A method for constructing a pneumonia model based on recombinant IL-6 and application of the pneumonia model

Abstract

The application discloses a kind of based on recombinant IL-6 pneumonia model construction method and the application of pneumonia model.The pneumonia model construction method is:S1, to the lung of animal delivery recombinant IL-6 solution several days, establish animal pneumonia model;S2, to animal pneumonia model is carried out lung function evaluation and pathological evaluation, judge whether lung cancer model is successfully constructed.The method for constructing animal pneumonia model of the application is simple, easy to operate, and the time required for modeling is short.The animal pneumonia model of the application can simulate lung inflammatory diseases with IL-6 inflammatory factor up-regulation characteristics, proves that only IL-6 this inflammatory factor pulmonary administration can cause pneumonia, and provides a new animal model for mechanism research, prevention, diagnosis and treatment of pneumonia characterized by IL-6 elevation.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing a pneumonia model based on recombinant IL-6 and the application of the pneumonia model. Background Technology

[0002] Pneumonia is one of the leading causes of death worldwide and one of the most common serious infections in children and infants. It can be caused by various microorganisms, including bacteria, viruses, and fungi, leading to infection of the alveoli and surrounding tissues in the lungs. Clinically, pneumonia can trigger changes in inflammatory markers, including white blood cell count, C-reactive protein, and IL-6. IL-6 is an important pro-inflammatory cytokine; high levels of IL-6 can lead to chronic inflammatory diseases and cytokine storms. Serum IL-6 levels have been shown to be closely related to disease severity and prognosis in COVID-19 patients, and can serve as a potential therapeutic target for critically ill cases. Furthermore, IL-6 is a good indicator for diagnosing the severity of community-acquired pneumonia in children.

[0003] Preclinical studies using animal models can help advance the diagnosis and drug development of pneumonia. Currently, commonly used animal models of pneumonia include bacterial pneumonia models, fungal pneumonia models, viral pneumonia models, and radiation-induced pneumonia models. No IL-6-based animal models of pneumonia have been found yet, and it remains unclear whether IL-6 alone is sufficient to induce pneumonia-related pathological changes.

[0004] Existing research on IL-6 mainly focuses on the effects of regulating IL-6 expression in cells in vitro or administering IL-6 to cells, as well as conducting disease or pathological studies on IL-6 knockout transgenic animals. However, whether IL-6 administration to the lungs induces inflammatory changes in the lungs of animals has not yet been studied, hindering the mechanistic research, prevention, diagnosis, and treatment of pneumonia characterized by elevated IL-6 levels. Summary of the Invention

[0005] Based on the above-mentioned prior art, the present invention provides a method for constructing a pneumonia model based on recombinant IL-6 and the application of the pneumonia model, filling the gap in IL-6-based animal pneumonia models and their application in pneumonia mechanism research, prevention, diagnosis and treatment.

[0006] The construction method of this invention is simple, easy to operate, and requires little time for model making.

[0007] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

[0008] A method for constructing a pneumonia model based on recombinant IL-6 includes the following steps:

[0009] S1. A pneumonia model was established by delivering recombinant IL-6 solution to the lungs of animals for several days.

[0010] S2. Perform lung function and pathological assessments on the animal pneumonia model to determine whether the pneumonia model has been successfully constructed.

[0011] Furthermore, the recombinant IL-6 is human or mouse recombinant IL-6.

[0012] Furthermore, the solvent of the recombinant IL-6 solution is phosphate buffer containing 0.1 v / v% bovine serum albumin, and the concentration of the recombinant IL-6 solution is 1 ng / μL.

[0013] Furthermore, the recombinant IL-6 solution is delivered over a period of 4-6 days, once daily, at a dose of 100 μL per animal.

[0014] Furthermore, the animal in question is a mouse.

[0015] Furthermore, the delivery method is any one of tracheal instillation, nasal inhalation, and nebulized inhalation.

