Biomarker for diagnosing pulmonary fibrosis and application of biomarker

Abstract

The invention discloses a biomarker for diagnosing pulmonary fibrosis and application of the biomarker, and belongs to the technical field of biomarkers in clinical medicine. The biological marker for preparing and diagnosing pulmonary fibrosis is a nerve growth factor NGF, the nerve growth factor NGF can remarkably promote differentiation of lung fibroblast MRC5 cells, and along with expression increase of a receptor TrkA of the lung fibroblast MRC5 cells, the migration capacity and the multiplication capacity of the lung fibroblast MRC5 cells are remarkably enhanced. Meanwhile, the NGF also aggravates the formation of the BLM-induced pulmonary fibrosis of the mouse. Therefore, the nerve growth factor NGF as the biomarker has accurate and specific detection and auxiliary diagnosis on pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF).

Description

Technical Field

[0001] This invention relates to the field of biomarker technology in clinical medicine, specifically to the preparation of biomarkers for diagnosing pulmonary fibrosis and their applications. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease affecting interstitial tissue. It can be detected by high-resolution computed tomography or biopsy as having characteristics of common interstitial pneumonia, but its specific cause remains unknown. The incidence of IPF has been increasing annually in recent years. Therefore, early detection and treatment of this disease are crucial for improving patient survival rates.

[0003] Increasing evidence suggests that immune dysregulation plays a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Identifying key inflammatory factors and immune cells that significantly influence the pathogenesis and progression of IPF would provide new diagnostic and therapeutic targets for the diagnosis and treatment of this disease, which would be of great benefit. Summary of the Invention

[0004] This invention proposes a biomarker for the preparation of diagnostic pulmonary fibrosis and its application.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: The first objective of this invention is to provide a biomarker for the preparation of diagnoses of pulmonary fibrosis, wherein the biomarker is nerve growth factor (NGF).

[0006] Furthermore, the pulmonary fibrosis includes one or more of the following: primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia. Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0007] The second objective of this invention is to provide an application of a biomarker in the preparation of products for detecting or diagnosing pulmonary fibrosis, wherein the biomarker is nerve growth factor (NGF), and the product for detecting or diagnosing pulmonary fibrosis is a detection reagent, and / or a detection kit, and / or a diagnostic reagent and / or a diagnostic kit.

[0008] Furthermore, the detection or diagnostic product for pulmonary fibrosis is used to detect the expression level of nerve growth factor (NGF) in biological samples.

[0009] Furthermore, when the product for detecting or diagnosing pulmonary fibrosis detects pulmonary fibrosis, the detection of pulmonary fibrosis is positively correlated with the expression level of nerve growth factor (NGF).

[0010] Furthermore, the biological samples are selected from peripheral blood and tissue samples.

[0011] This invention relates to the preparation of biomarkers for diagnosing pulmonary fibrosis and their applications, and its beneficial effects are as follows: (1) The biomarker of this invention is nerve growth factor (NGF). NGF can significantly enhance the differentiation of human lung fibroblasts MRC5 cells, and the induction of differentiation is accompanied by an increase in the expression of its receptor TrkA, which significantly enhances the migration and proliferation ability of lung fibroblasts. Therefore, NGF, as a biomarker, has the accuracy and specificity for the detection and auxiliary diagnosis of pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF).

