Application of TRBC2 in gout diagnosis

Abstract

The invention discloses an application of TRBC2 in diagnosis of gout. According to the application, a recognized moving meniscus tearing patient (equivalent to a completely healthy synovial tissue) without gout medical history is taken as a contrast, single cell transcriptome analysis is carried out on the basis of a clinical biological sample, and a marker TRBC2 of a T cell subset is found to have significant difference in gout patients; a Raman spectrum technology is further adopted to find that the TRBC2 protein is specifically and highly expressed in synovial tissues of gouty arthritis patients and has relatively high diagnosis efficiency, and the TRBC2 is relatively high in clinical value and wide in application prospect when being used as a marker for diagnosing gout.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of TRBC2 in diagnosis of gout. BACKGROUND

[0002] Gout is a crystal arthropathy caused by hyperuricemia due to purine metabolism disorder and uric acid excretion disorder, and the main pathological basis is deposition of monosodium urate crystals in joints and synovial membranes. In recent years, the global incidence of gout has continued to rise.

[0003] Gout can be divided into hyperuricemia period, acute gouty arthritis period, intermittent period and chronic tophus period according to the progression of the disease, and hyperuricemia is a prerequisite for the disease. With the continuous increase of uric acid concentration, urate crystals are gradually precipitated and deposited in the joint cavity, synovial membrane and other soft tissues. When it is recognized by the immune system, it will start a local inflammatory response, activate NLRP3 inflammasome and induce the release of IL-1β and other pro-inflammatory factors, and acute gouty arthritis will break out. Gouty arthritis usually occurs in the acute phase when first seen in clinic.

[0004] Gout acute attack period is manifested as red, swollen, hot and painful joints. Synovium is an important involved tissue, and synovial lesions are the core link of gouty arthritis lesions. Histopathologically, there is an inflammatory cell infiltration mainly composed of neutrophils in the synovium, and a large number of monosodium urate crystals exist in the joint cavity fluid. With the development of the disease, into the chronic gout stage, the urate crystals are wrapped by a large number of granulation tissue to form tophus, accompanied by chronic synovial hyperplasia, fibrosis and cartilage destruction, which can eventually cause joint deformity and dysfunction. The development of biomarkers has important clinical value for the diagnosis of acute gouty arthritis. SUMMARY

[0005] In order to make up for the shortcomings of the prior art, the application provides application of TRBC2 in diagnosis of gout.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] The first aspect of the present application provides application of a reagent for detecting the level of TRBC2 in the preparation of a product for diagnosing gout.

[0008] Further, the diagnosis of gout is to distinguish gout from meniscus tear.

[0009] Further, the gout is gouty arthritis.

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

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

[0012] Further, the reagent includes a binding agent that specifically binds to a TRBC2 protein, a primer that specifically amplifies TRBC2, and a probe that specifically recognizes TRBC2.

[0013] Further, the reagent further includes a reagent for detecting the expression level of TRBC2 gene or protein by PCR method, gene chip detection method, NGS detection method, nucleic acid probe method, ELISA method, immunoblotting method, mass spectrometry, immunohistochemistry, and Raman spectroscopy.

[0014] Further, the reagent further includes a detectable label.

[0015] Further, the detectable label includes a fluorescent dye, a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a chemiluminescent moiety, a magnetic particle, a bioluminescent moiety.

[0016] The second aspect of the present application provides a product for diagnosing gout, the product comprising a reagent for detecting the level of TRBC2.

[0017] Further, the product includes a kit, a test paper, a chip, and a nucleic acid film strip.

[0018] The third aspect of the present application provides the use of TRBC2 in the preparation of a model / system / device for diagnosing gout.

[0019] Further, the diagnosis of gout is to distinguish gout from meniscus tear.

[0020] Further, the gout is gouty arthritis.

