Construction method of pulmonary fibrosis disease model

Abstract

The invention provides a construction method and application of a pulmonary fibrosis disease model, and belongs to the field of animal models. The construction method of the animal model comprises the step of inducing a mouse to show typical pulmonary fibrosis disease characteristics through combination of BSO and BLM. The model mouse can be used as a pulmonary fibrosis disease model for research of pulmonary fibrosis diseases, can help clarify the pathogenesis process and mechanism of the pulmonary fibrosis diseases, and provides a new target for treatment or prevention of the diseases.

Description

Technical Field

[0001] The invention belongs to the field of animal models, and particularly relates to a method for constructing a pulmonary fibrosis disease model. Background Art

[0002] Pulmonary fibrosis (PF) is a fatal lung disease characterized by damage to alveolar epithelial cells, areas of type II cell proliferation, accumulation of fibroblasts and myofibroblasts, and deposition of extracellular matrix proteins. The result is a gradual loss of normal lung architecture and impaired gas exchange. Relevant features of the human disease include the temporal heterogeneity of fibrotic lesions, the progressive nature of the disease, and the development of fibrotic lesions. With the development of aging and the intensification of environmental pollution, the incidence of pulmonary fibrosis is increasing, especially in the elderly. Pulmonary fibrosis is progressive and irreversible, often leading to death, with a five-year survival rate of less than 40%. Survival rates decrease with age and are even lower in men.

[0003] Bleomycin (BLM) is a compound commonly used to induce pulmonary fibrosis in experimental animals. It causes the occurrence and development of fibrosis by inducing oxidative stress and inflammatory response. Intratracheal instillation of BLM induces pulmonary fibrosis by increasing inflammatory response and epithelial damage, which is the most commonly used pulmonary fibrosis modeling method in experiments. The invention patent application with publication number CN112568188A discloses a method for making a Brown Norway rat pulmonary fibrosis model, which uses C57BL / 6J mice to meet the target animal needs, reduces the experimental cost, and is conducive to conducting more in vivo experiments. However, many studies have confirmed that lung damage caused by intratracheal instillation of BLM can be relieved on its own. Moreover, in the C57BL / 6J mice of this model, PF was detected on the 14th day after the first administration, usually reaching a peak level on the 21st to 28th day, and returning to normal after about 6 weeks. The pulmonary fibrosis process in actual patients with pulmonary fibrosis is irreversible. Therefore, since intratracheal instillation of BLM alone is not an ideal animal model for inducing pulmonary fibrosis in mice, it is difficult to effectively study the pathogenesis of pulmonary fibrosis. In order to better screen and study the pathogenic mechanism of pulmonary fibrosis and screen out effective targeted therapeutic drugs, it is urgent to construct an animal model of continuous development of fibrosis. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a pulmonary fibrosis disease model.

[0005] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a method for constructing a pulmonary fibrosis disease model, before the experiment begins, the experimental animals are fed with food containing L-buthionine (S, R)-sulfenyl imine; at the beginning of the experiment, the trachea of ​​the experimental animals is instilled with bleomycin solution.

[0006] The preferred technical solution is: feeding the experimental animals with food containing L-buthionine (S, R)-sulfenyl imide 2-4 days before the start of the experiment.

[0007] The preferred technical solution is: when feeding the experimental animals with food containing L-buthionine (S, R)-sulfenyl imine, the dosage of L-buthionine (S, R)-sulfenyl imine is 30-8-mg / 100g food.

[0008] The preferred technical solution is: when the bleomycin solution is instilled into the trachea, the dosage is: 1.5 U / kg body weight, 50 μL / mouse.

[0009] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0010] The existing pulmonary fibrosis model only shows fibrosis in the short term and returns to normal levels within a period of time. It cannot simulate the progressive and irreversible characteristics of pulmonary fibrosis, which makes it difficult to effectively study the pathogenesis of pulmonary fibrosis. BSO can significantly promote the accumulation of ROS levels in mice, thereby continuously causing inflammatory responses in the body. The mouse pulmonary fibrosis model induced by BSO and BLM in the present invention shows a higher and irreversible level of fibrosis, which is more in line with the ideal pulmonary fibrosis disease model. The new application of BSO and BLM in the treatment of pulmonary fibrosis can expand its research in the field of medical technology and provide a new theoretical direction for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 BALF was collected for H&E staining and quantitative analysis of the total number of inflammatory cells.