[0012] (2) The biomarkers of the present invention are detected by peripheral blood, which has the advantages of relatively simple sampling, good stability and suitability for auxiliary diagnosis. They can provide new technical means for the early identification and clinical judgment of pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), and have good reproducibility and promotion prospects. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a schematic diagram showing the specific expression results of the biomarker nerve growth factor (NGF) of this invention on idiopathic pulmonary fibrosis (IPF). In this diagram, A represents the expression level of NGF detected by RT-qPCR after peripheral blood RNA extraction from 6 healthy individuals and 6 IPF patients. B is a volcano plot showing the differential expression of mRNA between different groups (IPF / Control) in the IPF dataset GSE70866. C is the differential expression analysis of NGF in bronchoalveolar lavage fluid (BALF) of controls and IPF patients in dataset GSE70866. D is the diagnostic ROC curve of NGF expression level in BALF in the GSE70866 dataset. E is the Kaplan-Meier survival analysis of the high NGF expression group and the low NGF expression group in dataset GSE70866. Figure 2This diagram illustrates the in vitro and in vivo experimental verification of the specific expression of nerve growth factor (NGF) on idiopathic pulmonary fibrosis (IPF). After bleomycin-induced pulmonary fibrosis in mice, A shows HE staining of lung tissue; B shows Masson staining of lung tissue; C shows the Ashcroft score used to assess the degree of pulmonary fibrosis in mice; D shows the hydroxyproline detection results in mouse lung tissue; E shows the protein expression levels of α-SMA, Collagen I, and Fibronectin 1 in mouse lung tissue detected by Western blotting; F shows the expression of NGF in mouse lung tissue detected by immunohistochemistry; G shows the protein expression levels of NGF, p75NTR, TrkA, p-ERK, ERK, p-FAK, and FAK in mouse lung tissue detected by Western blotting; TGF-β1 acts on human lung fibroblasts MRC5, leading to their activation; H shows the protein expression levels of α-SMA, Collagen I, and Fibronectin 1 in activated lung fibroblasts detected by Western blotting; I... Western blot analysis was performed to detect the protein expression levels of NGF, p75NTR, TrkA, p-ERK, ERK, p-FAK, and FAK in activated lung fibroblasts. Figure 3 Nerve growth factor (NGF) activates human lung fibroblasts by activating the TrkA / FAK / ERK signaling pathway. In this study, A represents the cell proliferation activity of MRC5 cells after treatment with different concentrations of NGF, as detected by CCK8 assay; B represents the mRNA expression levels of ACTA2, COL1A1, and FN1 in MRC5 cells after NGF treatment, as detected by RT-qPCR; C and D represent the protein expression levels of α-SMA, Collagen I, and Fibronectin 1 in MRC5 cells after NGF treatment, as detected by Western blotting; E, F, and G represent the protein expression levels of NGF, p75NTR, TrkA, p-ERK, ERK, p-FAK, and FAK in MRC5 cells after NGF treatment, as detected by Western blotting; H represents the cell migration ability of MRC5 cells after NGF treatment, as detected by cell scratch assay; and I represents the cell migration ability of MRC5 cells after NGF treatment, as detected by Transwell assay. Figure 4NGF can exacerbate bleomycin-induced pulmonary fibrosis in mice by activating the TrkA / FAK / ERK signaling pathway. A represents the mRNA expression level of acta2 in mouse lung tissue detected by RT-qPCR; B represents the mRNA expression level of col1a1 in mouse lung tissue detected by RT-qPCR; C represents the mRNA expression level of fn1 in mouse lung tissue detected by RT-qPCR; D represents the expression of TGF-β1 in mouse bronchoalveolar lavage fluid (BALF) detected by ELISA; E and F represent the protein expression levels of α-SMA, Collagen I, Fibronectin 1, p75NTR, TrkA, p-ERK, ERK, p-FAK, and FAK in mouse lung tissue detected by Western blotting; G represents the protein expression levels of α-SMA, Collagen I, Fibronectin 1, p75NTR, TrkA, p-ERK, ERK, p-FAK, and FAK detected by Western blotting. HE staining of mouse lung tissue was performed. H represents the Ashcroft score for assessing the degree of pulmonary fibrosis in mice. H represents Masson staining of mouse lung tissue. J represents the hydroxyproline detection result of mouse lung tissue. K represents the protein expression of α-SMA in mouse lung tissue detected by immunohistochemistry. L represents the protein expression of Collagen I in mouse lung tissue detected by immunohistochemistry. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0016] This invention provides the application of the biomarker nerve growth factor (NGF) in the preparation of diagnostic materials for pulmonary fibrosis.

[0017] In this invention, pulmonary fibrosis includes one or more of the following: primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia. In this embodiment, the detection and diagnostic markers for idiopathic pulmonary fibrosis are particularly specific and accurate.

[0018] The present invention also provides a diagnostic reagent or kit for detecting pulmonary fibrosis, wherein the diagnostic reagent or kit mainly includes at least the biomarker nerve growth factor (NGF).

[0019] The present invention also provides a diagnostic reagent or kit for assisting in the diagnosis of pulmonary fibrosis, wherein the diagnostic reagent or kit mainly includes at least the biomarker nerve growth factor (NGF).

[0020] In detection or diagnostic reagents or kits, the expression level of nerve growth factor (NGF) in peripheral blood of biological samples is measured for detection or diagnosis. A significant increase in the expression level of NGF in peripheral blood indicates the possibility of detecting or diagnosing pulmonary fibrosis.

[0021] Example 1

[0022] Validation of the specific expression of the biomarker nerve growth factor (NGF) in idiopathic pulmonary fibrosis (IPF) I. RT-qPCR assay: Peripheral blood samples were collected from 6 IPF patients and 6 healthy controls. The expression levels of the identified inflammatory cytokines were measured using real-time quantitative polymerase chain reaction (RT-qPCR) in the peripheral blood samples. The results are as follows: Figure 1 As shown in A, from Figure 1 As can be seen in A, the expression level of B-NGF is increased. The RT-qPCR detection method used in this method is a routine method well known to those skilled in the art, and its specific steps will not be elaborated here.

