Canine pulmonary fibrosis model as well as construction method and application thereof

Abstract

The invention relates to a method for noninvasive construction of a dog pulmonary fibrosis model and application thereof, the method comprises the following steps: a dog is subjected to nano-silica microsphere exposure, and the particle size of nano-silica microspheres is 400-600 nm. According to the construction method disclosed by the invention, the dogs are subjected to nano-silica microsphere exposure, and related inadaptation symptoms of the dogs caused by exposure operation can be effectively eliminated by selecting the particle sizes of the nano-silica microspheres, the exposure dosage and the exposure program, so that after the exposure operation is completed, the drinking water, appetite and mental states of the dogs can be recovered to normal levels; the canine pulmonary fibrosis model which can be used for pathological mechanism research of pulmonary fibrosis, screening of pulmonary fibrosis treatment targets or drugs and the like is successfully constructed and obtained. The method disclosed by the invention is simple to operate, controllable in exposure dose and good in repeatability.

Description

Technical Field

[0001] The invention belongs to the technical field of disease animal model construction, and specifically relates to a non-invasive canine pulmonary fibrosis model and a construction method and application thereof. Background Art

[0002] Silicosis is a serious occupational pneumoconiosis that seriously endangers the health of workers and has a high mortality rate. Although current protective measures have a certain effect on preventing dust from being inhaled into the lungs, nano-sized dust can pass through the protective barrier, invade the lungs and deposit, causing serious lung damage. Reports show that silicosis is associated with many biological processes such as abnormal interstitial pneumonia, macrophage inflammatory response, and severe progressive pulmonary fibrosis.

[0003] Although dust aerosol exposure is most consistent with the actual situation of inducing silicosis animal models, it will also cause certain potential exposure risks to researchers. In order to seek a safer method to complete the induction of silicosis models, some people have found that intratracheal instillation can induce pulmonary fibrosis in rats. At present, many toxicologists are more concerned about the molecular mechanism of silicosis exposure, and inflammation-mediated pulmonary fibrosis is a major consensus. Indicators that may be related to pulmonary fibrosis include α-SMA, TGF-β, and FN. α-Smooth muscle actin (α-SMA) causes fibrosis in various pathological conditions, thereby inducing target organ damage. Transforming growth factor-β (TGF-β), as the most common cytokine, induces fibroblast synthesis and promotes the adhesion and deposition of extracellular matrix, further inducing fibrosis. Fibronectin (FN) is a high-molecular glycoprotein with strong cell adhesion activity, which participates in the fibrosis process of target organs through high expression.

[0004] Occupational dust exposure causes irreversible lung damage to practitioners, which has attracted widespread attention from environmental toxicologists, and a large number of pulmonary fibrosis modeling methods have been reported. Lou Jianlin et al. successfully induced a rat pulmonary fibrosis disease model by airway instillation of ground quartz dust suspension for 30 days, which was manifested by the appearance of typical cellular silicosis nodules in rat lung tissue, accompanied by alveolar structure destruction and collagen fiber deposition. However, compared with small rodents, dogs have greater advantages in the study of pulmonary fibrosis models. Their lung tissue structure is more similar to that of humans and their individuals are larger, which is more convenient for airway instillation operations. Their longer life cycle is also more conducive to exploring long-term pathological changes after dust accumulation in the lungs. Yao Rong et al. successfully induced secondary pulmonary fibrosis in rhesus monkeys by a single oral administration of paraquat, accompanied by collagen fiber deposition in lung tissue. Compared with primates, dogs have superior reproductive performance and more suitable cost advantages, making canine pulmonary fibrosis disease models widely used. In addition, there is a need in the art for a canine model of pulmonary fibrosis that can be used to explore the pathogenesis of pulmonary fibrosis and provide effective support for interventional treatments. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a non-invasive canine pulmonary fibrosis model construction method and its application in the study of the pathological mechanism of pulmonary fibrosis and the screening of therapeutic targets or drugs for pulmonary fibrosis. The construction method is simple to operate, the exposure dose is controllable, and the repeatability is good.

