Use of trbc2 in the diagnosis of gout

Abstract

The application discloses application of TRBC2 in diagnosis of gout. In the application, patients with torn meniscus (equivalent to completely healthy synovial tissue) without a history of gout are used as a control, single-cell transcriptome analysis is carried out based on clinical biological samples, it is found that the marker TRBC2 of T cell subpopulation presents significant difference in gout patients, further Raman spectrum technology is used to find that TRBC2 protein is specifically highly expressed in synovial tissue of gouty arthritis patients, and has high diagnostic efficiency. The clinical value of TRBC2 as a marker for diagnosing gout is high, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of TRBC2 in the diagnosis of gout. Background Technology

[0002] Gout is a crystalline arthropathy caused by hyperuricemia resulting from purine metabolism disorders and impaired uric acid excretion. Its main pathological basis is the deposition of monosodium urate crystals in the joints and synovium. In recent years, the global incidence of gout has been steadily increasing.

[0003] Gout can be classified into four stages based on its progression: hyperuricemia, acute gouty arthritis, intercritical period, and chronic tophi. Hyperuricemia is a prerequisite for its development. As uric acid concentration continues to rise, urate crystals gradually precipitate and deposit in soft tissues such as the joint cavity and synovium. When these crystals are recognized by the immune system, they trigger a local inflammatory response, activating the NLRP3 inflammasome and inducing the release of pro-inflammatory factors such as IL-1β, leading to an acute gouty arthritis attack. Gouty arthritis patients presenting for the first time in the clinical setting are usually in the acute phase.

[0004] Acute gout attacks manifest as redness, swelling, heat, and pain in the joints. The synovium, as a crucial affected tissue, is a core component of gouty arthritis. Pathologically, the synovium shows inflammatory cell infiltration, predominantly neutrophils, and abundant monosodium urate crystals in the synovial fluid. As the disease progresses into chronic gout, urate crystals are encapsulated by extensive granulation tissue, forming tophi, accompanied by chronic synovial hyperplasia, fibrosis, and cartilage destruction, ultimately leading to joint deformities and functional impairment. Developing biomarkers has significant clinical value for the diagnosis of acute gouty arthritis. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides the application of TRBC2 in the diagnosis of gout.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides the use of a reagent for detecting TRBC2 levels in the preparation of products for diagnosing gout.

[0008] Furthermore, the diagnosis of gout is to differentiate gout from meniscus tears.

[0009] Furthermore, the gout mentioned is gouty arthritis.

[0010] Furthermore, the meniscus tear is a meniscus tear caused by sports injury.

[0011] Furthermore, the TRBC2 level includes any one or more of the characteristic peaks of TRBC2, namely 1002, 1332, 2878, and 3058.

[0012] Furthermore, the reagents include a binding agent that specifically binds to the TRBC2 protein, primers that specifically amplify TRBC2, and probes that specifically recognize TRBC2.

[0013] Furthermore, the reagents also include reagents for detecting the expression level of TRBC2 gene or protein by PCR, gene chip detection, NGS detection, nucleic acid probe method, ELISA, Western blotting, mass spectrometry, immunohistochemistry, and Raman spectroscopy.

[0014] Furthermore, the reagent also includes detectable markers.

[0015] Furthermore, the detectable markers include fluorescent dyes, radioactive isotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent components, magnetic particles, and bioluminescent components.

[0016] A second aspect of the invention provides a product for diagnosing gout, the product comprising a reagent for detecting TRBC2 levels.

[0017] Furthermore, the products include reagent kits, test strips, chips, and nucleic acid membrane strips.

[0018] A third aspect of the invention provides the use of TRBC2 in the preparation of models / systems / devices for diagnosing gout.

[0019] Furthermore, the diagnosis of gout is to differentiate gout from meniscus tears.

[0020] Furthermore, the gout mentioned is gouty arthritis.

[0021] Furthermore, the meniscus tear is a meniscus tear caused by sports injury.

[0022] A fourth aspect of the present invention provides a gout diagnostic system / device based on Raman spectroscopy, characterized in that the system / device comprises:

[0023] Acquisition module: Used to acquire Raman spectral data of the sample to be tested;

[0024] Extraction module: used to extract Raman spectral data of the target protein in the sample to be tested, wherein the target protein is TRBC2;

[0025] Prediction module: Based on the Raman spectral data of the target protein TRBC2, classification prediction is performed to obtain the classification result of whether the test sample has gout. If the level of any one or more of the characteristic peaks 1002, 1332, 2878, and 3058 of TRBC2 is high, the test sample is classified as having gout. If the level of any one or more of the characteristic peaks 1002, 1332, 2878, and 3058 of TRBC2 is low, the test sample is classified as not having gout.

