Chronic obstructive pulmonary disease mouse model as well as construction method and application thereof
By employing a synergistic induction strategy combining biofuel smoke exposure with elastase atomization, the problems of long processing time and unstable phenotypes in existing COPD mouse models have been solved, resulting in a more clinically relevant and stable COPD mouse model suitable for COPD research and drug screening.
Patent Information
- Application Number
- CN202511160709.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-19
- Publication Date
- 2025-12-19
AI Technical Summary
Existing COPD mouse models are difficult to capture the changes of chronic bronchitis and emphysema simultaneously from a pathophysiological perspective. Furthermore, existing methods are time-consuming, have unstable phenotypes, are highly harmful to animals, and have poor relevance to reality.
A mouse model of chronic obstructive pulmonary disease was established by using a synergistic induction strategy of biofuel smoke exposure combined with elastase atomization. The mouse model was established by exposing the mouse to smoke 2-3 times a day for 2-4 hours each time, 6 days a week, combined with weekly elastase atomization inhalation for 16 weeks.
The model achieved stability and real-world relevance in a shorter time, reduced harm to animals, shortened model construction time, and improved phenotypic stability and consistency with the pathological characteristics of human COPD.
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Figure CN121154797A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biotechnology, and particularly relates to a mouse model of chronic obstructive pulmonary disease and a construction method and application thereof. BACKGROUND
[0002] Due to the complex pathophysiology of chronic obstructive pulmonary disease (COPD), it is difficult to capture the changes related to chronic bronchitis or emphysema in a single model. The existing reported modeling method does not involve the use of biofuels combined with other methods for modeling of stable COPD. A widely used acute exacerbation period COPD modeling method at home and abroad is PM2.5 instillation combined with LPS model. This method significantly shortens the modeling time of COPD and improves the stability of the animal model, however, there are many influencing factors of this modeling method, including the difference in the time and dose of LPS administration, and the PM2.5 instillation has poor correlation with reality. The COPD animal model using elastase instillation is currently mainly combined with cigarette smoke exposure, and the modeling time is shorter. However, the cigarette smoke exposure combined with elastase instillation model has some limitations, for example, in the process of tracheal instillation of protease, tracheal incision is required, plus the complexity of smoke exposure, these operations may cause the model animals to suffer heavy trauma, difficult to recover, and even may induce infection; the method of airway instillation is easy to cause animals to cough, and the operation of airway incision can cause animals to be infected and the mortality rate to rise. It cannot guarantee that the drug reaches the small airways for induction, nor can it guarantee the consistency of the drug concentration in each airway, resulting in an unclear phenotype. Studies have shown that elastase is involved in the occurrence and development of COPD, but the disease pathogenesis of the single method of elastase injection is less understood, and the immune cells involved in this model are low, so the model using elastase alone is only an emphysema and lung tissue destruction model, and the real simulation degree of COPD is poor.
[0003] The existing biofuel related modeling method is limited to PM2.5 collection and direct instillation into the airway of animals or exposure to automobile exhaust, ignoring the impact of other biofuels and other toxic and harmful substances on the airway on the disease - an important environmental risk factor for COPD. In rural areas of China, a large number of people still use wood as fuel and there is a behavior of burning grasswood ash as fertilizer, especially women and children, who are plagued by the disease. In addition, the existing modeling method takes a long time of 6 months, and has the disadvantage of unstable phenotype. SUMMARY
[0004] The present application aims to overcome the shortcomings and deficiencies of the prior art and provide a mouse model of chronic obstructive pulmonary disease and a construction method and application thereof. The present application has the advantages of high practical relevance, short time, high stability and low harm to animals by exposing mice to biofuel smoke and atomizing protease for 4 months to induce the COPD disease model.
[0005] To achieve the above object, the technical scheme adopted by the present application comprises:
[0006] In a first aspect, the present application provides a construction method of a mouse model of chronic obstructive pulmonary disease, comprising the following steps:
[0007] The mouse model of chronic obstructive pulmonary disease is constructed by treating the mouse with biofuel smoke exposure and inducing the mouse with elastase atomization.
