Use of CITED4 and / or METRN in the differential diagnosis of intervertebral disc degeneration grades

Abstract

The present invention discloses the use of CITED4 and / or METRN in the differential diagnosis of the degree of intervertebral disc degeneration. CITED4 and METRN show significant differences in tissues with different degrees of intervertebral disc degeneration, and are highly valuable in the differential diagnosis of the degree of intervertebral disc degeneration. Therefore, they can be used in the differential diagnosis of intervertebral disc degeneration and the evaluation of the effectiveness of related intervention measures. This provides a new approach to the differential diagnosis of the degree of intervertebral disc degeneration in this field, and has important popularization and application value.

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedical technology, and specifically, the present invention relates to the use of CITED4 and / or METRN in the differential diagnosis of the degree of intervertebral disc degeneration.

[0002] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to an invention patent application entitled "USE OF CITED4 AND / OR METRN IN THE DIFFERENTIAL DIAGNOSIS OF INTERVERTEBRAL DISC DEGENERATION DEGREE," Application No. 2023102156761, filed March 8, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Intervertebral disc degeneration (IDD), also known as intervertebral disc degeneration, is a degenerative change caused by aging, decreased mobility, decreased lumbar spine stability, and increased stress on the intervertebral disc. It is commonly seen in middle-aged and elderly people. When the stress on the intervertebral disc exceeds its maximum capacity, the annulus fibrosus ruptures, eventually leading to the extrusion of the nucleus pulposus, resulting in lower back and leg pain. Intervertebral disc degeneration is one of the leading causes of lower back pain. Statistics show that approximately 84% of adults experience lower back pain at least once in their lifetime, with the majority of cases being due to disc degeneration. Severe disc degeneration can also lead to sensory impairment in the lower limbs, fecal incontinence, and urinary incontinence. Disc degeneration is a significant factor in permanent disability worldwide, and as the global population ages, disc degeneration poses an increasingly serious health threat. Therefore, early diagnosis of intervertebral disc degeneration and quantification of the degree of disc degeneration are of great clinical value for timely intervention and treatment of intervertebral disc degeneration and delaying further progression of intervertebral disc degeneration.

[0004] Currently, clinical diagnosis of intervertebral disc degeneration is still primarily based on imaging, using imaging techniques such as magnetic resonance imaging and X-rays to observe abnormal changes in the intervertebral disc and determine the degree of degeneration of the intervertebral disc tissue. However, compared to traditional histopathological diagnosis and histopathology-based molecular pathology diagnostic techniques, imaging diagnosis is highly specific, non-invasive, and safe. However, because changes in tissue imaging characteristics are slow, using imaging characteristics as markers of intervertebral disc degeneration still has certain limitations in early basic research exploring treatment plans, and in subsequent clinical diagnosis and optimization of treatment plans. Searching for diagnostic biomarkers of intervertebral disc degeneration at deeper cellular and molecular levels may be potentially meaningful and valuable for clinical diagnosis and treatment. Abnormal changes in molecular biology usually accompany or precede changes in the morphology of diseased tissue, and changes in gene and protein levels are usually more sensitive than changes in the morphology of diseased tissue. Research has shown that degeneration of nucleus pulposus tissue occurs earlier than that of other intervertebral disc tissues and is closely associated with cellular senescence, apoptosis, autophagy, changes in extracellular matrix homeostasis, and a decrease in the water content of the nucleus pulposus. Chondrocytes occupy the majority of the adult nucleus pulposus, bear significant loads, and are an important component in maintaining the biological activity of the intervertebral disc. Therefore, exploring the molecular characteristics of chondrocytes in degenerated nucleus pulposus tissue is a hot topic in this field.

[0005] Currently, there are no studies or reports on the use of CITED4 and / or METRN in the differential diagnosis of disc degeneration grades. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems currently existing in the field, the present invention provides the use of CITED4 and / or METRN in the differential diagnosis of the degree of intervertebral disc degeneration.

[0007] The present invention utilizes scRNA-seq and bioinformatics analysis techniques to analyze the single-cell gene expression profiles of clinically collected human intervertebral disc nucleus pulposus tissues. This allowed the inventors to isolate, characterize, and identify a chondrocyte subpopulation that can reflect the degenerative process of the intervertebral disc. Through further research, the inventors identified two new signature genes, CITED4 and METRN, expressed by this cell subpopulation. These signature genes have high diagnostic value in the differential diagnosis of the degree of degeneration of the intervertebral disc nucleus pulposus tissue and are important for indicating the occurrence and progression of pathological degeneration of the intervertebral disc nucleus pulposus tissue.

[0008] In order to achieve the above object, the present invention uses the following technical means.

[0009] A first aspect of the present invention provides the use of a reagent for detecting the expression level of CITED4 and / or METRN in a sample in the manufacture of a product for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the efficacy of a treatment for intervertebral disc degeneration.

[0010] Furthermore, the reagent a reagent for detecting the mRNA expression level of CITED4 and / or METRN in a sample; a reagent for detecting the protein expression level of CITED4 and / or METRN in a sample; or The present invention includes a reagent for detecting the number of CITED4 and / or METRN positive expressing cells in a sample.

[0011] Furthermore, the reagent for detecting the mRNA expression level of CITED4 and / or METRN in the sample comprises: Primers that specifically amplify CITED4 and / or METRN, or It includes probes that specifically recognize CITED4 and / or METRN.