[0016] Furthermore, the method for lung function assessment is as follows:

[0017] Dynamic hyperpolarization of lungs in animal pneumonia models 129 Xe magnetic resonance spectroscopy, for the acquisition of hyperpolarization 129 Xe magnetic resonance spectroscopy data were processed to obtain pulmonary gas-blood exchange function parameters of animal pneumonia models. Based on the obtained gas-blood exchange function parameters, it was assessed whether recombinant IL-6 had an impact on pulmonary gas-blood exchange function in animals.

[0018] Furthermore, the method for pathological evaluation is as follows:

[0019] S1. The lungs of an animal pneumonia model were lavaged to obtain bronchoalveolar lavage fluid. The number of cells in the bronchoalveolar lavage fluid was detected. Based on the number of cells, it was determined whether recombinant IL-6 had an effect on increasing the number of inflammatory cells exudated in the lung tissue.

[0020] S2. H&E staining was performed on the lung tissue of the animal pneumonia model to observe the lung histological morphology and determine whether recombinant IL-6 had an effect on the lung histological morphology of the animals.

[0021] S3. Immunohistochemical staining of lung tissue from an animal pneumonia model with CD68 and MPO was performed to observe the number of macrophages and neutrophils in the lung tissue, thereby determining whether recombinant IL-6 has an effect on increasing the number of macrophages and neutrophils in animal lung tissue.

[0022] The pneumonia model constructed using the above method can be applied to the study of pneumonia mechanisms and the screening of pneumonia drugs.

[0023] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0024] The method for constructing an animal pneumonia model according to this invention is simple, easy to operate, and requires a short time for model establishment. The animal pneumonia model of this invention can simulate lung inflammatory diseases characterized by upregulation of the IL-6 inflammatory factor, demonstrating that administration of IL-6 alone to the lungs can induce pneumonia. This provides a new animal model for the study of the mechanism, prevention, diagnosis, and treatment of pneumonia characterized by elevated IL-6. Attached Figure Description

[0025] Figure 1 The figure shows the test results of the lung air-blood exchange function parameters of the mouse pneumonia model constructed in Example 1.

[0026] Figure 2 The figure shows the results of the total cell count test in the bronchoalveolar lavage fluid of the mouse pneumonia model constructed in Example 1.

[0027] Figure 3 H&E staining image of lung tissue sections from the mouse pneumonia model constructed in Example 1. Figure 4 Immunohistochemical staining images of macrophages and neutrophils in lung tissue sections from the mouse pneumonia model constructed in Example 1. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments.

[0029] Example 1

[0030] 1. Construction of a mouse pneumonia model

[0031] Place approximately 20g of BALB / c nude mice in an anesthesia induction box and infuse them with 1-1.5% isoflurane. Once the mice are fully anesthetized, remove them from the box and suspend them at a 45° angle on the endotracheal intubation platform. Locate the tracheal opening under a cold light source; it will appear as a bright spot and will show lateral opening and closing of the vocal cords with each breath. Place a 20g cannula around the guidewire and guide it into the trachea. Remove the guidewire and push the cannula further into the trachea to complete the intubation.

[0032] Recombinant IL-6 was dissolved in PBS containing 0.1% BSA (endotoxin-free) to prepare a recombinant IL-6 solution with a concentration of 1 ng / μl. 100 μL of the recombinant IL-6 solution was injected into the endotracheal tube. Control group nude mice received 100 μL of PBS containing 0.1% BSA (endotoxin-free) via tracheal injection. Injections were given once daily for 5 days.

