Idiopathic pulmonary fibrosis plasma protein markers and their use in preparing detection reagents or diagnostic tools

Abstract

The present invention discloses a plasma protein marker for idiopathic pulmonary fibrosis and its application in the preparation of detection reagents or diagnostic tools, wherein the plasma protein markers are serine protease inhibitor family G1, kininogen 1, fibrin 3 and transthyretin, and also discloses the application of the plasma protein marker for idiopathic pulmonary fibrosis in the preparation of detection reagents or diagnostic tools. The results show that the area under the receiver operating characteristic curve (ROC) (AUC) values ​​of the four protein combinations for predicting pulmonary fibrosis are 0.799 and 0.848 (validation data set), and there are significant differences in the expression levels of the four proteins in normal people and patients with pulmonary fibrosis, and the coefficients of variation are all less than 0.5, which further confirms the robustness and accuracy of the four protein combinations in predicting idiopathic pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical diagnostic markers and tools for pulmonary fibrosis, and specifically relates to a plasma protein marker for idiopathic pulmonary fibrosis and its application in preparing detection reagents or diagnostic tools. Background Art

[0002] Pulmonary fibrosis is a terminal change of a large class of lung diseases characterized by fibroblast proliferation and accumulation of a large amount of extracellular matrix accompanied by inflammatory damage and tissue structure destruction. That is, the normal alveolar tissue is damaged and then repaired abnormally, resulting in structural abnormalities (scar formation). Signs and symptoms of pulmonary fibrosis include: shortness of breath (dyspnea), dry cough, fatigue, unexplained weight loss, muscle and joint pain, and widening and rounding of the tips of fingers or toes (clubbing). The progression of pulmonary fibrosis and the severity of symptoms vary from person to person. Some people quickly develop severe disease, others have moderate symptoms and worsen more slowly over months or years, and some people may have symptoms that worsen rapidly (acute exacerbations), such as severe shortness of breath, which may last for days to weeks. Currently, there are no biomarkers for the occurrence and outcome of idiopathic pulmonary fibrosis. Summary of the invention

[0003] The technical problem solved by the present invention is to provide a plasma protein marker for idiopathic pulmonary fibrosis and its application in the preparation of detection reagents or diagnostic tools. Studies have found that the protein concentrations of Serpin Family G Member 1 (gene name SERPING1), Kininogen 1 (gene name KNG1), Ficolin 3 (gene name FCN3), and Transthyretin (gene name TTR) in the plasma of normal people and patients with idiopathic pulmonary fibrosis are significantly different, and the coefficients of variation are all lower than 0.5, which further confirms the accuracy and robustness of a combination of multiple proteins in predicting idiopathic pulmonary fibrosis.

[0004] In order to solve the above technical problems, the present invention adopts the following technical scheme, which is an idiopathic pulmonary fibrosis plasma protein marker, characterized in that the idiopathic pulmonary fibrosis plasma protein marker is one or more of serine protease inhibitor family G1, kininogen 1, fibrin 3 or thyroxine transporter protein, preferably a combination of serine protease inhibitor family G1, kininogen 1, fibrin 3 and thyroxine transporter protein.

[0005] The use of an antibody that specifically binds to a protein in the preparation of a detection reagent or diagnostic tool for idiopathic pulmonary fibrosis is characterized in that the protein is one or more of serine protease inhibitor family G1, kininogen 1, fibrin 3 or thyroxine transporter protein, preferably a combination of serine protease inhibitor family G1, kininogen 1, fibrin 3 and thyroxine transporter protein.

[0006] It is further defined that the antibody is one or more of anti-SERPING1, anti-KNG1, anti-FCN3 or anti-TTR.

[0007] The idiopathic pulmonary fibrosis plasma protein marker is characterized in that the idiopathic pulmonary fibrosis plasma protein marker is the expression product of the following genes, wherein the genes are one or more of SERPING1, KNG1, FCN3 or TTR, preferably a combination of SERPING1, KNG1, FCN3 and TTR.

[0008] The use of a preparation for inhibiting gene expression in the preparation of a detection reagent or diagnostic tool for idiopathic pulmonary fibrosis is characterized in that the gene is one or more of SERPING1, KNG1, FCN3 or TTR, preferably a combination of SERPING1, KNG1, FCN3 and TTR.

[0009] The use of a preparation for inhibiting gene expression in the preparation of a detection reagent or diagnostic tool for detecting the transition from mild to severe cases of novel coronavirus disease, characterized in that the gene is one or more of SERPING1, KNG1, FCN3 or TTR, preferably a combination of SERPING1, KNG1, FCN3 and TTR.

[0010] It is further defined that the agent is one or more of an inhibitor of SERPING1 gene expression, an inhibitor of KNG1 gene expression, an inhibitor of FCN3 gene expression, or an inhibitor of TTR gene expression.