[0012] Furthermore, the reagent for detecting the protein expression level of CITED4 and / or METRN in the sample comprises: an antibody that specifically binds to a protein encoded by CITED4 and / or METRN, or It includes an affinity protein that specifically binds to a protein encoded by CITED4 and / or METRN.

[0013] Furthermore, the reagent for detecting the number of CITED4 and / or METRN positive expressing cells in the sample includes a reagent for detecting the number of CITED4 and / or METRN positive expressing cells by immunohistochemistry.

[0014] In the present invention, information on the CITED4 and METRN genes is as follows: the gene ID of the CITED4 gene (Cbp / p300 interacting transactivator with Glu / Asp-rich carboxy-terminal domain 4) is 163732, and the gene ID of the METRN gene (meteorin, glial cell differentiation regulator) is 79006. Detailed information on the genes can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0015] In the present invention, the CITED4 and METRN genes include the human CITED4 and METRN genes, as well as polynucleotides functionally equivalent to either the human CITED4 and METRN genes. These may be naturally occurring or artificially synthesized, or may be obtained by introducing a vector capable of expressing a DNA fragment of CITED4 or METRN into a cell. Examples of such vectors include viral vectors and eukaryotic cell expression vectors. Examples of such viral vectors include, but are not limited to, any suitable vector, such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpesvirus (e.g., herpes simplex virus, vaccinia virus, and EB virus) vectors, and alphavirus vectors. The eukaryotic cell expression vector may be any suitable expression vector, including, but not limited to, pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEF Bos expression vector, pTet expression vector, pTRE expression vector, or vectors modified based on known expression vectors such as pBin438 and pCAMBIA1301.

[0016] Furthermore, the primer is an amplification primer and refers to a nucleic acid fragment containing 5 to 100 nucleotides. Preferably, the primer or amplification primer contains 15 to 30 nucleotides capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction). In a specific embodiment of the present invention, the primer is a primer that specifically amplifies the gene CITED4 and / or METRN.

[0017] Furthermore, the term "probe" refers to a molecule capable of binding to a specific sequence, subsequence, or other portion of another molecule. In a specific embodiment of the present invention, the term "probe" refers to a probe that specifically recognizes CITED4 and / or METRN. Unless otherwise specified, the term "probe" generally refers to a polynucleotide probe capable of binding to another polynucleotide (often referred to as a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that does not have complete sequence complementarity with the probe. Hybridization methods include, but are not limited to, liquid-phase, solid-phase, mixed-phase, or in situ hybridization. Exemplary probes in the present invention include gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection probes, probes linked to molecular barcodes, and probes immobilized on beads.

[0018] The stringency of a hybridization reaction can be easily determined by one skilled in the art and is usually calculated empirically based on the probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for accurate annealing, while shorter probes require lower temperatures. Hybridization typically relies on the ability of denatured DNA to reanneal when a complementary strand is present in an environment below its melting point. The higher the required homology between the probe and the hybridizable sequence, the higher the usable relative temperature. Consequently, it is expected that higher relative temperatures result in more stringent reaction conditions, while lower relative temperatures result in more lenient reaction conditions.

[0019] Furthermore, reagents that specifically bind to proteins encoded by CITED4 and / or METRN include, but are not limited to, antibodies, affinity proteins, and further include peptides, aptamers and / or compounds that specifically bind to proteins encoded by CITED4 and / or METRN.

[0020] Furthermore, the term "antibody" is well known in the art and refers to an immunoglobulin specific to an antigenic site. The antibody of the present invention refers to an antibody that specifically binds to a protein encoded by CITED4 and / or METRN of the present invention, and can be produced according to a method commonly used in the art. Antibody forms include polyclonal or monoclonal antibodies, antibody fragments (Fab, Fab', F(ab')2, Fv fragments, etc.), single-chain Fv (scFv) antibodies, multispecific antibodies (bispecific antibodies, etc.), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, as long as they exhibit the desired biological binding activity.

[0021] Furthermore, the peptide has high binding affinity to target substances (proteins encoded by CITED4 and / or METRN described in the present invention) and is not denatured by heat or chemical treatment. Furthermore, due to its small size, it can be attached to other proteins to form fusion proteins. Specifically, because it can specifically attach to polymeric protein chains, it can be used as a diagnostic kit and drug delivery material.

[0022] Furthermore, the term "aptamer" refers to a polynucleotide consisting of a specific type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid), and the single-stranded nucleic acid itself has a stable tertiary structure and is capable of binding to a target molecule (a protein encoded by CITED4 and / or METRN described in the present invention) with high affinity and specificity. As described above, aptamers can specifically bind to antigenic substances like antibodies, but because they are composed of polynucleotides that are more stable, have a simpler structure, and are easier to synthesize than proteins, they can be used instead of antibodies.

