Construction method of recurrent chronic infection induced bronchiectasia mouse model

Abstract

The invention discloses a construction method of a bronchiectasia mouse model induced by recurrent chronic infection. The construction method comprises the following steps: S1, infecting a mouse with pseudomonas aeruginosa; s2.6-8 days later, the mice obtained in the step S1 are infected with the pseudomonas aeruginosa again; and S3, feeding the mouse obtained in the step S2 until the mouse is identified as bronchiectasis by pathology and imaging. According to the method, the agar beads coated with the low-concentration PAO1 pseudomonas aeruginosa are subjected to airway instillation for many times, a more stable, universal and repeatable infection-induced bronchiectasis animal model is constructed, the modeling success rate reaches up to 100%, death is avoided, the safety is higher, the representativeness is higher, model strains are easier to obtain, and the method is suitable for large-scale popularization and application. The in-depth study of bronchiectasis and the popularization and application of related treatment methods and treatment products are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of animal modeling, and in particular to a method for constructing a mouse model of bronchiectasis induced by repeated chronic infection. Background Art

[0002] Bronchiectasis (abbreviated as "bronchiectasis") is a clinical syndrome characterized by abnormal bronchial dilation and bronchial wall thickening shown in lung imaging, accompanied by clinical manifestations such as chronic cough, sputum and even hemoptysis. Bronchiectasis has complex heterogeneous symptoms, and its occurrence and development mechanism is also complex. The core development mechanism can be summarized as a "vicious vortex," in which infection, inflammation, airway dysfunction, and airway structural changes intertwine and develop progressively over time. Bronchiectasis is difficult to identify through general clinical manifestations. It was first described by French physician René Based on auscultation and autopsy reports, the invention of bronchography later allowed the imaging characteristics of the disease to be determined. Currently, the gold standard for the diagnosis of bronchiectasis is high-resolution CT (HRCT).

[0003] The exploration of animal models of bronchiectasis has also experienced many setbacks. There is no internationally unified standard for the modeling of animal models of bronchiectasis, and there is still a lack of suitable animal models. In addition, there is a lack of pathological and imaging evaluation standards, which hinders the exploration of the pathogenesis of bronchiectasis and the development of new drugs. Although many useful explorations have been made in chronic infection models and gene knockout mouse models, the lack of significant bronchiectasis characteristics, high modeling costs, long modeling cycles and low modeling rates have limited the further promotion of these models. Previous studies have explored a large number of animal modeling methods for bronchiectasis. Compared with the extremely difficult surgical airway ligation, bacterial infection-induced bronchiectasis is currently the most mainstream modeling method. Animals with chronic infection of Pseudomonas aeruginosa are often used, and agar-coated Pseudomonas aeruginosa is used for airway instillation, ultimately achieving the construction of a chronic lung infection model, in order to achieve chronic infection and persistent inflammation of the airways, and form secondary symptoms of bronchiectasis.

[0004] Existing bacterial infection-induced modeling methods all focus on whether animals have persistent lung infection. Usually, only one instillation can successfully construct a chronic infection model. However, in this state, animals cannot cause bronchiectasis in the short term, and cannot successfully form bronchiectasis 100%. Those skilled in the art often use the recognized Pseudomonas aeruginosa model strain PAO1 to construct a bronchiectasis animal model according to the traditional bacterial infection method, but its success rate is only about 20%. Although those skilled in the art have tried to increase the success rate of bronchiectasis modeling to 70% to 80% through strain optimization, most of these strains are the own strains of the research and development team, which are difficult for the public to obtain, and the modeling method is not easy to replicate and promote on a large scale, which is not conducive to promoting the study of bronchiectasis. In addition, in the bacterial infection-induced modeling method, there is a contradiction between the mortality rate of animals and the infection dose of the strain. If the infection dose is low, effective persistent infection cannot be produced, and if the infection dose is too high, it is easy to cause death. Therefore, it is urgent to develop a modeling method with a higher success rate, stronger safety, universal and repeatable, so as to provide a more reliable animal model for in-depth research on bronchiectasis. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides a method for constructing a mouse model of bronchiectasis induced by repeated chronic infection.

[0006] One object of the present invention is to provide a method for constructing a mouse model of bronchiectasis induced by repeated chronic infection.

[0007] Another object of the present invention is to provide application of the construction method.

[0008] Another object of the present invention is to provide an application of the bronchiectasis mouse model obtained by the construction method.

[0009] In order to achieve the above object, the present invention is implemented by the following scheme:

[0010] Based on the previous years of exploration of animal models of bronchiectasis, the applicant has explored the chronic Pseudomonas aeruginosa lung infection model, the chronic Pseudomonas aeruginosa lung infection rat model and the Pkd2 SM-CKOMouse model. The present invention proposes a method for constructing a mouse model of bronchiectasis induced by repeated chronic infection by improving the modeling method, and achieves directional infection of the airway through coated Pseudomonas aeruginosa PAO1 agar beads. Repeated multiple instillations achieve repeated and continuous infection and inflammation, simulate clinical bacterial infection, and simulate the process of infection-induced bronchiectasis formation. The animal model shows significant weight loss in the early stage of infection, and Pseudomonas aeruginosa PAO1 can effectively proliferate in mice. In the later stage, the body weight gradually increases to the initial level. Airway inflammation, bacterial colonization, and Micro-CT all determine that the mice have successfully formed airway dilation in a short period of time, providing a reliable animal model for the occurrence and development of bronchiectasis, which is suitable for the study of the mechanism of bronchiectasis caused by infection.