[0026] Furthermore, the diagnosis of gout is to differentiate gout from meniscus tears.

[0027] Furthermore, the gout mentioned is gouty arthritis.

[0028] Furthermore, the meniscus tear is a meniscus tear caused by sports injury.

[0029] Advantages and beneficial effects of the present invention:

[0030] This application uses patients with recognized no history of gout and sports-related meniscus tears (equivalent to completely healthy synovial tissue) as controls. Single-cell transcriptome analysis based on clinical biological samples revealed that the T cell subset marker TRBC2 showed significant differences in gout patients. Further Raman spectroscopy revealed that TRBC2 protein is specifically highly expressed in the synovial tissue of patients with gouty arthritis and has high diagnostic efficacy. Using TRBC2 as a marker for diagnosing gout has high clinical value and broad application prospects. Attached Figure Description

[0031] Figure 1 This is an ultrasound diagnostic image of a patient with gouty arthritis;

[0032] Figure 2 The training set consists of synovial tissue morphological features of patients with gouty arthritis and sports meniscus tears. Among them, 2A is a synovial tissue image of a patient with sports meniscus tears, and 2B is a synovial tissue image of a patient with gouty arthritis.

[0033] Figure 3 These are cell subpopulation clustering diagrams from single-cell transcriptome sequencing of synovial tissue. Among them, 3A is the tSNE cell clustering diagram and 3B is the UMAP cell clustering diagram.

[0034] Figure 4This is a graph showing the number of T cell subsets (number 4) in synovial tissue. Among them, 4A is the number of tSNE cells in the synovial tissue of the control group sample 1 with a torn meniscus due to movement; 4B is the number of tSNE cells in the synovial tissue of the control group sample 2 with a torn meniscus due to movement; 4C is the number of tSNE cells in the synovial tissue of the experimental group sample 1 with gouty arthritis; 4D is the number of tSNE cells in the synovial tissue of the experimental group sample 2 with gouty arthritis; 4E is the number of tSNE cells in the synovial tissue of the experimental group sample 3 with gouty arthritis; and 4F is the number of tSNE cells in the synovial tissue of the experimental group sample 4 with gouty arthritis.

[0035] Figure 5 This is a graph showing the mRNA expression level of TRBC2 as a marker of T cell subset (number 4) in synovial tissue obtained by single-cell transcriptome sequencing. Among them, 5A is a violin plot of cell subset distribution, 5B is a tSNE plot of cell subset distribution, and 5C is a bar graph of expression in synovial tissue of motor meniscus tear (control group) and gouty arthritis (experimental group).

[0036] Figure 6 These are images of synovial tissue morphology characteristics from patients with gouty arthritis and sports-related meniscus tears. Among them, 6A is an image of synovial tissue from a patient with sports-related meniscus tears, and 6B is an image of synovial tissue from a patient with gouty arthritis.

[0037] Figure 7 This is a diagram assessing the uniformity of Raman spectral characteristics of synovial tissue.

[0038] Figure 8 The image shows the Raman spectrum of the standard TRBC2 recombinant protein, with the main peak positions overlapping with the synovial tissue of patients with meniscus tears (control group: C) and patients with gouty arthritis (experimental group: T).

[0039] Figure 9 The average spectrum within the sample shows that the expression of TRBC2 protein in the synovial tissue of patients with gouty arthritis (experimental group: T) is significantly higher than that in the synovial tissue of patients with sports meniscus tears (control group: C). Among them, 9A is the expression difference map of characteristic peak 1002, 9B is the expression difference map of characteristic peak 1332, 9C is the expression difference map of characteristic peak 2878, and 9D is the expression difference map of characteristic peak 3058.

[0040] Figure 10 This is a single-spectrum graph showing that the expression of TRBC2 protein in the synovial tissue of patients with gouty arthritis (experimental group) is significantly higher than that in the synovial tissue of patients with sports meniscus tears (control group). Among them, 10A is the expression graph of characteristic peak 1002, 10B is the expression graph of characteristic peak 1332, 10C is the expression graph of characteristic peak 2878, and 10D is the expression graph of characteristic peak 3058.

