Pulmonary fibrosis marker and application thereof

Abstract

The invention relates to a pulmonary fibrosis marker and application thereof. The pulmonary fibrosis marker comprises a discoid structural domain receptor 2. The invention finds that the content of the discoid structural domain receptor 2 is related to the occurrence of the pulmonary fibrosis, and has the characteristics of high detection accuracy, convenience, rapidness, safety and noninvasive property when being used for diagnosis and disease evaluation of the pulmonary fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a pulmonary fibrosis marker and application thereof. Background Art

[0002] According to statistics, 45% of deaths from human diseases are related to organ fibrosis, with pulmonary fibrosis being a serious threat to patients' lives. To date, clinical trials for human fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), have been disappointing, with no drug proven to reverse fibrosis. Furthermore, current clinical diagnostic guidelines define a relatively late treatment window, and interventions for fibrosis that has already caused structural distortion and deformation are unlikely to be effective. Therefore, accurately identifying the nodes of localized fibrosis disorders and initiating interventions before significant morphological changes occur is key to the current treatment of fibrosis. Based on the principle of early diagnosis, identifying specific biomarkers is the best option to overcome this dilemma.

[0003] Interstitial lung disease (ILD) is a general term for a clinical-pathological entity consisting of a group of different diseases with diffuse lung parenchyma, alveolar inflammation and interstitial fibrosis as basic pathological lesions, and clinical manifestations such as active dyspnea, diffuse infiltration shadows on chest X-rays, restrictive ventilation disorders, decreased diffusion capacity and hypoxemia. Currently, according to clinical guidelines, the standard for diagnosing interstitial lung disease is chest HRCT (high resolution computer tomography) combined with surgical lung biopsy (SLB) or transbronchial lung cryobiopsy (TBLC). However, the above methods have the following problems: (1) Lung biopsy is an invasive examination and may cause patients to develop pneumothorax, bleeding, and infection; (2) Lung biopsy has contraindications, such as severe patients with severe organ dysfunction and coagulation disorders, which limits its application; (3) The chest HRCT lesions of patients with interstitial lung disease are diverse and sometimes difficult to distinguish from infection and pulmonary edema, requiring experienced radiologists. Secondly, lung biopsy has high requirements for surgeons and pathologists, and is difficult to perform in primary hospitals; (4) Interstitial lung disease lesions are diffuse and heterogeneous, and the pathological results of different lesions are different, making it difficult to comprehensively judge the patient's disease status; (5) The cost is high.

[0004] Discoidin domain receptor (DDR) is a typical single-pass transmembrane receptor tyrosine kinase (RTK). Unlike the "fast on, fast off" activation pattern of cytokine receptor RTKs, tyrosine phosphorylation of discoidin domain receptor 1 (DDR1) and discoidin domain receptor 2 (DDR2) requires several hours after ligand macromolecular collagen binding to be induced and can persist for dozens of hours, presenting a unique "slow on, slow off" pattern. This activation feature may be the molecular biological basis for DDRs to mediate the persistent biological effects of chronic fibrotic diseases.

[0005] Exosomes carry nucleic acids, proteins, lipids and metabolites, and are the mediators of short-range and long-range intercellular communication in health and disease. They are closely related to immune responses, viral pathogenicity, pregnancy, cardiovascular disease, central nervous system-related diseases and cancer progression. They are currently a research hotspot for non-invasive "liquid biopsy" methods to diagnose and even monitor patients' disease status and response to treatment. The proteins, metabolites and nucleic acids delivered to recipient cells may change their biological responses, thereby promoting or inhibiting disease progression.

[0006] Therefore, the development of non-invasive, highly sensitive and specific examination methods that can be used to diagnose interstitial lung disease and the progression of pulmonary fibrosis has become an urgent problem to be solved. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a pulmonary fibrosis marker and its application. The pulmonary fibrosis marker includes discoidin domain receptor 2. The present invention found that the content of discoidin domain receptor 2 is related to the occurrence of pulmonary fibrosis. It is used for the diagnosis and disease assessment of pulmonary fibrosis, and has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a pulmonary fibrosis marker, wherein the pulmonary fibrosis marker comprises discoidin domain receptor 2.

[0010] In the present invention, it is creatively discovered that the content of discoidin domain receptor 2 is related to the occurrence of pulmonary fibrosis. After experimental testing, the content of discoidin domain receptor 2 in patients with pulmonary fibrosis is significantly increased. It can not only be used for the diagnosis and disease assessment of pulmonary fibrosis, but also for scientific research, such as for exploring the pathogenesis of pulmonary fibrosis, or for studying drugs for treating or preventing pulmonary fibrosis, etc.