[0008] In the prior art, COPD models mostly rely on cigarette smoke exposure (simulating smoking triggers) or simple elastase / lipopolysaccharide (LPS) induction (simulating protease imbalance or infection triggers). However, clinical studies have shown that biofuel smoke is the main risk factor for COPD in non-smoking populations in developing countries, and its damage mechanism differs from that of cigarette smoke. Moreover, the inventors have also found in previous studies that compared with COPD patients exposed to cigarette smoke, COPD patients exposed to biomass smoke have slower decline in small airway lung function, increased lower lung emphysema, narrowed airway lumen size, and increased airway wall thickness (all P<0.05), which also demonstrates that there are obvious differences in the pathophysiological characteristics of COPD caused by biomass smoke exposure and cigarette smoke exposure. Therefore, the present application supplements the core cause of COPD, which is often overlooked, by exposing to biofuel smoke, making the model more representative of the real population and closer to the diversity of global COPD etiology.
[0009] In addition, the core pathological features of COPD include chronic airway inflammation, airway obstruction, and emphysema. Simple biofuel smoke exposure can induce chronic inflammation and airway obstruction, but the progression of emphysema is slow, at least more than 6 months; simple elastase atomization can quickly induce emphysema, but it is difficult to simulate the remodeling caused by chronic airway inflammation and long-term damage. The present application can simultaneously exhibit all the above core pathological features in a shorter time through the synergistic effect of "biofuel smoke (inducing chronic inflammation + remodeling) combined with elastase (accelerating emphysema)", and the degree is closer to the progressive damage process of human COPD, solving the problems of single pathology and long time consumption of existing models.
[0010] Therefore, the application is superior to the prior art in terms of etiology representation, pathology comprehensiveness and model stability, and provides an ideal model closer to the clinic for etiology research, mechanism exploration and drug screening and development of COPD.
[0011] Preferably, the specific method of the biofuel smoke exposure treatment is that the mouse is exposed to a smoke exposure box for biofuel smoke exposure, 2-3 times a day, each time lasting for 2-4 hours, and exposed 6 days a week.
[0012] Preferably, the smoke is generated by simmering biofuel, filtered through a filter, and then delivered to the smoke exposure box by a piston pump.
[0013] The application adopts the above-mentioned biofuel smoke exposure treatment method to expose the mouse to smoke, filters the smoke through a filter, which can reduce the interference of irrelevant impurities or large particles and ensure that the smoke composition entering the exposure box is more stable; and the exposure frequency and duration of 2-3 times a day, each time lasting for 2-4 hours, and 6 days a week form a continuous and regular stimulation, which can not only ensure a sufficient smoke exposure dose to effectively induce pathological changes, but also accelerate the formation of the disease model through an efficient cumulative effect, which helps to shorten the model construction time. Therefore, the smoke exposure operation described in the application provides support for constructing a stable, highly relevant and time-saving mouse model of chronic obstructive pulmonary disease from the aspects of realistic simulation, condition control and efficiency improvement.
[0014] More preferably, the specific method of the biofuel smoke exposure treatment is that the mouse is exposed to a smoke exposure box for biofuel smoke exposure, 2 times a day, each time lasting for 3 hours, and exposed 6 days a week.
[0015] Preferably, the biofuel comprises wood chips.
[0016] Preferably, the elastase is porcine pancreatic elastase.
[0017] Preferably, the specific method of the elastase aerosolization induction is that the mouse is subjected to elastase aerosolization inhalation treatment once a week, each time lasting for 25-30 min, and the treatment lasts for 16 weeks.
[0018] Preferably, the dose of the elastase is 5-7 U per mouse during the aerosolization inhalation treatment.