[0006] The first aspect of the present invention is to provide a method for constructing a canine pulmonary fibrosis model, comprising the following steps: exposing a canine to nano-silica microspheres, wherein the nano-silica microspheres have a particle size of 400nm to 600nm and a total dose of 200mg / kg to 600mg / kg.

[0007] In some of the embodiments, the total exposure dose of the nano-silica microspheres is 200 mg / kg to 400 mg / kg, and more preferably 200 mg / kg to 300 mg / kg.

[0008] In some embodiments, exposure is performed once every 5 to 10 days, for a total of 4 to 8 exposures.

[0009] In some embodiments, exposure is performed once every 6 to 8 days, for a total of 4 to 6 exposures.

[0010] In some embodiments, the exposure method includes airway instillation, airway atomization, nebulization inhalation, and nasal instillation.

[0011] In some embodiments, the exposure method is airway instillation; the nano-silica microspheres are dissolved in a solvent to obtain a nano-silica microsphere suspension, which is then instilled into the airway.

[0012] In some embodiments, the solvent is water.

[0013] In some embodiments, the concentration of the nano-silica microsphere suspension is 50 mg / mL to 150 mg / mL, preferably 100 mg / mL to 150 mg / mL.

[0014] In some embodiments, the nano-silica microsphere suspension further contains antibiotics; preferably, the antibiotics include at least one of penicillin and streptomycin.

[0015] In some embodiments, the dog is a beagle.

[0016] The second aspect of the present invention is to provide a canine pulmonary fibrosis model constructed by any of the above methods.

[0017] The third aspect of the present invention is to provide the use of the pulmonary fibrosis model obtained by the method or construction as described above in screening therapeutic targets or drugs for pulmonary fibrosis.

[0018] The invention provides application of the above method or pulmonary fibrosis model in the study of the pathological mechanism of pulmonary fibrosis.

[0019] The present invention provides a method for constructing a canine pulmonary fibrosis model. The method comprises the steps of exposing a canine to nano-silica microspheres, and controlling the particle size (400nm-600nm) of the nano-silica microspheres, as well as the exposure dose and the exposure procedure, so as to effectively eliminate the related maladaptive symptoms of the canine caused by the exposure operation, so that after the exposure operation is completed, the drinking water, appetite and mental state of the canine can return to normal levels, and a canine pulmonary fibrosis model which can be used for the study of the pathological mechanism of pulmonary fibrosis, the screening of therapeutic targets or drugs for pulmonary fibrosis, etc. is successfully constructed.

[0020] The method for constructing a canine pulmonary fibrosis model of the present invention has the advantages of simple operation, controllable exposure dose and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1 is the external appearance of the lungs of beagle dogs in each group in Example 1.

[0022] Figure 2 Schematic diagram of HE staining results of lung tissues of beagle dogs in each group in Example 1, wherein from left to right are control group and pulmonary fibrosis model group; yellow arrows: alveolar dilation; red arrows: alveolar wall capillary congestion; green arrows: bronchiolar mucosal epithelial hyperplasia, irregular arrangement, small round vacuoles appear in the cytoplasm of epithelial cells; black arrows: neutrophil infiltration.

[0023] Figure 3 Schematic diagram of Masson staining results of pathological sections of lung tissues of beagle dogs in each group in Example 1, from left to right, respectively, control group and pulmonary fibrosis model group.

[0024] Figure 4 Schematic diagram of the immunohistochemistry results of α-smooth muscle actin (α-SMA) in the lung tissues of dogs in each group in Example 1, from left to right are the control group, the pulmonary fibrosis model group and the PBS negative control group.

[0025] Figure 5 Schematic diagram of the immunohistochemical results of fibronectin (FN) in the lung tissue of each group of dogs in Example 1, from left to right are the control group, the pulmonary fibrosis model group and the PBS negative control group.

