Biomarkers for diagnosing systemic lupus erythematosus and use thereof

Abstract

The application provides a biomarker for diagnosing systemic lupus erythematosus and application thereof. It is found in the application that the expression of ferritin in neutrophil granulocytes of patients with systemic lupus erythematosus is significantly reduced and is increased after treatment, and the ferritin in neutrophil granulocytes has disease specificity and cell specificity, and the ferritin in neutrophil granulocytes has important significance for clinical diagnosis or auxiliary diagnosis of systemic lupus erythematosus and disease evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to biomarkers for diagnosing systemic lupus erythematosus and their applications. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs and systems. Currently, clinical diagnosis of SLE primarily relies on clinical manifestations and laboratory test results. However, due to the unclear pathogenesis of SLE and its complex and diverse clinical presentations, relying solely on a single symptom or abnormal laboratory indicator can easily lead to missed or misdiagnosis. Therefore, a deeper understanding of the pathogenesis of SLE and the search for novel biomarkers with better diagnostic efficacy are important research directions for improving the diagnostic level of SLE. Summary of the Invention

[0003] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0004] The first aspect of this invention provides the application of a reagent for detecting the expression level of a biomarker in neutrophils of a subject in the preparation of products for diagnosing or assisting in the diagnosis of systemic lupus erythematosus, wherein the biomarker is Ferritin.

[0005] Biomarkers used in this invention include proteins, protein subunits, genes encoding proteins, genes encoding protein subunits, or homologs, mutations, or isotypes of these proteins or genes. Biomarkers encompass full-length, unprocessed biomarkers, as well as any form of biomarker derived from cell processing. Naturally occurring variants of biomarkers (e.g., splice variants or alleles) are also included.

[0006] In different tissues, ferritin is a shell-like protein nanostructure with 24 subunits formed by copolymerization of ferritin heavy chain (FTH) and ferritin light chain (FTL) in different proportions.

[0007] In this invention, the subjects include humans or non-human mammals.

[0008] Non-human mammals refer to all members of the class Mammalia other than humans, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic rodent class (e.g., mice, rats, squirrels, beavers, groundhogs, voles, hamsters, guinea pigs, and spiny guinea pigs), including any offspring derived from them.

[0009] In one specific implementation, the subject is selected from human subjects.

[0010] In some implementations, the neutrophils are obtained from bone marrow, peripheral blood, or tissue.

[0011] In one specific implementation, the neutrophils are obtained from peripheral blood.

[0012] The term "expression level" as used in this invention generally refers to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., gene-encoded and / or epigenetic information) is transformed into structures present and functioning in the cell. Therefore, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotide fragments, translated polypeptide fragments, or polynucleotide and / or polypeptide modified fragments (e.g., post-translational modifications of a polypeptide) should also be considered expressed, regardless of whether they originate from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides (e.g., through proteolysis).

[0013] The term "diagnosis" or "auxiliary diagnosis" used in this invention refers to making a medical judgment on an individual's health status.

[0014] In this invention, the reagents include, but are not limited to, reagents used to detect the expression levels of biomarkers in neutrophils of subjects using sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, chromatography technology, or mass spectrometry technology.

[0015] In this invention, the sequencing technologies include (but are not limited to): first-generation sequencing, second-generation sequencing, and third-generation sequencing. First-generation sequencing, also known as Sanger sequencing, is a sequencing technology that utilizes DNA polymerase synthesis reactions. First-generation sequencing is a sequencing technology based on the Sanger method. Second-generation sequencing is based on massive parallel analysis (MPS), which can simultaneously synthesize the complementary strand of the sequencing template and acquire sequence data. Third-generation sequencing is based on single-molecule sequencing and massive parallel sequencing technologies.

[0016] In this invention, the nucleic acid hybridization technology refers to the process by which complementary nucleotide sequences (DNA and DNA, DNA and RNA, RNA and RNA, etc.) form non-covalent bonds through Watson-Crick base pairing, thereby forming stable homologous or heterologous double-stranded molecules, also known as nucleic acid hybridization.