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

[0022] The fourth aspect of the present application provides a Raman spectroscopy-based gout diagnosis system / device, characterized in that the system / device comprises:

[0023] An acquisition module for acquiring Raman spectrum data of a sample to be tested;

[0024] An extraction module for extracting Raman spectrum data of a target protein in the sample to be tested, the target protein being TRBC2;

[0025] The prediction module: based on the Raman spectrum data of the target protein TRBC2, the classification prediction is carried out, and the classification result of whether the sample to be tested is gout is obtained; if the level of any one or more of the characteristic peaks 1002, 1332, 2878 and 3058 of the TRBC2 is high, the classification result of the sample to be tested suffering from gout is obtained; if the level of any one or more of the characteristic peaks 1002, 1332, 2878 and 3058 of the TRBC2 is low, the classification result of the sample to be tested not suffering from gout is obtained.

[0026] Further, the diagnosis of gout is to distinguish gout from meniscus tear.

[0027] Further, the gout is gouty arthritis.

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

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

[0030] The present application adopts the sports meniscus tear patients with no gout history (equivalent to completely healthy synovial tissue) as the control, carries out single cell transcriptome analysis based on clinical biological samples, finds that the marker TRBC2 of T cell subpopulation presents significant difference in gout patients, further adopts Raman spectrum technology to find that the TRBC2 protein is specifically highly expressed in the synovial tissue of gouty arthritis patients, and has high diagnostic efficiency, so that the TRBC2 is used as the marker for diagnosing gout, and has high clinical value and wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is an ultrasonic diagnosis graph of a gouty arthritis patient;

[0032] Figure 2 is a synovial tissue morphological feature graph of the training set gouty arthritis and sports meniscus tear patients, wherein 2A is a synovial tissue graph of a sports meniscus tear patient, and 2B is a synovial tissue graph of a gouty arthritis patient;

[0033] Figure 3 is a synovial tissue single cell transcriptome sequencing cell subpopulation clustering graph, wherein 3A is a tSNE cell clustering graph, and 3B is a UMAP cell clustering graph;

[0034] Figure 4Figure 4 is a tSNE cell number plot of T cell subset (No. 4) in synovial tissue, wherein 4A is a tSNE cell number plot of synovial tissue of control sample 1 of sports meniscus tear, 4B is a tSNE cell number plot of synovial tissue of control sample 2 of sports meniscus tear, 4C is a tSNE cell number plot of synovial tissue of experimental sample 1 of gouty arthritis, 4D is a tSNE cell number plot of synovial tissue of experimental sample 2 of gouty arthritis, 4E is a tSNE cell number plot of synovial tissue of experimental sample 3 of gouty arthritis, and 4F is a tSNE cell number plot of synovial tissue of experimental sample 4 of gouty arthritis;

[0035] Figure 5 Figure 5 is a mRNA expression level plot of TRBC2 as a marker of T cell subset (No. 4) in single-cell transcriptome sequencing of synovial tissue, wherein 5A is a violin plot of cell subset distribution, 5B is a tSNE plot of cell subset distribution, and 5C is a column chart of expression in synovial tissue of sports meniscus tear (control group) and gouty arthritis (experimental group);

[0036] Figure 6 Figure 6 is a synovial tissue morphological feature plot of the validation set of gouty arthritis and sports meniscus tear patients, wherein 6A is a synovial tissue plot of sports meniscus tear patient, and 6B is a synovial tissue plot of gouty arthritis patient;

[0037] Figure 7 Figure 7 is a synovial tissue Raman spectrum feature uniformity evaluation plot;

[0038] Figure 8 Figure 8 is a plot of the coincidence of the main peak positions of the standard TRBC2 recombinant protein and the synovial tissue of sports meniscus tear patients (control group: C) and the synovial tissue of gouty arthritis patients (experimental group: T);

[0039] Figure 9 Figure 9 is a plot showing that the expression of TRBC2 protein in the synovial tissue of gouty arthritis patients (experimental group: T) is significantly higher than that in the synovial tissue of sports meniscus tear patients (control group: C), wherein 9A is a plot of expression difference of characteristic peak 1002, 9B is a plot of expression difference of characteristic peak 1332, 9C is a plot of expression difference of characteristic peak 2878, and 9D is a plot of expression difference of characteristic peak 3058;

[0040] Figure 10 Figure 10 is a plot showing that the expression of TRBC2 protein in the synovial tissue of gouty arthritis patients (experimental group) is significantly higher than that in the synovial tissue of sports meniscus tear patients (control group), wherein 10A is a plot of expression of characteristic peak 1002, 10B is a plot of expression of characteristic peak 1332, 10C is a plot of expression of characteristic peak 2878, and 10D is a plot of expression of characteristic peak 3058.