[0041] Figure 11 The ROC curves of the Raman characteristic peaks 1002, 1332, 2878, and 3058 of TRBC2 protein in synovial tissue for diagnosing gouty arthritis (experimental group) and sports meniscus tears (control group);

[0042] Figure 12 This is a peak importance analysis plot, where 12A is a regression coefficient plot reflecting the direct contribution and predictive power of each peak to the sample classification, and 12B is a score ranking plot of the peak variable importance projection reflecting the top 10 peaks of the two groups of samples.

[0043] Figure 13 The ROC curves of TRBC2 protein in synovial tissue in patients with gouty arthritis (experimental group) and patients with sports meniscus tears (control group) are shown.

[0044] Figure 14 This is a Raman image showing TRBC2 protein aggregation near urate crystals in the synovial tissue of gouty arthritis (yellow arrows indicate urate crystals; patients with gouty arthritis (experimental group) and patients with muscular meniscus tears (control group)). Detailed Implementation

[0045] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] This invention provides the application of a reagent for detecting TRBC2 levels in the preparation of products for diagnosing gout.

[0047] In some implementations, TRBC2 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed TRBC2, as well as any form of TRBC2 derived from cells and processed. The term encompasses naturally occurring variants of TRBC2 (e.g., splice variants or allelic variants). The term encompasses TRBC2 from, for example, human and any other vertebrate sources, including mammalian TRBC2 such as primates and rodents (e.g., mice and rats), gene ID: 28638.

[0048] The reagents include a binding agent that specifically binds to the TRBC2 protein, primers that specifically amplify TRBC2, and probes that specifically recognize TRBC2.

[0049] In some implementations, primers are short nucleic acid molecules, such as DNA oligonucleotides, that can form a hybrid between the primer and the target nucleic acid strand through nucleic acid hybridization and annealing with a complementary target nucleic acid molecule. The primer can be extended along the target nucleic acid molecule using polymerase. Therefore, primers can be used to amplify target nucleic acid molecules, where the primer sequence is specific to the target nucleic acid molecule; for example, the primer will hybridize with the target nucleic acid molecule under very high-tightness hybridization conditions.

[0050] In some embodiments, the probe can be DNA, RNA, a DNA-RNA chimera, PNA, or other derivatives. There is no limitation on the length of the probe; any length is acceptable as long as specific hybridization and binding to the target nucleotide sequence are achieved. The probe length can be as short as 25, 20, 15, 13, or 10 bases. Similarly, the probe length can be as long as 60, 80, 100, 150, 300 bases or longer, even encompassing the entire gene.

[0051] In some embodiments, primers or probes may be chemically synthesized using phosphorimide solid-phase support or other well-known methods. Modifications may also be performed using many techniques known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more analogs of natural nucleotides, and modifications between nucleotides. For example, modifications may be made to uncharged linkers (e.g., methyl phosphate, triphosphate, phosphorimide, carbamate, etc.) or charged linkers (e.g., thiophosphate, dithiophosphate, etc.).

[0052] The reagents also include reagents for detecting TRBC2 gene or protein expression levels using PCR methods, such as quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR); gene chip detection methods, such as DNA chips, RNA chips, and protein chips; NGS detection methods (Next Generation Sequencing, also known as high-throughput sequencing), such as RNA or DNA sequencing (RNAseq / DNAseq); nucleic acid probe methods, such as DNA probes, RNA probes, and oligonucleotide probes; ELISA (Enzyme-Linked Immunosorbent Assay); Western blotting (Immunoblotting, also known as protein blotting); mass spectrometry (MS); immunohistochemistry (IHC); and Raman spectroscopy methods, such as spontaneous Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), resonance Raman spectroscopy (RRS), and Fourier transform Raman spectroscopy (FT-Raman).

[0053] The reagent also includes a detectable marker.

[0054] In some embodiments, a detectable marker refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a sample. Suitable detectable markers include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components. Therefore, a marker is any composition detectable by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrochemical, optical, chemical detection devices, or any other suitable device. In some embodiments, the marker can be detected visually without the aid of a device.

[0055] Among them, radioactive isotopes include but are not limited to 3 H, 14 C 35 S, 125 I, 131 I.

[0056] Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and acetylcholinesterase.

[0057] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0058] The products include reagent kits, test strips, chips, and nucleic acid membrane strips.

[0059] In some implementations, a chip, also referred to as an array, refers to a solid support containing linked nucleic acid or peptide probes. Arrays typically contain a variety of different nucleic acid or peptide probes attached to a substrate surface at different known locations. These arrays, also known as “microarrays,” can typically be produced using mechanosynthesis or photoguided synthesis, which combines photolithography and solid-phase synthesis methods. Arrays can comprise flat surfaces or can be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. Arrays can be packaged in a manner that allows for diagnostic or other manipulation of a fully functional device.