[0017] In this invention, the nucleic acid amplification technology refers to a general term for a large class of technical methods. Currently, nucleic acid amplification technology includes conventional PCR, real-time fluorescence PCR, isothermal nucleic acid amplification technology, etc., which can specifically amplify extremely small amounts of target DNA by millions of times, thereby greatly improving the ability to analyze and detect DNA molecules. It can detect single DNA molecules or samples containing only one target DNA molecule per 100,000 cells.

[0018] In this invention, the protein immunoassay technique refers to a class of methods for detecting target analytes, including but not limited to radioimmunoassay, direct, indirect or comparative enzyme-linked immunosorbent assay, enzyme immunoassay, fluorescence immunoassay, Western blotting, immunoprecipitation, and immunoassays based on any particle (e.g., using gold particles, silver particles or latex particles, magnetic particles or quantum dots). Protein immunoassay can be performed in the form of microtiter plates or strips.

[0019] In this invention, the chromatographic technique refers to a method for separating and analyzing various components in a complex mixture. It utilizes the fact that different substances have different partition coefficients in a system composed of a stationary phase and a mobile phase. When the two phases move relative to each other, these substances move together with the mobile phase and undergo repeated partitioning between the two phases, thereby achieving separation of the substances.

[0020] In this invention, the mass spectrometry technique refers to a method of separating and detecting moving ions (charged atoms, molecules or molecular fragments, isotopic ions, fragment ions, rearranged ions, multiply charged ions, metastable ions, negative ions, and ions generated by ion-molecule interactions) according to their mass-to-charge ratio using electric and magnetic fields. Determining the accurate mass of the ions allows for the determination of their compound composition.

[0021] In some embodiments, the reagents include, but are not limited to, reagents for detecting the expression levels of biomarker mRNA in neutrophils of subjects, and reagents for detecting the expression levels of biomarker proteins and / or peptides in neutrophils.

[0022] In some implementations, the reagents used to detect the expression level of biomarker mRNA in the subject's neutrophils include, but are not limited to, probes that specifically recognize biomarkers and primers that specifically amplify biomarkers.

[0023] As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific, intended target biomarker. In some embodiments, the term "probe" refers to any molecule or its association with any substrate and / or reaction product disclosed herein, whether indirectly or directly, covalently or nonvalently, and such association or binding can be detected using the methods disclosed herein. In some embodiments, the probe is a fluorescent probe, an antibody, or an absorbance-based probe. In some embodiments, the probe may be a nucleic acid sequence comprising a fluorescent molecule or substrate that becomes fluorescent upon exposure to an enzyme, and the nucleic acid sequence is complementary to a fragment of a nucleic acid sequence.

[0024] The primers described in this invention can be prepared by chemical synthesis, appropriately designed using methods well known to those skilled in the art and with reference to known information, and prepared by chemical synthesis.

[0025] In some embodiments, the reagents used to detect the expression levels of biomarker proteins and / or peptides in neutrophils of the subject include, but are not limited to, antibodies or fragments thereof that specifically bind to the biomarkers, and affinity proteins.

[0026] As used in this article, the term "peptide" refers to a compound composed of amino acids linked by peptide bonds, including the full-length polypeptide or amino acid fragments.

[0027] The antibodies or fragments thereof described in this invention can be of any structure, size, immunoglobulin class, origin, etc., as long as they bind to the target protein. The antibodies or fragments thereof described in this invention can be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody that retains its antigen-binding activity or a peptide containing a portion of an antibody. Antibody fragments may include F(ab')2, Fab', Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V regions (biantibodies), or peptides containing CDRs. Antibodies can be obtained by methods known to those skilled in the art. For example, a mammalian cell expression vector containing a polypeptide that retains whole or part of the target protein or integrating polynucleotides encoding them can be prepared as an antigen. After immunizing an animal with the antigen, immune cells are obtained from the immunized animal and fused with cancer cells to obtain a hybridoma. Antibodies are then collected from the hybridoma culture. Finally, monoclonal antibodies against the marker protein can be obtained by antigen-specific purification of the obtained antibody using the marker protein or a portion thereof used as the antigen.