[0041] Figure 11 Figure 12A and 12B are the peak importance analysis graphs, wherein 12A is the regression coefficient graph reflecting the direct contribution and predictive force of each peak to sample classification, and 12B is the peak variable importance projection score ordering graph reflecting the top 10 peaks of the two groups of samples;

[0042] Figure 12 Figure 12A and 12B are the peak importance analysis graphs, wherein 12A is the regression coefficient graph reflecting the direct contribution and predictive force of each peak to sample classification, and 12B is the peak variable importance projection score ordering graph reflecting the top 10 peaks of the two groups of samples;

[0043] Figure 13 Figure 12A and 12B are the peak importance analysis graphs, wherein 12A is the regression coefficient graph reflecting the direct contribution and predictive force of each peak to sample classification, and 12B is the peak variable importance projection score ordering graph reflecting the top 10 peaks of the two groups of samples;

[0044] Figure 14 Figure 12A and 12B are the peak importance analysis graphs, wherein 12A is the regression coefficient graph reflecting the direct contribution and predictive force of each peak to sample classification, and 12B is the peak variable importance projection score ordering graph reflecting the top 10 peaks of the two groups of samples; DETAILED DESCRIPTION

[0045] The following provides definitions for some of the terms used in this specification. Unless otherwise defined, 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 application belongs.

[0046] The present application provides the use of a reagent for detecting the level of TRBC2 in the preparation of a product for diagnosing gout.

[0047] In some embodiments, TRBC2 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed TRBC2, as well as any form of TRBC2 that results from processing in cells. The term encompasses naturally occurring variants of TRBC2 (e.g., splice variants or allelic variants). The term encompasses TRBC2 of human and any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats), for example, GenBank ID: 28638.

[0048] The reagent includes a binding agent specifically binding to TRBC2 protein, a primer specifically amplifying TRBC2, a probe specifically recognizing TRBC2.

[0049] In some embodiments, a primer refers to a short nucleic acid molecule, such as a DNA oligonucleotide, that can anneal to a complementary target nucleic acid molecule by nucleic acid hybridization, forming a hybrid between the primer and the target nucleic acid strand. The primer can be extended by a polymerase along the target nucleic acid molecule. Thus, a primer can be used to amplify a target nucleic acid molecule, where the sequence of the primer is specific for the target nucleic acid molecule, e.g., the primer will hybridize to the target nucleic acid molecule under very high stringency hybridization conditions.

[0050] In some embodiments, a probe can be DNA, RNA, DNA-RNA chimera, PNA, or other derivatives. The length of the probe is not limited, as long as specific hybridization is achieved, i.e., specific binding to the nucleotide sequence of interest, any length can be used. The length of the probe can be as short as 25, 20, 15, 13, or 10 bases in length. Likewise, the length of the probe can be as long as 60, 80, 100, 150, 300 bases or longer, even an entire gene.

[0051] In some embodiments, a primer or probe can be chemically synthesized using phosphoramidite solid support methods or other well known methods. Modifications can also be used using a number of means known in the art. Non-limiting examples of these modifications include methylation, capping, substitution with one or more analogs of natural nucleotides, and modifications between nucleotides. For example, uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) can be modified, or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.) can be modified.

[0052] The reagent also includes a reagent for detecting the expression level of TRBC2 gene or protein by PCR method, such as quantitative PCR assay (qPCR), reverse transcriptase quantitative PCR (RT-qPCR); gene chip detection method, such as DNA chip, RNA chip, protein chip; NGS detection method (Next Generation Sequencing, also known as high-throughput sequencing technology), such as RNA or DNA sequencing (RNAseq / DNAseq); nucleic acid probe method, such as DNA probe, RNA probe, oligonucleotide probe; ELISA method (Enzyme-Linked Immunosorbent Assay); immunoblotting method (Immunoblotting, also known as Western Blotting); mass spectrometry method (Mass Spectrometry, MS); immunohistochemistry (Immunohistochemistry, IHC); Raman spectroscopy, such as spontaneous Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), resonance Raman spectroscopy (RRS), Fourier transform Raman spectroscopy (FT-Raman) and the like.