[0011] A method for constructing a mouse model of bronchiectasis induced by repeated chronic infection, comprising the following steps:

[0012] S1. Infect mice with Pseudomonas aeruginosa;

[0013] S2. After 6 to 8 days, the mice obtained in step S1 are infected again with the Pseudomonas aeruginosa, that is, the mice are infected twice with the Pseudomonas aeruginosa;

[0014] S3. raising the mice obtained in step S2 until the mice are identified as having bronchiectasis by pathology and imaging;

[0015] The single injection dose of Pseudomonas aeruginosa in mice was 10 5 CFU / pc~5×10 5 CFU / unit; the strain of Pseudomonas aeruginosa is PAO1.

[0016] Preferably, step S2 includes the following steps:

[0017] S21. After 6 to 8 days, the mice obtained in step S1 are infected again with the Pseudomonas aeruginosa;

[0018] S22. Repeat step S21 0 to 4 times, that is, infect mice 2 to 6 times using the Pseudomonas aeruginosa.

[0019] More preferably, in step S21, 7 days later, the mice obtained in step S1 are infected again with the Pseudomonas aeruginosa.

[0020] More preferably, in step S22, step S21 is repeated 2 or 4 times, that is, the mice are infected with the Pseudomonas aeruginosa 4 or 6 times in common.

[0021] Further preferably, in step S22, step S21 is repeated twice, that is, the mice are infected four times with the Pseudomonas aeruginosa.

[0022] Preferably, before the treatment in step S1, the mice are adaptively fed.

[0023] Preferably, before the treatment in step S1, the weight of the mouse is 28 g to 32 g.

[0024] Preferably, before the treatment in step S1, the mice are 12 weeks old.

[0025] Preferably, the strain of the mouse is C57BL / 6.

[0026] Preferably, the mouse is male.

[0027] Preferably, the strain number of the Pseudomonas aeruginosa is: ATCC 47085.

[0028] Preferably, the single infection dose of Pseudomonas aeruginosa is 2×10 5 CFU / piece.

[0029] Preferably, the pathological detection method includes HE staining.

[0030] Preferably, the imaging detection method includes CT.

[0031] More preferably, the CT comprises Micro-CT.

[0032] Preferably, the method of infecting mice with Pseudomonas aeruginosa comprises: injecting Pseudomonas aeruginosa agar beads into the airways of mice.

[0033] More preferably, the method of injecting Pseudomonas aeruginosa agar beads into the airway of a mouse comprises the following steps: fixing an anesthetized mouse, injecting the Pseudomonas aeruginosa agar beads into the airway of the mouse through the glottis via an indwelling tube, removing the indwelling tube, and keeping the mouse in a head-up, tail-down posture to prevent backflow.

[0034] Further preferably, after removing the indwelling tube, the mouse is kept in a posture with its head up and its tail down, and the mouse is gently shaken laterally several times to allow the Pseudomonas aeruginosa agar beads to be more evenly distributed in the lungs.

[0035] More preferably, the mouse is gently shaken laterally 5 times.

[0036] More preferably, the Pseudomonas aeruginosa agar beads contain 10 5 CFU / 50μl~5×10 5 CFU / 50 μl agar beads of Pseudomonas aeruginosa.

[0037] Further preferably, the Pseudomonas aeruginosa agar beads contain 2×10 5CFU / 50 μl agar beads of Pseudomonas aeruginosa.

[0038] More preferably, the diameter of the Pseudomonas aeruginosa agar beads is 100 μm to 300 μm.

[0039] More preferably, the method for preparing the Pseudomonas aeruginosa agar beads comprises the following steps: fully mixing the Pseudomonas aeruginosa with TSA culture medium and mineral oil, and performing solid-liquid separation to obtain the agar beads.

[0040] Further preferably, the preparation method of the Pseudomonas aeruginosa agar beads comprises the following steps: culturing the Pseudomonas aeruginosa in TSA culture medium to obtain a monoclonal colony, then culturing the monoclonal colony in TSB liquid culture medium to obtain a bacterial culture solution, collecting the bacterial cells by centrifugation, and resuspending the bacterial cells in PBS to obtain a bacterial solution; fully mixing the bacterial solution with liquid TSA culture medium and mineral oil in a volume ratio of 1: (8-10): 100 to obtain an agar bead-heavy mineral oil mixture, and performing solid-liquid separation to obtain the product.