[0033] 2. Lung function assessment

[0034] Nude mice were anesthetized with isoflurane gas, endotracheally intubated to control their breathing, and fixed in a supine position on an animal bed. Once their breathing became slow and stable, a 7T animal MRI scanner (Bruker Biospec 70 / 20USR; Germany) was used for imaging. 129 Acquisition of Xe CSSR pulse sequences. In the CSSR pulse sequence acquisition experiment, it was used to acquire Xe CSSR pulse sequences in dissolved states. 129 The lengths of the two Gaussian pulses for Xe signal saturation and excitation were 0.5 ms and 0.3 ms, respectively. Twenty-five exchange time points were set between 1.4 and 200 ms to acquire dynamic magnetic resonance spectra of the lungs (1.4, 3, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, and 200 ms). Before the formal breath-hold sampling, each nude mouse underwent two xenon pre-breathing cycles to improve dissolved oxygen levels. 129 Xe signal, CSSR acquisition experiment was repeated 5 times for each mouse. The raw FID data was subjected to one-dimensional Fourier transform using Matlab to obtain hyperpolarization at 25 different exchange times. 129 Xe magnetic resonance spectroscopy was further used to fit and normalize the Xe dissolved and gaseous signals, and finally the gas-blood exchange function parameters were obtained by fitting the MOXE model.

[0035] Gas-blood exchange parameters include: capillary residence time (Tau), an increase of which indicates prolonged blood exchange time in the alveolar capillaries; alveolar septal thickness (d), an increase of which indicates thickening of the alveolar septa; and the lung parenchyma / alveolar volume ratio (Vs / Va), an increase of which indicates increased lung parenchyma volume and / or decreased alveolar volume. Increased capillary residence time, alveolar septal thickness, and lung parenchyma / alveolar volume ratio indicate impaired gas-blood exchange function in the lungs.

[0036] Comparison of capillary blood residence time, alveolar septal thickness, and lung parenchyma / alveolar volume ratio in pneumonia model group (IL-6) and control group (PBS) nude mice. Figure 1 As shown, by Figure 1 The results showed that the Tau, d, and Vs / Va levels in the pneumonia model group (IL-6) were significantly higher than those in the control group (PBS), with P values ​​of 0.009, 0.041, and 0.018, respectively. This indicates that lung injection of recombinant IL-6 in nude mice significantly prolongs the blood exchange time in the alveolar capillaries, thickens the alveolar septa, increases the lung parenchyma volume, and / or decreases the alveolar volume, thereby affecting the pulmonary gas-blood exchange function of nude mice.

[0037] 3. Pathological assessment

[0038] 3.1 Total Cell Count Test in Bronchoalveolar Lavage Fluid

[0039] After anesthetizing nude mice, endotracheal intubation was performed to fully expose the lungs. The left hilum was ligated, and 300 μL of pre-cooled sterile PBS was injected into the intubation tube. The tube was then slowly aspirated, and this process was repeated three times to obtain bronchoalveolar lavage fluid (BALF) from the right lung. The BALF was centrifuged at 4°C and 3000 rpm for 5 min. The resulting cell pellet was resuspended in PBS and then counted using a cell counter.

[0040] The total cell count in the bronchoalveolar lavage fluid of nude mice in the pneumonia model group (IL-6) and the control group (PBS) was compared to that of other mice. Figure 2 As shown, by Figure 2 The results showed that the total cell count in the bronchoalveolar lavage fluid of the pneumonia model group (IL-6) was significantly higher than that in the control group (PBS), with a P value of 0.024. This indicates that injection of recombinant IL-6 into the lungs of nude mice leads to a significant increase in inflammatory cells in the alveoli.

[0041] 3.2 H&E staining treatment of lung tissue

[0042] The left lung of the nude mouse in step 3.1 was removed, fixed in formalin, and then subjected to routine H&E staining to observe the lung tissue morphology.

[0043] H&E staining images of lung tissue sections from nude mice in the pneumonia model group (IL-6) and the control group (PBS) are shown below. Figure 3 As shown, by Figure 3 It was found that the alveolar structure of nude mice in the pneumonia model group (IL-6) was disordered, with thickened alveolar walls, narrowed alveolar cavities, inflammatory cells and exudates in some alveoli, and fusion of a small number of alveoli. This indicates that injection of recombinant IL-6 into the lungs of nude mice leads to alveolitis, thickening of alveolar septa, and disordered alveolar structure.