[0026] Figure 6Schematic diagram of the immunohistochemistry results of transforming growth factor-β (TGF-β) in the lung tissue of each group of dogs in Example 1, from left to right are the control group, the pulmonary fibrosis model group and the PBS negative control group.

[0027] Figure 7 HE staining of lung tissue of dogs in the model group in Comparative Example 1.

[0028] Figure 8 This is Masson staining of lung tissue of dogs in the model group in Comparative Example 1.

[0029] Fig. 9 This is the serum AST detection of dogs in the model group in Comparative Example 3.

[0030] Fig.10 This is the serum ALT detection of dogs in the model group in Comparative Example 3.

[0031] Fig.11 This is Masson staining of lung tissue of dogs in the model group in Comparative Example 3 (40×).

[0032] Fig.12 This is the linear monitoring of the weight of the dogs in the model group in Comparative Example 4.

[0033] Fig.13 This is the linear monitoring of the weight of the dogs in the model group in Comparative Example 5. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0037] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0038] Some embodiments of the present invention relate to a method for constructing a canine pulmonary fibrosis model, comprising the following steps: exposing the canine to nano-silica microspheres, wherein the particle size of the nano-silica microspheres is 400nm to 600nm.

[0039] The inventors have found through research that the particle size of nano-silica microspheres will significantly affect the state of the dog during the modeling process. It is critical to use nano-silica microspheres with a particle size of 400nm to 600nm, and to apply a suitable total exposure dose to ensure the success rate of modeling and the good physiological state of the experimental dogs during the modeling process. Within 2 to 3 days after the exposure operation, the discomfort symptoms caused by the exposure operation will disappear, and the drinking water, appetite, and mental state will return to normal.

[0040] In some preferred embodiments, the total exposure dose of the nano-silica microspheres is 200 mg / kg to 600 mg / kg, preferably 200 mg / kg to 400 mg / kg, and more preferably 200 mg / kg to 300 mg / kg.

[0041] We found that even the exposure dose of nano-silica microspheres with appropriate particle size would affect the success rate of modeling the canine pulmonary fibrosis model and the status of the model dogs. An exposure dose that was too low could not successfully establish a canine pulmonary fibrosis model, while an exposure dose that was too high could cause the death of the dog, or produce related adverse symptoms, such as abnormal drinking, appetite and mental state, and the dog could not be used for subsequent research.

[0042] During the exposure operation, the number of days between each exposure and the dose of a single exposure will also affect the effect of canine pulmonary fibrosis modeling. Preferably, exposure is performed once every 5 to 10 days, for a total of 4 to 8 exposures; more preferably, exposure is performed once every 6 to 8 days, and most preferably, the interval is 7 days, for a total of 4 to 6 exposures. We found that a single exposure operation with a short interval (e.g., 4 days or less) can easily cause the death of the dog, or after completing all exposure operations, the dog's drinking water, appetite, and mental state cannot return to normal, which does not meet the requirements of subsequent research applications.

[0043] In some of the preferred embodiments, the exposure methods include airway instillation, airway atomization, aerosol inhalation, nasal instillation, and more preferably airway instillation. Airway instillation can better control the exposure dose, has strong operability, and good repeatability.

[0044] By controlling the particle size of the nano-silica microspheres (400 nm to 600 nm) and further controlling the exposure dose and each exposure procedure, the present invention can effectively eliminate the related discomfort symptoms of dogs caused by the exposure operation, so that after the exposure operation is completed, the drinking water, appetite, and mental state of the dogs can return to the normal level, meeting the requirements of subsequent research applications.

[0045] The present invention will be further described in detail below in conjunction with specific embodiments.

[0046] Example 1

[0047] 1. Experimental materials

[0048] (1) Basic characteristics of nano-silica microspheres

[0049] Nano-silica microspheres were selected: Xi'an Aopep Biotechnology Co., Ltd., with a particle size normally distributed between 400 - 600 nm and an average particle size of 500 nm.