[0023] II. Data Analysis Methods: The IPF dataset GSE70866 was obtained from the Gene Expression Comprehensive Database (GEO) to identify differentially expressed mRNAs. The thresholds applied were log fusion change (logFC) > 0.5 and adjusted p-value (P.adj) < 0.05 (Figure 1B). This dataset included 20 healthy controls and 212 IPF bronchoalveolar lavage fluid (BALF) samples. Analysis of the GSE70866 dataset showed elevated nerve growth factor (NGF) gene expression levels in the IPF group (Figure 1C). ROC curve analysis of the GSE70866 dataset revealed that NGF expression had good diagnostic efficacy for IPF (Figure 1D). Kaplan-Meier survival analysis showed that patients with high NGF expression had a decreasing overall survival rate. Figure 1 E).

[0024] The GSE70866 dataset analysis and Kaplan-Meier survival analysis used in this method are standard graphical analysis methods for those skilled in the art, and the specific steps and principles will not be elaborated here.

[0025] Example 2

[0026] I. Animal Experiments (1) Animal model establishment: 6-8 week old C57BL / 6 mice were selected and acclimatized for 1 week, and a control group was set up ( Figure 2Six mice were divided into two groups: the Saline group and the BLM group. Each group received an intraperitoneal injection of 1% sodium pentobarbital solution. After anesthesia, the mice were fixed supine on an operating table. The angle of the operating table was adjusted, and the mice's tongues were gently pulled out with forceps to fully expose the larynx. Bleomycin (BLM) (5 mg / kg, 50 μl total volume) was injected into the trachea using a head-mounted spotlight. The control group received the same treatment with an equal volume of saline (Saline). Twenty-one days later, the mice were euthanized by cervical dislocation under sodium pentobarbital solution anesthesia. Lung tissue was collected for pathological sections and Western blotting to detect the expression of relevant molecular proteins.

[0027] (2) Lung tissue acquisition Expose the mouse's heart and lungs, cut open the left atrium, and perform right ventricular puncture. Inject 5 ml of physiological saline to flush the lungs until they turn white. Cut off the right lung lobe, remove surrounding tissue and adipose tissue, and place it in a cryovial for cryopreservation in liquid nitrogen for later use. Cut off the left lung, similarly remove surrounding tissue and adipose tissue, and fix it in 4% paraformaldehyde solution at room temperature for 48-72 hours for pathological section analysis.

[0028] (3) Staining analysis of mouse lung tissue pathological sections 1) Preparation of lung pathological histological specimens A. Paraffin embedding a. Dehydration and clearing: The dehydration program was set using a Leica TP1020. Ethanol dehydration was performed in the following sequence: 30% ethanol for 60 min, 50% ethanol for 60 min, 70% ethanol for 60 min, 80% ethanol for 60 min, 90% ethanol for 60 min, 100% ethanol I for 30 min, and then 100% ethanol II. Xylene clearing: Xylene I and Xylene II, 10 min each. b. Paraffin embedding: Both paraffin I and paraffin II are used to prepare embedding blocks at 56-60℃ for 2 hours.

[0029] B. Slicing and Patching a. Slicing: After trimming the cut surface of the paraffin block into a right-angled quadrilateral of appropriate size, fix it on the microtome, adjust the blade angle and the angle of the paraffin block, and slice it into thin slices with a thickness of 3-5µm; b. Spreading: Float the cut slices in deionized water at 37℃ – 42℃ to fully spread them out; c. Mounting: Insert the glass slide vertically into the water, and then mount it close to the fully flattened wax slide; d. Baking: Place the pasted pieces in a 37℃ constant temperature oven and bake for 3 days.

[0030] 2) HE staining A. Dewaxing and hydration: Xylene I 10 min, Xylene II 10 min, anhydrous ethanol 5 min, anhydrous ethanol 5 min, 95% ethanol 5 min, 85% ethanol 5 min, 70% ethanol 5 min, pure water 5 min; B. Staining: Hematoxylin for 15 min, rinse with running water and drain, differentiate with hydrochloric acid ethanol for 30 s, rinse with tap water for 10 min, and eosin for 2 min; C. Dehydration, permeation and mounting: Soak in 95% ethanol, anhydrous ethanol I and anhydrous ethanol II for 5 min each, soak in xylene I, xylene II and xylene III for 10 min each, and mount with neutral resin.

