Construction method and application of COPD (chronic obstructive pulmonary disease) and senescence mouse model

Abstract

The invention discloses a construction method and application of a COPD (chronic obstructive pulmonary disease) combined aging mouse model, and belongs to the technical field of animal model construction. The construction method comprises the following steps: performing subcutaneous injection of D-galactose on the neck and back of a mouse at the beginning of mouse chronic obstructive pulmonary model building; the injection dosage of the D-galactose is 140-160mg / kg, one time per day and 7-9 weeks continuously, and then the injection dosage is changed to 2 days per time until modeling is finished. The model constructed by the invention more truly reflects the natural pathogenesis process of COPD, the COPD combined aging mouse model overcomes the defects of a traditional COPD animal model, and a more efficient and more reliable animal model is provided for basic research of COPD. Establishment of the model not only has important economic value, but also has profound significance for basic and clinical research, and the research risk and cost are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and in particular relates to a method for constructing a COPD combined with aging mouse model and its application. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung disease characterized by chronic respiratory symptoms (dyspnea, cough, and sputum production) caused by persistent abnormalities in the airways (bronchitis and bronchiolitis), alveoli (emphysema), and / or pulmonary vasculature, resulting in persistent and progressive airflow limitation. COPD is highly prevalent and the third leading cause of death worldwide, impacting both the health and socioeconomic burden of the entire population. COPD results from a complex, dynamic, cumulative, and recurrent interaction of genes and the environment throughout life, which alters normal lung development or aging processes (GETomics). COPD is considered an age-related chronic disease, and its prevalence increases with age. Naturally aging lungs also exhibit structural changes similar to those seen in COPD, such as decreased pulmonary ventilation function, decreased lung elasticity, and increased airway and alveolar diameters. COPD patients also exhibit senescence in multiple cellular systems, including airway and alveolar epithelial cells. Currently, exposure to tobacco smoke (CS) is considered a major risk factor for COPD. Existing animal models of COPD that simulate long-term smoking primarily include the CS exposure model and the CS exposure combined with lipopolysaccharide (LPS) model. The CS exposure mouse model is widely used and effectively mimics the natural course of COPD with high similarity. However, it is time-consuming, exhibits a subtle inflammatory secretory phenotype, and is easily reversible, significantly hindering research progress. Combining CS exposure with nasal or intratracheal LPS instillation can shorten the course of the disease, but it struggles to simulate the natural course and early stages of COPD. Summary of the Invention

[0003] This application addresses the shortcomings of existing tobacco smoke exposure models, adds age as an independent risk factor, and proposes an optimized COPD mouse model, as follows:

[0004] The present invention provides a method for constructing a COPD combined with aging mouse model, which comprises the following steps: at the beginning of modeling of the COPD mouse model, subcutaneously injecting D-galactose into the back of the mouse neck; the injection dose of the D-galactose is 140-160 mg / kg, once / day, for 7-9 weeks, and then changed to 2 times / day until the modeling is completed.

[0005] In one embodiment of the present invention, the mouse COPD model is constructed using cigarette smoke.

[0006] In one embodiment of the present invention, the injection dose of D-galactose is 150 mg / kg.

[0007] In one embodiment of the present invention, the injection duration of D-galactose is 8 weeks.

[0008] In one embodiment of the present invention, the concentration of D-galactose is 25 mg / mL, and the injection dose is 6 mL / kg.

[0009] In one embodiment of the present invention, the modeling time of the COPD combined with aging mouse model is 3-6 months.

[0010] The present invention also provides application of the above construction method in constructing a COPD combined with aging mouse model.

[0011] The present invention also provides the application of the mouse model constructed by the above construction method in basic research on COPD.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) The present invention found that the COPD combined with aging mouse model showed more severe lung function decline compared with the traditional tobacco smoke exposure COPD mouse model, as manifested by increased functional residual capacity (FRC), resistance index (RI), and static lung compliance (Cchord), and decreased dynamic lung compliance (Cdyn), 50 millisecond forced expiratory volume (FEV50) / forced vital capacity (FVC) values, and 100 millisecond forced expiratory volume (FEV100) / FVC values. HE staining showed that emphysema was more severe, and the mean linear intercept changed significantly. These results indicate that the COPD phenotype is more obvious in the COPD combined with aging mouse model.

[0014] (2) The present invention found that the COPD combined with aging mouse model had an increased cell classification cell count in BALF compared with the traditional tobacco smoke exposure COPD mouse model, indicating that the inflammatory infiltration was more obvious.

[0015] (3) The present invention found that the COPD combined with aging mouse model combines age-related characteristics and more realistically reflects the natural pathogenesis of COPD. The COPD combined with aging mouse model has the same effect of being exposed to tobacco smoke for 3 months as that of being exposed to traditional tobacco smoke for 6 months. It can shorten the COPD modeling time, reduce time and manpower and material costs, and can also simulate early COPD without the need for additional operations such as nasal drops or injections.

