Construction method of sicca syndrome combined pulmonary fibrosis animal model

Abstract

The invention provides a method for constructing an animal model with sicca syndrome combined with pulmonary fibrosis, and relates to the technical field of animal model construction.The method comprises the following steps that 8-week-old NOD / Ltj mice qualified in quarantine are purchased and fed for 12 weeks; in 12 weeks, bleomycin (BLM) is injected into the mouse in the trachea, the injection dosage of bleomycin is 3 mg / kg, and construction of the sicca syndrome combined pulmonary fibrosis animal model is completed 21 days after injection. The SS-PF mouse model disclosed by the invention has high repeatability and stability, can be constructed on a large scale, and is suitable for various research requirements. The model is not only suitable for basic research, but also can be applied to drug screening and curative effect evaluation, and has a wide application prospect. Through the model, large-scale drug screening experiments can be carried out, the effectiveness and safety of drugs can be evaluated, a new treatment choice is provided for SS-PF patients, and the clinical treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to a method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis. Background Art

[0002] Sjögren's syndrome is an autoimmune disease characterized by the body's immune system attacking its own tissues, mainly manifested as chronic inflammation and destruction of exocrine glands, especially salivary glands and lacrimal glands, leading to symptoms of dry eyes and dry mouth. In addition, SS can also affect other organs, including the lungs, kidneys, skin and nervous system. The pathogenesis of SS is not yet fully understood, but it is known to be closely related to abnormal activation of the immune system and the production of autoantibodies.

[0003] Pulmonary fibrosis (PF) is a progressive and irreversible lung disease characterized by the gradual scarring and hardening of lung tissue, leading to decreased lung function and dyspnea. The pathological process of PF includes the proliferation of fibroblasts, excessive deposition of collagen, and remodeling of lung tissue structure. Currently, the exact cause of PF is not fully understood, but it is known to be closely related to genetics, environmental factors, and some specific diseases (such as SS).

[0004] Among patients with Sjögren's syndrome, about 20%-30% will develop pulmonary fibrosis, a complication known as Sjögren's syndrome with pulmonary fibrosis (SS-PF). The pathological process of SS-PF is complex and variable, involving the participation of multiple immune cells and inflammatory factors. SS generally precedes PF, but in some cases, PF can precede SS, or both can occur simultaneously. The prognosis of patients with SS-PF is poor, and there is currently no effective treatment.

[0005] Currently, research on SS and PF mainly focuses on their respective disease models:

[0006] Animal models of Sjögren's syndrome: The most commonly used is the spontaneous Sjögren's syndrome model mouse NOD / Ltj, which develops pathological features similar to human SS under natural conditions, including lymphocyte infiltration of salivary and lacrimal glands. However, these mice do not naturally develop pulmonary fibrosis, which limits the study of the mechanism of SS combined with PF.

[0007] Animal models of pulmonary fibrosis: The bleomycin (BLM)-induced mouse pulmonary fibrosis model is the most commonly used PF model. Bleomycin is an antitumor antibiotic that can induce obvious fibrotic features in mouse lung tissue by intratracheal or intraperitoneal injection.

[0008] Currently, researchers are trying to induce pulmonary fibrosis based on classic autoimmune disease model mice (such as rheumatoid arthritis model mice CIA) to construct a comorbidity model. For example, bleomycin is used to induce a rheumatoid arthritis-related pulmonary fibrosis model. However, studies on constructing a pulmonary fibrosis model in Sjögren's syndrome model mice are still rare.

[0009] Therefore, developing a mouse model of Sjögren's syndrome with pulmonary fibrosis (SS-PF) based on NOD / Ltj mice and inducing pulmonary fibrosis with bleomycin will provide an effective experimental platform for the study of the pathological mechanism and drug screening of SS-PF. This model can not only reflect the pathological characteristics of SS and PF, but also simulate the progression of the disease, providing an important basis for the development of new drugs and the evaluation of efficacy. Summary of the invention

[0010] The purpose of the present invention is to solve the existing technical problems raised in the above background technology and to propose a method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis.

[0011] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0012] The present invention proposes a method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis, comprising the following steps:

[0013] S1: Purchase 8-week-old NOD / Ltj mice that have passed quarantine and raise them until 12 weeks old;

[0014] S2: At 12 weeks, mice were injected with intratracheal bleomycin (BLM) at a dose of 3 mg / kg. The animal model of Sjögren's syndrome combined with pulmonary fibrosis was established 21 days after the injection.