[0023] Furthermore, the reagents for detecting the number of CITED4 and / or METRN-positive cells by immunohistochemistry include any reagents necessary for detecting the number of CITED4 and / or METRN-positive cells by immunohistochemistry, such as, but not limited to, fixatives, buffers, coloring solutions, adhesives, mounting media, enzyme digestion solutions, and sucrose solutions. Fixatives include, but are not limited to, formaldehyde, glutaraldehyde, paraformaldehyde, ethanol, HneFIX, and acetone. Buffers include, but are not limited to, PBS buffer, citrate buffer, EDTA buffer, and TBS buffer. Coloring solutions include, but are not limited to, DAB coloring solution, 4-chloro-1-naphthol (4-Cl-1-Naphthol) coloring solution, 3-amino-9-ethylcarbozole (AEC) coloring solution, TMB coloring solution, and NBT coloring solution. The adhesives include, but are not limited to, gelatin, resin adhesives, polylysine, and commercially available adhesives. The mounting medium includes, but is not limited to, non-fat dry milk, BSA, serum, and Fab fragment single-chain secondary antibodies. The enzyme digestion solution includes, but is not limited to, trypsin digestion solution and pepsin digestion solution.

[0024] Furthermore, the differential diagnosis of the degree of intervertebral disc degeneration refers to the differential diagnosis of the degree of intervertebral disc degeneration in a subject. In a specific embodiment of the present invention, the differential diagnosis of the degree of intervertebral disc degeneration particularly refers to the differential diagnosis of moderate and severe degenerated tissue, mild and severe degenerated tissue, and mild, moderate, and severe degenerated tissue using CITED4 and / or METRN. Experimental verification has shown that the AUC values ​​of CITED4 and / or METRN in the differential diagnosis of tissues with different degeneration degrees are all above 0.9, demonstrating high sensitivity, specificity, and diagnostic efficiency.

[0025] Furthermore, evaluating the therapeutic effect of intervertebral disc degeneration refers to evaluating the effect of a specific treatment or therapeutic drug on a subject. In a specific embodiment of the present invention, evaluating the therapeutic effect of intervertebral disc degeneration refers to differentially diagnosing moderate and severe degeneration, mild and severe degeneration, and mild, moderate, and severe degeneration in a subject with degenerated intervertebral disc, particularly using CITED4 and / or METRN, to further evaluate the effect of a specific treatment or therapeutic drug on the subject, which has good prospects for clinical use.

[0026] Furthermore, the expression level refers to the absolute or relative amount of CITED4 and / or METRN described in the present invention, and the expression level of CITED4 and / or METRN described in the present invention can be determined by various techniques well known to those skilled in the art, in particular, the absolute or relative amount of CITED4 and / or METRN described in the present invention can be detected using immunohistochemical detection methods well known to those skilled in the art.

[0027] Furthermore, the term "sample" refers to a composition obtained from or derived from a subject of interest, including cellular and / or other molecular entities characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological properties. The sample can be obtained from the subject's blood, other liquid samples of biological origin, and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The tissue sample may be derived from, for example, fresh, frozen, and / or preserved organ or tissue samples, solid tissues such as biopsy tissue or aspirates, blood or any blood component, bodily fluids, cells from an individual at any time during pregnancy or development, or plasma. The term "sample" also includes biological samples that have been processed in any way after collection, such as by treatment with reagents, stabilization, enrichment for specific components (e.g., proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning.

[0028] Furthermore, the sample includes, but is not limited to, tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural effusion, peritoneal fluid, bladder washings, secretions (e.g., mammary secretions), oral washes, swabs (e.g., oral swabs), imprint cytology specimens, fine needle aspiration samples, cell extracts, and combinations thereof. In a specific embodiment of the present invention, the sample is preferably a tissue sample from a subject, and more preferably a tissue sample of a degenerated intervertebral disc from a subject.

[0029] Furthermore, the differential diagnosis refers to detecting, determining, differentiating, or recognizing an individual's health condition or status based on one or more symptoms, data, or other information about the individual. In a specific embodiment of the present invention, the differential diagnosis of the degree of intervertebral disc degeneration refers to distinguishing between individuals with different degrees of intervertebral disc degeneration, particularly between individuals with moderate and severe intervertebral disc degeneration, mild and severe intervertebral disc degeneration, and mild, moderate, and severe intervertebral disc degeneration.

[0030] Additionally, the reagents detect CITED4 and / or METRN expression levels in a sample from a subject by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques, protein immunological techniques, immunohistochemical techniques.

[0031] Furthermore, the sequencing technology is a nucleic acid sequencing technology, including chain terminator (Sanger) sequencing technology and dye terminator sequencing technology.Those skilled in the art will understand that because RNA is less stable in cells and more susceptible to nuclease attack in experiments, it is common to reverse transcribe RNA into DNA before sequencing.In addition, the sequencing technology also includes next-generation sequencing technology (i.e., deep sequencing / high-throughput sequencing technology).

[0032] Additionally, said nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarrays, and Southern or Northern blotting.

[0033] Furthermore, the nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

[0034] Furthermore, the protein immunological techniques include sandwich immunoassays, such as sandwich ELISA, which detect the marker using two antibodies that recognize different epitopes on CITED4 and / or METRN, radioimmunoassays (RIA), direct, indirect or control enzyme-linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), fluorescence immunoassays (FIA), Western blotting, immunoprecipitation, and any particle (e.g., gold particles, silver particles, latex particles, magnetic particles or quantum dots)-based immunoassays.

[0035] A second aspect of the present invention provides a product for the differential diagnosis of the degree of disc degeneration or for the evaluation of the effectiveness of a treatment for disc degeneration.

[0036] Additionally, the article of manufacture includes reagents for detecting the expression level of CITED4 and / or METRN in a sample.