[0016] In summary, this invention is the first to utilize age, a key factor in the pathogenesis of COPD, in a COPD animal model, creating a novel and superior model for simulating COPD. COPD model evaluation experiments have demonstrated that this COPD combined with aging mouse model offers the advantages of shortened modeling time and more comprehensive simulation results. This COPD combined with aging mouse model overcomes the shortcomings of traditional COPD animal models and provides a more efficient and reliable modeling method for basic COPD research. The establishment of this model not only has significant economic value but also has far-reaching implications for basic and clinical research, reducing research risks and costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figures show the lung function of the COPD combined with aging mouse model and the traditional tobacco smoke exposure mouse model in Example 1, where Air-3m, CS-3m, D-gal-Air-3m, and D-gal-CS-3m represent 3 months of air exposure, 3 months of smoke exposure, 3 months of air exposure + 2 months of D-galactose injection, and 3 months of smoke exposure + 2 months of D-galactose injection, respectively: (A) FRC value; (B) Cdyn value; (C) cChord value; (D) RI value; (E) FEV50 / FVC value; (F) FEV100 / FVC value; n = 6-8 per group, *P < 0.05 and **P < 0.01.

[0018] Figure 2 The severity of emphysema in the COPD combined with aging mouse model and the traditional tobacco smoke exposure mouse model in Example 1, including: (A) HE image; (B) mean linear intercept statistical graph; n = 6-8 per group, *P < 0.05 and **P < 0.01.

[0019] Figure 3 The BALF cell classification and counts of the COPD combined with aging mouse model and the traditional tobacco smoke exposure mouse model in Example 1, including: (A) macrophages; (B) lymphocytes; (C) neutrophils; *P<0.05 and **P<0.01. DETAILED DESCRIPTION

[0020] Example 1

[0021] 1 Experimental methods

[0022] 1.1 Research subjects

[0023] In this study, 2-month-old C57BL / 6J male mice were injected subcutaneously with D-galactose in the back of the neck and exposed to tobacco smoke for 3 and 6 months. Lung function was tested, and lung tissue, peripheral blood, and bronchoalveolar lavage fluid were extracted.

[0024] 1.2 COPD combined with aging mouse model

[0025] Wild-type, 2-month-old C57BL / 6J male COPD mice (6-8 weeks old) were purchased from Weitong Lihua Animal Experimental Technology Co., Ltd. Animals were housed in a specific pathogen-free facility, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. All methods were performed in accordance with the guidelines and regulations approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. The COPD mouse model was established using Hongmei brand filtered cigarettes, each containing 11 mg tar, 1.0 mg nicotine, and 13 mg carbon monoxide. Two-month-old mice were exposed to CS using a whole-body exposure system. Briefly, mice were exposed to CS (exposure chamber size 60*50*50 cm, 9 cigarettes / h, 2 hours / time, twice daily, 6 days / week) for 12 and 24 weeks. A control group was exposed only to filtered room air (RA). After the experiment, all mice underwent lung function testing and were sacrificed. At the same time, at the beginning of tobacco smoke exposure modeling, D-galactose was injected subcutaneously in the back of the neck at a dose of 150 mg / kg, once a day for 8 weeks. After 8 weeks, it was changed to 2 times a day, and continued until the mice died due to intolerance or the tobacco smoke exposure model was ended.

[0026] 1.3 COPD mouse model

[0027] Mice were anesthetized with intraperitoneal injection of avertin (20 μl / g body weight), tracheotomized, intubated, and placed in the systemic cavity. Mechanical ventilation was performed using a small animal spirometer. Instrument parameters were set as follows: ventilator pressure ±10 cm H2O, inspiratory flow rate 0.8 ml / s, expiratory flow rate 0.5 ml / s, maximum inspiratory and expiratory pressures ±40 cm H2O, and respiratory rate 140 breaths / min. Three semiautomatic maneuvers were performed: Boyle's law FRC, quasi-static pressure-volume (PV), and rapid flow-volume (FV) maneuvers. Dynamic lung compliance (Cdyn) and static lung compliance (Cchord) were measured. At the last FV maneuver, the resistive index (RI), forced expiratory volume in 50 milliseconds (FEV50), and forced expiratory volume in 100 milliseconds (FEV100) were measured.

[0028] 1.4 Bronchoalveolar lavage fluid (BALF) analysis

[0029] Inject 0.6 ml of ice saline into the left lung trachea three times. Centrifuge all collected BALF at 4°C for 5 minutes and weigh 500 g. Take the supernatant and store it in a -80°C refrigerator for cytokine detection. Resuspend the cell pellet in 1 mL of normal saline and take 10 μl for cell counting to calculate 1×10 5The volume required for the number of cells was adjusted to 1 ml with normal saline, and the cells were spun using a cytospin. The BALF cells were stained with hematoxylin-eosin (HE) and the neutrophils, macrophages and lymphocytes were classified and counted.