[0011] Preferably, the source of the discoidin receptor 2 includes exosomes.

[0012] In the present invention, experimental detection showed that the content of discoidin domain receptor 2 in the exosomes of patients with pulmonary fibrosis was significantly increased, and exosomes were widely distributed in various body fluids. The acquisition of exosomes is relatively simple. Compared with lung biopsy, the operation is simple, non-invasive and has little damage, and can be widely used in various usage scenarios.

[0013] Preferably, the source of the exosomes includes any one of blood, saliva, urine, cerebrospinal fluid or milk, or a combination of at least two thereof.

[0014] Preferably, the exosomes are derived from human.

[0015] In a second aspect, the present invention provides a use of the pulmonary fibrosis marker according to the first aspect in the preparation of a product for detecting pulmonary fibrosis.

[0016] Preferably, the product comprises any one of a kit, a chip, an analysis device or an analysis model, or a combination of at least two of them.

[0017] In a third aspect, the present invention provides a kit for detecting pulmonary fibrosis, wherein the kit comprises reagents and / or devices for detecting the content of the pulmonary fibrosis marker described in the first aspect.

[0018] In a fourth aspect, the present invention provides an analytical model for predicting human pulmonary fibrosis, wherein the input variable of the model is the absorbance value of the pulmonary fibrosis marker described in the first aspect, and the output variable is the difference multiple.

[0019] Preferably, the calculation formula of the analysis model is:

[0020]

[0021] The present invention constructs a difference multiple, and according to the above formula, the risk of human pulmonary fibrosis can be predicted and play a warning role.

[0022] Preferably, the absorbance value of the marker in the normal sample is 0.251-0.5601. The 0.251-0.5601 can be, for example, 0.251, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56 or 0.0.5601.

[0023] Preferably, the judgment standard of the difference multiple is: when the difference multiple is greater than 1, it is judged that there is a risk of pulmonary fibrosis.

[0024] In the present invention, if the absorbance value is greater than the absorbance value range of a normal sample defined in the present invention, it can be determined that the sample has a risk of developing pulmonary fibrosis.

[0025] In a fifth aspect, the present invention provides a device for detecting pulmonary fibrosis, wherein the device comprises an analyzing unit and an evaluating unit.

[0026] Preferably, the analysis unit is used to perform the following steps: detecting the content of the pulmonary fibrosis marker described in the first aspect in the sample to be tested.

[0027] The evaluation unit is used to perform the following steps: inputting the content detected by the analysis unit into the human pulmonary fibrosis prediction analysis model described in the fourth aspect for computational analysis.

[0028] In a sixth aspect, the present invention provides a computer-readable storage medium having a computer program or instruction stored thereon, which, when executed by a processor, implements the functions of the device for detecting pulmonary fibrosis described in the fifth aspect.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] The present invention discovered for the first time that the content of plasma exosome DDR2 is related to the occurrence of pulmonary fibrosis. The content of plasma exosome DDR2 in patients with pulmonary fibrosis is significantly higher than that in healthy controls. The content of DDR2 in plasma exosomes of bleomycin model monkeys is related to the disease progression. The content of plasma exosome DDR2 in patients with pulmonary fibrosis is related to the disease progression. It is used for the diagnosis and disease assessment of pulmonary fibrosis, and has the characteristics of high detection accuracy, convenience, speed, safety and non-invasiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the test results of the monkey model of unilateral bleomycin-induced pulmonary fibrosis. Figure a shows the test results of positron emission tomography-CT-fibroblast activation protein inhibitor imaging (PET-CT-FAPI) and HRCT, and Figure b shows the results of protein blotting.

[0032] Figure 2 Figure 2 shows the results of Western blot detection of plasma exosomes from patients with interstitial lung disease and healthy subjects.

[0033] Figure 3 This is a bar graph showing the expression levels of DDR2 in plasma exosomes from patients with interstitial lung disease and healthy controls.

[0034] Figure 4ROC curve of DDR2 expression in plasma exosomes of patients with interstitial lung disease

[0035] Figure 5 Figure 2 shows the results of Western blot detection of plasma exosomes in patients with different disease states.

[0036] Figure 6 Figure 2 shows the detection results of high-resolution computed tomography (HRCT) in patients with different disease states.

[0037] Figure 7 The results of Western blot detection of plasma exosomes from another batch of clinical samples for further diagnosis of interstitial disease patients.