[0053] The reagent also includes a detectable label.

[0054] In some embodiments, the detectable label refers to a composition capable of producing a detectable signal indicating the presence of a target polynucleotide in an assay sample. Suitable detectable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Thus, a label is any composition capable of being detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection devices or any other suitable device. In some embodiments, the label can be detected visually without the aid of a device.

[0055] Among them, the radioisotope includes but is not limited to 3 H, 14 C, 35 S, 125 I, 131 I.

[0056] The enzyme includes but is not limited to horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

[0057] The fluorescent molecule includes but is not limited to FITC, rhodamine, lanthanide phosphor.

[0058] The product includes a kit, a test paper, a chip, a nucleic acid membrane strip.

[0059] In some embodiments, a chip, also referred to as an array, refers to a solid support comprising attached nucleic acid or peptide probes. An array typically comprises a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also referred to as "microarrays", can typically be produced using mechanical synthesis methods or light-directed synthesis methods, which incorporate a combination of photolithographic methods and solid-phase synthesis methods. Arrays can comprise a flat surface, or can be nucleic acids or peptides on beads, gels, polymeric 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 chip includes a gene chip, a protein chip.

[0061] In some embodiments, the gene chip includes a solid support and probes immobilized on the solid support, the probes including oligonucleotide probes for TRBC2 gene for detecting the transcription level of TRBC2 gene; the protein chip includes a solid support and antibodies specific to TRBC2 protein immobilized on the solid support; the gene chip can be used to detect the expression level of multiple genes including human TRBC2 gene (e.g., multiple genes associated with gout). The protein chip can be used to detect the expression level of multiple proteins including human TRBC2 protein (e.g., multiple proteins associated with gout). By simultaneously detecting multiple markers associated with gout, the accuracy of diagnosing gout can be greatly improved.

[0062] In some embodiments, the kit can further include a fluorescent dye, and various known fluorescent dyes can be used. For example, methods using an intercalator having a labeling function, methods using a probe that binds a fluorescent substance on a nucleotide that specifically hybridizes to a relatively amplified DNA sequence, and the like can be listed. As the intercalator, ethidium bromide, SYBR Green I, Resolight, and EvaGreen, which are unsaturated fluorescent dyes, can be listed. As for the amount of use, the recommendations of the manufacturer of the fluorescent dye used are followed.

[0063] The kit can further include an instruction manual, a buffer. The instruction manual can include guidance on obtaining a sample, processing the sample. In addition, the kit can contain genomic DNA of bacteria used as a positive control for PCR, and sterile water used as a negative control.

[0064] In some embodiments, the components of the kit can be packaged in an aqueous medium or in a lyophilized form. Suitable containers for the kits are known to those of skill in the art and are available from commercial suppliers. Generally, a kit will include at least one vial, test tube, flask, bottle, syringe or other container, into which a component can be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit can also contain a second, third or additional containers to maintain separate the components. However, different components of the kit can be contained in a single vial. The kits of the application will also typically include a container into which the reagents can be contained, sealed for commercial sale. Such containers can include injection or blow molded plastic containers into which the desired vials are retained.

[0065] The solid support of the kit can be, for example, plastic, silicon wafer, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide. The biological sample can be, for example, a 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 comprises a substrate and probes immobilized on the substrate; the substrate can be any substrate suitable for immobilizing probes, including but not limited to nylon membrane, nitrocellulose membrane, polypropylene membrane, glass slide, silicon wafer, microbead.

[0067] The present application provides a gout diagnosis system / device based on Raman spectroscopy.

[0068] In some embodiments, the disclosed system / device can be implemented in other manners. For example, the division of the system / device embodiments described above is merely an example, and there can be other division manners. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. In addition, the display or discussion of relative coupling or direct coupling or communication connection between the units can be indirect coupling or communication connection through some interfaces, devices or units, and can be electrical, mechanical or other forms.