[0060] The chips include gene chips and protein chips.

[0061] In some embodiments, the gene chip includes a solid-phase carrier and probes immobilized on the solid-phase carrier, the probes including oligonucleotide probes targeting the TRBC2 gene for detecting the transcriptional level of the TRBC2 gene; the protein chip includes a solid-phase carrier and a specific antibody against the TRBC2 protein immobilized on the solid-phase carrier; the gene chip can be used to detect the expression levels of multiple genes, including the human TRBC2 gene (e.g., multiple genes associated with gout). The protein chip can be used to detect the expression levels of multiple proteins, including the human TRBC2 protein (e.g., multiple proteins associated with gout). By simultaneously detecting multiple gout-related biomarkers, the accuracy of gout diagnosis can be greatly improved.

[0062] In some embodiments, the kit may also include a fluorescent dye, and a variety of known fluorescent dyes may be used. Examples include methods using an intercalator with a marking function, and methods using probes that bind fluorescent substances to nucleotides that specifically hybridize to the relatively amplified DNA sequence. Examples of intercalators include ethidium bromide and SYBR Green I as unsaturated fluorescent dyes, and Resolight and EvaGreen as saturated fluorescent dyes. The dosage should be as recommended by the manufacturer or distributor of the fluorescent dye used.

[0063] The kit also includes instructions and buffer solutions. The instructions may include guidance on obtaining and processing samples. Additionally, the kit may contain bacterial genomic DNA as a positive control for PCR and sterile water as a negative control.

[0064] In some embodiments, the components of the kit may be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container in which one component can be placed, and preferably, appropriately aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kit of the present invention will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0065] The solid support of the kit may be, for example, plastic, silicon wafer, metal, resin, glass, membrane, particles, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide. The biological sample may be, for example, cell culture, cell line, tissue, oral tissue, gastrointestinal tissue, organ, organelle, biological fluid, plasma sample, urine sample, or skin.

[0066] In some embodiments, the nucleic acid membrane strip includes a substrate and probes fixed on the substrate; the substrate can be any substrate suitable for fixing the probes, including but not limited to nylon membranes, nitrocellulose membranes, polypropylene membranes, glass slides, silicone wafers, and micro-magnetic beads.

[0067] This invention provides a gout diagnostic system / device based on Raman spectroscopy.

[0068] In some embodiments, the disclosed system / device can be implemented in other ways. For example, the system / device embodiments described above are merely illustrative. For instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection between devices or units through some interfaces, and may be electrical, mechanical, or other forms.

[0069] Those skilled in the art will understand that all or part of the steps in the various methods of the above embodiments can be implemented by a program instructing related hardware. The program can be stored in a computer-readable storage medium, which may include: read-only memory (ROM), random access memory (RAM), disk or optical disk, etc.

[0070] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0071] Example 1: Collection of Training Set Cases

[0072] 1. Experimental materials

[0073] 1) Inclusion and exclusion criteria for clinical patients and control group patients

[0074] Inclusion and exclusion criteria for patients with gouty arthritis:

[0075] Inclusion criteria

[0076] a. Individuals diagnosed with gout according to the 2015 American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) classification criteria (clinical score ≥8).

[0077] b. Ultrasound confirms double-track sign, synovial thickening (≥4mm), Doppler blood flow signal (grade ≥2), and / or joint effusion and urate crystal deposition.

[0078] c. Ages 18 to 65, gender not limited.

[0079] d. The patient was informed and gave consent to participate in this study.

[0080] Exclusion criteria

[0081] a. Individuals with severe joint deformities, multiple tophi throughout the body, and local ulceration.

[0082] b. Individuals with severe lesions of vital organs or mental illnesses.

[0083] c. Pregnant or breastfeeding patients.

[0084] d. Patients with gouty kidney injury.

[0085] Inclusion and exclusion criteria for patients with meniscus tears due to sports injuries:

[0086] Inclusion criteria

[0087] a. There is a clear history of sports injury, which may have been accompanied by knee twisting, excessive flexion and extension, or direct impact.

[0088] b. If the diagnosis is confirmed by imaging examination as traumatic meniscus tear, the diagnostic criteria refer to the "Consensus and Practice Guidelines for Lateral Meniscus Injury" formulated by the Chinese Society of Sports Medicine in 2022.

[0089] c. Ages 18 to 45, gender not limited.

[0090] d. The patient was informed and gave consent to participate in this study.