[0041] More preferably, the bacterial liquid is fully mixed with liquid TSA culture medium and mineral oil in a volume ratio of 1:9:100.

[0042] More preferably, the bacterial liquid is first fully mixed with liquid TSA culture medium to obtain a TSA-PAO1 mixture, and then the TSA-PAO1 mixture is fully mixed with the mineral oil.

[0043] Still more preferably, the TSA-PAO1 mixture and the mineral oil are first stirred at 20°C to 30°C at a rotation speed of 1000 to 2000 rpm for 4 to 6 min, and then stirred at 0°C to 4°C at a rotation speed of 200 to 400 rpm for 10 to 30 min.

[0044] Still more preferably, the TSA-PAO1 mixture and the mineral oil are first stirred at 25° C. at a rotation speed of 1500 rpm for 5 min, and then stirred on ice at a rotation speed of 300 rpm for 20 min.

[0045] More preferably, the liquid TSA culture medium is a TSA culture medium at 40°C to 55°C.

[0046] Still more preferably, the liquid TSA medium is 50°C TSA medium.

[0047] More preferably, before mixing, the mineral oil is preheated to 40°C to 55°C.

[0048] Still further preferably, the mineral oil is preheated to 50°C before mixing.

[0049] Further preferably, the solid-liquid separation method comprises screening.

[0050] More preferably, the sieves used for screening include sieves with a diameter of 300 μm and a diameter of 100 μm.

[0051] Further preferably, the solid-liquid separation method includes centrifugation and washing.

[0052] More preferably, the centrifugal conditions include centrifugation at 3500 g to 4500 g for 10 min to 20 min.

[0053] Still further preferably, the centrifugal conditions include centrifugation at 4000 g for 15 min.

[0054] More preferably, the washing method comprises: centrifugal washing with PBS, washing 4 to 6 times.

[0055] Still more preferably, the washing method comprises: washing by centrifugation with PBS, washing 5 times.

[0056] Further preferably, the solid-liquid separation method includes sufficient standing.

[0057] More preferably, the standing time is 10 min to 30 min.

[0058] Still more preferably, the standing time is 30 min.

[0059] Specifically, the preparation method of the Pseudomonas aeruginosa agar beads comprises the following steps: culturing the Pseudomonas aeruginosa in TSA medium to obtain a monoclonal colony, culturing the monoclonal colony in TSB liquid medium to obtain a bacterial culture solution, collecting the bacterial cells by centrifugation, and resuspending the bacterial cells in PBS to obtain a bacterial solution; fully mixing the bacterial solution with 50°C TSA medium and mineral oil in a volume ratio of 1:9 to obtain a TSA-PAO1 mixture, and then stirring the TSA-PAO1 mixture and the 50°C mineral oil at 25°C at a rotation speed of 1500 rpm for 5 minutes, and then evaporating on ice. The mixture was stirred at a speed of 300 rpm for 20 min to obtain an agar bead-heavy mineral oil mixture; the mixture was centrifuged at 4000 g for 15 min, the precipitate was collected, and the mixture was washed by centrifugation with PBS for 5 times, the precipitate was collected and resuspended with PBS to obtain an agar bead suspension; the agar bead suspension was filtered with sieves with diameters of 300 μm and 100 μm, the agar beads trapped on the sieve with a diameter of 100 μm were collected by washing with PBS to obtain a suspension containing Pseudomonas aeruginosa PAO1 agar beads with a diameter of 100 to 300 μm, the mixture was allowed to stand for 30 min, the supernatant was removed by aspiration, and the agar beads settled in the lower layer were collected to obtain the mixture.

[0060] The application of any of the construction methods in any of the following aspects (1) to (4) should also be within the scope of protection of the present invention:

[0061] (1) Study the pathogenesis of bronchiectasis;

[0062] (2) Study the treatment of bronchiectasis;

[0063] (3) preparing or screening products for preventing and / or treating bronchiectasis;

[0064] (4) Testing the safety and / or efficacy of drugs.

[0065] Preferably, the bronchiectasis is complicated by infection.

[0066] More preferably, the infection comprises a bacterial infection.

[0067] The application of the bronchiectasis mouse model obtained by any of the construction methods in any of the following aspects (1) to (4) should also be within the scope of protection of the present invention:

[0068] (1) Study the pathogenesis of bronchiectasis;

[0069] (2) Study the treatment of bronchiectasis;

[0070] (3) preparing or screening products for preventing and / or treating bronchiectasis;

[0071] (4) Testing the safety and / or efficacy of drugs.

[0072] Preferably, the bronchiectasis is complicated by infection.

[0073] More preferably, the infection comprises a bacterial infection.

[0074] Compared with the prior art, the present invention has the following beneficial effects:

[0075] The present invention constructs a more stable, universal and repeatable infection-induced bronchiectasis animal model by multiple airway instillations of agar beads coated with low-concentration PAO1 Pseudomonas aeruginosa. The modeling success rate is as high as 100%, there is no mortality, the modeling is safer and more representative, and the model strain is easier to obtain, which is conducive to the in-depth study of bronchiectasis and the promotion and application of related treatment methods and treatment products. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Schematic diagram of the process for constructing a mouse model of bronchiectasis induced by recurrent chronic infection.