[0044] 3.3 Immunohistochemical staining of lung tissue

[0045] The fixed left lung from step 3.2 was subjected to immunohistochemical staining for CD68 (a marker of macrophages) and MPO (a marker of neutrophils) to observe the number of macrophages and neutrophils in the left lung of nude mice.

[0046] Immunohistochemical staining images of macrophages and neutrophils in lung tissue sections of nude mice in the pneumonia model group (IL-6) and the control group (PBS) are shown below. Figure 4 As shown, by Figure 4 It was found that increased macrophages and neutrophils were observed in the alveolar septa and alveolar cavities of nude mice in the pneumonia model group (IL-6).

[0047] In summary, injection of recombinant IL-6 into the lungs of nude mice successfully induced pulmonary inflammatory changes, manifested as an increase in infiltrating inflammatory cells in the lungs, thickening of alveolar septa, and disordered alveolar structure. 129 Xe NMR showed impaired lung function in nude mice, indicating the successful establishment of a pneumonia model.

Claims

1. A method for constructing a BALB / c nude mouse pneumonia model based on recombinant IL-6, characterized in that... Includes the following steps: S1. A BALB / c nude mouse pneumonia model was established by delivering recombinant IL-6 solution to the lungs of BALB / c nude mice for several days. The delivery method can be any one of endotracheal instillation, nasal inhalation, or nebulized inhalation; S2. Perform lung function and pathological evaluation on the BALB / c nude mouse pneumonia model to determine whether the pneumonia model has been successfully constructed. The method for assessing lung function is as follows: Hyperpolarization of lung tissue collected from BALB / c nude mouse pneumonia model 129 Xe magnetic resonance spectroscopy, for the acquisition of hyperpolarization 129 Xe magnetic resonance spectroscopy data were processed to obtain pulmonary gas-blood exchange function parameters of the BALB / c nude mouse pneumonia model. Based on the obtained gas-blood exchange function parameters, it was evaluated whether recombinant IL-6 had an impact on pulmonary gas-blood exchange function of BALB / c nude mice. The method for pathological evaluation is as follows: S2-1. The lungs of the BALB / c nude mouse pneumonia model were lavaged to obtain bronchoalveolar lavage fluid. The number of cells in the bronchoalveolar lavage fluid was detected. Based on the number of cells, it was determined whether recombinant IL-6 had an effect on increasing the number of inflammatory cells exudated in the lung tissue. S2-2. H&E staining was performed on the lung tissue of the BALB / c nude mouse pneumonia model to observe the lung histological morphology and determine whether recombinant IL-6 had an effect on the lung histological morphology of BALB / c nude mice. S2-3. Immunohistochemical staining of lung tissue from the BALB / c nude mouse pneumonia model with CD68 and MPO was performed to observe the number of macrophages and neutrophils in the lung tissue, thereby determining whether recombinant IL-6 has an effect on increasing the number of macrophages and neutrophils in the lung tissue of BALB / c nude mice.

2. The method for constructing a BALB / c nude mouse pneumonia model based on recombinant IL-6 according to claim 1, characterized in that: The recombinant IL-6 mentioned is human or mouse recombinant IL-6.

3. The method for constructing a BALB / c nude mouse pneumonia model based on recombinant IL-6 according to claim 1, characterized in that: The solvent for the recombinant IL-6 solution is phosphate buffer containing 0.1 v / v% bovine serum albumin, and the concentration of the recombinant IL-6 solution is 1 ng / μL.

4. The method for constructing a BALB / c nude mouse pneumonia model based on recombinant IL-6 according to claim 1, characterized in that: The recombinant IL-6 solution was delivered over a period of 4-6 days, once a day, at a dose of 100 μL per BALB / c nude mouse.

5. The application of the pneumonia model constructed using the construction method described in any one of claims 1 to 4 in the study of pneumonia mechanisms and the screening of pneumonia drugs.

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