[0037] Additionally, the products include detection kits and biochips.

[0038] The detection kit further comprises primers and probes that specifically bind to CITED4 and / or METRN, The biochip comprises a solid phase carrier and a probe attached to the solid phase carrier and specifically recognizing CITED4 and / or METRN.

[0039] Furthermore, the detection kit further comprises one or more substances selected from the group consisting of a container, an instruction manual, a positive control, a negative control, a buffer, an auxiliary agent, or a solvent.

[0040] Furthermore, the detection kit includes an RT-PCR detection kit, an ELISA detection kit, a protein chip detection kit, a rapid detection kit, a DNA chip detection kit, an immunohistochemistry detection kit, or an MRM (multiple reaction monitoring) detection kit.

[0041] The detection kit may further include elements necessary for reverse transcription-polymerase chain reaction. The RT-PCR detection kit includes a pair of primers specific to a gene encoding a marker protein. Each primer is a nucleotide having a nucleic acid sequence specific to the gene, and its length may be approximately 7 bp to 50 bp, more specifically, approximately 10 bp to 39 bp. The kit may also include a primer specific to the nucleic acid sequence of a control gene. Preferably, the RT-PCR detection kit may further include a test tube or appropriate container, a reaction buffer (with different pH values ​​and magnesium concentrations), deoxynucleotides (dNTPs), enzymes (e.g., Taq polymerase and reverse transcriptase), a deoxyribonuclease inhibitor, a ribonuclease inhibitor, DEPC-water, and sterile water.

[0042] Furthermore, the detection kit may include elements necessary for operating a DNA chip. The DNA chip kit may include a substrate that binds to an oligonucleotide corresponding to a gene, cDNA, or a fragment thereof, and reagents, drugs, and enzymes for constructing a fluorescently labeled probe. The substrate may also include an oligonucleotide corresponding to a control gene, cDNA, or a fragment thereof.

[0043] In some embodiments, the detection kit of the present invention may include elements necessary for performing ELISA. The ELISA detection kit may include an antibody specific to a protein (a protein encoded by CITED4 and / or METRN described in the present invention). The antibody has high selectivity and affinity for the marker protein and does not cross-react with other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. The ELISA detection kit may also include an antibody specific to a control protein. The ELISA detection kit may also include a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, a chromophore, an enzyme (e.g., bound to the antibody) and its substrate, or a substance capable of binding to the antibody.

[0044] Furthermore, the biochip is also referred to as an array, which refers to a solid support containing linked nucleic acid or peptide probes. Arrays typically contain multiple different nucleic acid or peptide probes linked to different known locations on a substrate surface. These arrays, also known as microarrays, are typically fabricated by mechanical synthesis or light-directed synthesis, which combines photolithography and solid-phase synthesis. Arrays may include flat surfaces, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or other suitable substrates. Arrays may be packaged in a manner that allows for diagnostic or other operation of a fully functional device.

[0045] A microarray is an ordered arrangement of hybrid array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The substrate may be a solid substrate, such as a glass or silica slide, or a semi-solid substrate, such as beads, fiber optic adhesive, or nitrocellulose membrane. The nucleotide sequences may be DNA, RNA, or any sequence thereof.

[0046] In the present invention, the biochip includes a gene chip and a protein chip, the gene chip including a solid support and oligonucleotide probes immobilized on the solid support in an orderly manner and specifically corresponding to a part or all of the sequences represented by CITED4 and / or METRN, and the protein chip including a solid support and an antibody or ligand specific to a protein encoded by CITED4 and / or METRN immobilized on the solid support.

[0047] Furthermore, the antibodies expressly include chimeric antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as long as the desired biological activity is exhibited, and fragments of such antibodies.

[0048] Furthermore, the ligand may include a peptide, antibody or fragment thereof, aptamer, or oligonucleotide capable of specifically binding to CITED4 and / or METRN. The antibody against the protein encoded by CITED4 and / or METRN used in the present invention is used in the broadest sense and specifically includes, for example, monoclonal antibodies, polyclonal antibodies, multiepitope-specific antibodies, multispecific antibodies, and antibody fragments. Such antibodies may be chimeric antibodies, humanized antibodies, human antibodies, and synthetic antibodies. The fragment may be any antibody as long as it retains the ability to bind to the protein encoded by CITED4 and / or METRN.

[0049] A third aspect of the present invention provides the use of a reagent for detecting the expression level of CITED4 and / or METRN in a sample in the manufacture of a system and / or device for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the effectiveness of a treatment for intervertebral disc degeneration.

[0050] A fourth aspect of the present invention provides a system and / or device for differential diagnosis of the degree of disc degeneration or evaluation of the effectiveness of a treatment for disc degeneration.

[0051] The system / apparatus further includes a processor, an input module, and an output module; The processor performs logical operations on the input information using bioinformatics methods, the input module is used to input the expression levels of CITED4 and / or METRN in the subject's sample, the computer-readable medium includes instructions that, when executed by the processor, execute an algorithm on the input expression levels of CITED4 and / or METRN, and the output module outputs the degree of disc degeneration or the treatment effect of disc degeneration in the subject.

[0052] Furthermore, the subject according to the present invention refers to any animal, including humans and non-human animals. The term non-human animals includes all vertebrates, for example, mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and any livestock or pets, and non-mammals such as chickens, amphibians, and reptiles. In a specific embodiment of the present invention, the subject is preferably a human.