[0077] Figure 2 This is the morphology of Pseudomonas aeruginosa PAO1 agar beads.

[0078] Figure 3 Survival curves of mice instilled with different concentrations of PAO1 agar beads in the airways, n=10.

[0079] Figure 4 Schematic diagram of the nomenclature for the mouse airway grades.

[0080] Figure 5 The figure is the statistical graph of the body weight of mice in each group.

[0081] Figure 6 It is a line graph of the weight changes of mice in each group.

[0082] Figure 7 are the lung pathology evaluation results of mice in each group; A is the coronal lung pathology HE results of mice in each group (including objective lens 1X and objective lens 10X); B is the pathology score, n=10, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0083] Figure 8 The three-dimensional reconstruction images of the lung airways of mice in each group and the coronal airway projection images viewed under the lung window (WL=-400, WW=1500) using the minimum density projection of 10.0 mm.

[0084] Fig. 9 Statistical bar graphs of the airway volume and the mid-airway cross-sectional circumference of LMB3, MiRMB3, CaRMB3, AcRMB3, and Cr RMB3 in each group of mice, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0085] Fig.10 Bar graph showing lung statistics of mice in each group, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0086] Fig.11 For Pkd2 SM-CKO Gross appearance of mice.

[0087] Fig.12 For Pkd2 SM-CKO HE staining results of multiple cysts in the lungs, liver and kidneys of mice (including objective lens 1X and objective lens 10X).

[0088] Fig.13 For Pkd2 SM-CKO Imaging performance of mice. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0090] Example 1 Construction method of a mouse model of bronchiectasis induced by recurrent chronic infection and evaluation of modeling effect

[0091] The process of constructing a bronchiectasis mouse model and evaluating the modeling effect in this example is as follows: Figure 1 As shown, the method mainly includes the following steps: preparation of the instillation agent, instillation into the mouse airway and modeling identification.

[0092] 1. Preparation of Infusion

[0093] 1. Pseudomonas aeruginosa PAO1 culture

[0094] Pseudomonas aeruginosa PAO1 (ATCC 47085, stored in the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health) was inoculated into TSA medium for plate streak culture, and then a single colony was picked and inoculated into 5 ml of TSB liquid medium, and cultured overnight at 220 rpm and 37°C for 18 h to obtain the first culture solution.

[0095] The first culture solution was inoculated into the new TSB liquid culture medium and shaken again. The first culture solution and the new TSB liquid culture medium were mixed at a volume ratio of 1:20, and shaken again at 220 rpm and 37° C. for 18 hours to obtain a second culture solution.

[0096] 2. Bacterial liquid coating

[0097] All the second culture solution was taken, centrifuged at 4°C, 2500g for 10 min, the supernatant was discarded, the bacteria were resuspended with 1 mL of sterile PBS, and then 9 mL of TSA solid culture medium preheated to 50°C was added and mixed evenly to obtain a TSA-Pseudomonas aeruginosa PAO1 mixture, recorded as TSA-PAO1 mixture.

[0098] While hot, quickly add 10 mL of the TSA-PAO1 mixture into a conical flask containing 100 mL of mineral oil (the mineral oil product number is 8042-47-5, also known as liquid paraffin) preheated to 50°C, and rapidly stir with a stirrer at room temperature (25°C) for 5 min at a speed of 1500 rpm, then move to ice and stir at a low speed for 20 min at a speed of 300 rpm to obtain an agar bead-heavy mineral oil mixture.

[0099] 3. Agar bead separation

[0100] The agar beads-heavy mineral oil mixture was transferred to a 50 ml centrifuge tube and separated by low-temperature high-speed centrifugation, the centrifugal force was 4000 g, and the centrifugation time was 15 min. The upper mineral oil was sucked off, and sterile PBS was added for washing and centrifugation to separate the mineral oil and agar beads, the centrifugal force was 4000 g, the centrifugation time was 15 min, and the number of elution centrifugation was 5 times. The precipitate was collected to obtain Pseudomonas aeruginosa PAO1 agar beads, and then resuspended with sterile PBS to obtain an agar bead suspension.

[0101] The agar bead suspension was filtered through 300 μm and 100 μm diameter sieves, and the agar beads trapped on the 100 μm diameter sieve were washed with sterile PBS to obtain a suspension containing Pseudomonas aeruginosa PAO1 agar beads with a diameter of 100 to 300 μm. The suspension was allowed to stand for 30 min, the supernatant was removed, and the agar beads that settled in the lower layer were collected and recorded as PAO1 agar beads. They were stored at 4°C for future use. The morphology of PAO1 agar beads observed under a microscope was as follows: Figure 2 As shown, its diameter is 100 to 300 μm.