[0015] As a preferred technical solution of the present invention, the preparation method of the bleomycin solution in S2 is:

[0016] Sterile physiological saline was used to dissolve the bleomycin dry powder. The concentration of bleomycin after dissolution was 1.5 mg / mL.

[0017] As a preferred technical solution of the present invention, the specific operation method in S2 is:

[0018] The surface skin, subcutaneous tissue and muscles of the mouse neck trachea were cut open layer by layer to fully expose the trachea. The syringe needle was gently inserted into the trachea at a 45-degree angle at the position of the ring hose. When it entered about 1 / 4 of the length, the needle was stopped and the bleomycin solution was quickly injected at one time.

[0019] As a preferred technical solution of the present invention, the bleomycin injection method in S2 comprises:

[0020] After injection, the mouse was shaken upright from side to side to allow the bleomycin solution to enter both lungs and be evenly distributed in the lungs.

[0021] As a preferred technical solution of the present invention, the mouse anesthesia method in S2 is:

[0022] Use isoflurane for anesthesia, which includes placing the mouse's mouth and nose at the gas anesthesia machine, adjusting the anesthetic concentration to a high concentration, and performing the operation after the mouse is fully anesthetized.

[0023] As a preferred technical solution of the present invention, the feeding conditions of the mice in S1 are:

[0024] They were maintained under standard laboratory conditions, including appropriate temperature, humidity, and photoperiod.

[0025] The present invention also provides a mouse model of Sjögren's syndrome combined with pulmonary fibrosis, which is constructed by the following method:

[0026] Purchase quarantine-qualified 8-week-old NOD / Ltj mice and raise them until they are 12 weeks old.

[0027] At 12 weeks, mice were injected intratracheally with 3 mg / kg bleomycin, 21 days after injection.

[0028] The present invention also provides a mouse model of Sjögren's syndrome combined with pulmonary fibrosis, which exhibits the following characteristics:

[0029] The submandibular glands were infiltrated with numerous foci of mononuclear cells, consistent with the features of Sjögren's syndrome.

[0030] Inflammatory infiltration and collagen fiber deposition in lung tissue are consistent with the characteristics of pulmonary fibrosis.

[0031] A mouse model of Sjögren's syndrome combined with pulmonary fibrosis, which can be used for the following purposes:

[0032] Study on pathological mechanism.

[0033] Drug screening and efficacy evaluation.

[0034] The present invention also provides an application of an animal model of Sjögren's syndrome combined with pulmonary fibrosis, wherein the model is used to study the pathophysiological mechanism of Sjögren's syndrome combined with pulmonary fibrosis and a treatment method thereof.

[0035] Beneficial effects:

[0036] Providing an animal model that truly simulates the pathological characteristics of SS-PF: The present invention successfully constructed a mouse model of Sjögren's syndrome combined with pulmonary fibrosis (SS-PF) by using bleomycin to induce pulmonary fibrosis in NOD / Ltj mice. This model has the typical pathological characteristics of both SS and PF, including lymphocyte infiltration of salivary glands and lacrimal glands, as well as fibrosis and inflammatory infiltration of lung tissues, which can truly reflect the pathological process of human SS-PF and provide an important experimental tool for studying the pathological mechanism of SS-PF.

[0037] Promoting SS-PF mechanism research and drug development: The lack of a suitable SS-PF animal model currently limits the pathological mechanism research and new drug development of SS combined with PF. The SS-PF mouse model provided by the present invention can be used to explore the pathogenesis, immune response and fibrosis process of SS-PF, helping scientists to deeply understand the complex pathology of the disease. At the same time, the model can also be used to screen and evaluate potential therapeutic drugs, accelerating the development of new drugs and clinical transformation.

[0038] Improve the breadth of research and clinical applications: The SS-PF mouse model of the present invention has high repeatability and stability, can be constructed on a large scale, and is suitable for a variety of research needs. This model is not only suitable for basic research, but can also be used for drug screening and efficacy evaluation, and has broad application prospects. Through this model, large-scale drug screening experiments can be carried out to evaluate the effectiveness and safety of drugs, provide new treatment options for SS-PF patients, and improve clinical treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 HE staining of mouse submandibular gland and scoring of lymphocyte infiltration foci. Magnification: 40 times. Arrows indicate lymphocyte infiltration foci.