[0011] It is further defined that the diagnostic tool is a chip, a test strip or a test kit.

[0012] An idiopathic pulmonary fibrosis disease detection reagent is characterized by comprising antibodies that specifically bind to proteins and / or preparations that inhibit gene expression, wherein the proteins are a combination of serine protease inhibitor family G1, kininogen 1, fibrin 3 and thyroxine transporter protein, and the genes are a combination of SERPING1, KNG1, FCN3 and TTR.

[0013] A diagnostic tool for idiopathic pulmonary fibrosis, characterized in that it includes antibodies that specifically bind to proteins and / or preparations that inhibit gene expression, wherein the proteins are a combination of serine protease inhibitor family G1, kininogen 1, fibrin 3 and thyroxine transporter protein, and the genes are a combination of SERPING1, KNG1, FCN3 and TTR.

[0014] Changes in human plasma proteins represent indicators of pathophysiological changes under disease conditions caused by various reasons. Combining proteomics with machine learning technology can analyze the differences in plasma protein expression between normal people and patients with idiopathic pulmonary fibrosis, establish a complete protein database, analyze and compare the changes in proteome expression of the disease, and discover specific proteins related to the disease process as potential biomarkers for the diagnosis of idiopathic pulmonary fibrosis.

[0015] The present invention provides a group of plasma protein markers for diagnosing idiopathic pulmonary fibrosis, including Serpin Family G Member 1 (SERPING1), Kininogen 1 (KNG1), Ficolin 3 (FCN3) and Transthyretin (TTR). The results showed that the area under the receiver operating characteristic curve (ROC) of the combination of these four proteins for predicting idiopathic pulmonary fibrosis was 0.799 (cohort 1) and 0.848 (cohort 2). There were significant differences in the expression levels of the four proteins in normal people and patients with pulmonary fibrosis, and the coefficients of variation were all less than 0.5, which further confirmed the robustness and accuracy of multiple protein combinations in predicting idiopathic pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To verify the plasma protein profile results of the normal group and pulmonary fibrosis group in the dataset;

[0017] Figure 2 The plasma protein spectrum results of four proteins: SERPING1, KNG1, FCN3, and TTR;

[0018] Figure 3 is the confusion matrix;

[0019] Figure 4 For ROC curve analysis;

[0020] Figure 5 To verify the plasma protein profile results of the normal group and pulmonary fibrosis group in the dataset;

[0021] Figure 6The plasma protein spectrum results of four proteins: SERPING1, KNG1, FCN3, and TTR;

[0022] Figure 7 is the confusion matrix;

[0023] Figure 8 This is the ROC curve analysis. DETAILED DESCRIPTION

[0024] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0025] The raw materials and reagents used in the idiopathic pulmonary fibrosis plasma protein markers and idiopathic pulmonary fibrosis detection reagents or diagnostic tools provided by the present invention can all be purchased from the market.

[0026] The present invention will be further described below in conjunction with embodiments:

[0027] Example 1

[0028] Mass spectrometry analysis of plasma samples from normal people and patients with pulmonary fibrosis

[0029] 1. Experimental Materials

[0030] Plasma samples from patients with pulmonary fibrosis were obtained from a hospital, including whole blood collected from 30 normal people and 30 patients with pulmonary fibrosis, totaling 60 whole blood samples. According to the 2018 version of the clinical practical guidelines for the diagnosis of IPF developed by the American Thoracic Society (ATS), the European Respiratory Society (ERS), the Japanese Respiratory Society (JRS), and the Association of Latin American Thorax (ALAT), diagnostic measures are mainly implemented from two aspects: chest high-resolution CT and lung biopsy histopathological examination: (1) Chest CT: High-resolution CT features of patients with pulmonary fibrosis include honeycombing, stretching bronchi or bronchiolar dilatation (mainly appearing in ground-glass shadows and fine grid shadows). In acute exacerbation, high-resolution CT shows ground-glass shadows or consolidation shadows in both lungs on the basis of fibrosis. (2) Pathological phenotype of lung tissue biopsy: Patients with pulmonary fibrosis specifically present with unevenly distributed, dense fibrotic lesions under low-power microscopy, and the surrounding lung tissue structure may be relatively normal, accompanied by lung structural remodeling and honeycomb formation. The histopathological manifestations of patients with acute exacerbation are the above combined with diffuse alveolar damage, which may be accompanied by hyaline membrane formation.

[0031] 2. Experimental Procedure

[0032] (1) The following experimental processes (1) to (3) are all carried out in a biosafety laboratory.

[0033] (2) Incubate the whole blood sample in a 37°C water bath for 1 h until the blood cells are completely coagulated.

[0034] (3) Centrifuge the sample at 4°C, 3000 rpm for 15 min, and aspirate the upper plasma. Place the plasma in a 56°C water bath for 60 min to completely inactivate it, and then thoroughly disinfect it for later use.