[0050] 2. Experimental animals

[0051] 6 healthy male beagle dogs with uniform body weight, weighing 9 - 11 kg (average about 10 kg), were provided by Guangzhou Institute of Pharmaceutical Research Co., Ltd., with the production license number [SCXK(Yue)2023 - 0007].

[0052] 3. Animal grouping

[0053] After 14 days of adaptive conventional feeding, the above-mentioned beagle dogs were randomly divided into 2 groups, with 3 dogs in each group: the control group (Control Group) and the model group (Model Group).

[0054] 4. Main instruments and reagents for the experiment

[0055] (1) Automatic high-pressure sterilizer: Shanghai Boxun Industry Co., Ltd., Medical Equipment Factory;

[0056] (2) Large animal laryngoscope: Jiangsu Yongle Medical Technology Co., Ltd.;

[0057] (3) Pathological slicer: Ernst Leitz Company;

[0058] (4) Microscope: Ernst Leitz Company;

[0059] (5) Nano-silica microspheres: Xi'an Tai'ao Biotechnology Co., Ltd.

[0060] 5. Establishment of a canine pulmonary fibrosis model

[0061] (1) Preparation of Nanosilica Microsphere Suspension

[0062] Nano-silica microspheres (average particle size of 500nm) were mixed with deionized water to prepare a suspension with a concentration of 100mg / mL, and 2v / v% penicillin-streptomycin solution (a dual-antibody solution containing 10,000IU penicillin and 10,000μg / mL streptomycin) was added. The suspension was divided into 10mL glass tubes, sealed and sterilized, and shaken thoroughly before the experiment.

[0063] (2) Establishment of pulmonary fibrosis model

[0064] The control group was routinely fed and instilled with the same volume of normal saline as the model group. The beagle dogs in the model group were instilled with the nano-silica microsphere suspension in the airways, 7 days / time, 5 mL each time, a total of 4 instillations, each exposure dose was about 50 mg / kg, the experimental period was 28 days, and the total exposure dose was about 200 mg / kg.

[0065] The specific operation includes the following steps:

[0066] The beagles in the model group were first anesthetized with thiazine (0.03 mL / kg BW) and proporfin (0.05 mL / kg BW), and the anesthesia performance of the dogs was observed at any time. It is generally believed that the corneal reflex, limbs, and abdominal wall muscle tension are the observation indicators. When the beagles' breathing is stable, blood pressure is normal, abdominal wall muscles are relaxed, and corneal reflex is sluggish, tracheal intubation can be performed with the assistance of laryngoscope.

[0067] The operator places the dog in a supine position on the operating table and pulls the dog's tongue to one side. The dog's mouth is then opened to fully expose the epiglottis cartilage. With the help of a large animal laryngoscope, the tongue is pressed down and the airway is opened. Then, a respiratory anesthesia tube of appropriate length is slowly inserted into the base of the dog's airway and then pulled out about 1 cm.

[0068] After the intubation of the organ tube is completed, 5 mL of the nano-silica microsphere suspension is extracted with a syringe, the scalp needle (50 cm) is extended, the needle is cut off, and the length of the scalp needle hose is cut to be slightly longer than the endotracheal tube by 1.5 cm. The scalp needle tube is slowly inserted along the endotracheal tube to the predetermined depth, and the suspension is slowly injected. After the airway instillation is completed, the endotracheal tube and the instillation hose are pulled out. The dog is then released from restraint, and the dog is evenly turned left and right with force so that the suspension is evenly distributed in the lungs. All operations were approved by the Experimental Animal Management Committee of Guangzhou General Institute of Pharmaceutical Research Co., Ltd. (No.: 2024A0003).

[0069] (3) Confirmation of pulmonary fibrosis model

[0070] After the experimental period, the dogs in the control group and the model group were anesthetized with thiazine (0.03 mL / kg BW) and proporfin (0.05 mL / kg BW), and then euthanized by intravenous injection of 10% potassium chloride. Lung tissues were then collected for hematoxylin-eosin (HE) staining, Masson's trichrome staining, and immunohistochemistry (IHC) staining of fibrosis-related factors.