[0053] The present invention further provides a computer-readable storage medium having stored thereon a computer program which, when executed by a processor, implements the system and / or apparatus according to the fourth aspect of the present invention.

[0054] Furthermore, the term "system / apparatus" is a way to distinguish between different components, elements, parts, portions, or assemblies at different levels. However, other terms may be used instead of the above terms if they can achieve the same purpose. As known to those skilled in the art, the present invention can be embodied as an apparatus, a method, or a computer program product. Therefore, the disclosed subject matter can be specifically embodied in either entirely hardware, entirely software (including firmware, resident software, microcode, etc.), or a combination of hardware and software. In some specific embodiments, the present invention may be embodied in the form of a computer program product on one or more computer-readable mediums, the computer-readable mediums including computer-readable program code.

[0055] Any combination of one or more computer-readable media can be used. The computer-readable medium may be a computer-readable signal medium or a computer-readable storage medium. The computer-readable storage medium may be, for example, but not limited to, an electric, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination thereof. In the present invention, the computer-readable storage medium may be any tangible medium that contains or stores a program, and the program may be used by or in combination with an instruction execution system, apparatus, or device.

[0056] A fifth aspect of the present invention provides a biomarker for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the effectiveness of a treatment for intervertebral disc degeneration.

[0057] Further, the biomarker is CITED4 and / or METRN.

[0058] A sixth aspect of the present invention provides the use of the biomarkers CITED4 and / or METRN in the differential diagnosis of the degree of disc degeneration or in assessing the efficacy of a treatment for disc degeneration.

[0059] In the present invention, as long as the biomarkers CITED4 and / or METRN described in the present invention are used to differentially diagnose the degree of intervertebral disc degeneration or to evaluate the therapeutic effect of intervertebral disc degeneration, all such methods are within the scope of protection of the present invention, and are not limited to the specific method of using the biomarkers CITED4 and / or METRN, as long as the purpose of differentially diagnosing the degree of intervertebral disc degeneration or evaluating the therapeutic effect of intervertebral disc degeneration can be achieved or basically achieved, all such methods are within the scope of protection of the present invention.

[0060] A seventh aspect of the present invention provides a method for differential diagnosis of the degree of disc degeneration or evaluation of the effectiveness of a treatment for disc degeneration.

[0061] Furthermore, the method comprises: (1) collecting a sample from a subject; (2) detecting the expression level of CITED4 and / or METRN in a sample derived from a subject; and (3) differentially diagnosing the degree of disc degeneration in the subject or evaluating the therapeutic effect of the subject on disc degeneration based on the detected expression levels of CITED4 and / or METRN.

[0062] An eighth aspect of the present invention provides the use of the biomarkers CITED4 and / or METRN in the treatment and / or prevention of intervertebral disc degeneration.

[0063] In the present invention, as long as the biomarkers CITED4 and / or METRN described in the present invention are used to treat and / or prevent intervertebral disc degeneration, all such methods are within the scope of protection of the present invention, and are not limited to specific methods of using the biomarkers CITED4 and / or METRN. As long as the purpose of treating and / or preventing the degree of intervertebral disc degeneration can be achieved or basically achieved using CITED4 and / or METRN, all such methods are within the scope of protection of the present invention.

[0064] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0065] (1) We have discovered for the first time that CITED4 and METRN are differentially expressed in intervertebral disc tissues with different degrees of degeneration, are highly correlated with the degree of degeneration, and demonstrate high accuracy, sensitivity, and specificity in the clinical samples collected in this study. The AUC values ​​for CITED4 in the differential diagnosis of moderate vs. severe, mild vs. severe, and mild-moderate vs. severe degeneration were 0.917, 0.961, and 0.938, respectively. The AUC values ​​for METRN in the differential diagnosis of moderate vs. severe, mild vs. severe, and mild-moderate vs. severe degeneration were 0.911, 0.929, and 0.92, respectively. The AUC values ​​for CITED4 and METRN in the differential diagnosis of moderate vs. severe, mild vs. severe, and mild-moderate vs. severe degeneration were 0.933, 0.953, and 0.949, respectively. This indicates that CITED4 and METRN can be used as biomarkers for the differential diagnosis of disc degeneration, and that their effective use in the differential diagnosis of disc degeneration can provide timely and effective intervention and treatment at an early stage.

[0066] (2) CITED4 and / or METRN according to the present invention have high diagnostic efficiency for the differential diagnosis of the degree of intervertebral disc degeneration and have good prospects for development as a diagnostic method. Furthermore, the present invention provides a new solution strategy to solve the technical problems present in the field: imaging diagnosis in clinical settings relies on tissue imaging characteristics, but changes in tissue imaging characteristics are slow, resulting in delayed diagnostic results; and changes in diseased tissue morphology often lag behind abnormal changes in molecular biology, resulting in low sensitivity of related diagnostic methods based on tissue morphology. The signature genes identified in the present invention at the molecular level can provide a certain theoretical basis for the discovery, promotion, and use of biomarkers for intervertebral disc degeneration, and are of great utility for the differential diagnosis of intervertebral disc degeneration, the evaluation of the effectiveness of related interventions, and understanding the pathogenesis and progression of the disease. [Brief explanation of the drawings]