[0038] Figure 8 This is a bar chart showing the expression levels of DDR2 in plasma exosomes from another batch of clinical samples. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0040] Sources of reagents and equipment used in the following examples:

[0041] Huixin "S" type chip: Shenzhen Huixin Biomedical Technology Co., Ltd.;

[0042] Tim 4 magnetic beads: Fujifilm Wako Trading Co., Ltd.

[0043] Example 1

[0044] This example provides a method for detecting the content of discoidin receptor 2 in exosomes

[0045] (1) Extraction of exosomes

[0046] 1.5 mL of venous whole blood was added to an EDTA anticoagulant tube and centrifuged at 3000 rpm for 10 minutes. The upper plasma layer was collected. 5 mL of PBS was added to 200 μL of plasma for dilution, and plasma exosomes were extracted using a Huixin "S" chip. 400 μL of PBS was used to recover the exosomes from the "S" chip for subsequent experiments. 10 μL of exosomes was mixed with 2.5 μL of 5× loading buffer, boiled at 100°C for 10 minutes to denature the protein, and then analyzed by Western blotting (WB).

[0047] (2) Magnetic beads capture plasma exosomes and perform ELISA detection

[0048] Thoroughly vortex Tim 4 magnetic beads to mix them evenly, with 30 μL of magnetic beads per tube. Add 300 μL of washing solution to each tube to wash the magnetic beads, centrifuge at 7000 rpm for 30 seconds, and then continue magnetic aspiration to remove the supernatant and wash the magnetic beads three times. Mix 10 μL of sample with magnetic beads and add PBS to 100 μL. Incubate at room temperature for 1 hour. Vortex every 20 minutes during incubation to mix thoroughly. After magnetic aspiration, remove the supernatant and wash the magnetic beads three times with washing solution. Add 100 μL of blocking solution to each tube and block the magnetic beads at room temperature for 1 hour. Vortex every 20 minutes to mix thoroughly. After magnetic aspiration, remove the supernatant and wash the magnetic beads three times with washing solution.

[0049] Add antibodies to each tube. Different antibodies are added according to the specific experiment. 1 μg of each antibody is added to a total volume of 100 μL and incubated at room temperature for 1 hour. Vortex every 20 minutes to mix it thoroughly. After magnetic absorption, remove the supernatant and use the cleaning solution to wash the magnetic beads 3 times. Dilute the secondary antibody 1:5000, incubate at room temperature for 1 hour in a total volume of 100 μL. Vortex every 20 minutes to mix it thoroughly. After magnetic absorption, remove the supernatant and use the cleaning solution to wash the magnetic beads 3 times. Add 100 μL of TMB to each tube. After the color change occurs, use 50 μL of H2SO4 to terminate the experiment and detect the OD in the enzyme reader. 450 The numerical value of .

[0050] Example 2

[0051] This example uses animal model verification

[0052] The animal model used was a monkey model of unilateral bleomycin-induced pulmonary fibrosis, and the specificity of peripheral blood plasma exosome DDR2 content as a diagnostic biomarker for identifying disease progression was verified in this animal model. A monkey model of left-sided bleomycin-induced pulmonary fibrosis was constructed and subjected to positron emission tomography-CT-fibroblast activation protein inhibitor imaging (PET-CT-FAPI). Venous blood was obtained from the model and the plasma exosome DDR2 content was measured and analyzed using the method in Example 1.

[0053] A bleomycin pulmonary fibrosis model was established in the left lung of a monkey using fiberoptic bronchoscopy. First, general anesthesia was administered with a dose of anesthetic drugs selected according to body weight, and vital signs were monitored. The monkey was then placed in an appropriate position to ensure airway patency. Local anesthetics were then used to anesthetize the throat, and a fiberoptic bronchoscope was slowly inserted through the mouth into the left lung. A sterile syringe was used to draw an appropriate amount of bleomycin solution and inject it into the left lung, and the reaction was monitored. Finally, after the injection was completed, the fiberoptic bronchoscope was withdrawn, and the monkey was placed in a recovery room to wait for the anesthetic drugs to be metabolized and vital signs to be continuously monitored. Supportive treatment was given if necessary, and positron emission tomography-CT-fibroblast activation protein inhibitor imaging (PET-CT-FAPI) imaging and peripheral blood collection were performed on the 4th and 54th days after bleomycin modeling, respectively.