[0030] 1.5 Pathological HE staining of mouse lung tissue

[0031] Hematoxylin-eosin (HE) staining: The left lung of the mouse was fixed with 10% formalin solution for 24 hours, then embedded in paraffin, and the paraffin block was cut into 4 μm thick sections. The dewaxed tissue sections were stained with a HE staining kit for histological examination.

[0032] 1.15 Statistical Analysis

[0033] GraphPad Prism 10.0 software was used to analyze the experimental results and plot them. All data are presented as mean ± standard error (SEM). Comparisons between two groups were performed using the independent sample t-test; comparisons between multiple groups were performed using one-way ANOVA, with pairwise comparisons between groups performed. P < 0.05 indicated statistical significance, and P < 0.01 and P < 0.001 indicated statistically significant differences.

[0034] 2 Experimental results

[0035] 2.1 COPD combined with aging model with decreased lung function

[0036] By comparing the differences between the traditional tobacco smoke exposure model and the COPD combined with aging mouse model, we compared the lung function of the combined 3- and 6-month models. Figure 1 The specific experimental method is as follows: mice were exposed to CS for 6 months (9 CS per hour, 2 hours per group, 2 groups per day, 6 days per week), and the forced pulmonary operation system was used to measure lung function parameters ( Figure 1 AF), including FRC, Cdyn, Cchord, RI, FEV50 / FVC value and FEV100 / FVC value, with 6-10 mice in each group; lung function results showed that compared with the traditional tobacco smoke exposure model, the COPD combined with aging model had more severe lung function decline, as shown by functional residual capacity (FRC) ( Figure 1 A) Static lung compliance (Cchord) Figure 1 C) and resistance index (RI) ( Figure 1 D) increases, lung dynamic compliance ( Figure 1 B), and forced expiratory volume in 50 milliseconds (FEV50) / FVC value forced vital capacity (FVC) ( Figure 1 E) and 100 millisecond forced expiratory volume (FEV100) / FVC value ( Figure 1 F) decrease.

[0037] 2.2 COPD combined with aging model has more obvious emphysema

[0038] In the models of tobacco smoke exposure for 3 and 6 months, the average linear intercept in the lungs of COPD combined with aging model mice was significantly greater than that of the traditional COPD mouse model, suggesting that the COPD combined with aging model can simulate the alveolar destruction and alveolar cavity expansion of COPD better. In COPD combined with aging model mice, HE staining microscopy showed that the alveolar arrangement was more severely damaged ( Figure 2 A). Compared with the traditional COPD mouse model, the average linear intercept of the alveoli in the COPD combined with aging model mice showed significant changes ( Figure 2 B) These results suggest that age increases the susceptibility to COPD.

[0039] 2.3 Inflammatory infiltration is more pronounced in COPD combined with aging models

[0040] CS-mediated recruitment of inflammatory cells into the lung is a major cause of chronic bronchitis and emphysema. To determine the role of CS in lung inflammation in a model of COPD combined with aging, we assessed the influx of inflammatory cells into the BALF using hematoxylin and eosin staining.

[0041] The results showed that the cell classification counts in BALF of mice exposed to air and tobacco smoke for 3 and 6 months, including macrophages ( Figure 3 A), lymphocytes ( Figure 3 B) and neutrophils ( Figure 3 C) There was a statistically significant difference. Compared with the COPD mouse model at 3 and 6 months, the COPD combined with aging mouse model had an increased number of classified cell counts in BALF ( Figure 3 AC).

[0042] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing a COPD combined with aging mouse model, characterized in that: The construction method includes the following steps: at the beginning of modeling of COPD in mice, subcutaneously injecting D-galactose into the back of the mouse's neck; the injection dose of D-galactose is 140-160 mg / kg, once / day, for 7-9 weeks, and then changed to once every 2 days until the modeling is completed.

2. The construction method according to claim 1, characterized in that The mouse COPD model was established using cigarette smoke.

3. The construction method according to claim 2, characterized in that The injection dose of D-galactose is 150 mg / kg.

4. The construction method according to claim 3, characterized in that The injection duration of D-galactose was 8 weeks.

5. The construction method according to claim 4, characterized in that: The concentration of the D-galactose is 25 mg / mL, and the injection dose is 6 mL / kg.

6. The construction method according to claim 5, characterized in that: The modeling time of the COPD combined with aging mouse model is 3-6 months.

7. Use of the construction method according to any one of claims 1 to 6 in constructing a COPD combined with aging mouse model.

8. Use of the mouse model constructed by the construction method according to any one of claims 1 to 6 in basic research on COPD.

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