[0028] In one specific implementation, the reagent used to detect the expression levels of biomarker proteins and / or peptides in the subject's neutrophils is an antibody that specifically binds to the biomarker.

[0029] In some implementations, a subject is diagnosed with systemic lupus erythematosus if the level of Ferritin expression in neutrophils is significantly lower than that in healthy individuals.

[0030] In this invention, the healthy subject is a subject who does not have systemic lupus erythematosus or has a low risk of having systemic lupus erythematosus, while excluding those with other chronic or acute infections or serious underlying diseases.

[0031] A second aspect of the present invention provides a product for diagnosing or assisting in the diagnosis of systemic lupus erythematosus, the product comprising a reagent for detecting the expression level of a biomarker in neutrophils of a subject, the biomarker being Ferritin.

[0032] In some implementations, the product includes, but is not limited to, chips, reagent kits, test strips, or high-throughput sequencing platforms.

[0033] In some embodiments, the chip can be prepared using conventional biochip preparation methods known to those skilled in the art, including (but not limited to): using a solid-phase support of a modified glass slide or silicon wafer, with the 5' end of the probe containing an amino-modified polydT string, preparing the oligonucleotide probe into a solution, and then spotting it onto the modified glass slide or silicon wafer using a spotting instrument, arranging it into a predetermined sequence or array, and then fixing it by leaving it overnight to obtain the chip of the present invention.

[0034] In some embodiments, the kit further includes instructions for use or a label, a positive control, a negative control, a buffer, an adjuvant, or a solvent, and one or more containers for containing the compositions contained in the kit. The compositions may be in liquid form or lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, including glass or plastic. The instructions for use or label details how to use the kit to test samples and the use of the kit for the diagnosis or auxiliary diagnosis of systemic lupus erythematosus.

[0035] In some embodiments, the kit may also include a variety of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed in this invention. Any reagent that is based on the detection of Ferritin or Ferritin and GAPDH to diagnose or assist in the diagnosis of systemic lupus erythematosus is included within the scope of protection of this invention.

[0036] In some implementations, the kits include, but are not limited to, qPCR kits, immunoblotting kits, immunochromatographic kits, flow cytometry kits, immunohistochemical kits, ELISA kits, or electrochemiluminescence kits.

[0037] In some implementations, a subject is diagnosed with systemic lupus erythematosus if the level of Ferritin expression in neutrophils is significantly lower than that in healthy individuals.

[0038] In some embodiments, the product also includes reagents for detecting the expression levels of endogenous control genes / proteins in neutrophils.

[0039] In one specific implementation, the endogenous control gene / protein is selected from GAPDH.

[0040] In some embodiments, the reagents include, but are not limited to, reagents for detecting the expression level of GAPDH mRNA in neutrophils of a subject, and reagents for detecting the expression level of GAPDH protein and / or peptide in neutrophils.

[0041] In some implementations, the reagents used to detect the expression level of GAPDH mRNA in the subject's neutrophils include, but are not limited to, probes that specifically recognize GAPDH and primers that specifically amplify GAPDH.

[0042] In some embodiments, the reagents used to detect the expression levels of GAPDH protein and / or peptides in the subject's neutrophils include, but are not limited to, antibodies or fragments thereof that specifically bind to GAPDH, and affinity proteins.

[0043] In one specific implementation, the reagent used to detect the expression levels of GAPDH protein and / or peptides in the subject's neutrophils is an antibody that specifically binds to GAPDH.

[0044] In some implementations, the product diagnoses whether a subject has systemic lupus erythematosus by measuring the Ferritin to GAPDH ratio in the subject's neutrophils.

[0045] In one specific implementation, if the Ferritin to GAPDH ratio is less than 0.771, the subject is diagnosed with systemic lupus erythematosus.