[0091] Exclusion criteria

[0092] a. There are contraindications to surgery.

[0093] b. Accompanied by congenital malformation of the meniscus.

[0094] c. Degenerative meniscus injury.

[0095] d. Accompanied by knee fracture, cruciate ligament injury, and collateral ligament injury.

[0096] e. Accompanied by dysfunction of vital organs.

[0097] f. Accompanied by rheumatic diseases.

[0098] 2) Staining reagents

[0099] Dewaxing solution (Kyushu Berlin, BLB-01), clearing solution (Kyushu Berlin, BLB-07), differentiation solution (Kyushu Berlin, BLB-04), hematoxylin (Kyushu Berlin, BLB-03).

[0100] 2. Experimental Methods

[0101] 1) Ultrasound diagnosis

[0102] Before the examination, the patient's medical history (history of acute attacks, chronic course, history of tophi), symptoms (joint redness, swelling, heat, and pain), signs (joint tenderness, limited range of motion), and serum uric acid levels should be obtained. Joint ultrasound examination should be performed to confirm the "double track sign" of the knee joint, synovial thickening (≥4mm), Doppler blood flow signal (grade ≥2), and / or joint effusion and urate crystal deposition. The diagnostic criteria must meet the 2015 American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) gout classification criteria (clinical score ≥8 points).

[0103] 2) Pathological tissue preparation and diagnosis

[0104] Patients with meniscus tears due to sports injuries were assigned to the control group (C), and patients with gouty arthritis were assigned to the experimental group (T). Synovial tissue was collected from patients with meniscus tears due to sports injuries or gouty arthritis after arthroscopic surgery for subsequent pathological preparation. The collected synovial tissue was immediately fixed in 10% neutral formaldehyde solution for approximately 24-48 hours to prevent autolysis and denaturation. Subsequently, the tissue was dehydrated by sequentially immersing it in ethanol solutions of different concentrations for 30 minutes to 1 hour per concentration. The tissue was then cleared using xylene. After embedding, the cleared tissue was placed in paraffin wax and repeatedly impregnated until completely saturated. The tissue was then sectioned to a thickness of 2.5 μm.

[0105] HE staining is performed on the sections. First, hematoxylin staining is applied and the sections are soaked for 3-5 minutes. Then, they are washed with water. Differentiation is performed using 1% hydrochloric acid alcohol for 10-30 seconds. Then, they are washed with water. For blueing, the sections are soaked in weakly alkaline water (e.g., 0.5% sodium bicarbonate solution) for 1-2 minutes until the nuclei turn blue. Then, eosin staining is applied and the sections are soaked for 1-2 minutes. Finally, they are washed with tap water. The sections are then dehydrated, cleared, and mounted. Dehydration is performed by soaking in 95% ethanol for 30 seconds, followed by soaking in 100% ethanol for 30 seconds. Clearing is performed by soaking in xylene I for 2 minutes, followed by soaking in xylene II for 2 minutes. The sections are then mounted with neutral resin or mounting medium. HE sections are independently interpreted by two mid-career pathologists. If there is disagreement, the sections are re-examined, with a focus on the areas of disagreement. Discussions are held, and a third senior pathologist provides their opinion and makes the final decision.

[0106] 3. Experimental Results

[0107] 1) Ultrasound diagnosis

[0108] This study was approved by the hospital's medical ethics committee and informed consent was obtained from the subjects or their families (guardians). A total of 6 patients were included as subjects for subsequent single-cell transcriptome sequencing. Data collection was conducted at Wangjing Hospital of the China Academy of Chinese Medical Sciences in Beijing, China, from January 2022 to January 2023. The control group consisted of 2 patients, and the experimental group consisted of 4 patients. Figure 1 ).

[0109] 2) Histopathological diagnosis of synovial tissue in gouty arthritis

[0110] In the control group, patients with meniscus tears showed 1-2 layers of synovial cells on the synovial cavity surface, with loose connective tissue and adipose tissue beneath the synovium. No significant fibrovascular proliferation, inflammatory cell infiltration, or giant cell reaction was observed. In the experimental group, patients with gouty arthritis showed synovitis with diffusely distributed urate crystals of varying sizes in non-cellular clusters. No giant cell reaction was observed around the cell clusters. Interstitial fibrosis was present, with predominantly lymphocyte-based inflammatory cell infiltration. Figure 2 ).

[0111] Example 2: Single-cell transcriptome sequencing of training set cases

[0112] 1. Experimental materials

[0113] Cell capture and cDNA synthesis were performed using the Single Cell 3′ Library and Gel Bead Kit V3.1; Single Cell B-Chip Kit (10x Genomics, 1000074).