[0012] Figure 2 To quantitatively analyze the number of macrophages, neutrophils and lymphocytes in inflammatory cells.

[0013] Figure 3 Lung tissue sections were stained with DHE, and the mean fluorescence intensity (MFI) was quantified.

[0014] Figure 4 H&E staining of mouse lung tissue sections. The Ashcroft fibrosis score was calculated based on the H&E staining results of mouse lung sections.

[0015] Figure 5 Sirius red staining of mouse lung tissue sections.

[0016] Figure 6 The figure shows the changes in mouse body weight. DETAILED DESCRIPTION

[0017] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in the embodiments.

[0018] See also Figure 1-6 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance, and any structural modification, change in proportional relationship or adjustment of size. The following examples are provided to better understand the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0019] Example 1: Combination of BLM and BSO induces sustained recruitment of inflammatory factors in mouse BLAF

[0020] 1. Grouping of mice

[0021] Twenty-four SPF 8-week-old male C57BL / 6J mice were randomly divided into three groups, namely, saline control group (control group), BLM group (model group), and BLM combined with BSO group (model + BSO group). At the beginning of the experiment, the three groups were simultaneously subjected to tracheal instillation experiments, in which the mice in the model group and the BLM combined with BSO group were instilled with BLM solution (1.5U / kg body weight, 50μL / mouse), and the mice in the control group were instilled with saline (0.9% NaCl, 50μL / mouse). In addition, 3 days before the start of the experiment, the mice in the BLM combined with BSO group were fed with food containing BSO (0.25mmol / kg (body weight) / day, and the dose of BSO fed in the food was estimated to be 50mg / 100g food), and the mice in the other groups were fed with normal feed. At the fourth week of the experiment, 4 mice were randomly selected from each group, anesthetized with tribromoethanol and killed, and lung tissues and bronchoalveolar lavage fluid (BALF) were collected. At the 8th week of the experiment, the remaining 4 mice in each group were taken, anesthetized with tribromoethanol and sacrificed, and the lung tissues and BALF were collected.

[0022] 2. Collect BALF and detect inflammatory cells

[0023] After killing, collect the BALF of the mice. First, fix the mouse on the operating table, cut the neck skin with scissors, and use forceps to bluntly separate the neck muscles to expose the main trachea. Use a syringe needle to poke a small hole in the main bronchus near the head end, insert a 10mL syringe with a flat needle into the trachea through the previously punctured small hole, and then tighten the needle inserted into the trachea with surgical sutures. Use a syringe to inhale 0.5mL of sterile PBS, then gently push it into the mouse lungs, gently press the mouse chest cavity with your thumb several times, and then slowly withdraw the lavage fluid. Repeat this operation 3 times, and collect the BALF solution in a 1.5mL enzyme-free test tube (a total of 1.5mL is lavaged, and the recovery rate must not be less than 80%). The collected BALF is placed at 4°C and centrifuged at 1000rpm for 10 minutes. Finally, discard the supernatant after centrifugation, add 0.2mL of red blood cell lysis solution to the precipitate, mix it with a pipette, and let it stand for 2 minutes. Add 0.4 mL of lavage fluid (0.5 mM EDTA solution, 1 mL and 100 mg bovine serum albumin (BSA) completely dissolved in 99 mL PBS to make 100 mL of lavage fluid, stored at 4°C for use), gently pipette to mix, centrifuge at 100 g for 6 minutes, discard the supernatant, then add 1 mL of lavage fluid to the precipitate and mix to make a cell suspension, aspirate 10 μL of cell suspension and add it to a hemacytometer, count the total number of cells in the suspension under a microscope and calculate the cell density of the suspension, count each sample 4 times and take the average value.

[0024] The remaining cell suspension was applied to a clean glass slide, air-dried, stained with hematoxylin and eosin (H&E), and then mounted with neutral resin. Images were taken by a Zeiss Axio Scope.A1 fluorescence microscope. All cells in the field of view were counted continuously, and the proportions of neutrophils, lymphocytes, and macrophages were calculated separately, and then the density of inflammatory cells in mouse BALF samples was calculated.