[0071] The specific operation is as follows: the lung tissue is fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, and then sliced ​​into sections with a thickness of 5 μm, and the above-mentioned staining operation is performed.

[0072] HE staining: Paraffin sections were dewaxed, dehydrated, and stained with hematoxylin-eosin. Next, neutral resin was applied, covered with glass, and after curing, scanned using Panoramic MIDI (3DHISTECH, Hungary). Case-viewer 2.4 (3DHISTECH, Hungary) was used to capture and analyze the microstructural field of view.

[0073] Masson staining: Paraffin sections are dewaxed to water, followed by potassium dichromate staining, iron hematoxylin staining, and Ponceau staining. Next, they are treated with phosphomolybdic acid aqueous solution and then aniline blue staining, followed by aniline blue staining. Finally, glacial acetic acid differentiation, dehydration, sealing, and microscopic examination are performed, and image acquisition and analysis are performed.

[0074] IHC staining: The preparation method of tissue sections is the same as above, the only difference is that charged slides are used to carry lung tissue sections. The tissue sections were placed in 10mM sodium citrate buffer (pH=6.0) and heated in a microwave oven for 10 minutes for antigen dissociation. After cooling, the sections were immersed in 3% hydrogen peroxide solution for 15 minutes. The sections were washed with PBS and then blocked with 10% horse serum at 37°C for 60 minutes. After blocking, the sections were incubated overnight at 4°C with primary antibodies against α-SMA, FN, TGF-β, and COL-3 (Sigma-Aldrich, USA, 1:200). Then, biotin-labeled goat anti-rabbit IgG was used for incubation at 37°C for 60 minutes. Finally, all sections were incubated with DAB (Beyotime, China) and then observed under a microscope (Leica, Germany). Five areas were randomly selected on each section for observation, and the total optical density values ​​were quantitatively analyzed using an image analyzer (Image-Pro Plus 4.5, Media Cybernetics).

[0075] After the experiment in this example, the dogs were in good spirits, and there were no abnormalities in appetite and drinking water, which met the requirements of animal welfare and subsequent experiments.

[0076] like Figure 1 As shown, no obvious lung damage was observed in the control group, but compared with the control group, extensive blood circulation disorders were clearly observed in the lung group in the model.

[0077] like Figure 2 As shown, compared with the control group, the alveoli in the model group were significantly dilated, the alveolar wall capillaries were congested, the bronchiolar mucosal epithelium proliferated and arranged irregularly, small round vacuoles appeared in the cytoplasm of epithelial cells, and neutrophil infiltration was present.

[0078] like Figure 3 As shown, the fibrinogen deposition in the model group was significantly increased compared with the control group.

[0079] like Figure 4 Figure 5 and Figure 6 As shown in the figure, compared with the control group, the positive expressions of fibrosis-related factors α-SMA, FN, and TGF-β in the lung tissue of the model group were significantly enhanced.

[0080] (4) Conclusion

[0081] The above results show that the method of the present invention can successfully induce canine pulmonary fibrosis. A large number of alveolar fusions and fibrinogen deposition occur in the lung tissue of the canine model, accompanied by positive expression of fibrosis-related factors (α-SMA, FN, TGF-β).

[0082] We also conducted corresponding comparative experiments.

[0083] Comparative Example 1

[0084] The method for constructing the canine pulmonary fibrosis model in this comparative example is the same as that in Example 1 except for the particle size of the nano-silicon dioxide used. The specific experimental conditions are as follows:

[0085] (1) Preparation of Nanosilica Microsphere Suspension

[0086] Nano-silica microspheres (average particle size 1000nm, Xi'an Ao Peptide Biotechnology Co., Ltd.) were mixed with deionized water to prepare a suspension with a concentration of 100mg / mL, and 2v / v% penicillin-streptomycin solution (a dual-antibody solution containing 10,000IU penicillin and 10,000μg / mL streptomycin) was added. The suspension was divided into 10mL glass tubes, sealed and sterilized, and shaken thoroughly before the experiment.

