergillosis by a carefully designed phased and complex sensitization and challenge strategy, especially the introduction of live Aspergillus fumigatus spores airway colonization. The model has important application value in the field of pathogenesis research and drug development.

[0022] The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the principles and substance of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. A method for constructing an APBA animal model, characterized in that, include: An ABPA animal model was established by intratracheal injection of a suspension of live Aspergillus fumigatus spores, including: S1. Select mice for adaptive feeding; S2, Initial sensitization stage: Aspergillus fumigatus protein was dissolved in PBS buffer and mixed with incomplete Freund's adjuvant to prepare a sensitization working solution; mice were initially sensitized by a combination of intraperitoneal injection and subcutaneous injection in the neck. S3, Repeated Challenge Phase: At a specific time after the initial sensitization phase, mice were repeatedly challenged by nasal drops using Aspergillus fumigatus protein dissolved in PBS buffer. S4. Colonization and activation stage of live Aspergillus fumigatus spores: After the repeated activation stage, a predetermined number of live Aspergillus fumigatus spores were suspended in PBS buffer containing surfactant, i.e., live Aspergillus fumigatus spore suspension, and delivered to the lungs of mice by intratracheal injection to enable Aspergillus fumigatus to colonize in the lungs of mice. S5. Evaluation of Model Construction: After the colonization and stimulation phase of live Aspergillus fumigatus spores, samples were collected from mice, including blood, bronchoalveolar lavage fluid, and lung tissue. The successful construction of the model was comprehensively evaluated, with the following criteria for successful construction: ① Elevated serum total IgE and Aspergillus fumigatus-specific IgE levels; or ② The total number of inflammatory cells and the proportion of eosinophils in the bronchoalveolar lavage fluid were significantly increased, as were the levels of IFN-γ (Th1 type), IL-17 (Th17 type), IL-5, IL-4, IL-13, IL-33, and TSLP (Th2 type); or ③ Lung tissue pathological sections showed eosinophilic infiltration and increased mucus secretion in the airways.

2. The method for constructing an APBA animal model according to claim 1, characterized in that, The selection of mice for adaptive feeding in step S1 specifically includes: Eight-week-old female C57BL / 6J mice, free from specific pathogen infection, were selected. The C57BL / 6J female mice were placed in a standardized breeding environment for one week for acclimatization feeding. The breeding environment was controlled with stable temperature, suitable humidity, day and night alternating lighting, and good ventilation. During the rearing period, provide sufficient sterile drinking water and standard feed, and observe the emotional state of the C57BL / 6J female mice daily to ensure that the C57BL / 6J female mice are free from disease infection and have stable emotions.

3. The method for constructing an APBA animal model according to claim 1, characterized in that, In step S2, the amount of Aspergillus fumigatus protein used is 10 μg per mouse, the amount of PBS buffer used is 100 μL, the amount of incomplete Freund's adjuvant used is 100 μL, and the amount of sensitizing working solution used for intraperitoneal injection and subcutaneous injection in the neck is 100 μL each. In step S3, the amount of Aspergillus fumigatus protein used for each stimulation is 20 μg per mouse, and the amount of PBS buffer used is 40 μL. The PBS buffer containing surfactant mentioned in step S4 is a PBS buffer containing 0.1% Tween-80 by volume, with a suspension volume of 30 μL.

4. The method for constructing an APBA animal model according to claim 1, characterized in that, The number of live Aspergillus fumigatus spores mentioned in step S4 is 5 × 10⁶ per mouse. 6 indivual.

5. The method for constructing an APBA animal model according to claim 1, characterized in that, The blood sample collection described in step S5 specifically includes: Blood was collected from mice by enucleation. The blood was collected in a sterile container, left at room temperature for a period of time, and then centrifuged to obtain serum. Serum was aliquoted into sterile cryovials, sealed, and stored in a low-temperature freezer for later use in serological marker testing.

6. The method for constructing an APBA animal model according to claim 1, characterized in that, In step S4, the bronchoalveolar lavage fluid and lung tissue were collected, and the mice were divided into two groups, specifically: ① In the first group, bronchoalveolar lavage fluid was collected via endotracheal intubation. A certain amount of PBS buffer was injected first, and after a brief retention period, the lavage fluid was slowly recovered. This process was repeated multiple times, and the recovered lavage fluids were combined. The combined bronchoalveolar lavage fluid was then centrifuged at an appropriate speed to separate the supernatant and basal cells. The supernatant was aliquoted into cryovials and cryopreserved for later use. The basal cells were added to fixative, gently pipetted to mix, and fixed for a period of time before use. After collecting the bronchoalveolar lavage fluid, the mice were dissected, and the intact lung tissue was removed and placed in cryovials for cryopreservation. ②The second group directly dissected and removed lung tissue. One side of the lung tissue was placed in a fixative solution and completely immersed for pathological fixation. The other side of the lung tissue was quickly placed in liquid nitrogen for flash freezing. After flash freezing, it was transferred to a low-temperature freezer for long-term cryopreservation for subsequent molecular biological detection.

7. A method for constructing an APBA animal model according to any one of claims 1 to 6, characterized in that, The animal construction method described herein is used in the preparation of tools for studying the pathogenesis of ABPA or for screening and evaluating therapeutic drugs.

Images