[0046] The third aspect of the present invention provides any of the following methods:

[0047] (1) A method for screening candidate drugs for the treatment of systemic lupus erythematosus, the method comprising the following steps:

[0048] a) Contact the test substance with neutrophils containing or expressing Ferritin;

[0049] b) Detect the expression level of Ferritin in the neutrophils;

[0050] c) Select substances that can increase ferritin levels in neutrophils as candidate drugs for the treatment of systemic lupus erythematosus;

[0051] (2) A method for evaluating the therapeutic effect of a drug for treating systemic lupus erythematosus, the method comprising:

[0052] a) Administering the drug to the subject;

[0053] b) Detect the expression level of Ferritin in the neutrophils of the subjects;

[0054] c) Assess the ability of the drug to increase ferritin in the neutrophils of the subjects.

[0055] In one specific implementation, the method in (1) includes:

[0056] a) Contact the test substance with neutrophils containing or expressing Ferritin and GAPDH;

[0057] b) Detect the expression levels of Ferritin and GAPDH in the neutrophils;

[0058] c) Select substances that can increase the Ferritin to GAPDH ratio in neutrophils as candidate drugs for the treatment of systemic lupus erythematosus.

[0059] In one specific implementation, the method in (2) includes:

[0060] a) Administering the drug to the subject;

[0061] b) Detect the expression levels of Ferritin and GAPDH in the neutrophils of the subjects;

[0062] c) Assess the ability of the drug to increase the Ferritin to GAPDH ratio in neutrophils of subjects.

[0063] The fourth aspect of the present invention provides any of the following systems:

[0064] (1) A diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus, the diagnostic system comprising:

[0065] Data acquisition unit: used to obtain the expression level of Ferritin in neutrophils of the target subject;

[0066] Results output unit: The obtained Ferritin expression level is compared with a threshold. If the Ferritin expression level is lower than the threshold, the computer determines that the subject is diagnosed with systemic lupus erythematosus.

[0067] (2) A diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus, the diagnostic system comprising:

[0068] Data acquisition unit: used to obtain the expression levels of Ferritin and endogenous control genes / proteins in neutrophils of the target subjects;

[0069] Data processing unit: Used to calculate the ratio of expression levels of Ferritin and endogenous control genes / proteins based on the obtained data, and obtain the calculated value;

[0070] The result output unit is used to compare the calculated value obtained by the data processing unit with the threshold. If the calculated value is lower than the threshold, the computer determines that the subject is diagnosed with systemic lupus erythematosus.

[0071] In one specific implementation, the endogenous control gene / protein is selected from GAPDH.

[0072] In one specific implementation, the threshold is 0.771.

[0073] The fifth aspect of the present invention provides a device / apparatus for computer-aided diagnosis or assisted diagnosis of systemic lupus erythematosus, comprising:

[0074] The system includes a memory for storing program instructions and a processor for calling the program instructions. When the program instructions are executed, they implement the unit functions of the diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus as described in the fourth aspect of the present invention.

[0075] As used in this invention, the term "device" refers to any suitable computing or processing device, or other device constructed or modified to store data or information. Examples of devices suitable for use with embodiments of this invention include stand-alone computing devices; networks, including local area networks (LANs), wide area networks (WANs), the Internet, intranets, and extranets; electronic devices such as personal digital assistants (PDAs), mobile phones, page management programs, etc.; and local and distributed processing systems.

[0076] The sixth aspect of the present invention provides a computer-readable storage medium storing a computer program, which, when executed by a processor, implements the unit functions of the diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus as described in the fourth aspect of the present invention.

[0077] The seventh aspect of the present invention provides the application of the biomarkers described in the first aspect of the present invention in screening candidate drugs for the treatment of systemic lupus erythematosus (SLE), evaluating the therapeutic effects of drugs for the treatment of SLE, and preparing computer-aided diagnostic systems / devices / apparatus / readable storage media for the diagnosis of SLE.