[0069] Those of ordinary skill in the art can understand that all or part of the steps of the above-mentioned various methods can be completed by a program instructing related hardware, and the program can be stored in a computer readable storage medium, such as a read only memory (ROM), a random access memory (RAM), a magnetic disk or an optical disk.

[0070] The application will be further described in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations. The principal features of the present application can be employed in various embodiments without departing from the scope of the application.

[0071] Example 1 Training set case collection

[0072] 1. Experimental materials

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

[0074] Inclusion criteria for patients with gouty arthritis:

[0075] Inclusion criteria

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

[0077] b. Those with definite double contour, synovial thickening (≥ 4 mm), Doppler blood flow signal (grade ≥ 2), and / or joint effusion and urate crystal deposition.

[0078] c. Those aged 18 to 65 years, regardless of gender.

[0079] d. Those who are informed and agree to participate in the study.

[0080] Exclusion criteria

[0081] a. Those with severe joint deformity, multiple gouty tophi with local ulceration.

[0082] b. Those with serious lesions of important organs or mental diseases.

[0083] c. Pregnant or lactating patients.

[0084] d. Patients with gouty kidney damage.

[0085] Inclusion and exclusion criteria for patients with meniscal tear caused by sports injury:

[0086] Inclusion criteria

[0087] a. Those with a clear history of sports injury, which may be accompanied by knee joint twisting, overflexion or direct impact when the injury occurs.

[0088] b. Imaging examination confirmed as traumatic meniscus tear, the diagnostic criteria refer to the Consensus and Practice Guidelines for Lateral Meniscus Injury formulated by the Sports Medicine Branch of the Chinese Medical Association in 2022.

[0089] c. 18-45 years old, male and female.

[0090] d. The patient is informed and agrees to participate in this study.

[0091] Exclusion criteria

[0092] a. Surgical contraindication.

[0093] b. Accompanied by congenital deformity of meniscus.

[0094] c. Degenerative meniscus injury.

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

[0096] e. Accompanied by important organ dysfunction.

[0097] f. Accompanied by rheumatic diseases.

[0098] 2) Staining reagent

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

[0100] 2. Experimental method

[0101] 1) Ultrasonic diagnosis

[0102] Understand the patient's history (acute attack history, chronic course, gout stone history), symptoms (joint red, swollen, hot, painful), signs (joint tenderness, limited activity), blood uric acid level, etc. before examination. Joint ultrasound examination to determine knee joint "double track sign", synovial thickening (≥4mm), Doppler blood flow signal (grade ≥2), and / or joint effusion and urate crystal deposition. The diagnostic criteria meet the 2015 American College of Rheumatology and European League Against Rheumatism (ACR / EULAR) gout classification criteria (clinical score ≥8).

[0103] 2) Pathological tissue section and diagnosis

[0104] According to the standard of emission reduction, patients with meniscus tear caused by sports injury are taken as the control group (C), and patients with gouty arthritis are taken as the experimental group (T). Synovial tissue left after arthroscopic surgery of patients with meniscus tear caused by sports injury or gouty arthritis is taken for subsequent pathological tissue sectioning. The left synovial tissue is immediately placed in a 10% neutral formaldehyde solution for fixation, usually for about 24-48 hours to prevent autolysis and denaturation of the tissue. Then, the fixed tissue is dehydrated by placing it in different concentrations of ethanol solution, with each concentration of ethanol being soaked for 30 minutes to 1 hour. The tissue is transparentized. The dehydrated tissue needs to be transparentized with xylene. The tissue is embedded by placing the transparentized tissue in paraffin, and the tissue is completely infiltrated with paraffin through multiple wax immersion, and then the tissue is embedded in a paraffin block. The tissue is sectioned, and the section thickness is 2.5 pm.