[0114] 2. Experimental Methods

[0115] 1) Single-cell transcriptome sequencing

[0116] Cell suspensions (300-600 live cells per μL) were loaded onto Chromium Single Cell Controllers (10xGenomics) to prepare single-cell gel beads. In short, single cells were suspended in PBS containing 0.04% BSA. Approximately 6000 cells were loaded per channel, covering approximately 3000 target cells. Captured cells were lysed, and the released RNA was labeled by reverse transcription in individual GEMs. Reverse transcription was performed at 53°C for 45 min on an S1000™ Touch Thermal Cycler (BioRad), followed by 5 min at 85°C, and stored at 4°C. cDNA was generated, amplified, and quality assessed using an Agilent 4200. Single-cell RNA-Seq library preparation was performed using a single-cell 3′ library and gel bead kit V3.1. Sequencing was performed using an Illumina Nova Seq 6000 sequencer, with a sequencing depth of at least 100,000 reads per cell and a 150 bp bp end.

[0117] 2) Bioinformatics data analysis

[0118] For cell subpopulation clustering, Cell Ranger software was obtained from the 10x Genomics website: https: / / support.10xgenomics.com / single-cell-gene expression / software / downloads / latest. The Cell Ranger counting module was used to generate filter, barcode counting, and UMI counting feature barcode matrices to determine clusters. Principal component analysis (PCA) and the top ten dimensionality were used for dimensionality reduction, and K-means and graph-based algorithms were used to generate clusters. Additionally, Seurat 3.0 (R package) was used for clustering. Genes with fewer than 200 genes, genes ranking in the top 1%, or mitochondrial genes exceeding 25% were considered outliers and filtered. Principal component analysis was used for dimensionality reduction, and tSNE and UMAP were performed.

[0119] Enrichment analyses were performed, including GO enrichment, KEGG enrichment, Reactome enrichment, and disease enrichment. Cluster markers were adjusted using Benjamin-Hochberg multiple tests with KOBAS software; the top 20 marker genes in each cluster were used. Results were visualized using R packages.

[0120] Cell types are annotated using SingleR (https: / / bioconductor.org / packages / devel / bioc / html / SingleR.html). SingleR enables unbiased cell type identification from single-cell RNA sequencing data by independently inferring the source cell for each single cell using a pure cell type reference transcriptome dataset. For human samples, Blueprint_Encode or HPCA is used.

[0121] 3. Experimental Results

[0122] 1) Single-cell transcriptome sequencing

[0123] A total of 61,215 cells were captured from 6 human synovial tissues, with 87% reads and a median gene count of 2,206 per cell. Based on differential gene expression and GOBP enrichment analysis, 22 cell subpopulations were identified. Figure 3 ).

[0124] 2) T cell subset analysis

[0125] Compared with the synovial tissue of the control group (patients with meniscus tears due to sports injuries), the number of T cell subsets (number 4) in the synovial tissue of gouty arthritis was significantly increased. Figure 4The TRBC2 gene is a characteristic marker of this subset, and it is highly expressed in T cell subsets and synovial tissue of gouty arthritis. Figure 5 TRBC2 is involved in T cell receptor signaling pathways; adaptive immune responses; and α-β T cell activation. TRBC2 is part of the α-β T cell receptor complex and is a constant region of the β chain of the T cell receptor (TR). The α-β T cell receptor is an antigen-specific receptor essential for immune responses and is present on the cell surface of T lymphocytes. It recognizes the peptide-major histocompatibility complex (pMHC) displayed by antigen-presenting cells (APCs), a prerequisite for effective T cell adaptive immunity against pathogens. Binding of α-β TR to pMHC triggers the aggregation of TR-CD3 clusters on the cell surface and intracellular activation of LCK. LCK phosphorylates the ITAM motif of CD3G, CD3D, CD3E, and CD247, thereby enabling the recruitment of ZAP 70. ZAP 70, in turn, phosphorylates LAT, which recruits numerous signaling molecules to form the LAT signaling body. The LAT signaling body propagates signaling branches to three major signaling pathways: calcium, mitogen-activated protein kinase (MAPK) kinase, and nuclear factor NF-κB (NF-κB) pathway, leading to the mobilization of transcription factors that are essential for gene expression and for T cell growth and differentiation.