[0067] [Figure 1]FIG. 1 shows the results of quality control in the preparation of a single-cell suspension of human intervertebral disc nucleus pulposus tissue. [Figure 2] FIG. 1 shows the results of single cell subpopulations of human intervertebral disc nucleus pulposus tissue. [Figure 3] 1 is a violin plot showing the mRNA expression of marker molecules CITED4 and METRN in subpopulation 8 of chondrocytes in human intervertebral disc nucleus pulposus tissue. [Figure 4] This figure shows the results of identifying cells in human intervertebral disc nucleus pulposus tissue that are positive for Aggrecan (ACAN) expression as chondrocytes. The left image is magnified 100x, and the right image is magnified 400x. [Figure 5] This figure shows the results of the difference in CITED4+-expressing chondrocytes in human intervertebral disc nucleus pulposus tissues of different Pfirrmann grades after immunohistochemical staining, magnified 100 times. [Figure 6] This figure shows the statistical results of the difference in integrated optical density between the number of CITED4+ cells and METRN+ cells in human intervertebral disc nucleus pulposus tissues from different groups in the training set, where panel A shows CITED4+ cells and panel B shows METRN+ cells. [Figure 7] Figure 1 shows ROC curves obtained using CITED4+ cells for human intervertebral disc nucleus pulposus pathological tissues with different degrees of degeneration in the training set. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 8] Figure 1 shows the ROC curves obtained using METRN+ cells for human intervertebral disc nucleus pulposus pathological tissues with different degrees of degeneration in the training set. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 9]Figure 1 shows the ROC curves obtained using METRN+ and CITED4+ cells for human intervertebral disc nucleus pulposus pathological tissues with different degrees of degeneration in the training set. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 10] This figure shows the statistical results of the difference in integrated optical density between the number of CITED4+ cells and METRN+ cells in human intervertebral disc nucleus pulposus tissues from different groups in the validation set, where Panel A shows CITED4+ cells and Panel B shows METRN+ cells. [Figure 11] Figure 1 shows the ROC curves obtained using CITED4+ cells for human intervertebral disc nucleus pulposus pathological tissues with different degrees of degeneration in the validation set. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 12] Figure 1 shows the ROC curves obtained using METRN+ cells for human intervertebral disc nucleus pulposus pathological tissues with different degrees of degeneration in the validation set. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 13] Figure 1 shows the ROC curves obtained using METRN+ and CITED4+ cells for human intervertebral disc nucleus pulposus pathological tissues with different degrees of degeneration in the validation set. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 14]This figure shows the statistical results of the difference in the number of CITED4+ cells in human intervertebral disc nucleus pulposus tissue from different groups, where L is mild degeneration, n=17, M is moderate degeneration, n=18, S is severe degeneration, n=15, LM is mild to moderate degeneration, n=35, MS is moderate to severe degeneration, n=33, *p<0.05, **p<0.01, ***p<0.001, NSp>0.05. [Figure 15] Figure 1 shows ROC curves obtained using CITED4+ cells for human intervertebral disc nucleus pulposus pathological tissue with different degrees of degeneration. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 16] This figure shows the results of the difference in METRN+-expressing chondrocytes in human intervertebral disc nucleus pulposus tissues of different Pfirrmann grades after immunohistochemical staining, magnified 100 times. [Figure 17] This figure shows the statistical results of the difference in integrated optical density of METRN+ cells in human intervertebral disc nucleus pulposus tissue from different groups, where L is mild degeneration, n=17, M is moderate degeneration, n=18, S is severe degeneration, n=15, LM is mild-moderate degeneration, n=35, MS is moderate-severe degeneration, n=33, *p<0.05, **p<0.01, ***p<0.001, NSp>0.05. [Figure 18] Figure 1 shows ROC curves obtained using METRN+ cells for human intervertebral disc nucleus pulposus pathological tissue with different degrees of degeneration. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). [Figure 19]Figure 1 shows ROC curves obtained using METRN+ and CITED4+ cells for human intervertebral disc nucleus pulposus pathological tissue with different degrees of degeneration. Figure A is L / M (comparison of mild degeneration with moderate degeneration), Figure B is M / S (comparison of moderate degeneration with severe degeneration), Figure C is L / S (comparison of mild degeneration with severe degeneration), Figure D is LM / S (comparison of mild-moderate degeneration with severe degeneration), and Figure E is L / MS (comparison of mild degeneration with moderate-severe degeneration). DETAILED DESCRIPTION OF THE INVENTION

[0068] As a result of extensive and thorough research, the present inventors have discovered for the first time that CITED4 and METRN show significantly different expression levels in intervertebral disc tissues with different degrees of degeneration, and are highly correlated with the degree of intervertebral disc degeneration. In clinical samples collected in this invention, they have shown high differential diagnostic efficiency for the degree of intervertebral disc degeneration, with high accuracy, sensitivity, and specificity. CITED4 and METRN can therefore be used as biomarkers for the differential diagnosis of the degree of intervertebral disc degeneration in clinical settings.

[0069] Unless otherwise specified, technical terms used herein are generally used in accordance with their common practice. To further explain the present invention, some of the terms used in the present invention are explained below.

[0070] As used herein, the terms "comprise" or "containing" include one or more of the stated elements or components but do not exclude other elements or components.