[0102] 4. Bacterial content adjustment

[0103] Take 0.5 mL of PAO1 agar beads and make a homogenate with a sterile homogenizer, and then determine the bacterial content. The method is as follows: dilute the homogenate in multiples, apply it on a solid TSA culture plate, incubate at 37°C overnight, and calculate the bacterial content of PAO1 agar beads the next day. According to the calculation results, adjust the bacterial content of PAO1 agar beads with sterile PBS.

[0104] After the instillation pilot experiment, PAO1 agar beads with different bacterial contents were instilled into the mouse airways, and the following results were obtained: Figure 3 The survival curve shown in the figure shows that when the bacterial count of PAO1 agar beads is 10 5 CFU / 50μl and 5×10 5 No mice died during the 7-day observation period. 6 CFU / 50μl, one mouse died on the first day after instillation, and the final survival rate was 80%; when the bacterial content of PAO1 agar beads was 5×10 6 CFU / 50μl, the final survival rate of mice was only 40%; when the bacterial content of PAO1 agar beads was 10 7 CFU / 50μl, the final survival rate of mice was only 20%; when the bacterial content of PAO1 agar beads was 5×10 7 CFU / 50μl, all the bacteria died on the 5th day. 5 CFU / 50μl~5×10 5CFU / 50μl is a safe instillation concentration.

[0105] In this example, the bacterial content is 2×10 5 PAO1 agar beads / 50 μL were prepared to obtain a dripping agent for constructing an animal model.

[0106] 2. Airway Instillation in Mice

[0107] 1. Grouping of mice

[0108] Fifty C57BL / 6 mice (12 weeks old, male, body weight stabilized at 30±2g after adaptive feeding before modeling) were randomly divided into 5 groups, 10 mice in each group, respectively recorded as blank control group (CTL), 1 instillation group (1W), 2 instillation group (2W), 4 instillation group (4W) and 6 instillation group (6W).

[0109] 2. Instillation treatment

[0110] Except for the blank control group which was drip-injected with sterile PBS, the mice in the other four groups were drip-injected with the drip preparation prepared in this example. The drip method was as follows:

[0111] The mice were anesthetized with 2% Avertin (2,2,2-tribromoethanol, catalog number T48402-25G) and weighed. The mice were fixed on the small animal operation fixing plate, the upper incisors of the mice were fixed with a thin wire, and the fixing plate was tilted 70°. The 22G indwelling tube was passed through the glottis into the mouse airway and fixed, and 50 μl of instillation agent was injected through the indwelling tube. After staying for 20 seconds and the instillation agent was completely inhaled, the indwelling tube was removed, and then the fixing plate was erected, the mouse was kept head up and tail down, and gently shaken horizontally 5 times to allow the instillation agent to be more evenly distributed in the lungs and prevent backflow. After the instillation is completed, the mouse was put back in the cage, and attention was paid to keeping warm. After waking up, continue to feed normally. The day of the first instillation was taken as the 0th day of modeling.

[0112] The mice in the one-instillation group received only one instillation on the 0th day of modeling; the mice in the two-instillation group received one instillation on the 0th day and the 7th day of modeling; the mice in the four-instillation group received one instillation on the 0th day, the 7th day, the 14th day and the 21st day of modeling; the mice in the six-instillation group received one instillation on the 0th day, the 7th day, the 14th day, the 21st day, the 28th day and the 35th day of modeling; the blank control group received one instillation on the 0th day, the 7th day, the 14th day, the 21st day, the 28th day and the 35th day of modeling.

[0113] 3. Modeling effect evaluation

[0114] 1. Basic situation monitoring

[0115] During the modeling period, the weight changes of the five groups of mice were monitored daily, and the weight changes were expressed as weight after modeling / weight before modeling. The survival of mice was counted, and the survival rate of mice (%) was calculated as follows: number of survivors / total number of models × 100%.

[0116] 2. Pathological and imaging evaluation

[0117] Ten mice in the instillation group were treated once on the 6th day of modeling: 5 mice were randomly selected for Micro-CT (super NovaMicro CT SNC-100, resolution 35μm) scanning, and then the left lung was taken for pathological sectioning and stained with HE. The whole lungs of the remaining 5 mice were extracted under a sterile environment, and 1ml of sterile PBS was used for homogenization of each 500mg lung tissue, and then gradient dilution was performed for plating, and the live bacterial load in the lungs was counted.

[0118] On the 14th day of modeling, 10 mice in the double instillation group were first subjected to Micro-CT scanning according to the same method, and then the left lung was taken for pathological sections and stained with HE.

[0119] On the 21st day of modeling, 10 mice in the group treated with four instillations were first subjected to Micro-CT scanning according to the same method, and then the left lung was taken for pathological sections and stained with HE.

[0120] On the 35th day of modeling, 10 mice in the 6-times instillation group were first subjected to Micro-CT scanning according to the same method, and then the left lung was taken for pathological sections and stained with HE.

[0121] On the 35th day of modeling, 10 mice in the blank control group were treated and first subjected to Micro-CT scanning according to the same method, and then the left lung was taken for pathological sections and stained with HE.