[0126] Example 3: Collection of Validation Set Cases

[0127] 1) Clinical patients

[0128] Synovial tissue samples were collected from 30 patients with meniscus tears due to sports injuries (control group) and 30 patients with gouty arthritis (experimental group). Data were collected at Wangjing Hospital of the China Academy of Chinese Medical Sciences in Beijing, China, from January 2022 to January 2023. This study was approved by the hospital's medical ethics committee.

[0129] 2) Main reagents

[0130] Standard: Human recombinant TRBC2 protein, 20 μl (1 μg / μl). Company: www.ABsea.bio. CAT No. PP-12225. Lot No. 240527040R.

[0131] 2. Experimental Methods

[0132] Clinical inclusion and exclusion criteria

[0133] Inclusion and exclusion criteria for patients with gouty arthritis:

[0134] Inclusion criteria

[0135] a. Individuals diagnosed with gout according to the 2015 American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) classification criteria (clinical score ≥8).

[0136] b. Ultrasound confirms double-track sign, synovial thickening (≥4mm), Doppler blood flow signal (grade ≥2), and / or joint effusion and urate crystal deposition.

[0137] c. Ages 18 to 65, gender not limited.

[0138] d. The patient was informed and gave consent to participate in this study.

[0139] Exclusion criteria

[0140] a. Individuals with severe joint deformities, multiple tophi throughout the body, and local ulceration.

[0141] b. Individuals with severe lesions of vital organs or mental illnesses.

[0142] c. Pregnant or breastfeeding patients.

[0143] d. Patients with gouty kidney injury.

[0144] Inclusion and exclusion criteria for patients with meniscus tears due to sports injuries:

[0145] Inclusion criteria

[0146] a. There is a clear history of sports injury, which may have been accompanied by knee twisting, excessive flexion and extension, or direct impact.

[0147] b. If the diagnosis is confirmed by imaging examination as traumatic meniscus tear, the diagnostic criteria refer to the "Consensus and Practice Guidelines for Lateral Meniscus Injury" formulated by the Chinese Society of Sports Medicine in 2022.

[0148] c. Ages 18 to 45, gender not limited.

[0149] d. The patient was informed and gave consent to participate in this study.

[0150] Exclusion criteria

[0151] a. There are contraindications to surgery.

[0152] b. Accompanied by congenital malformation of the meniscus.

[0153] c. Degenerative meniscus injury.

[0154] d. Accompanied by knee fracture, cruciate ligament injury, and collateral ligament injury.

[0155] e. Accompanied by dysfunction of vital organs.

[0156] f. Accompanied by rheumatic diseases.

[0157] 2) Spontaneous Raman spectroscopy detection and imaging

[0158] All samples were prepared into paraffin sections. After complete dewaxing, spontaneous Raman spectroscopy was used for detection and imaging. All spectra were acquired using a WITec alpha 300R+ Raman confocal microspectrophotometer (Ulm, Germany), equipped with a piezoelectric platform (UHTS 300, WITec, GmbH), a 100x air objective (Zeiss EC EPIPLAN, NA = 0.90), a green solid-state excitation laser (λ = 532 nm, 32 mW, WITec, GmbH), and an imaging spectrograph (Newton, Andorra Technologies Ltd., UK). The spectrograph was equipped with a 600-groove / mm grating and a thermoelectrically cooled (60°C) charge-coupled device (CCD) detector. The grating and detector were optically connected to the objective via a 10 μm diameter single-mode silicon fiber cable. The laser excitation spot size was 350 nm, enabling the acquisition of wavelengths in the range of 0–3600 cm⁻¹. -1 The spectral data were obtained. In all cases, the excitation laser intensity remained constant between sample scans, the integration time was 0.1 seconds, the rapid scan area was 8μm*8μm, and then the average of 100 spectra was used to represent one unit. Fifty single spectra were acquired for each sample, and the sample was imaged.

[0159] 3) Raman data processing

[0160] All model analyses were performed using Python 3.9. Libraries used included, but were not limited to: scipy for statistical tools, sklearn for machine learning models, tensorflow and pytorch for deep learning models, and matplotlib and seaborn for plotting. In non-image or sequence models, eigenvalue data underwent dimensionality reduction using PCA while maintaining variable independence, reducing the dimensionality to a level that preserves at least 99% interpretable variance. In image or sequence models (GRU, CNN), full-spectrum data was used for eigenvalue data.