[0071] As used herein, the term "biomarker" refers to an indicator of a patient's phenotype (specifically, in the present invention, a patient with degenerative disc disease), such as an indicator of a pathological condition or responsiveness to a therapeutic agent, that can be detected in a biological sample from the patient, and examples of such biomarkers include, but are not limited to, DNA, RNA, proteins, small molecule metabolites, carbohydrates, glycolipid molecules, etc. In a specific embodiment of the present invention, the biomarker is CITED4 and / or METRN.

[0072] The term "expression level" as used herein means "level" and refers to the absolute or relative amount of the biomarkers CITED4 and / or METRN according to the present invention. The expression level of the biomarkers CITED4 and / or METRN according to the present invention can be determined by various techniques, in particular the absolute or relative amount of the biomarkers CITED4 and / or METRN according to the present invention can be detected using methods well known to those skilled in the art.

[0073] In some embodiments, the expression level of a gene (i.e., the biomarkers CITED4 and / or METRN) can be measured by various techniques known in the art. Typically, the expression level of a gene can be determined by measuring the amount of mRNA. Methods for measuring mRNA amount are well known in the art. For example, nucleic acids contained in a sample (e.g., a blood, cell, or tissue sample taken from a patient) are first extracted according to standardized methods, such as using cellular enzymes or chemical solutions or using nucleic acid-binding resins according to the manufacturer's instructions. The extracted mRNA is then detected by in situ hybridization (e.g., Northern blotting analysis, in situ hybridization) and / or amplification (e.g., RT-PCR). Other amplification methods include, but are not limited to, ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

[0074] The term "significantly different" as used herein refers to a comparison of the expression levels of one or more biomarkers described in the present invention in another sample, where the content or concentration of the biomarkers is measured, and refers to the difference in the content or concentration of one or more biomarkers of the present invention in one sample. As used herein, "significantly different genes" can be measured using the ratio of the level of a particular biomarker (gene) to the average level of the particular biomarker in the control, where this ratio is not equal to 1.0. The difference can also be measured using a p-value. When using a p-value, if the p-value is less than 0.1, the biomarker is identified as being different between the first and second groups. More preferably, the p-value is less than 0.05. Even more preferably, the p-value is less than 0.01. Even more preferably, the p-value is less than 0.005. Most preferably, the p-value is less than 0.001. When determining the difference based on the ratio, if the ratio of the levels of the first sample and the second sample is greater or less than 1.0, the biomarker shows a difference. For example, the ratio is greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or less than 1, such as 0.8, 0.6, 0.4, 0.2, 0.1, 0.05.

[0075] As used herein, the term "diagnosis" refers to the detection, determination, or recognition of an individual's health condition or status based on one or more symptoms, data, or other information related to the individual. An individual's health condition may be diagnosed as healthy / normal (i.e., free of disease or disorder), unhealthy / abnormal (i.e., with disease or disorder), or a particular severity of illness (e.g., mild, mild-moderate, moderate, moderate-severe, severe). The terms diagnosis, early diagnosis, making a diagnosis, and variations of these terms include early detection of a disease / disorder associated with a particular disease or disorder (specifically, in the present invention, patients with degenerative disc disease), the nature or classification of the disease, detection of disease progression, cure, or recurrence, and detection of an individual's response to treatment or post-treatment of the disease.

[0076] In some embodiments, the detection probes provided by the present invention may further comprise one or more detectable markers. Specific examples of such detectable markers include fluorescent molecules (or fluorescent dyes), fluorescent nanoparticles, radioisotopes, detectable enzymes, etc. In some embodiments, the fluorescent molecules can be selected from the "Instruction Manual - Fluorescent Probe and Labeling Technology Guide." Special fluorescent groups that can be bound to nucleic acid molecules include, but are not limited to, 4-acetylamino-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine, isothiocyanatoacridine, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid, 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate, N-(4-anilino-1-naphthyl)maleimide, o-aminobenzamide, brilliant yellow, coumarin, and derivatives thereof. In some embodiments, the fluorescent nanoparticles include, but are not limited to, semiconductor nanocrystals, quantum dots, etc. In some embodiments, the radioisotopes include, but are not limited to, radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt, etc. In some embodiments, the detectable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, β-lactamase, etc.

[0077] The term "area under the curve" or "AUC" as used herein refers to the area under a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measurements are useful for comparing the accuracy of classifiers across a range of data. A classifier with a high AUC is more capable of correctly classifying unknowns between two or more subject groups (e.g., intervertebral disc nucleus tissue samples from patients with different degrees of degeneration). ROC curves are useful for describing the effectiveness of a particular feature (e.g., any biomarker described herein and / or any item of other biomedical information) in distinguishing between two groups. Typically, feature data are selected across groups in ascending order based on the value of a single feature. Then, true positive and false positive rates are calculated for each feature value. The true positive rate is determined by counting the number of cases exceeding the feature value and dividing by the total number of cases. The false positive rate is determined by counting the number of controls exceeding the feature value and dividing by the total number of controls. ROC curves may be generated for a single feature or for other single outputs. For example, a combination of two or more features may be combined mathematically (e.g., by addition, subtraction, multiplication, etc.) to provide a single sum value, which is plotted on an ROC curve. Any combination of multiple features that result in a single output value may also be plotted on an ROC curve. These feature combinations may comprise a test. An ROC curve is a graph of a test's true positive rate (sensitivity) against its false positive rate (1-specificity), and can be used to analyze diagnostic accuracy.