[0122] The pathological condition of the mouse lung tissue was evaluated according to the scoring criteria shown in Table 1, and the total lung tissue pathological score was calculated as A+3(B+C)+E+D.

[0123] Table 1 Lung histopathological scoring criteria

[0124]

[0125] 3. Evaluation of airway dilation

[0126] According to the Micro-CT images of mouse lungs, reference was made to the existing technology "CT manifestations of bronchiectasis: Expert consensus writing group on bronchiectasis, Infection Group of the Respiratory Disease Branch of the Chinese Medical Association. Expert consensus on the diagnosis and treatment of bronchiectasis in adults in China. Chinese Journal of Tuberculosis and Respiratory Diseases, 2021, 44(04):311-321.; Aliberti S, Goeminne PC, O'Donnell AE, et al. Criteria and definitions for the radiological and clinical diagnosis of bronchiectasis in adults for use in clinical trials: international consensus recommendations. Lancet Respir Med. 2022; 10(3):298-306. doi:10.1016 / S2213-2600(21)00277-0; Tiddens HAWM, Meerburg JJ, van der Eerden MM, Ciet P. The radiological diagnosis of bronchiectasis: what's in a name? .Eur Respir Rev. 2020; 29(156): 190120. Published 2020Jun17. doi: 10.1183 / 16000617.0120-2019” to assess the airway dilation in mice.

[0127] In addition, based on the Micro-CT image data of the mouse lung, multi-planar reconstruction was performed using DICOM viewing software (RadiAntDICOMViewer 2023.1), and the coronal airway projection was viewed using a minimum density projection of 10.0 mm under the lung window (WL = -400, WW = 1500), and the cross-sectional perimeter of the middle airway segment (the midpoint between the previous airway bifurcation and the next airway bifurcation) such as LMB3, Ca RMB3, MiRMB3, AcRMB3, and CrRMB3 was measured in the cross section. Avizo 3D was used to reconstruct the airway 3D of the Micro-CT image data, and Label analysis was used to measure the airway volume. All data are shown as mean ± standard deviation (SD). Analysis of variance was followed by Bonferroni multiple comparison test (GraphPad Prism) for analysis.

[0128] Mouse airway classification and related nomenclature refer to the prior art "Navarro, M., J. Ruberte and A. Carretero, 6-Respiratory apparatus, in Morphological Mouse Phenotyping, J. Ruberte, A. Carretero and M. Navarro, J. Ruberte, A. Carretero and M. Na varro^Editors. 2017, Academic Press. p. 147-178." and "Thiesse J, Namati E, Si eren JC, Smith AR, Reinhardt JM, Hoffman EA, McLennan G. Lung structure phenotype variation in inbred mouse strains revealed through in vivo micro-CT imaging. J Appl Physiol (1985). 2010 Dec; 109 (6): 1960-8." For details, see Figure 4 shown.

[0129] 4. Evaluation results

[0130] (1) Survival

[0131] During the modeling process, no deaths occurred in the five groups of mice, and the survival rate was 100%, indicating that the lower concentration of instilled bacteria effectively avoided the inflammatory storm caused by acute infection and inflammation, thereby reducing the mortality rate caused by modeling.

[0132] (2) Weight changes

[0133] The weight changes of mice in each group from day 0 to day 7 of modeling are shown in Figure 5 and Figure 6 As shown, except for the blank control group, the body weights of the mice in the other four groups gradually decreased after the first infusion, slowly recovered after the second day of modeling, and basically returned to the initial level on the seventh day of modeling.

[0134] (3) Pathological results

[0135] HE staining results of lung pathological sections of mice in each group are shown in Figure 7As shown in A, compared with the blank control group, the four groups of mice instilled with PAO1 agar beads showed different degrees of inflammation. In the mice in the one-time instillation group, about 25% of the bronchioles and bronchi were infiltrated by inflammatory cells, and the infiltration sites were often accompanied by short ring-shaped or moderately complete ring-shaped infiltrations. The thickness of the infiltrating inflammatory cells was less than 5, and perivascular infiltration was occasionally seen, accompanied by a small amount of interstitial pneumonia. About 50% of the mice in the two-time instillation group were infiltrated by inflammatory cells around the bronchioles and bronchi. The infiltration sites were often accompanied by discontinuous or moderately complete rings, and the thickness of the infiltrating cells was less than 5. More than 5, perivascular infiltration was common, accompanied by a small amount of interstitial pneumonia; in the 4-instillation group, about more than 50% of the mice in the bronchioles and bronchi were infiltrated by inflammatory cells, and a complete inflammatory cell infiltration ring was formed at the infiltration site. The thickness of the inflammatory cell infiltration was more than 10, and most of the inflammatory infiltrations were perivascular, accompanied by a small amount of interstitial pneumonia; in the 6-instillation group, about more than 75% of the mice in the bronchioles and bronchi were infiltrated by inflammatory cells, and a complete inflammatory cell infiltration ring was formed at the infiltration site. The thickness of the inflammatory cell infiltration was more than 10, and most of the inflammatory infiltrations were perivascular, accompanied by a small amount of interstitial pneumonia.