[0040] Figure 2 HE staining of mouse lungs.

[0041] Figure 3 Masson staining of mouse lung.

[0042] Figure 4 Ashcroft score of mouse lung. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] See also Figure 1-4Example 1: Construction of a mouse model of Sjögren's syndrome combined with pulmonary fibrosis

[0045] Materials and Reagents

[0046] NOD / Ltj mice (8 weeks old)

[0047] Bleomycin (BLM)

[0048] Sterile saline

[0049] Anesthetic isoflurane

[0050] Surgical tools: syringes, tweezers, surgical scissors, etc.

[0051] step

[0052] Mouse breeding: 8-week-old NOD / Ltj mice that passed quarantine were purchased and raised to 12 weeks old under standard laboratory conditions including: temperature 22±2°C, relative humidity 50±10%, and a photoperiod of 12 h light / 12 h dark.

[0053] Preparation of bleomycin solution: Weigh 15 mg of bleomycin dry powder and dissolve it in 10 mL of sterile saline to prepare a solution with a concentration of 1.5 mg / mL. Place the solution on ice for later use and store the remaining portion at -20°C.

[0054] Mouse anesthesia: Use isoflurane for anesthesia, place the mouse's mouth and nose toward the gas anesthesia machine, adjust the anesthetic concentration to a high concentration, and after the mouse is fully anesthetized, fix it in a supine position on a mouse board, and adjust the anesthetic concentration to a low concentration to maintain the anesthesia state.

[0055] Bleomycin injection:

[0056] Weigh the mouse and record the body weight. Calculate the dosage of bleomycin according to the formula: required volume of bleomycin solution (μL) = mouse body weight (g)*2.

[0057] Use forceps to gently hold the mouse trachea with your left hand, hold a 1mL syringe in your right hand with the needle bevel facing upward, insert it at a 45-degree angle to the trachea, stop inserting the needle after it has entered about 1 / 4 of the length, and quickly inject the calculated volume of bleomycin solution.

[0058] After injection, the mouse was shaken upright from side to side to allow the bleomycin solution to be evenly distributed in both lungs.

[0059] Postoperative care: The mouse's neck incision was sutured and disinfected with alcohol. After the mouse woke up from anesthesia and its body dried and rewarmed, it was returned to the cage for feeding.

[0060] Model evaluation: The mice were treated on days 7, 14, and 21, and the submandibular glands and lungs of the mice were evaluated.

[0061] HE staining of the submandibular gland was used to evaluate the formation of lymphocyte infiltration foci in the submandibular gland of the model mice.

[0062] HE staining and Masson staining of mouse lungs were used to evaluate the degree of lung inflammation and fibrosis.

[0063] Example 2: Model repeatability and stability verification

[0064] In order to verify the repeatability and stability of the constructed model, another batch of 8-week-old NOD / Ltj mice was selected and the steps in Example 1 were repeated. The submandibular gland lymphocyte infiltration and pulmonary fibrosis of mice from different batches were compared after 21 days to ensure the repeatability and stability of the model.

[0065] Example 3: Effects of different doses of bleomycin on model construction

[0066] Materials and Reagents

[0067] Same as Example 1

[0068] Bleomycin solutions of different concentrations (1 mg / mL and 2 mg / mL)

[0069] step

[0070] Mice were raised according to the method of Example 1, and 1 mg / mL and 2 mg / mL bleomycin solutions were prepared.

[0071] Bleomycin was injected into the low-dose group (2 mg / kg), standard-dose group (3 mg / kg) and high-dose group (4 mg / kg).

[0072] The other steps are the same as in Example 1.

[0073] Result evaluation: Compare the pathological changes of submandibular gland and lung tissues of mice on the 21st day after injection of different doses of bleomycin, and evaluate the effects of different doses on model construction.

[0074] Example 4: Application of the model in drug screening

[0075] 12-week-old NOD / Ltj mice were selected and SS combined with PF mouse models were constructed according to the method of Example 1. The model mice were randomly divided into a drug treatment group and a control group. The drug treatment group was given the drug to be screened (such as anti-fibrosis drugs), and the control group was given an equal volume of normal saline.