[0031] 3) Masson staining Dewaxing and hydration are the same as the HE staining described above; immersion in Weigert iron hematoxylin staining for 10 min; differentiation in acidic ethanol solution for 15 s, followed by rinsing with running water; re-blueing with Masson's blue solution for 5 min, followed by rinsing with running water; staining with Ponceau S staining solution for 5 min, followed by rinsing with weak acid working solution for 1 min; rinsing with phosphomolybdic acid solution for 1 min, followed by rinsing with weak acid working solution for 1 min; staining with aniline blue staining solution for 2 min, followed by rinsing with weak acid working solution for 1 min; dehydration, permeabilization, and mounting are the same as the HE staining described above.

[0032] (4) Ashcroft fibrosis score of mouse lung tissue After scanning HE-stained pathological sections, the Ashcroft score was used as the standard for determining pulmonary fibrosis (see Table 1). Three pathologists conducted a blind review, randomly selecting 30 fields of view for each scanned pathological section image. The mean score of all field scores was used as the fibrosis grade for that sample. The degree of fibrosis was graded from 0 to 8. Observers first determined whether the lung tissue in the field was normal or fibrotic. A score of 0 was given if normal lung tissue predominated; if fibrosis predominated, the score was set to an odd number. If two adjacent odd numbers could not be determined, an even number was used. Fields of view mostly occupied by large blood vessels or the trachea were ignored; if only a small portion of the field was occupied, the degree of fibrosis in the remaining field was assessed.

[0033]

[0034] (5) Immunohistochemistry (IHC) of mouse lung tissue pathological sections 1) Paraffin sectioning A. Baking: The slices are vertically inserted into a glass jar and baked in an oven at 60 ℃ for 1 h, followed by the addition of xylene; B. Dewaxing and hydration are the same as the HE staining described above; C. Inactivation of endogenous peroxidase: 3% H2O2, approximately 200µl per tablet, in a humidified chamber for 10-15 min, followed by washing with PBS for 5 min × 3 times.

[0035] 2) Antigen retrieval: Boil alkaline antigen retrieval solution (10mM Tris buffer, 1mM EDTA, pH adjusted to 9.0 with 1M NaOH) on high in a microwave oven, add the slides, heat on high for 2 min, cool for 5 min, heat on high for 1 min, cool for 5 min (the cycle can be increased according to the retrieval effect), cool to room temperature for 15-20 min; wash with PBS for 5 min × 3 times; 3) Blocking: Add 50µl of 10% sheep serum to each sheet and incubate in a humidified chamber at room temperature for 1 hour; 4) Antibody incubation: After removing the blocking agent, add 50µl of primary antibody and incubate at 37°C for 1 hour in a humidified chamber; wash the slides with PBS for 5 minutes each time (3 times); add 50µl of secondary antibody with HRP and incubate at 37°C for 30 minutes in a humidified chamber; wash the slides with PBS for 5 minutes each time (3 times). 5) DAB staining and hematoxylin counterstaining: Add 50µl of freshly prepared DAB staining solution, control the staining under a microscope for 3-10 min, stop the staining with PBS, add hematoxylin staining solution, rinse with tap water, and separate the colors with the separating solution. 6) Dehydration, permeation, and mounting are the same as for HE staining.

[0036] (6) Extraction of protein from mouse lung tissue Protein extraction: After weighing the wet weight of the mouse lung tissue (about the size of a mung bean), add BeyoLytic™ mammalian active protein extraction reagent at a ratio of 200-400 μl per 20 mg of tissue. Depending on the specific experimental purpose, add appropriate amounts (1:50 = inhibitor: protein extraction reagent, 4-8 µl each) of protease inhibitor and phosphatase inhibitor, followed by 0.52-1.04 µl of BeyoZonase™ super nuclease. Then, use an ultrasonic homogenizer to thoroughly lyse the lung tissue. After centrifugation at 12000 g for 10 min at 4°C, transfer the supernatant to a new EP tube.

[0037] (7) Protein sample quantification 1) Preparation of protein standards: Prepare protein standards to a concentration of 25 mg / ml, aliquot, and store at -20ºC for long-term use. When measuring protein sample concentration, further dilute to 0.5 mg / ml before use.

[0038] 2) Preparation of BCA working solution: Prepare BCA working solution according to the ratio of reagent A: reagent B = 50:1.

[0039] 3) Protein sample concentration detection: Add 0.5 mg / ml standard to each well of a 96-well plate at volumes of 0, 1, 2, 4, 8, 12, 16, and 20 µl, respectively, while simultaneously adding standard diluent to each well to bring the volume to 20 µl. Add 1 µl of the sample to be tested to each well of the 96-well plate, again adding standard diluent to bring the volume to 20 µl, thus diluting the sample 20-fold. Add 200 µl of prepared BCA working solution to both the sample and standard wells and incubate at 37ºC for 30 min. Measure the absorbance at a wavelength of A562 nm using a microplate reader, and calculate the sample protein concentration based on the standard curve.