[0078] The eighth aspect of the present invention provides a method for diagnosing or assisting in the diagnosis of systemic lupus erythematosus, the method comprising detecting the expression level of Ferritin in neutrophils of a subject, wherein if the expression level of Ferritin in neutrophils of a subject is significantly reduced compared with that of a healthy person, the subject is diagnosed with systemic lupus erythematosus.

[0079] In one specific implementation, the method includes detecting the expression levels of Ferritin and GAPDH in the neutrophils of the subject, calculating the ratio of the expression levels of Ferritin and GAPDH, and if the Ferritin to GAPDH ratio is less than 0.771, the subject is diagnosed with systemic lupus erythematosus.

[0080] The advantages and beneficial effects of this invention are as follows:

[0081] This invention provides biomarkers for diagnosing systemic lupus erythematosus (SLE) and their applications. This invention reveals that Ferritin expression in neutrophils of SLE patients is significantly decreased and recovers after treatment, exhibiting both disease-specific and cell-specific characteristics. Ferritin in neutrophils is of significant importance for the clinical diagnosis or auxiliary diagnosis of SLE and for disease assessment. Attached Figure Description

[0082] Figure 1 Heatmap of differentially expressed proteomic proteins in neutrophils from healthy individuals and SLE patients.

[0083] Figure 2 The graph shows the results of Ferritin protein expression levels in neutrophils before and after treatment in SLE patients.

[0084] Figure 3 The graph shows the results of Ferritin expression levels in different cell types in healthy individuals and SLE patients.

[0085] Figure 4 The graph shows the results of Ferritin protein expression levels in neutrophils of healthy individuals and patients with various autoimmune diseases.

[0086] Figure 5 The ROC curve of relative protein expression of Ferritin in neutrophils. Detailed Implementation

[0087] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0088] Example 1: Neutrophil proteomic analysis in SLE patients and healthy individuals (HC)

[0089] Five healthy individuals and four SLE patients were selected.

[0090] 1. Isolation of peripheral blood neutrophils from healthy individuals and SLE patients: Peripheral blood was collected using K2E (EDTA) tubes (BD Vacutainer). After centrifugation at 2000 rpm for 10 min at 24°C, the supernatant plasma was aspirated, and an equal volume of PBS was added and mixed in a pipette. 5 ml of human lymphocyte separation medium (Daiyo) was added to a 15 ml centrifuge tube. The peripheral blood was slowly spread onto the lymphocyte separation medium, and centrifuged rapidly at 800 g, 3 L / 3 F L, 20 min, 4°C. After centrifugation, the cloudy cells in the center were PBMCs, and the thin layer of white cells on top of the red blood cells were neutrophils. An equal volume of PBS was added to the PBMCs, and centrifugation was performed at 1800 rpm for 5 min at 4°C. The supernatant was discarded after centrifugation. 15 ml of red blood cell lysis buffer (BD Biosciences) was added to the neutrophils, and the mixture was shaken for 5 min. Then centrifuge at 1800 rpm for 5 min at 4°C. After centrifugation, discard the supernatant, add 7 ml of cell lysis buffer to the pellet, resuspend, shake on a shaker at room temperature for 5 min, and centrifuge again at 1800 rpm for 5 min at 4°C. Discard the supernatant after centrifugation. The cells are now ready for experiments or can be frozen at -80°C.