[0105] HE section staining is performed. First, hematoxylin staining is soaked for 3-5 minutes. Water washing. Differentiation with 1% hydrochloric acid alcohol for 10-30 seconds. Water washing. Blueing: soak in weak alkaline water (such as 0.5% sodium bicarbonate solution) for 1-2 minutes to make the cell nucleus blue. Eosin staining is soaked for 1-2 minutes. Water washing: rinse with tap water. Dehydration, transparentization and mounting are performed again, with dehydrating using 95% ethanol for 30 seconds and 100% ethanol for 30 seconds. Transparentization is performed using xylene I for 2 minutes and xylene II for 2 minutes, and mounting is performed using neutral gum or mounting agent to store the section. The HE sections are independently interpreted by two middle-aged pathologists. When there is a difference of opinion, the sections are re-examined and the areas of difference are focused on, and a third senior pathologist is consulted to provide a third-party opinion and make a final decision.

[0106] 3. Experimental results

[0107] 1) Ultrasonic diagnosis

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

[0109] 2) Synovial pathological diagnosis of gouty arthritis

[0110] The synovial tissue of the control group of patients with torn meniscus showed 1-2 layers of synoviocytes on the surface of the cavity, loose connective tissue and adipose tissue under the synovium, and no obvious fibrovascular hyperplasia, inflammatory cell infiltration and giant cell reaction. The synovial tissue of the experimental group of patients with gouty arthritis showed synovitis, with diffuse distribution of urate crystals of different sizes without cell mass, no giant cell reaction around the cell mass, and interstitial fibrosis, mainly with lymphocyte infiltration Figure 2 ).

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

[0112] 1. Experimental materials

[0113] Cell capture and cDNA synthesis used 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 suspension (300-600 live cells per microliter) was loaded onto a Chromium single-cell controller (10x Genomics) to prepare single-cell gel beads. Briefly, single cells were suspended in PBS containing 0.04% BSA. About 6000 cells were loaded per lane, covering about 3000 target cells. The captured cells were lysed, and the released RNA was labeled by reverse transcription in single GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (BioRad) at 53°C for 45 minutes, then at 85°C for 5 minutes, and stored at 4°C. cDNA was generated, then amplified, and quality assessment was performed using an Agilent 4200. Single-cell RNA-Seq library preparation used single-cell 3' library and gel bead kit V3.1 to construct the library. Sequencing was performed using an Illumina Nova Seq 6000 sequencer, with a sequencing depth of at least 100,000 reads per cell, and 150 bp for both ends.

[0117] 2) Bioinformatics data analysis

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

[0119] Enrichment analysis was performed, including GO enrichment, KEGG enrichment, Reactome enrichment, and disease enrichment. Cluster markers were adjusted by Benjamin-Hochberg multiple test using KOBAS software; the top 20 marker genes were used. The R package was used to visualize the results.

[0120] Cell types were annotated by SingleR (https: / / bioconductor.org / packages / devel / bioc / html / SingleR.html). SingleR can independently infer the source cell of each single cell by using reference transcriptome data sets of pure cell types, so as to perform unbiased cell type identification from single cell RNA sequencing data. For human samples, Blueprint_Encode or HPCA was used.

[0121] 3. Experimental results

[0122] 1) Single cell transcriptome sequencing

[0123] 6 human synovial tissues were captured 61215 cells, cell reads 87% Reads, and the median number of genes per cell was 2206. According to the differential gene expression of subpopulation and GO BP enrichment analysis, 22 cell subpopulations were identified (Table 1) Figure 3 ).