[0040] 4) Protein denaturation: After mixing the protein sample with 5× loading buffer, heat in a 100ºC metal bath for 5-10 min, cool to room temperature, aliquot and store at -80℃.

[0041] (8) Western blot (WB) assay to detect protein levels in lung tissue 1) SDS-PAGE gel preparation and electrophoretic separation of proteins A. Based on the molecular size of the protein, 10% and 6% separating gels are mainly prepared. The specific reagents and volumes required for preparation are shown in Table 2.

[0042]

[0043] Prepare the liquid in a 50ml centrifuge tube according to the volume in the table above. Let it stand at room temperature for a few minutes to degas. Carefully pour the gel into the gap between the glass plates. Use 500µl of anhydrous ethanol to press the gel into place. Wait for the gel to solidify before proceeding with subsequent operations.

[0044] B. Preparation of 5% stacking gel: Take another 50ml centrifuge tube and add 2.7ml deionized water, 0.67ml 30% polyacrylamide solution, 0.5ml 1.0M Tris-HCl (pH 6.8), 0.04ml 10% SDS, 0.04ml 10% AP and 0.004ml TEMED in sequence. After mixing, discard the isopropanol in the glass plate where the separating gel has solidified. Then quickly pour in the prepared stacking gel solution, slowly insert the matching sample comb, and wait for the upper layer of gel to solidify.

[0045] C. Electrophoretic separation of proteins Sample loading: Clamp the glass plate carrying the SDS-PAGE gel with electrode plates and place it in the electrophoresis tank. Add electrophoresis buffer and check for leakage in the inner tank. Slowly and vertically pull out the sample comb, and then add an appropriate amount of protein sample and protein marker to the lane. Electrophoresis: Maintain a constant voltage of 80V for about half an hour for the stacking gel, and a constant voltage of 120V for the separating gel until the color of the loading buffer almost reaches the bottom of the gel (about 1-2 hours). 2) Transfer: Sandwich Preparation: First, cut a PVDF membrane of appropriate size and activate it in methanol for 10 seconds. Then, immerse it and filter paper in transfer buffer. Next, prepare the sandwich in the following order from bottom to top: sponge, filter paper, PVDF membrane, gel, filter paper, sponge, removing air bubbles after each layer. Fix the sandwich in a sieve plate, and insert the sieve plate into the transfer tank in the following order: white to red, black to black. Transfer Conditions: Submerge the transfer tank in an ice-water mixture, and fill the tank with transfer buffer. Proteins under 70 kDa can be electrotransferred at 200 mA for 1.5 hours. For larger proteins, electrotransfer overnight at 4 degrees Celsius with a low current. 3) Blocking: Pour an appropriate amount of 5% or 7% milk into the antibody incubation box, place the PVDF membrane after transfer into the milk, and block at room temperature for 1 hour, or at 4°C overnight. 4) Primary antibody incubation: After removing the blocking solution, add the primary antibody directly and incubate overnight at 4°C. Wash the membrane with TBST for 5 min × 4 times. 5) Secondary antibody incubation: Incubate HRP-labeled secondary antibody at room temperature for 1 hour, then wash the membrane with TBST for 5 minutes each time (4 times). 6) ECL colorimetric analysis: Prepare the working solution according to the ECL colorimetric kit used. Remove the membrane from the TBST, absorb excess liquid on clean absorbent paper, and evenly drop the prepared ECL working solution onto the membrane. Incubate in the dark for 5 minutes, then expose using an imaging system. Quantitative analysis is performed after measuring the optical density of the bands using GEL Pro Analyzer software.

[0046] (9) Detection of hydroxyproline content in mouse lung tissue 1) Weigh 30-50 mg of mouse lung tissue into a 15 ml glass tube, add 1 ml of hydrolysate, and place in a 95℃ water bath for 20 min to hydrolyze; 2) After rinsing the glass tube with running water, add 10µl of indicator and mix well; 3) Add 1 ml of pH-adjusting solution A to the glass tube and mix well. At this point, the solution turns red. 4) Slowly add pH-adjusting solution B dropwise to the glass tube, mixing well after each drop, until the liquid changes from red to yellow-green, adjusting the pH to approximately 6.0-6.8. Continue adding deionized water to 10ml and mix thoroughly. 5) Pipette 4 ml of the above-treated liquid into a new 10 ml test tube, add 30 mg of activated carbon, centrifuge at 3500 rpm / min for 10 min, and then pipette 1 ml of the supernatant into a new EP tube to be used as the detection solution; 6) Take new 5ml test tubes and label them A550 blank tube, A550 standard tube, and A550 test tubes for each sample. Add 1ml of deionized water to the blank tube, 1ml of 5μg / ml standard working solution to the standard tube, and 1ml of test solution to each sample test tube; at the same time, add 0.5ml of Reagent I to each tube, mix well, and let stand at room temperature for 10min. Then add 0.5ml of Reagent II to each tube, mix well, and let stand at room temperature for 5min. 7) Continue to add 0.5 ml of reagent 3 to each tube and mix well. Incubate at 60°C for 15 min, centrifuge at 3500 rpm / min for 10 min, and transfer 200 μl of the supernatant to a 96-well plate. Use a multi-functional microplate reader to detect the absorbance value at an A550 nm wavelength. 8) Calculate the hydroxyproline content (μg / mg wet weight) of each sample = (A550 test tube – A550 blank tube) / (A550 standard tube – A550 blank tube) × standard tube concentration × total volume of hydrolysate / wet weight of lung tissue.