[0091] 2. Proteomic Profile: Protein Extraction and Digestion: Samples were added to 300 μL of 8M urea lysis buffer (containing 10% protease inhibitor), centrifuged (14, 100×g, 20 min), and the supernatant was collected. Concentration was determined using the Bradford method, and the samples were stored at -80℃. 50 μg of protein was reduced with 200 mM DTT (37℃, 1 h), diluted 8-fold, and then digested overnight at 37℃ with trypsin (1:25). Reverse-phase Separation: After digestion, the C18 column was washed sequentially with acetonitrile and 0.1% formic acid. Samples were loaded and eluted with a pH 10 aqueous gradient (6%-50% acetonitrile). The samples were combined into three groups and lyophilized. LC-MS / MS analysis: Lyophilized samples were reconstituted in 2% methanol / 0.1% formic acid, centrifuged, and loaded onto mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) for gradient separation (4%-99% B, 120 min). Mass spectrometry parameters included full scan (250-1450 m / z, resolution 120,000), CID fragmentation (collision energy 30%), and dynamic exclusion for 18 seconds. Data were processed using Maxquant (Uniprot_HUMAN database). Identification criteria: precursor ion / fragment ion mass error ±15 ppm / ±0.5 Da. Cysteine ​​alkylation fixation modification and methionine oxidation variable modification were performed. Differential protein analysis and intersection with genes related to the ferroptosis pathway were conducted, revealing that FTH1 was the most significantly reduced. Figure 1 ).

[0092] Example 2: Comparison of Ferritin expression levels in neutrophils before and after treatment in SLE patients

[0093] Peripheral blood samples were collected from 5 SLE patients before and after treatment, and neutrophils were isolated according to the method in Example 1.

[0094] 1. Protein extraction: Resuspend cells in 100 μl of RIPA (Solarbio), vortex for 30 seconds, let stand for 30 minutes, centrifuge at 13000g for 10 minutes at 4℃, transfer the supernatant to an EP tube, and use 2 μl for BCA protein quantification (ThermoFisher Scientific). Add 1 / 4 volume of 5× protein loading buffer (Solarbio), mix well, boil in a metal bath at 100℃ for 10 minutes, cool on ice, and then immediately incubate.

[0095] 2. Immunoblot Assay: Western blot: Place the pre-prepared SDS-PAGE gel (ACE) into the electrophoresis tank, pour in the electrophoresis buffer, and add the marker, sample, and marker in sequence. Add more electrophoresis buffer and perform electrophoresis at a constant voltage of 100-120V. Cut and activate the PVDF membrane, prepare the transfer buffer (Bio-Rad), and moisten the thick filter paper with the transfer buffer. After electrophoresis, rinse the electrophoresis tank with tap water. From top to bottom, place a single sheet of thick filter paper (Bio-Rad), PVDF membrane, gel, and another single sheet of thick filter paper. Place the tank in a Trans-Blot Turbo (Bio-Rad) semi-dry transfer apparatus, set to constant current, 1.2A, and perform transfer for 25 minutes. After transfer, block with 5% skim milk powder solution for 60 minutes, and rinse with 1×TBST for 5 minutes each time, 3 times. Cut the membrane and add appropriate amounts of antibodies: Ferritin (dilution 1:1000, abcam, ab75973); GAPDH (dilution 1:10000, abcam, ab128915), and incubate overnight on a shaker at room temperature. The next day, wash with 1×TBST for 10 min three times. Add secondary antibody (dilution 1:5000, easybio, BE0101-100ul), incubate at room temperature for 40 min, and wash with 1×TBST for 10 min three times. Mix chemiluminescence buffer A and B 1:1, and perform detection. Save the white light image. Grayscale analysis of the chemiluminescence image and the merged image was performed using ImageJ software. Statistical analysis was performed using Prism software.

[0096] The results showed that, compared with before treatment, the ratio of Ferritin to GAPDH in neutrophils of SLE patients increased after treatment. Figure 2 ).

[0097] The above results suggest that Ferritin may serve as a clinical biomarker for identifying healthy individuals, evaluating the efficacy of SLE treatment, and screening candidate drugs for SLE treatment.

[0098] Example 3: Comparison of Ferritin expression levels in PBMCs and neutrophils in healthy individuals and SLE patients

[0099] Six healthy individuals and six SLE patients were selected. Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated using the method described in Example 1. After protein extraction, ferritin expression levels were detected by Western blotting. The results showed that, in both healthy individuals (HC) and SLE patients, the relative ferritin / GAPDH protein expression level was significantly lower in neutrophils compared to PBMCs. Figure 3This suggests that the decreased expression level of Ferritin protein is cell-specific in neutrophils.