[0019] The present application can be closer to the pathological exposure process of human respiratory diseases by comparing the nebulization inhalation of elastase with the traditional instillation, and make the damage of elastase to the airway structure more uniform, consistent, and the operation more simple, and the selection of porcine pancreatic elastase is more suitable for the simulation of related pathological mechanism, thereby making the model more relevant to the clinical features of human chronic obstructive pulmonary disease. In addition, the specific dose of elastase (5-7 U / each) and the nebulization time (25-30 min / time) can effectively induce the characteristic pathological changes of chronic obstructive pulmonary disease, and reduce the problems of excessive damage or insufficient induction, and ensure the reliability and repeatability of the finally constructed model.
[0020] More preferably, the dose of elastase is 6 U / each during the nebulization inhalation treatment.
[0021] In the second aspect, the present application provides a chronic obstructive pulmonary disease mouse model obtained by the construction method.
[0022] In the third aspect, the present application provides the application of the chronic obstructive pulmonary disease mouse model in the pathological mechanism research of chronic obstructive pulmonary disease, or the screening of drugs for treating chronic obstructive pulmonary disease.
[0023] Compared with the prior art, the present application has the following beneficial effects:
[0024] (1) The present application greatly shortens the induction time of the traditional COPD model from 6 months to 4 months;
[0025] (2) The present application obtains a stable phenotype, a good biological fuel exposure related COPD animal disease model with good practical relevance;
[0026] (3) The material used in the present application is wood chips, which has lower cost, does not need to be extracted into PM2.5, and has lower technical difficulty;
[0027] (4) The present application takes into account the animal welfare, greatly reduces the infection rate and mortality rate of animals. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 The operation flow chart of the mouse model of chronic obstructive pulmonary disease induced by biological dye smoke exposure combined with elastase nebulization;
[0029] Figure 2 The lung function change result chart of the COPD mouse model (BME) constructed in Example 1 and the control group mice (CON);
[0030] Figure 3 The pathological change result chart of the COPD mouse model (BME) constructed in Example 1 and the control group mice (CON);
[0031] Figure 4 Figure showing lung tissue inflammation results of COPD mouse model (BME) constructed for Example 1 and control group mice (CON);
[0032] Figure 5 Figure showing systemic inflammation results of COPD mouse model (BME) constructed for Example 1 and control group mice (CON);
[0033] Figure 6 Figure showing lung function changes results of COPD mouse model (BM) constructed by biological dye smoke exposure alone and control group mice (Control);
[0034] Figure 7 Figure showing pathological changes results of COPD mouse model (BM) constructed by biological dye smoke exposure alone and control group mice (Control). DETAILED DESCRIPTION
[0035] In order to make the objectives, technical solutions and effects of the present application clearer and more explicit, the present application is further described in detail below with reference to examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.
[0036] Unless otherwise specified, the reagents used in the examples are conventional reagents in the art and can be purchased through commercial channels. Unless otherwise specified, the experimental operations in the examples are conventional operations in the art or can be understood or known by those skilled in the art according to their mastery of existing technology or common knowledge.
[0037] The mice used in the examples are 6-8 week old C57BL / 6J mice, purchased from Guangdong Yaoke Biotechnology Co., Ltd.
[0038] Example 1
[0039] The present embodiment provides a method for constructing a mouse model of chronic obstructive pulmonary disease, and the specific method is as follows:
[0040] The mouse model of chronic obstructive pulmonary disease is induced by biological dye smoke exposure combined with elastase atomization, i.e. the mouse is placed in a whole-body exposure system for biological fuel smoke exposure, 2 times a day, each time for 3 hours, and exposed 6 days a week. Elastase atomization is performed once a week on the first day. The specific operation flow chart is shown in Figure 1The animal whole body exposure inhalation system of TSE Company in Germany was used. The exposure device was composed of three parts: fuel furnace, piston pump and smoke exposure box. The fuel furnace had 8 trays, each tray contained 10 g of sawdust, and the biofuel smoke was generated by simmering the sawdust. The harmful particles were delivered to the smoke exposure box containing the mouse cage by the piston pump after being filtered by the filter. The PARI BOY of PARI Company in Germany was used for aerosol inhalation of mice using porcine pancreatic elastase (LS002292) purchased from Worthington-biochem Company in the United States. The dose was 6 U per mouse, and the aerosol inhalation time was 30 min each time, lasting for 16 weeks, thereby constructing the mouse model of chronic obstructive pulmonary disease.