[0047] Experimental results are as follows Figure 2 As shown, firstly, HE staining, Masson staining, Ashcroft scoring, hydroxyproline detection, and Western blotting were performed on mouse lung tissue to detect the expression of extracellular matrix markers α-SMA, Collagen I, and Fibronectin 1 fibrosis-related proteins in the mouse lung group, assessing the successful establishment of the BLM-induced mouse pulmonary fibrosis model. Secondly, immunohistochemistry and Western blotting were performed on mouse lung tissue to detect NGF expression, revealing its upregulation in mouse pulmonary fibrosis. Finally, Western blotting was performed on mouse lung tissue to detect the expression of NGF downstream signaling pathway molecules p75NTR, TrkA, p-ERK, and p-FAK, indicating that the signaling pathway was activated in the mouse pulmonary fibrosis model.

[0048] II. Cell Experiments (1) Construction of lung fibroblast activation model: Human lung fibroblast MRC5 cells were treated with 10 ng / ml TGF-β1 for 48 h to construct lung fibroblast activation model. Cells were seeded in 60 mm culture dishes when extracting protein.

[0049] (2) Extraction of total cell protein 1) Vaccination 2×10 5 After culturing MRC5 cells in a 60 mm dish for 24 h, 10 ng / ml TGF-β1 was added and the cells were treated for 48 h before total cell protein was extracted. 2) Take 200-400 µl of BeyoLytic™ mammalian active protein extraction reagent per 60 mm dish. Add appropriate amounts (4-8 µl each of inhibitor and protein extraction reagent at a 1:50 ratio) of protease inhibitor and phosphatase inhibitor, and add BeyoZonase™ super nuclease to a final concentration of 50 U / ml (0.52-1.04 µl) to reduce the viscosity of the extraction product. Place in an ice bath for later use.

[0050] 3) Discard the culture medium in the 60mm dish, wash 3 times with pre-cooled PBS, add the solution prepared above, and place on ice on a shaker for lysis for 2-20 minutes.

[0051] 4) Use a pipette to suspend the cell extract and transfer it to a centrifuge tube. Centrifuge at 14,000g at 4ºC for 5-10 minutes and collect the supernatant.

[0052] (3) Protein sample quantification is the same as described in animal experiments. (4) The WB experiment was performed to detect cell protein levels as described in the animal experiment.

[0053] like Figure 2 As shown, after TGF-β1 treatment of MRC5 cells, the expression of its activation markers α-SMA, COL-1, and FN1 proteins was upregulated. Further investigation showed that the expression of NGF and its downstream proteins p75NTR, TrkA, p-ERK, and p-FAK was also upregulated in activated MRC5 cells.

[0054] Example 3 I. Experiment on the effect of nerve growth factor (NGF) on the proliferation of lung fibroblasts (1) Cell culture: 1×10⁶ cells per well 3 MRC5 cells were evenly seeded in 96-well plates, with control and experimental groups established. The experimental groups were treated with different concentrations of NGF (0, 25, 50, 100, 200, and 300 ng / ml) for 1, 2, and 3 days, respectively. The results were analyzed using a CCK8 assay, with the specific steps as follows: 1) The cells were divided into a blank group, a control group, and a drug group. MRC5 cells were digested with trypsin to prepare a cell suspension and seeded in 96-well plates. The drug groups were treated with different concentrations of NGF (0, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, and 300 ng / ml) for 1 day, 2 days, and 3 days, respectively. The blank group was not seeded with cells, and the control group was not treated with drugs. The same volume of DMEM high-glucose medium was added.

[0055] 2) Remove the supernatant and add CCK 8 reagent to each well at a mass ratio of (CCK 8 reagent: serum-free DMEM = 1:10); 3) After incubating in a 37℃ incubator for 0.5 hours and 1 hour respectively, the culture plate was removed and the absorbance (OD value) at 450nm was measured using an ELISA reader to calculate cell viability.