[0136] The lung tissue pathological scores of mice in each group are shown in Figure 7 As shown in B, it can be seen that the pathological score showed an upward trend with the increase in the number of infusions, and the lung tissue pathological scores of mice in the 4-infusion group and the 6-infusion group were basically close.

[0137] The above results show that mice that underwent repeated airway instillation of PAO1 agar beads all had varying degrees of peri-airway inflammatory cell infiltration, and a small amount of peri-alveolar inflammatory cell infiltration. The intra-group differences of 2, 4, and 6 instillations were relatively reduced. Multiple instillations made the infection site more uniform and reduced accidental errors.

[0138] (4) Imaging results

[0139] like Figure 8 As shown, after more than two instillations, the cross-sectional circumferences of the middle airway segment (the midpoint between the previous airway bifurcation and the next airway bifurcation) of mouse LMB3, CaRMB3, MiRMB3, AcRMB 3, CrRMB3, etc. were significantly increased compared with the blank control group and the one-instillation group, and the data of the four-instillation group and the six-instillation group were stable and close.

[0140] Avizo 3D was used to reconstruct the mouse airway. Fig. 9 As shown, the airway volume was measured, and the results also showed that the airway volume of mice in the two-instillation group was increased compared with the blank control group, and the airway volume of mice instilled 4 and 6 times was significantly increased, and the two were stable and close.

[0141] (5) Lung viable bacterial load

[0142] like Fig.10 As shown, no live bacteria were detected in the lung tissue of mice in the blank control group, the live bacterial load in the lungs of mice in the one-time instillation group was 53200±14305CFU / Lung, the live bacterial load in the lungs of mice in the two-time instillation group was 83200±24670CFU / Lung, the live bacterial load in the lungs of mice in the four-time instillation group was 79200±14786CFU / Lung, and the live bacterial load in the lungs of mice in the six-time instillation group was 98000±25298CFU / Lung. This indicates that compared with the one-time instillation, the bacterial load in the lungs of mice in the two-time instillation group or more was significantly increased and maintained at a relatively close level.

[0143] (6) Modeling success rate

[0144] The success rate of modeling was calculated based on the results of pathological and imaging evaluation: 0% in the blank control group, 50% in the one-time infusion group, and 100% in the two-time infusion group, the four-time infusion group, and the six-time infusion group.

[0145] The above results show that mice that have been repeatedly instilled more than twice can successfully develop typical bronchiectasis 100% successfully and can objectively simulate the airway dilation of clinical bronchiectasis.

[0146] Example 2 A method for constructing a mouse model of bronchiectasis induced by recurrent chronic infection

[0147] The construction method provided in this embodiment is basically the same as that in embodiment 1, except that: the bacterial content is 1×10 5 PAO1 agar beads / 50 μL were prepared to obtain a dripping agent for constructing an animal model.

[0148] The same method was used for 2 instillations, 4 instillations and 6 instillations. The modeling effect was evaluated and the results showed that none of the mice in this example died. The weight changes, pathological results, imaging results and lung viable bacterial load were all in the same trend as in Example 1. The mice that had undergone repeated airway instillation of PAO1 agar beads all had varying degrees of peri-airway inflammatory cell infiltration, a small amount of peri-alveolar inflammatory cell infiltration, uniform infection sites, and 100% successful formation of typical bronchiectasis, which could objectively simulate the airway dilation of clinical bronchiectasis.

[0149] Example 3 A method for constructing a mouse model of bronchiectasis induced by recurrent chronic infection

[0150] The construction method provided in this embodiment is basically the same as that in embodiment 1, except that: the bacterial content is 5×10 5 PAO1 agar beads / 50 μL were prepared to obtain a dripping agent for constructing an animal model.

[0151] The same method was used for 2 instillations, 4 instillations and 6 instillations. The modeling effect was evaluated and the results showed that none of the mice in this example died. The weight changes, pathological results, imaging results and lung viable bacterial load were all in the same trend as in Example 1. The mice that had undergone repeated airway instillation of PAO1 agar beads all had varying degrees of peri-airway inflammatory cell infiltration, a small amount of peri-alveolar inflammatory cell infiltration, uniform infection sites, and 100% successful formation of typical bronchiectasis, which could objectively simulate the airway dilation of clinical bronchiectasis.

[0152] Comparative Example 1: Construction method of a gene knockout bronchiectasis mouse model and evaluation of modeling effect

[0153] 1. Construction of Pkd2 conditional knockout mice

[0154] The mouse strain name is T009630Pkd2 paired with T006862Tagln-Cre, with a strain background of C57BL / 6JGpt, which was constructed and provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The construction technology is CRISPR-Cas9 CKO and CRISPR-Cas9 KI technology. The breeding scheme is: Pkd2: Fl / wt paired with Fl / wt; Tagln-Cre: ki / wt paired with wt / wt. Pkd2 is a gene related to polycystic kidney disease; Tagln-Cre is a Cre recombinase expressed by vascular smooth muscle; Fl means floxed, that is, the gene is surrounded by loxP sites and can be knocked out under the action of Cre recombinase. Finally, Pkd2 was obtained. Fl / Fl ,Tagln-Cre ki / wt This is the target mouse, named Pkd2 SM-CKO Mouse.