[0124] 2) T cell subpopulation analysis

[0125] Compared with the synovial tissue of the control group (patients with meniscus tear caused by sports injury), the number of T cell subpopulation (No. 4) in the synovial tissue of gouty arthritis was significantly increased Figure 4). The TRBC2 gene is a hallmark of this subgroup and is highly expressed in T cell subgroups and synovial tissue of gouty arthritis (Gout) Figure 5 ). TRBC2 is involved in the T cell receptor signaling pathway; adaptive immune response; and alpha-beta T cell activation. TRBC2 is part of the alpha-beta T cell receptor complex and is the T cell receptor (TR) beta chain constant region. The alpha-beta T cell receptor is an antigen-specific receptor that is essential for the immune response and is present on the cell surface of T lymphocytes. Recognition of peptide-major histocompatibility complex (pMHC) displayed by antigen-presenting cells (APCs) is a prerequisite for effective T cell adaptive immunity against pathogens. Binding of the alpha-beta TR to pMHC triggers TR-CD 3 clustering on the cell surface and intracellular activation of LCK, which phosphorylates the ITAM motifs of CD 3G, CD 3D, CD 3E, and CD 247, enabling the recruitment of ZAP 70. In turn, ZAP 70 phosphorylates LAT, which recruits many signaling molecules to form the LAT signalosome. The LAT signalosome propagates the signal branch to three major signaling pathways, the calcium, mitogen-activated protein kinase (MAPK) kinase, and nuclear factor NF-κB (NF-κB) pathways, leading to the mobilization of transcription factors that are critical for gene expression and essential for T cell growth and differentiation.

[0126] Example 3 Validation set case collection

[0127] 1) Clinical patients

[0128] Synovial tissues of 30 patients with meniscus tear caused by sports injury (control group) and 30 patients with gouty arthritis (experimental group) were collected, respectively. The collection site was Wanging Hospital of China Academy of Chinese Medical Sciences in Beijing, China, and the collection time was from January 2022 to January 2023. This study was approved by the hospital 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] Gouty arthritis patient inclusion and exclusion criteria:

[0134] Inclusion criteria

[0135] a. Meet the 2015 American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) classification criteria for gout (clinical score ≥8 points).

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

[0137] c. 18-65 years old, male and female.

[0138] d. The patient is informed and agrees to participate in this study.

[0139] Exclusion criteria

[0140] a. Patients with severe joint deformity, multiple gouty tophi with local ulceration.

[0141] b. Patients with serious lesions of important organs or mental diseases.

[0142] c. Pregnant or lactating patients.

[0143] d. Patients with gouty kidney damage.

[0144] Inclusion criteria for meniscal tear caused by sports injury:

[0145] Inclusion criteria

[0146] a. There is a clear history of sports injury, which may be accompanied by knee joint twisting, overflexion or direct impact when injury occurs.

[0147] b. Diagnosed as traumatic meniscal tear by imaging examination, the diagnostic criteria refer to the "Consensus and Practice Guidelines for Lateral Meniscus Injury" formulated by the Sports Medicine Branch of the Chinese Medical Association in 2022.

[0148] c. 18-45 years old, male and female.

[0149] d. The patient is informed and agrees to participate in this study.

[0150] Exclusion criteria

[0151] a. Contraindication for surgery.

[0152] b. Associated with congenital deformity of meniscus.

[0153] c. Degenerative meniscal injury.

[0154] d. Associated with knee fracture, cruciate ligament injury, collateral ligament injury.

[0155] e. with major organ dysfunction.

[0156] f. with rheumatic disease.

[0157] 2) Spontaneous Raman spectroscopy detection and imaging

[0158] All samples were prepared into paraffin sections, after the sections were completely deparaffinized, spontaneous Raman spectroscopy detection and imaging were used. All spectra were collected using a WITec alpha 300R + Raman confocal microspectrometer (Ulm, Germany), which was equipped with a piezoelectric stage (UHTS 300, WITec, GmbH.), a 100x air objective (Zeiss EC EPIPLAN, N.A. = 0.90), a green solid-state excitation laser (λ = 532 nm, 32 mW, WITec, GmbH.), and an imaging spectrometer (Newton, Andor Technology Ltd., UK) equipped with a 600 groove / mm grating and a thermoelectrically cooled (60 °C) charge-coupled detector. The grating and the detector were optically connected to the objective through a single-mode silica fiber cable with a diameter of 10 μm. The size of the laser excitation spot was 350 nm, and spectral data in the wavelength range of 0-3600 cm -1 were obtained. In all cases, the excitation laser intensity remained unchanged between sample scans, the integration time was 0.1 seconds, and the fast scan area was 8 μm*8 μm, and then the average of 100 spectra was used to represent a unit. 50 single spectra were collected for each sample, and the sample was imaged.