[0056] .

[0057] The results obtained are as follows Figure 3 As shown in Figure A, on day 2, the use of 100, 200, and 300 ng / ml significantly enhanced the proliferation of MRC5 cells. Therefore, a concentration of 200 ng / ml was chosen for subsequent experiments.

[0058] II. Effects of NGF on fibroblast differentiation (1) MRC5 cells were divided into a blank group ( Figure 2 MRC5 cells were divided into two groups: the Saline group and the NGF experimental group. The NGF experimental group was exposed to 200 ng / ml NGF for 48 hours to obtain the treated MRC5 cells.

[0059] (2) The MRC5 cells obtained in step (1) were analyzed using RT-qPCR. The results are shown in Figure 3B. Figure 3 As can be seen from B, the mRNA expression of ACTA2, COL1A1, and FN1 was significantly upregulated.

[0060] (3) The MRC5 cells obtained in step (1) were analyzed by Western blot, and the results are shown in Figure 3C-D. As can be seen from Figure 3C-D, the protein levels of COL-1, α-SMA and FN1 in the NGF experimental group were increased compared with those in the control group, indicating that NGF promoted the differentiation of MRC5 cells. WB detection revealed that NGF-induced differentiation was accompanied by increased expression of its receptors p75NTR and TrkA, and the expression of downstream signaling pathway molecules p-FAK and p-ERK was also increased (Figure 3E, F, G), indicating that the TrkA / FAK / ERK signaling pathway was activated during the differentiation of MRC5 cells induced by nerve growth factor (NGF).

[0061] III. Effects of NGF on the migration of lung fibroblasts (a) Cell scratch test (1) Cell seeding: After treating MRC5 cells with 200 ng / ml NGF for 48 h, prepare a single-cell suspension of MRC5 cells. Mark the bottom back of the 6-well plate with a cross mark using a marker pen. Seed each well with 1 x 10 cells. 5 Each cell.

[0062] (2) Scratching: Using a 20 μl pipette tip, scrape the cells along the marked "+" pattern in a 6-well plate. Then wash the cells with PBS and take pictures under a microscope at 0h. The cells were then cultured in basal culture for another 24h.

[0063] (3) Taking pictures: After discarding the culture medium and washing with PBS, take pictures with a microscope and compare them with the pictures taken at 0h.

[0064] (4) Results analysis: The area and height of the scratch were calculated using ImageJ software. The scratch width = scratch area / scratch height. The cell migration rate was calculated using the following formula: Cell migration rate = BE / ×100%, Where B is the initial width and E is the final width.

[0065] (II) Transwell Experiment (1) Using a 24-well plate as the lower chamber, add 500 μl of 20% FBS complete culture medium to the lower chamber beforehand, and place the transwell chamber in the 24-well plate; (2) Cell sample preparation: After treating MRC5 cells with 200 ng / ml NGF for 48 h, the cells were digested with 0.25% trypsin and the cells were collected for counting; (3) Cell counting: After resuspending the cells in the basal medium, the cell concentration was adjusted to 2×10⁻⁶ cells / mL for counting. 5 (4) Plating: Take 200 μl of the prepared single-cell suspension and add it to the upper layer of the transwell chamber. Make 3 replicates per group and place them in a cell culture incubator to continue culturing for 48 h. (5) Staining: Remove the 24-well plate, discard the liquid from the upper and lower layers of the chamber, wash with PBS, and gently wipe away the cells on the bottom membrane of the chamber with a cotton swab. Wash twice with PBS, and fix with 500 μl of 4% paraformaldehyde for 30 min. Discard the paraformaldehyde, stain with crystal violet staining solution for 20 min, and wash the bottom membrane of the chamber with PBS twice for 10 min each time. (6) Taking pictures: Take multiple pictures of the bottom membrane of the chamber under a microscope, count them using ImageJ software, and take the average value for statistical analysis.

[0066] The results are shown in Figure 3 H and Figure 3 As shown in Figure I, NGF significantly enhanced cell migration ability, indicating that NGF plays a key regulatory role in the activation of lung fibroblasts.

[0067] Example 4 NGF exacerbates BLM-induced pulmonary fibrosis in mice. (1) C57 / BL6 mice were randomly divided into a saline group, a bleomycin (BLM) group, and a bleomycin + nerve growth factor (BLM+NGF) group. The treatment of the BLM group and the Saline group was the same as in Example 2; the mice in the BLM + NGF group were simultaneously instilled with 5 mg / kg BLM and 25 μg NGF intratracheally, with a total volume of 50 μl, in a single administration, and the mice were sacrificed 21 days later.