[0087] Other experimental methods were carried out with reference to Example 1.

[0088] The control group is the same as the control group in Example 1. The following comparative examples are all the same.

[0089] (2) Establishment of pulmonary fibrosis model

[0090] The nano-silica microsphere suspension was dripped into the airways of 3 beagle dogs, 7 days / time, 5 mL each time, a total of 4 drips, the test period was 28 days, and the total exposure dose was about 200 mg / kg.

[0091] The beagles in the model group were first anesthetized with thiazine (0.03 mL / kg BW) and proporfin (0.05 mL / kg BW), and the anesthesia performance of the dogs was observed at any time. It is generally believed that the corneal reflex, limbs, and abdominal wall muscle tension are the observation indicators. When the beagles' breathing is stable, blood pressure is normal, abdominal wall muscles are relaxed, and corneal reflex is sluggish, tracheal intubation can be performed with the assistance of laryngoscope.

[0092] The modeling operation is the same as that in Example 1.

[0093] During and after the comparative test, the animals had obvious symptoms of maladaptation, their drinking water, appetite and mental state were poor, and they could not return to normal levels for a long time, which did not meet the subsequent application requirements.

[0094] The lung tissue samples were subsequently stained with HE and Masson staining, and the results are detailed in Figure 7 and Figure 8 The results showed that the lung tissue was severely damaged and had extensive bleeding, but the accumulation of collagen fibers was not obvious. Figure 7 HE staining of the lung tissue of dogs in the model group showed a large amount of red blood cell accumulation and destruction of the lung tissue structure. Figure 8 Masson staining of the lung tissue of the model dog shows deformation of the lung structure, but no obvious accumulation of collagen fibers.

[0095] Comparative Example 2

[0096] The method for constructing the canine pulmonary fibrosis model in this comparative example is the same as that in Example 1 except for the particle size of the nano-silicon dioxide used, and is specifically as follows:

[0097] (1) Preparation of Nanosilica Microsphere Suspension

[0098] Nano-silica microspheres (average particle size of 20 nm, purchased from Xi'an Ao Peptide Biotechnology Co., Ltd.) were mixed with deionized water to prepare a suspension with a concentration of 100 mg / mL, and 2 v / v% penicillin-streptomycin solution (a dual-antibody solution containing 10,000 IU penicillin and 10,000 μg / mL streptomycin) was added. The suspension was divided into 10 mL glass tubes, sealed and sterilized, and shaken thoroughly before the experiment.

[0099] (2) Establishment of pulmonary fibrosis model

[0100] The nano-silica microsphere suspension was dripped into the airways of 3 beagle dogs, 7 days / time, 5 mL each time, a total of 4 drips, the test period was 28 days, and the total exposure dose was about 200 mg / kg.

[0101] The beagles in the model group were first anesthetized with thiazine (0.03 mL / kg BW) and proporfin (0.05 mL / kg BW), and the anesthesia performance of the dogs was observed at any time. It is generally believed that the corneal reflex, limbs, and abdominal wall muscle tension are the observation indicators. When the beagles' breathing is stable, blood pressure is normal, abdominal wall muscles are relaxed, and corneal reflex is sluggish, tracheal intubation can be performed with the assistance of laryngoscope.

[0102] The modeling operation is the same as that in Example 1.

[0103] After testing, this comparative example could not successfully construct a canine pulmonary fibrosis model. This may be because when the particle size of nano-silica microspheres is too small, they are transported and deposited to other organs during the exposure operation, causing damage to other organs. Therefore, the constructed model is not a simple pulmonary fibrosis model. The figure below shows the linear detection of serum AST and ALT in the model group dogs. It was found that the serum AST and ALT levels increased in the later stage of the experiment, indicating liver damage. For details, see Fig. 9 and Fig.10 .