[0025] 3. Experimental results

[0026] In the process of pulmonary fibrosis progression, the recruitment of inflammatory cells leads to the continuous development of inflammatory response, which has been considered as the main cause of pulmonary fibrosis. In order to clarify the additive effect of BSO and BLM in pulmonary fibrosis, we first detected the inflammatory level in the lungs of mice. Figure 1 and 2As shown in the figure, we extracted the BALF of mice, then counted the total number of inflammatory cells and classified them by H&E staining. It was found that compared with the Ctrl group mice, the total number of inflammatory cells and the number of macrophages, neutrophils and lymphocytes in the BALF of the BLM group mice increased at week 4, while the number of these cells in the BALF of the BLM combined with BSO group mice increased more. However, after the 8th week of modeling, the number of these inflammatory cells in the BALF of the BLM group mice had largely decreased to normal levels, while the number of these inflammatory cells in the BALF of the BLM combined with BSO group mice still remained at a high level.

[0027] Example 2 Combination of BSO and BLM induces continuous progression of pulmonary fibrosis in mice, promotes continuous accumulation of ROS levels in mice and continuous accumulation of collagen in lung tissue

[0028] 1. After the experiment, the lung tissue was fixed with 4% paraformaldehyde overnight. Before the lung tissue sections were stained, the lung tissue was dehydrated, then fixed, dehydrated and embedded in paraffin. A paraffin slicer was used to prepare 7 μm lung tissue sections.

[0029] 2. Determination of ROS levels

[0030] To determine the ROS level in lung tissue, lung tissue sections were measured by DHE staining and finally photographed using a Zeiss AxioScope.A1ZEISS Scope A1 fluorescence microscope to quantify the DHE fluorescence intensity statistics in lung tissue.

[0031] 3. H&E and Sirius red staining of lung tissue

[0032] After deparaffinization and rehydration, H&E and Sirius red staining were performed. All images were taken using a Zeiss Axio Scope.A1 fluorescence microscope. Table 1 shows the Ashcroft scoring criteria for lung tissue fibrosis pathology.

[0033] Rating level Scoring Criteria 0 Normal lung tissue 1 Microfibrous thickening of the alveolar or bronchiolar walls 2-3 Moderate thickening of alveolar walls, no obvious damage to lung tissue structure 4-5 The degree of fibrosis worsens, the lung structure is significantly damaged, and fibrous bands or small fibrous masses are formed 6-7 Severe destruction of lung tissue structure, large area of ​​fibrosis, and may be accompanied by honeycomb lung formation 8 The whole field of vision is filled with fibrous tissue, and the entire lung tissue is completely occluded and necrotic.

[0034] 4. Experimental results

[0035] Redox imbalance is an important factor inducing pulmonary fibrosis. Figure 3 The results of DHE staining showed that compared with Ctrl, the ROS level in the BLM group was significantly enhanced 4 weeks after tracheal injection, but then at the 8th week, the ROS level was almost restored to normal. Compared with the BLM group, the BLM combined with BSO group showed a stronger accumulation of ROS levels in the fourth week, and the ROS level hardly decreased at the 8th week. This shows that the combination of BSO and BLM continuously reduced the antioxidants in the lung tissue of mice, leading to a continuous imbalance of redox.

[0036] like Figure 4 As shown in Table 1, PF was histologically analyzed by H&E staining, and the Ashcroft score was calculated. Compared with the Ctrl group, the BLM group induced the development of fibrosis in the lung tissue of mice to a great extent at week 4. In addition, mice in the BLM and BSO combined group showed a greater degree of fibrosis. However, we found that at week 8, the degree of pulmonary fibrosis in mice in the BLM group was significantly reduced, and even returned to normal levels. However, mice in the BLM and BSO combined group showed a sustained level of fibrosis.

[0037] It is well known that pulmonary fibrosis is associated with collagen deposition. Figure 5 As shown in the figure, collagen deposition in the lung tissue of mice was evaluated by Sirius red staining. The results showed that at the 4th week of the experiment, there was a certain accumulation of collagen in the lung tissue of mice in the BLM group, while mice in the combined BSO and BLM group showed more collagen accumulation. Consistent with the results of H&E staining, we found that at the 8th week, the content of collagen in the lung tissue of mice in the BLM group was significantly reduced. In the mice in the combined BLM and BSO group, collagen still existed at a high level.