[0068] (2) Acquisition and processing of bronchoalveolar lavage fluid: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital and then fixed supine on the operating table to expose the trachea. A small incision was made in the trachea, an indwelling intravenous catheter was inserted and ligated with silk suture. The trachea was lavaged three times with 1×PBS at doses of 0.8, 0.8 and 0.5 ml. The bronchoalveolar lavage fluid (BALF) was collected into a 5 ml centrifuge tube and centrifuged at 5000 rpm for 10 min within 2 h after the operation. The supernatant was frozen at -80℃ and TGF-β1 was detected by ELISA.

[0069] (3) ELISA detection of TGF-β1 expression in mouse bronchoalveolar lavage fluid 1) Standard addition: Set up standard wells and sample wells. Add 50 μl of standard at different concentrations to each standard well. TGF-β1: Standard concentrations are as follows: 2000, 1000, 500, 250, 125, 62.5, 0 pg / ml; 2) Sample addition: Set up blank wells and test sample wells separately. Dilute the sample 5 times before testing. That is, add 40 μl of sample diluent to the test sample well first, and then add 10 μl of the test sample. 3) Incubation after enzyme addition: Except for blank wells, add 100 μl of enzyme-labeled reagent to each well and incubate at 37℃ for 60 min; 4) Washing: After discarding the liquid in the microplate, shake it dry, fill each well with washing buffer, let it stand for 30 seconds, then discard it. Repeat 5 times. 5) Color development and termination: Add 50 μl of color developer A to each well, followed by 50 μl of color developer B, and develop the color at 37°C in the dark for 15 min; add 50 μl of stop solution to each well to terminate the reaction. 6) Measurement and Calculation: Measure the absorbance (OD value) of each well at a wavelength of 450 nm using a multi-functional microplate reader. Obtain the linear regression equation of the standard curve using the concentration and OD value of the standard. Substitute the OD value of the sample into the equation to calculate the sample concentration, then multiply by a 5-fold dilution factor to obtain the actual concentration of the sample.

[0070] Experimental results are as follows Figure 4As shown, firstly, mouse lung tissue was collected and RT-qPCR was used to detect the mRNA expression of acta2, col1a1, and fn1. It was found that the expression of all three was upregulated after simultaneous BLM+NGF treatment compared to the BLM-only treatment group. BALF from mice was collected and ELISA was used to detect that TGF-β1 was upregulated in the BLM+NGF group compared to the BLM-only treatment group. Western blotting was used to detect the expression of α-SMA, Collagen I, and Fibronectin 1 fibrosis-related proteins in mouse lung tissue, and it was found that their expression was upregulated after BLM+NGF treatment compared to the BLM-only treatment group. Western blotting was used to detect the expression of p75NTR, TrkA, p-FAK, and p-ERK in mouse lung tissue, and it was found that their expression was upregulated after BLM+NGF treatment compared to the BLM-only treatment group. HE staining and Ashcroft scoring of mouse lung tissue revealed that the degree of fibrosis was more severe in mice treated with BLM+NGF than in the BLM-only treatment group. Masson staining and hydroxyproline detection in mouse lung tissue revealed that collagen deposition was more severe in the lung tissue of mice treated with BLM+NGF compared to the BLM-only treatment group. After pathological sectioning of mouse lung tissue, IHC was used to detect the expression of α-SMA and Collagen I in the lung tissue. The results showed that their expression was upregulated in mice treated with BLM+NGF compared to the BLM-only treatment group.

[0071] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A biomarker for preparing a diagnosis of pulmonary fibrosis, characterized in that: The biomarker is nerve growth factor (NGF).

2. The biomarker for preparing diagnostic pulmonary fibrosis according to claim 1, characterized in that: The pulmonary fibrosis includes one or more of the following: primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia.

3. The biomarker for preparing diagnostic pulmonary fibrosis according to claim 1, characterized in that: The pulmonary fibrosis mentioned is idiopathic pulmonary fibrosis.

4. The application of a biomarker in the preparation of products for detecting or diagnosing pulmonary fibrosis, characterized in that: The biomarker is nerve growth factor (NGF), and the product for detecting or diagnosing pulmonary fibrosis is a detection reagent and / or a detection kit and / or a diagnostic reagent and / or a diagnostic kit.

5. The application according to claim 4, characterized in that: The product for detecting or diagnosing pulmonary fibrosis is used to detect the expression level of nerve growth factor (NGF) in biological samples.

6. The application according to claim 4, characterized in that: When the product for detecting or diagnosing pulmonary fibrosis was mentioned, the detection of pulmonary fibrosis was positively correlated with the expression level of nerve growth factor (NGF).

7. The application according to claim 4, characterized in that: The biological samples were selected from peripheral blood and tissue samples.

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