[0104] Comparative Example 3

[0105] The method for constructing the canine pulmonary fibrosis model in this comparative example is the same as that in Example 1 except for the total exposure dose, and is specifically as follows:

[0106] (1) Preparation of Nanosilica Microsphere Suspension

[0107] Nano-silica microspheres (average particle size of 500nm) were mixed with deionized water to prepare a suspension with a concentration of 100mg / mL, and 2v / v% penicillin-streptomycin solution (a dual-antibody solution containing 10,000IU penicillin and 10,000μg / mL streptomycin) was added. The suspension was divided into 10mL glass tubes, sealed and sterilized, and shaken thoroughly before the experiment.

[0108] (2) Establishment of pulmonary fibrosis model

[0109] The control group was fed conventionally, and the beagle dogs in the model group were dripped with the nano-silica microsphere suspension into the airway, 7 days / time, 5 mL each time, a total of 2 drips, the test period was 14 days, and the total exposure dose was about 100 mg / kg.

[0110] The beagles in the model group were first anesthetized with thiazine (0.03 mL / kg BW) and proporfin (0.05 mL / kg BW), and the anesthesia performance of the dogs was observed at any time. It is generally believed that the corneal reflex, limbs, and abdominal wall muscle tension are the observation indicators. When the beagles' breathing is stable, blood pressure is normal, abdominal wall muscles are relaxed, and corneal reflex is sluggish, tracheal intubation can be performed with the assistance of laryngoscope.

[0111] The modeling operation is the same as that in Example 1.

[0112] After testing, the model animal's lungs did not reach the pulmonary fibrosis index after the end of the comparative test period, and the model could not be successfully established. The weight of the dog used in the experiment in this comparative example was about 10 kg, and the exposure operation was: 100 mg / mL suspension, 7 days / time, 5 mL each time, a total of 2 instillations, and the total exposure dose was about 100 mg / kg. The dose was too small, and the fiber accumulation in the lung tissue was not obvious, so the pulmonary fibrosis model dog could not be obtained. The results of Masson staining of its lung tissue are detailed in Fig.11 .

[0113] Comparative Example 4

[0114] The method for constructing the canine pulmonary fibrosis model in this comparative example is the same as that in Example 1 except for the total exposure dose, and is specifically as follows:

[0115] (1) Preparation of Nanosilica Microsphere Suspension

[0116] Nano-silica microspheres (average particle size of 500nm) were mixed with deionized water to prepare a suspension with a concentration of 200mg / mL, and 2v / v% penicillin-streptomycin solution (a dual-antibody solution containing 10,000IU penicillin and 10,000μg / mL streptomycin) was added. The suspension was divided into 10mL glass tubes, sealed and sterilized, and shaken thoroughly before the experiment.

[0117] (2) Establishment of pulmonary fibrosis model

[0118] The control group was fed conventionally, and the beagle dogs in the model group were dripped with the nano-silica microsphere suspension into the airway, 5 days / time, 5 mL each time, a total of 7 drips, the test period was 35 days, and the total exposure dose was about 700 mg / kg.

[0119] The beagles in the model group were first anesthetized with thiazine (0.03 mL / kg BW) and proporfin (0.05 mL / kg BW), and the anesthesia performance of the dogs was observed at any time. It is generally believed that the corneal reflex, limbs, and abdominal wall muscle tension are the observation indicators. When the beagles' breathing is stable, blood pressure is normal, abdominal wall muscles are relaxed, and corneal reflex is sluggish, tracheal intubation can be performed with the assistance of laryngoscope.

[0120] The modeling operation is the same as that in Example 1.