[0155] 2. Modeling effect evaluation

[0156] Pkd2 SM-CKO The overall survival rate of mice is low, with an average lifespan of only 20±5 days. Due to the decrease in smooth muscle tension and reduced vascular permeability, the anatomical results show that Fig.11 As shown, Pkd2 SM-CKO All mice developed multiple ascites; HE staining showed Fig.12 As shown, Pkd2 SM-CKO Multiple cysts were observed in the lung, kidney, and liver tissues of mice, indicating that Pkd2 SM-CKO The mice had multiple cysts all over their bodies. All of the above indicated that Pkd2 SM-CKO The mice were in poor physical condition and could hardly support further experimental treatment.

[0157] Micro-CT scanning showed Fig.13 As shown, although Pkd2 SM-CKOMice can show signs of bronchiectasis on imaging, but the degree of airway dilation varies greatly among mice. Considering that the mice are 20 days old at this time, their lungs are not fully developed. The imaging manifestation of the spherical dilation of the airways is considered to be physiological dilation, which may be repaired later, and it is uncertain whether a stable bronchiectasis phenotype can be formed. The above results show that although Pkd2 gene-deficient mice can show a bronchiectasis phenotype, the bronchiectasis phenotype is not stable, and the experimental cycle is long, the knockout technology is demanding, the price is high, and there are many complications caused by gene defects, making it difficult to conduct more in-depth exploration, which seriously hinders the establishment of a bronchiectasis mouse model and cannot meet research needs.

[0158] Table 2 Current animal models

[0159]

[0160] As shown in Table 2, although the existing animal models can simulate some phenotypes of bronchiectasis to a certain extent, the general modeling success rate is low, and the operating cost required is more or less high. The present invention uses low bacterial concentration and repeated airway instillation of Pseudomonas aeruginosa PAO1 agar beads to simulate the occurrence and development of bronchiectasis induced by repeated infection, and evaluates it through lung pathology, Micro-CT and lung bacterial load, to construct a bronchiectasis mouse model with low mortality, high success rate, repeatability and evaluability, which provides a more effective and convenient way for in-depth research on bronchiectasis, and helps to further clarify the mechanism of bronchiectasis and the effect of drugs.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for constructing a mouse model of bronchiectasis induced by repeated chronic infection, characterized in that: The following steps are involved: S1. Infect mice with Pseudomonas aeruginosa; S2. After 6 to 8 days, the mice obtained in step S1 are infected again with the Pseudomonas aeruginosa; S3. raising the mice obtained in step S2 until the mice are identified as having bronchiectasis by pathology and imaging; The single infection dose of Pseudomonas aeruginosa is 10 5 CFU / pc~5×10 5 CFU / piece; The strain of Pseudomonas aeruginosa is PAO1.

2. The construction method according to claim 1, characterized in that: Step S2 includes the following steps: S21. After 6 to 8 days, the mice obtained in step S1 are infected again with the Pseudomonas aeruginosa; S22. Repeat step S21 0 to 4 times.

3. The construction method according to claim 2, characterized in that: In step S22, step S21 is repeated 2 times or 4 times.

4. The construction method according to any one of claims 1 to 3, characterized in that: The method of infecting mice with Pseudomonas aeruginosa comprises: injecting Pseudomonas aeruginosa agar beads into the airways of mice.

5. The construction method according to claim 4, characterized in that: The diameter of the Pseudomonas aeruginosa agar beads is 100 μm to 300 μm.

6. The construction method according to claim 4, characterized in that: The preparation method of the Pseudomonas aeruginosa agar beads comprises the following steps: culturing the Pseudomonas aeruginosa with TSA culture medium and fully mixing with mineral oil, and performing solid-liquid separation to obtain the agar beads.

7. The construction method according to claim 1, characterized in that: The pathological detection method includes HE staining.

8. The construction method according to claim 1, characterized in that: The imaging detection method includes CT.

9. Application of the construction method according to any one of claims 1 to 8 in any one of the following aspects (1) to (4): (1) Study the pathogenesis of bronchiectasis; (2) Study the treatment of bronchiectasis; (3) preparing or screening products for preventing and / or treating bronchiectasis; (4) Testing the safety and / or efficacy of drugs.

10. Use of the bronchiectasis mouse model obtained by the construction method according to any one of claims 1 to 8 in any one of the following aspects (1) to (4): (1) Study the pathogenesis of bronchiectasis; (2) Study the treatment of bronchiectasis; (3) preparing or screening products for preventing and / or treating bronchiectasis; (4) Testing the safety and / or efficacy of drugs.

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