[0035] (4) Commission a platform to label each group of plasma proteins and conduct relative quantitative mass spectrometry analysis. The results are as follows: Figure 1 shown.

[0036] Example 2

[0037] Machine learning to screen for pulmonary fibrosis markers

[0038] 1. Experimental Materials

[0039] Mass spectrometry data of 30 plasma samples (see Example 1)

[0040] 2. Experimental Procedure

[0041] (1) Screening of differentially expressed proteins: The absolute value of the fold change (FC) between the control group and the IPF group was greater than 1.5 (|log2FC|>1.5), and the difference was statistically significant less than 0.05 (P<0.05).

[0042] (2) No more than four proteins were randomly selected from the differentially expressed proteins to form a potential optimal marker combination (OBC). The initial weight value of each protein was set to 1, and 1,000 OBC candidates were set.

[0043] (3) For each candidate OBC, we randomly divided the original dataset into a training dataset and a test dataset with a ratio of 4:1. The test dataset was only used to evaluate the performance and was not used for training. The machine learning algorithm penalized logistic regression (PLR) was used to iteratively optimize the weight values ​​of the selected proteins using the least absolute shrinkage and selection operator penalty and ridge regression (L2 regularization) penalty.

[0044] (4) Through comparison, we found that the combination of these four proteins: SerpinFamily G Member 1 (SERPING1), Kininogen 1 (KNG1), Ficolin 3 (FCN3), and Transthyretin (TTR) as OBC can obtain better prediction results. The expression levels of the four proteins in the mass spectrometry data are shown in Figure 2. Figure 2 As shown. Using mass spectrometry data, the number of true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN) of each protein combination in the identified OBC to distinguish between normal people and patients with pulmonary fibrosis was calculated. The confusion matrix of TP, TN, FP, and FN is shown in Figure 3 shown.

[0045] (5) A 5-fold cross validation was performed, and the OBC receiver operating characteristic (ROC) curve was plotted based on the Sn and 1-Sp scores, and the area under the ROC curve (AUC) value was calculated. When the area under the ROC curve AUC is greater than 0.5, the closer it is to 1, the better the effect. The results are shown in Figure 4 As shown, the AUC value of the OBC is close to 0.8, which has a high accuracy, indicating that the combination has excellent performance in distinguishing non-fibrosis patients from fibrosis patients.

[0046] Example 3

[0047] Verifying OBC prediction accuracy

[0048] (1) A pulmonary fibrosis plasma mass spectrometry dataset was obtained from a public database, including 19 normal subjects and 17 patients with pulmonary fibrosis.

[0049] (2) Plasma mass spectrometry results such as Figure 5 shown.

[0050] (3) The expression levels of the four proteins in the OBC group: Serpin Family GMember 1 (gene name SERPING1), Kininogen 1 (gene name KNG1), Ficolin 3 (gene name FCN3), and Transthyretin (gene name TTR) in the mass spectrometry data are as follows: Figure 6 shown.

[0051] (4) The logistic regression model constructed using the above four proteins SERPING1, KNG1, FCN3, and TTR was validated on the dataset to obtain its confusion matrix and receiver operating characteristic (ROC) curve, and the area under the ROC line (AUC) value was calculated. The results are as follows: Figure 7 , Figure 8 shown.

[0052] (5) The AUC of the OBC combination in the validation data set can also reach above 0.84, which also has a high accuracy, and further confirms the accuracy of the OBC combination in distinguishing non-fibrotic people from patients with pulmonary fibrosis.

[0053] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. Idiopathic pulmonary fibrosis plasma protein marker, characterized by: The plasma protein markers of idiopathic pulmonary fibrosis are serine protease inhibitor family G1, kininogen 1, fibrin 3 and transthyretin.

2. Use of an antibody that specifically binds to a protein in the preparation of a detection reagent or diagnostic tool for idiopathic pulmonary fibrosis, characterized in that: The proteins are serine protease inhibitor family G1, kininogen 1, ficolin 3 and transthyretin.

3. The use according to claim 2, characterized in that: The antibodies are anti-SERPING1, anti-KNG1, anti-FCN3 and anti-TTR.

4. The use according to claim 2, characterized in that: The diagnostic tool is a chip, a test strip or a test kit.

5. Idiopathic pulmonary fibrosis plasma protein marker, characterized in that: The idiopathic pulmonary fibrosis plasma protein marker is the expression product of the following genes, wherein the genes are SERPING1, KNG1, FCN3 and TTR.

6. Idiopathic pulmonary fibrosis disease diagnostic tool, characterized in that Included are antibodies that specifically bind to a protein that is a combination of serpin family G1, kininogen 1, ficolin 3, and transthyretin.

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