[0121] After the end of the comparative test period, the animals showed obvious symptoms of discomfort, including obvious breathing difficulties, poor drinking, appetite and mental state, and had not fully recovered until the second experimental stage, which did not meet the subsequent application requirements. The weight of the dog used in the comparative experiment was about 10 kg, and the exposure operation was: 200 mg / mL suspension, 5 days / time, 5 mL each time, a total of 7 drips, the test period was 35 days, and the total exposure dose was about 700 mg / kg. The dose was too large, which affected the state of the model dog and was not conducive to subsequent application. The weight monitoring results are detailed in Fig.12 .

[0122] Comparative Example 5

[0123] The method for constructing the canine pulmonary fibrosis model in this comparative example is the same as that in Example 1 except for the interval between each exposure, and is specifically as follows:

[0124] (1) Preparation of Nanosilica Microsphere Suspension

[0125] Nano-silica microspheres (average particle size of 500nm) were mixed with deionized water to prepare a suspension with a concentration of 100mg / mL, and 2v / v% penicillin-streptomycin solution (a dual-antibody solution containing 10,000IU penicillin and 10,000μg / mL streptomycin) was added. The suspension was divided into 10mL glass tubes, sealed and sterilized, and shaken thoroughly before the experiment.

[0126] (2) Establishment of pulmonary fibrosis model

[0127] The nano-silica microsphere suspension was dripped into the airways of three beagle dogs, 4 days / time, 5 mL each time, 4 times in total, the test period was 16 days, and the total exposure dose was about 200 mg / kg.

[0128] The beagles in the model group were first anesthetized with thiazine (0.03 mL / kg BW) and proporfin (0.05 mL / kg BW), and the anesthesia performance of the dogs was observed at any time. It is generally believed that the corneal reflex, limbs, and abdominal wall muscle tension are the observation indicators. When the beagles' breathing is stable, blood pressure is normal, abdominal wall muscles are relaxed, and corneal reflex is sluggish, tracheal intubation can be performed with the assistance of laryngoscope.

[0129] The modeling operation is the same as that in Example 1.

[0130] During the comparative test, the animals showed obvious symptoms of discomfort; after the test, their drinking water, appetite and mental state were poor, and they could not return to normal levels for a long time, which did not meet the subsequent application requirements. In this comparative test, the interval between each exposure was 4 days, which was too short, and the animals' discomfort symptoms were not effectively eliminated. Such an interval exposure operation resulted in the experimental dogs' drinking water, appetite and mental state not being able to return to normal levels after the test. For details of their weight gain, see Fig.13 .

[0131] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a canine pulmonary fibrosis model, characterized in that: The following steps are involved: Dogs were exposed to nano-silica microspheres having a particle size of 400 nm to 600 nm and a total dose of 200 mg / kg to 600 mg / kg.

2. The method according to claim 1, characterized in that The total exposure dose of the nano-silica microspheres is 200 mg / kg to 400 mg / kg.

3. The construction method according to claim 1 or 2, characterized in that: Exposure was performed every 5 to 10 days, for a total of 4 to 8 exposures.

4. The construction method according to claim 3, characterized in that: Exposure was performed every 6 to 8 days, for a total of 4 to 6 exposures.

5. The construction method according to any one of claims 1 to 4, characterized in that: The exposure methods include airway instillation, airway atomization, atomization inhalation, and nasal instillation.

6. The construction method according to claim 5, characterized in that: The exposure method is airway instillation; the nano-silica microspheres are dissolved in a solvent to obtain a nano-silica microsphere suspension, which is then instilled into the airway.

7. The construction method according to claim 6, characterized in that: The solvent is water; and / or, The concentration of the nano-silica microsphere suspension is 50 mg / mL to 150 mg / mL; and / or, the dog is a beagle.

8. The construction method according to claim 6, characterized in that: The nano-silica microsphere suspension also contains antibiotics; preferably, the antibiotics include at least one of penicillin and streptomycin.

9. A canine pulmonary fibrosis model constructed according to the method of any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 or the canine pulmonary fibrosis model according to claim 9 in screening therapeutic targets or drugs for pulmonary fibrosis.

Images