[0054] Positron emission tomography-CT-fibroblast activation protein inhibitor imaging (PET-CT-FAPI) results are as follows Figure 1 As shown in Figure a, on the 4th day, obvious radioactive accumulation foci were seen in the inner band of the left upper lung, and corresponding high-resolution computed tomography (HRCT) showed consolidation shadows, confirming the successful bleomycin modeling. On the 54th day, no obvious radioactive accumulation foci were seen in the whole lung, and the corresponding HRCT showed a reduction in the volume of the left upper lung and consolidation damage. It is speculated that there were no active fibroblasts on the 54th day, and the patient had entered the late stage of pulmonary fibrosis.

[0055] The method of Example 1 was used to detect DDR2, tumor suppressor gene 101 (TSG101), and cell differentiation antigen (CD81) in the plasma exosomes of monkeys with unilateral bleomycin-induced pulmonary fibrosis. The WB results are as follows Figure 1 As shown in Figure b, magnetic beads were used to capture plasma exosomes and detect OD 450 We found that the expression of DDR2 in exosomes first increased and then decreased. The content of DDR2 in exosomes on the 4th day was significantly higher than that on the 0th day, indicating that the content of DDR2 in plasma exosomes increased in the state of pulmonary fibrosis, and the decrease in the content of DDR2 in plasma exosomes from the 4th day to the 54th day also corresponded to the late stage of the disease. The decrease in DDR2 content was consistent with the results of PET-CT-FAPI, indicating that the content of DDR2 in plasma is closely related to the disease status.

[0056] Table 1

[0057] sample Absorbance Day 0 Sample 0.0878 Day 4 Sample 0.8391 Day 54 sample 0.2410 Magnetic beads (background) 0.0698

[0058] Example 3

[0059] This example uses patient blood for verification

[0060] Peripheral blood plasma was collected from 7 clinical interstitial lung disease patients and named (ILD1, ILD2, ILD3, ILD4, ILD5, ILD6 and ILD7). The DDR2 content in plasma exosomes was detected by the method of Example 1 and compared with the content of DDR2 in plasma exosomes of 3 healthy controls (WYN, LQX and ZZY). The interstitial lung disease patients in this embodiment include 2 types of interstitial lung diseases, namely idiopathic pulmonary fibrosis (IPF) unrelated to rheumatic immunity, and interstitial pneumonia (IPAF) with autoimmune characteristics related to rheumatic immunity. The specific plasma volume used for the diagnosis and extraction of exosomes from each patient and the total volume of exosomes collected are shown in Table 2.

[0061] Table 2

[0062]

[0063]

[0064] The method of Example 1 was used to detect DDR2, cell differentiation antigen 9 (CD9), tumor suppressor gene 101 (TSG101), cell differentiation antigen (CD81) and heat shock protein (HSP70) in plasma exosomes. Figure 2 As shown in Table 3 and Table 4, the WB results of plasma exosomes showed that the expression of plasma DDR2 in patients with ILD2, ILD5, and ILD6 was significantly higher than that in the control group. Figure 3 As shown in Figure 2, plasma exosomes were captured and it was found that the expression level of DDR2 in plasma exosomes of patients with interstitial lung disease was significantly higher than that of the control group. The ROC curve was drawn, as shown in Figure 2. Figure 4 The area under the curve was 100% (p=0.017), and the CUT-OFF value was 0.6610 (sensitivity 100%, specificity 100%), which can be used to diagnose interstitial lung disease.

[0065] Table 3

[0066]

[0067]

[0068] Example 4

[0069] This example detects the DDR2 content in plasma exosomes of patients with different disease states.

[0070] In this example, peripheral blood plasma was collected from four patients with interstitial lung disease in the stable phase and the progressive fibrosis phase (progressive phase), and the experiment was conducted together with two healthy controls. DDR2, lysosomal associated membrane protein 3 (CD63), programmed cell death 6 interacting protein (ALIX), and cell differentiation antigen 81 (CD81) in plasma exosomes were detected according to the method of Example 1. The basic information of each patient is shown in Table 4. Progressive pulmonary fibrosis (PPF) is defined as patients with ILD other than IPF of known or unknown etiology with radiological evidence of pulmonary fibrosis, who meet at least two of the following three criteria within the past year: (1) worsening respiratory symptoms; (2) physiological evidence of disease progression (a. absolute decrease in forced vital capacity (FVC) ≥5% within 1 year of follow-up; b. diffusion capacity for carbon monoxide (DL) within 1 year of follow-up). CO absolute decrease ≥10%); (3) radiographic evidence of one or more disease progressions.

[0071] Table 4

[0072]

[0073]

[0074] The results are as follows Figure 5 and Figure 6 As shown, the content of DDR2 in plasma exosomes of patients 1, 2, and 4 increased significantly when they developed PPF.