[0038] Previous studies have shown that pulmonary fibrosis can lead to weight loss. Figure 6 As shown in the figure, the weight of mice in the BSO and BLM combination group decreased more significantly than that in the BLM group at week 4. However, at week 8, the weight of mice in the BLM group largely recovered to normal levels. However, the weight of mice in the BLM and BSO combination group still failed to recover to normal levels.

[0039] In summary, the above research results show that both the BLM group and the BLM and BSO combined group showed characteristics of pulmonary fibrosis at the 4th week of the experiment. However, the fibrosis degree was limited when BLM was used alone to induce the mouse pulmonary fibrosis model, and almost no signs of pulmonary fibrosis were observed at the 8th week, which did not meet the pathological characteristics of clinical pulmonary fibrosis patients. In this design, BLM and BSO were used together. Through a single tracheal instillation of BLM and continuous feeding of BSO-containing food, the mouse model constructed showed a stronger level of fibrosis than the mice induced by BLM alone, and at the 8th week of the experiment, the fibrosis level continued to develop, indicating that the pulmonary fibrosis experimental model designed in this design is irreversible.

[0040] The present invention discloses a method for inducing pulmonary fibrosis by combining BSO and BLM, which can also be used to study the mechanism of action of oxidative stress in pulmonary fibrosis, especially in the fibrosis model induced by BLM. In addition, the method can also be used as an experimental platform for screening antioxidants or other anti-fibrotic drugs, providing a new research approach for studying the pathogenesis and mechanism of pulmonary fibrosis, and providing a scientific basis for developing new treatment strategies for pulmonary fibrosis.

[0041] The construction method of the present invention induces target animals to exhibit typical pulmonary fibrosis disease characteristics by combining BSO with BLM.

[0042] The target animals induced by the combination of BSO and BLM are animals in which BSO promotes the accumulation of ROS in the target animals and BLM is injected into the trachea at the same time.

[0043] The target animal is a mouse, a rat, a horse, a pig, a monkey, a dog or an ape.

[0044] The method of inducing pulmonary fibrosis is drug-induced.

[0045] The method of promoting ROS accumulation in the target animal by BSO is to feed the target animal with food containing BSO.

[0046] The method of intratracheal injection of BLM is to administer it directly into the trachea.

[0047] The animal model is a mouse model, and the construction steps are as follows:

[0048] (1) feeding mice with food containing BSO, wherein the dosage of BSO was 0.25 mmol / kg (body weight) / d;

[0049] (2) BLM was administered to mice by tracheal instillation to induce pulmonary fibrosis, with the dosage of BLM being 1.5 U / kg (body weight);

[0050] (3) Regularly monitor lung function and tissue pathological changes, and use this model to evaluate the degree of lung fibrosis and study pathological mechanisms.

[0051] The present invention also provides the use of the animal model of pulmonary fibrosis disease constructed by the aforementioned method in screening therapeutic drugs for pulmonary fibrosis disease.

[0052] The present invention also provides the use of the animal model of pulmonary fibrosis disease constructed by the aforementioned method in the study of pulmonary fibrosis disease, wherein the purpose of the application is non-disease diagnosis or treatment.

[0053] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for constructing a pulmonary fibrosis disease model, characterized in that: Before the experiment, the experimental animals were fed with food containing L-buthionine (S,R)-sulfenyl imine; at the beginning of the experiment, bleomycin solution was instilled into the trachea of ​​the experimental animals.

2. The method for constructing a pulmonary fibrosis disease model according to claim 1, characterized in that: The experimental animals were fed a diet containing L-buthionine (S,R)-sulfenyl imide 2-4 days before the start of the experiment.

3. The method for constructing a pulmonary fibrosis disease model according to claim 1, characterized in that: When the experimental animals were fed a diet containing L-buthionine (S,R)-sulfenyl imine, the dose of L-buthionine (S,R)-sulfenyl imine was 30-8-mg / 100 g of diet.

4. The method for constructing a pulmonary fibrosis disease model according to claim 1, characterized in that: When bleomycin solution was instilled into the trachea, the dose was: 1.5 U / kg body weight, 50 μL / mouse.

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