[0041] Example 2
[0042] The difference between this embodiment and Example 1 is that the biofuel smoke exposure treatment is performed 3 times a day, each time for 2 hours, 6 days a week; and the elastase aerosol inhalation induction treatment is performed at a dose of 5 U per mouse, 30 min each time, for 16 weeks.
[0043] Example 3
[0044] The difference between this embodiment and Example 1 is that the biofuel smoke exposure treatment is performed 2 times a day, each time for 4 hours, 6 days a week; and the elastase aerosol inhalation induction treatment is performed at a dose of 7 U per mouse, 25 min each time, for 16 weeks.
[0045] Test Example 1
[0046] The COPD mouse model obtained by the construction method of Example 1 was used as a sample to determine the pathological changes, lung function changes and expression of inflammatory factors, so as to comprehensively evaluate the effectiveness, stability and consistency with the characteristics of human COPD disease of the model. The mean lung interstitial space (MLI) and basement membrane perimeter standardization index (Wat / Pbm) were used to evaluate the pathological changes of mouse lung tissue; the lung function detection was used to check the lung function changes of mice; the RT-PCR technique was used to evaluate the inflammation of mouse lung tissue; and the ELISA method was used to confirm the systemic inflammation of mice. The specific detection results are as follows:
[0047] 1. Lung function changes
[0048] Compared with the untreated control group (CON), the model group significantly increased functional residual capacity (FRC, P < 0.0001), quasi-static compliance (Cchord, P < 0.01), static compliance (Cfvc50, P < 0.001), and forced vital capacity (FVC, P = 0.0001), while dynamic lung compliance (Cdyn, P < 0.05) was significantly decreased. Figure 2 ).
[0049] The forced expiratory volume (FEV20, P < 0.05) in the model group before expiration was significantly different. Airway obstruction was present from 20 ms to 400 ms of expiration, which was reflected in the significantly reduced ratio of forced expiratory volume to forced vital capacity at each time point, indicating that there was airway obstruction throughout the expiration process.
[0050] The above data indicate that the modeling scheme of the present invention can significantly impair lung function, exhibiting characteristics of emphysema combined with airway obstruction.
[0051] 2. Pathological changes
[0052] HE staining was performed on lung tissue sections for pathological examination. The results showed that the modeling method of this invention could disrupt alveolar septa and fuse small alveoli to form large air-filled sacs in mice. Figure 3 A) The mean alveolar linear intercept (MLI) was significantly increased (P < 0.0001). Figure 3 D) suggests the formation of emphysema.
[0053] In addition, HE staining showed that the cilia of the mouse bronchial epithelium were flattened and lost, and the walls of the small airways were thickened. Figure 3 B). Quantitative analysis showed a significant increase in small airway wall thickness (WAt / Pbm) (P < 0.05). Figure 3 C, E) indicate the presence of bronchial inflammatory changes.
[0054] 3. Upregulates the expression of inflammatory factors
[0055] RT-qPCR detection of inflammatory factors in lung tissue revealed significantly increased expression of IL-1β, IL-10 (P < 0.01), and TNF-α (P < 0.001) in the model group. Figure 4 ).
[0056] 4. Systemic inflammation
[0057] COPD is not only a lung disease but also a systemic inflammatory process. Results showed that plasma IL-2 levels in the model group were significantly higher than those in the control group (P < 0.05). Figure 5 ).