[0075] Example 5

[0076] This example uses the fold difference test to determine the accuracy of diagnosing patients with interstitial diseases.

[0077] Peripheral blood plasma was collected from 9 patients with clinical interstitial lung disease and named (ILD1, ILD2, ILD3, ILD4, ILD5, ILD6, ILD7, ILD8 and ILD9). The DDR2 content in plasma exosomes was detected by the method of Example 1 and compared with the content of DDR2 in plasma exosomes of 2 healthy controls (Control 1 and Control 2). The interstitial lung disease patients in this example include 2 types of interstitial lung diseases, namely idiopathic pulmonary fibrosis (IPF) unrelated to rheumatic immunity and interstitial pneumonia (IPAF) with autoimmune characteristics related to rheumatic immunity. The specific plasma volume used for the diagnosis and extraction of exosomes from each patient and the total volume of exosomes collected are shown in Table 5.

[0078] Table 5

[0079]

[0080]

[0081] The method of Example 1 was used to detect DDR2, tumor suppressor gene 101 (TSG101), programmed cell death 6 interacting protein (ALIX), and cell differentiation antigen 81 (CD81) in plasma exosomes. Figure 7 As shown in Table 6, the WB results of plasma exosomes showed that the expression levels of plasma DDR2 in the 9 patients with ILD1 to ILD9 were significantly higher than those in the control group. Figure 7 、 Figure 8 As shown, plasma exosomes were captured and the expression of DDR2 in plasma exosomes from patients with interstitial lung disease was found to be significantly higher than that in controls. Both the CUT-OFF value and the multiple difference were used to diagnose interstitial lung disease in all nine patients. The multiple difference was calculated based on the average of the maximum and minimum absorbance values of the marker in the normal sample. The results indicate that the test accuracy of this application is high, and a value greater than the absorbance value of the marker in the normal sample indicates that the sample is at risk of pulmonary fibrosis.

[0082] Table 6

[0083]

[0084]

[0085] In summary, the present invention discovered for the first time that the content of DDR2 in plasma exosomes is associated with pulmonary fibrosis. The content of DDR2 in plasma exosomes of animal models of pulmonary fibrosis and patients with interstitial lung disease is significantly different from that of healthy controls. Its expression is closely related to the disease progression. It is used to diagnose interstitial lung disease (pulmonary fibrosis) and identify disease progression, with high detection accuracy, convenience, speed, safety and non-invasiveness.

[0086] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A pulmonary fibrosis marker, characterized in that: The pulmonary fibrosis markers include discoidin domain receptor 2.

2. The pulmonary fibrosis marker according to claim 1, characterized in that The source of the discoidin domain receptor 2 includes exosomes.

3. The pulmonary fibrosis marker according to claim 2, characterized in that The source of the exosomes includes any one of blood, saliva, urine, cerebrospinal fluid or milk, or a combination of at least two thereof; The sources of the exosomes include human.

4. Use of the pulmonary fibrosis marker and / or detection reagent thereof according to any one of claims 1 to 3 in the preparation of a product for detecting pulmonary fibrosis.

5. The use according to claim 4, characterized in that The product includes any one of a kit, a chip, an analysis device or an analysis model, or a combination of at least two of them.

6. A kit for detecting pulmonary fibrosis, characterized in that: The kit comprises reagents and / or devices for detecting the content of the pulmonary fibrosis marker according to any one of claims 1 to 3.

7. A prediction model for human pulmonary fibrosis, characterized in that: The input variable of the model is the absorbance value of the pulmonary fibrosis marker according to any one of claims 1 to 3, and the output variable is the difference multiple; The calculation formula of the difference multiple is:

8. The human pulmonary fibrosis prediction analysis model according to claim 7, characterized in that: The absorbance value of the marker in the normal sample is 0.251-0.5601; Preferably, the judgment standard of the difference multiple is: when the difference multiple is greater than 1, it is judged that there is a risk of pulmonary fibrosis.

9. A device for detecting pulmonary fibrosis, characterized in that: The device comprises an analysis unit and an evaluation unit; The analysis unit is used to perform the following operations: detecting the content of the pulmonary fibrosis marker according to any one of claims 1 to 3 in the sample to be tested; The evaluation unit is used to perform the following steps: inputting the content detected by the analysis unit into the human pulmonary fibrosis prediction analysis model according to claim 7 or 8 for calculation and analysis.

10. A computer-readable storage medium, characterized in that The computer-readable storage medium stores a computer program or instructions, which, when executed by a processor, implement the functions of the device for detecting pulmonary fibrosis according to claim 9.

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