[0076] Drug administration method: Administer the drug at regular intervals every day, via a gastric tube or intraperitoneal injection, for 21 consecutive days.

[0077] Efficacy evaluation:

[0078] Submandibular gland assessment: HE staining was used to assess lymphocyte infiltration.

[0079] Lung evaluation: HE staining and Masson staining were used to evaluate the degree of lung inflammation and fibrosis.

[0080] Physiological indicators: Detect physiological indicators such as weight changes and respiratory rate of mice.

[0081] The therapeutic effect of drugs on the SS combined with PF model was evaluated by comparing the pathological changes between the drug treatment group and the control group.

[0082] Example 5: Application of the model in pathological mechanism research

[0083] Twelve-week-old NOD / Ltj mice were selected and a mouse model of SS combined with PF was constructed according to the method in Example 1. The pathological mechanism of SS combined with PF was studied by the following methods:

[0084] Immunohistochemical staining: Detect the expression of fibroblast markers (such as α-SMA) in lung tissue.

[0085] Western blot analysis: Detect the expression levels of fibrosis-related proteins (such as collagen I and collagen III) in lung tissue.

[0086] RT-qPCR: Analysis of gene expression of inflammatory factors (such as TNF-α, IL-6) in lung tissue.

[0087] Through the above experimental methods, the pathological mechanism of SS combined with PF is deeply studied to provide a theoretical basis for disease treatment.

[0088] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0089] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis, characterized in that: The following steps are involved: S1: Purchase 8-week-old NOD / Ltj mice that have passed quarantine and raise them until 12 weeks old; S2: At 12 weeks, mice were injected with intratracheal bleomycin (BLM) at a dose of 3 mg / kg. The animal model of Sjögren's syndrome combined with pulmonary fibrosis was established 21 days after the injection.

2. The method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis according to claim 1, characterized in that: The preparation method of the bleomycin solution described in S2 is: Sterile physiological saline was used to dissolve the bleomycin dry powder. The concentration of bleomycin after dissolution was 1.5 mg / mL.

3. The method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis according to claim 2, characterized in that: The specific operation method in S2 is: The surface skin, subcutaneous tissue and muscles of the mouse neck trachea were cut open layer by layer to fully expose the trachea. The syringe needle was gently inserted into the trachea at a 45-degree angle at the position of the ring hose. When it entered about 1 / 4 of the length, the needle was stopped and the bleomycin solution was quickly injected at one time.

4. The method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis according to claim 3, characterized in that: The bleomycin injection method in S2 includes: After injection, the mouse was shaken upright from side to side to allow the bleomycin solution to enter both lungs and be evenly distributed in the lungs.

5. The method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis according to claim 1, characterized in that: The method for anesthetizing mice in S2 is: Use isoflurane for anesthesia, which includes placing the mouse's mouth and nose at the gas anesthesia machine, adjusting the anesthetic concentration to a high concentration, and performing the operation after the mouse is fully anesthetized.

6. The method for constructing an animal model of Sjögren's syndrome combined with pulmonary fibrosis according to claim 1, characterized in that: The housing conditions of mice described in S1 were: They were maintained under standard laboratory conditions, including appropriate temperature, humidity, and photoperiod.

7. A mouse model of Sjögren's syndrome combined with pulmonary fibrosis, characterized in that: The model is constructed by: Purchase 8-week-old NOD / Ltj mice that have passed quarantine and raise them until they are 12 weeks old; At 12 weeks, mice were injected intratracheally with 3 mg / kg bleomycin, 21 days after injection.

8. The mouse model of Sjögren's syndrome combined with pulmonary fibrosis according to claim 7, characterized in that: The model exhibits the following characteristics: There were numerous foci of mononuclear cell infiltration in the submandibular gland, consistent with the features of Sjögren's syndrome; Inflammatory infiltration and collagen fiber deposition in lung tissue are consistent with the characteristics of pulmonary fibrosis.

9. A mouse model of Sjögren's syndrome combined with pulmonary fibrosis according to claim 8, characterized in that: This model can be used for the following purposes: Research on pathological mechanisms; Drug screening and efficacy evaluation.

10. An application of an animal model of Sjögren's syndrome combined with pulmonary fibrosis, characterized in that: The model is used to study the pathophysiological mechanism and treatment methods of Sjögren's syndrome combined with pulmonary fibrosis.

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