[0100] Example 4: Comparison of Ferritin expression in neutrophils of healthy individuals and patients with different autoimmune diseases

[0101] To verify the disease specificity of decreased Ferritin levels in neutrophils, we selected autoimmune diseases with high plasma Ferritin levels, namely adult-onset Still's disease (AOSD) and rheumatoid arthritis (RA), as comparisons. Six healthy individuals, three AOSD patients, three RA patients, and six SLE patients were included. Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated using the method described in Example 1. Neutrophil proteins were extracted, and Ferritin expression levels were detected by immunoblotting. The results showed that only in SLE patients was the relative Ferritin / GAPDH protein expression level significantly decreased in neutrophils, suggesting that decreased Ferritin protein expression has disease specificity. Figure 4 ).

[0102] Example 5: Sensitivity and specificity of relative Ferritin expression in neutrophils for diagnosing SLE

[0103] Ninety-eight subjects were randomly assigned in a 7:3 ratio to a training set (67 cases, including 39 in the SLE group and 28 in the HC group) and a validation set (31 cases, including 18 in the SLE group and 13 in the HC group). Figure 5 As shown, ROC curves for diagnosing SLE were plotted based on the relative protein expression levels of Ferritin / GAPDH in neutrophils. The results showed that the AUC of the training set was 0.9231 (P<0.0001), and the AUC of the validation set was 0.8632 (P=0.0007). At a cutoff value of 0.7711, the diagnostic specificity of the training set was 82.14%, and the sensitivity was 92.31%.

[0104] The above results suggest that the expression level of Ferritin in neutrophils has potential application value in the clinical diagnosis of SLE.

[0105] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The application of a reagent for detecting the expression level of a biomarker in human peripheral blood neutrophils in the preparation of products for the diagnosis or auxiliary diagnosis of systemic lupus erythematosus, wherein the biomarker is Ferritin heavy chain.

2. The application according to claim 1, characterized in that, The reagents include those used to detect the expression levels of biomarkers in human peripheral blood neutrophils using protein immunoassay techniques.

3. The application according to claim 2, characterized in that, The reagents include those for detecting the expression levels of biomarker proteins and / or peptides in human peripheral blood neutrophils.

4. The application according to claim 3, characterized in that, The reagents used to detect the expression levels of biomarker proteins and / or peptides in human peripheral blood neutrophils include antibodies, antibody fragments, and affinity proteins that specifically bind to biomarkers.

5. A diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus, characterized in that, The diagnostic system includes: Data acquisition unit: used to obtain the expression level of Ferritin heavy chain in human peripheral blood neutrophils; Output unit: The expression level of the obtained Ferritin heavy chain is compared with a threshold. If the expression level of the Ferritin heavy chain is lower than the threshold, the computer determines that the person is diagnosed with systemic lupus erythematosus.

6. A diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus, characterized in that, The diagnostic system includes: Data acquisition unit: used to obtain the expression levels of Ferritin heavy chain and endogenous control protein in human peripheral blood neutrophils; Data processing unit: Used to calculate the ratio of expression levels of Ferritin heavy chain and endogenous control protein based on the obtained data, and obtain the calculated value; Result output unit: Used to compare the calculated value obtained by the data processing unit with the threshold. If the calculated value is lower than the threshold, the computer determines that the person is diagnosed with systemic lupus erythematosus. The endogenous control protein is GAPDH.

7. A device / apparatus for computer-aided diagnosis or assisted diagnosis of systemic lupus erythematosus, characterized in that, include: The system includes a memory for storing program instructions and a processor for calling the program instructions. When the program instructions are executed, they perform the unit functions of the diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus as described in any one of claims 5-6.

8. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program that, when executed by a processor, implements the unit functions of the diagnostic system for diagnosing or assisting in the diagnosis of systemic lupus erythematosus as described in any one of claims 5-6.

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