[0159] 3) Raman data processing

[0160] All model analyses were performed using Python 3.9 language. Libraries used include but are 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-based models, the feature value data was reduced by PCA to ensure variable independence and reduce the dimension to keep the explainable variance not less than 99%. In image or sequence models (GRU, CNN), the feature value data used the full spectrum data.

[0161] 3, Experimental results

[0162] 1) Accuracy quality control of synovial tissue Raman spectroscopy sample and TRBC2 protein detection

[0163] Raman spectroscopy provides evidence for identification and characterization of materials by analyzing the vibrational modes of molecules. Different substances have their unique "fingerprint" Raman spectra, reflecting their chemical structure, functional groups and interactions. All 60 samples were subjected to HE staining and histopathological evaluation consistent with the training set described above ( Figure 6 ), and sample quality control of Raman spectroscopy. The results showed that all samples had similar Raman spectral characteristics, with good sample uniformity ( Figure 7 ). Human recombinant TRBC2 protein was used as a standard to identify and compare the synovial tissue samples to be tested. The Raman spectrum showed that TRBC2 protein and protein-related functional groups were present in the synovial tissue, and there was no difference in peak, ensuring the accuracy of the qualitative and quantitative results ( Figure 8 ). Further analysis of the intensity difference of the main peaks of TRBC2 protein in the sample is needed to perform quantitative analysis.

[0164] 2) Analysis of TRBC2 protein single spectral peak

[0165] The standard TRBC2 protein had 4 main peaks, and the detection results of the 4 main peaks showed that the expression of TRBC2 protein in the synovial tissue of 30 patients with gouty arthritis (experimental group) was significantly stronger than that in the synovial tissue of 30 patients with meniscus tear (control group) ( Figure 9 and Figure 10 ). The diagnostic efficiency of the 4 main characteristic peaks was analyzed, and the results are shown in Table 1 and Figure 11 , the AUC of characteristic peaks 1002, 2878 and 3058 were all greater than 0.7, with good diagnostic efficiency. Further based on OPLS-DA classification, the top 10 Raman peaks of markers in gouty arthritis synovial tissue were analyzed according to the size of regression coefficient, and the variable importance projection score sorting of the same peak values was presented, and TRBC2 characteristic peaks 1002 and 2878 were located in the front row of distinguishing markers ( Figure 12 ). Binary Logistic regression analysis was performed on the 4 main characteristic peaks to calculate the prediction probability value, and the ROC curve of TRBC2 protein was drawn. The AUC value was 0.996, the specificity was 100%, and the sensitivity was 93.3% ( Figure 13 ). The results showed that TRBC2 protein had good diagnostic efficiency for gouty arthritis.

[0166] Table Diagnostic efficiency of TRBC2 protein Raman main characteristics for synovial tissue of gouty arthritis patients (experimental group)

[0167]

[0168] 3) Analysis of TRBC2 protein Raman imaging

[0169] Raman imaging analysis was performed on synovial tissue of patients with gouty arthritis (experimental group) and patients with torn sports meniscus (control group), respectively. The results showed that TRBC2 protein was enriched around the urate crystals in the synovial tissue of patients with gouty arthritis, and showed obvious specific aggregation (P < 0.05) Figure 14 ).

[0170] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. Application of reagents for detecting TRBC2 levels in the preparation of products for diagnosing gout.

2. The application according to claim 1, characterized in that, The TRBC2 level includes any one or more of the characteristic peaks of TRBC2, namely 1002, 1332, 2878, and 3058.

3. The application according to claim 1, characterized in that, The reagents include a binding agent that specifically binds to the TRBC2 protein, primers that specifically amplify TRBC2, and probes that specifically recognize TRBC2.

4. The application according to claim 3, characterized in that, The reagents also include those for detecting the expression level of TRBC2 gene or protein by PCR, gene chip detection, NGS detection, nucleic acid probe method, ELISA, immunoblotting, 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, substrates, chemiluminescent components, magnetic particles, and bioluminescent components.

7. A product for diagnosing gout, characterized in that, The product includes reagents for detecting TRBC2 levels.

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

9. Application of TRBC2 in the preparation of models / systems / devices for diagnosing gout.

10. 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 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.

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