[0161] 3. Experimental Results

[0162] 1) Quality control of synovial tissue Raman spectroscopy samples and accuracy control of TRBC2 protein detection

[0163] Raman spectroscopy provides evidence for the identification and characterization of substances by analyzing the vibrational modes of molecules. Different substances have unique "fingerprint" Raman spectra that reflect their chemical structure, functional groups, and interactions. All 60 samples underwent HE staining and histopathological evaluation consistent with the training set described above. Figure 6 The samples were then subjected to Raman spectroscopy quality control. The results showed that all samples possessed similar Raman spectral characteristics, indicating good sample homogeneity. Figure 7 Human recombinant TRBC2 protein was used as a standard for feature identification and comparison of synovial tissue samples. Raman spectroscopy peak positions showed that TRBC2 protein and its related functional groups were present in synovial tissue without significant differences, ensuring the accuracy of qualitative and quantitative results. Figure 8 Further analysis is needed to determine the intensity differences of the main elution peaks of the TRBC2 protein in the samples for quantitative analysis.

[0164] 2) TRBC2 protein single-spectrum peak analysis

[0165] The standard TRBC2 protein showed four major elution peaks. The results of all four major peaks indicated that TRBC2 protein expression in the synovial tissue of 30 patients with gouty arthritis (experimental group) was significantly higher than that in the synovial tissue of 30 patients with sports-induced meniscus tears (control group). Figure 9 and Figure 10 Diagnostic efficacy analysis was performed on the four main characteristic peaks, and the results are shown in Table 1 and 2. Figure 11 As shown, the AUCs of characteristic peaks 1002, 2878, and 3058 are all greater than 0.7, indicating good diagnostic efficacy. Further analysis based on OPLS-DA classification, ranked by regression coefficient, reveals the top 10 Raman peaks of biomarkers in the synovial tissue of gouty arthritis, and presents the variable importance projection scores of these peaks. TRBC2 characteristic peaks 1002 and 2878 are among the top distinguishing biomarkers. Figure 12 Binary logistic regression analysis was performed on the four main characteristic peaks to calculate the predicted probability values, and the ROC curve of the TRBC2 protein was plotted. The AUC value was 0.996, the specificity was 100%, and the sensitivity was 93.3%. Figure 13 The results showed that TRBC2 protein has good diagnostic efficacy for gouty arthritis.

[0166] surface Diagnostic efficacy of TRBC2 protein Raman spectroscopy features on synovial tissue (experimental group) of patients with gouty arthritis.

[0167]

[0168] 3) TRBC2 protein Raman imaging analysis

[0169] Raman imaging analysis was performed on the synovial tissues of patients with gouty arthritis (experimental group) and patients with sports-induced meniscus tears (control group). The results showed that TRBC2 protein was enriched around urate crystals in the synovial tissues of patients with gouty arthritis, exhibiting significant specific aggregation. Figure 14 ).

[0170] 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. Application of reagents for detecting TRBC2 protein expression levels in the preparation of products for diagnosing gout.

2. The application according to claim 1, characterized in that, The TRBC2 protein expression level includes any one or more of the characteristic peaks of TRBC2, namely 1002, 2878, and 3058.

3. The application according to claim 1, characterized in that, The reagent includes a binding agent that specifically binds to the TRBC2 protein.

4. The application according to claim 3, characterized in that, The reagents also include those for detecting TRBC2 protein expression levels using ELISA, Western blotting, mass spectrometry, immunohistochemistry, and Raman spectroscopy.

5. The application according to claim 4, characterized in that, The reagent also includes detectable markers.

6. The application according to claim 5, characterized in that, The detectable markers include fluorescent dyes, radioactive isotopes, nucleotide chromophores, enzymes, and magnetic particles.

7. The application according to claim 1, characterized in that, The products include reagent kits, test strips, chips, and nucleic acid membrane strips.

8. Application of TRBC2 protein expression level detection reagents in the preparation of systems / devices for diagnosing gout.

9. A gout diagnostic system / device based on Raman spectroscopy, characterized in that, The system / device includes: Acquisition module: Used to acquire Raman spectral data of the sample to be tested; Extraction module: used to extract Raman spectral data of the target protein in the sample to be tested, wherein the target protein is TRBC2; Prediction module: Based on the Raman spectral data of the target protein TRBC2, classification prediction is performed to obtain a classification result of whether the test sample has gout. If the level of any one or more of the characteristic peaks 1002, 2878, and 3058 of TRBC2 is high, the test sample is classified as having gout; if the level of any one or more of the characteristic peaks 1002, 2878, and 3058 of TRBC2 is low, the test sample is classified as not having gout. The level of any one or more of the characteristic peaks 1002, 2878, and 3058 of TRBC2 is judged to be high or low relative to the level of healthy controls.

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