[0058] The above results show that the COPD mouse model constructed by the synergistic induction strategy of biological fuel smoke exposure combined with elastase atomization is highly consistent with the core characteristics of COPD in terms of lung function damage, pathological characteristics (emphysema + bronchitis) and inflammatory response (local + systemic), indicating that it successfully constructed an animal model that can be used for COPD research.
[0059] Test Example 2
[0060] This test example explores the influence of a mouse model of chronic obstructive pulmonary disease induced by biological dye smoke exposure alone, and the specific exploration method is as follows:
[0061] The purchased 12 male 6-8 week old C57BL / 6J mice were divided into groups: clean air group (referred to as Control group, n = 6) and biological fuel PM2.5 exposure group (referred to as BM group, n = 6) (the above mice are not the same batch as the mice in the examples, and the basic values are different). The BM group was placed in the whole body exposure system and exposed to biological fuel PM2.5 twice a day, each for 3 hours, 6 days a week, for 6 months. The fuel stove has 8 trays, each tray contains 10g of wood chips, and the specific operation is shown in Example 1. The clean air group of mice was raised in a SPF clean air environment for 6 months. After 6 months of biological fuel exposure, the lung function of the mice in the two groups was detected and the corresponding sampling operation was performed.
[0062] The results show that biological fuel smoke exposure can cause the functional residual capacity (FRC), static lung compliance (Cchord), and compliance at 50% vital capacity (Cfvc50) of the mice to increase significantly, and the forced expiratory volume in 20 milliseconds (FEV20) and 20 millisecond rate (FEV20 / FVC (%)) to decrease significantly, with statistical significance compared with the control group (P<0.05) Figure 6 In terms of pathological changes, simple modeling for 6 months can cause the mouse alveoli to fuse to form gas-containing large cystic cavities Figure 7 A), and the mean alveolar linear intercept (MLI) to increase (P<0.05) Figure 7 B).
[0063] The above results show that biological fuel smoke exposure alone requires 6 months to induce typical COPD characteristics, while the synergistic induction strategy of biological fuel smoke exposure combined with elastase atomization described in the present application can achieve a model effect highly consistent with the core characteristics of COPD in only 4 months, which shortens the time by nearly 1 / 3, significantly reduces the experimental period, and accelerates the research process, especially suitable for scenarios that require rapid hypothesis verification or follow-up intervention experiments.
[0064] In conclusion, the construction method of the synergistic induction realizes multiple optimization of experimental efficiency and result reliability by significantly shortening the modeling time without reducing the model effect, and has obvious practical advantages.
[0065] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.
Claims
1. A method for constructing a mouse model of chronic obstructive pulmonary disease, characterized by, The method comprises the following steps: The mouse is exposed to biofuel smoke and induced by elastase atomization to construct the mouse model of chronic obstructive pulmonary disease.
2. The construction method of claim 1, wherein, The specific method of the biofuel smoke exposure treatment is that the mouse is exposed to biofuel smoke in a smoke exposure box, 2-3 times per day, each time lasting 2-4 hours, and exposed 6 days per week.
3. The construction method of claim 2, wherein, The smoke is generated by burning biofuel, filtered by a filter, and then delivered to the smoke exposure box by a piston pump.
4. The construction method of claim 1, wherein, The biofuel comprises wood chips.
5. The construction method of claim 1 wherein, The elastase is porcine pancreatic elastase.
6. The construction method of claim 1 wherein, The specific method of the elastase atomization induction is that the mouse is subjected to elastase atomization inhalation treatment once a week, each time lasting 25-30 min, and the treatment lasts for 16 weeks.
7. The construction method of claim 6, wherein, The dose of the elastase is 5-7 U per mouse during the atomization inhalation treatment.
8. The mouse model of chronic obstructive pulmonary disease obtained by the construction method according to any one of claims 1-7.
9. The mouse model of chronic obstructive pulmonary disease according to claim 8 is applied to the research on the pathological mechanism of chronic obstructive pulmonary disease or the screening of drugs for treating chronic obstructive pulmonary disease.