[0078] The term "sensitivity" as used herein refers to the number (%) of true-positive patients relative to the total number of target patients (100%), and is calculated using the formula: sensitivity = TP / (TP + FN) (TP = true positive, FN = false negative).

[0079] The term "specificity" as used herein refers to the number of true negative individuals (%) relative to the total number of healthy subjects (100%), and is calculated using the formula: specificity = TN / (TN + FP) (TN = true negative, FP = false positive).

[0080] As used herein, the term "sample" refers to a composition obtained from or derived from a subject of interest, including cellular and / or other molecular entities characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological properties. The sample can be obtained from the subject's blood, other biologically derived liquid samples, and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The tissue sample may be derived from, for example, fresh, frozen, and / or preserved organ or tissue samples, solid tissues such as biopsy tissue or aspirates, blood or any blood component, bodily fluids, cells from an individual at any time during pregnancy or development, or plasma. The term sample includes biological samples that have been processed in any way after collection, such as by treatment with reagents, stabilization, enrichment for specific components (e.g., proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning. Samples according to the present invention include, but are not limited to, tissue samples, blood samples, blood-derived cell samples, serum samples, plasma samples, lymph samples, synovial fluid samples, cell extract samples, or any combination thereof.

[0081] The present invention will be further described below with reference to specific examples, which are merely for the purpose of illustrating the present invention and should not be understood as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to these examples without departing from the principle and spirit of the present invention, and the scope of the present invention is limited by the claims and their equivalents. In the following examples, experimental methods without specific conditions are usually carried out according to conventional conditions or conditions recommended by manufacturers.

[0082] Example 1 scRNA-seq analysis experiment 1. Experimental materials Five cases of human degenerated intervertebral disc nucleus pulposus tissue, Chromium TMSingle Cell 3' Solution microfluidic platform, 10X Genomics single cell sequencing platform.

[0083] 2. Experimental method (1) Collection of human intervertebral disc nucleus pulposus tissue samples Five cases of human degenerative disc nucleus pulposus tissue were collected. The samples were pathological disc tissue samples taken from patients with traumatic lumbar burst fractures or degenerative disc herniations who required surgical resection treatment based on actual clinical diagnosis and treatment conditions. This study was approved by the hospital's ethics committee and conducted with informed consent from the subjects or their families (guardians).

[0084] (2) Collection of chondrocytes from intervertebral disc nucleus pulposus tissue and preparation of single-cell suspension The nucleus pulposus tissue was washed with PBS and then cut with ophthalmic scissors. The cut tissue was transferred to a 5 mL centrifuge tube containing tissue dissociation solution, digested with trypsin at 37°C for 0.5 h, and then digested with type II collagenase at 37°C for 2.5–3 h by rotation. The tissue was then passed through a 70 μm cell filter. After incubation, red blood cells were lysed, and the tissue was passed through a 40 μm cell filter to remove dead cells. The tissue was then subjected to gradient centrifugation at 4°C and resuspended in an appropriate volume of PBS.

[0085] (3) Sequencing Chromium, based on the 10X Genomics platform TM The cells were isolated at once using Single Cell 3' Solution, and all single cells within the range of 500-10,000 were labeled to detect gene expression at the single-cell level. The specific process is as follows: TMThe Single Cell 3' Solution is a microfluidic platform based on GemCode technology. Single cells and gel beads bearing barcodes and primers were encapsulated within oil droplets. The gel beads were then dissolved within each oil droplet, lysing the cells to release mRNA, which was then reverse-transcribed to generate barcoded cDNA for sequencing. After disrupting the liquid oil layer, a cDNA library was constructed. The library was then sequenced using an Illumina sequencing platform, enabling the acquisition of large amounts of single-cell gene expression data at once, thereby achieving expression sequencing at the single-cell level.

[0086] (4) Data processing and identification of cell subpopulations All samples were analyzed using cluster analysis to identify cell subpopulations, and rare cell subpopulations were discovered. Data from the experimental and control groups were combined and subjected to cluster analysis. The different distributions of the two samples in cell clusters and the different proportions of different cell types were compared. Differential gene expression analysis was performed based on the results of cell clustering to identify significantly different genes within each subpopulation as signature genes for that subpopulation, thereby helping to identify cell types. Differential gene function annotation and enrichment analysis, including enrichment analysis using GO annotations, KEGG annotations, and disease annotations, transcription factor annotations, and protein interaction networks, helped to identify cell functions. Using these techniques, subpopulation identification was performed on human intervertebral disc nucleus pulposus cells.

[0087] 3. Experimental results Prior to single-cell sequencing, quality control was performed on the prepared single-cell suspension. Using one human intervertebral disc nucleus pulposus tissue sample as an example, the results are shown in Figure 1. The cell viability was 84.17%, the cell aggregation rate was 26.09%, the viable cell concentration was 730 cells / µL, the average cell diameter was 12.26 µm, and the viable cell particle ratio was 77.05%.

[0088] Chromium, based on the 10X Genomics platform TM Single cells from human intervertebral disc nucleus pulposus tissue were isolated using Single Cell 3' Solution, and gene expression was detected at the single-cell level. Data processing and bioinformatics analysis identified single-cell subpopulations of human intervertebral disc nucleus pulposus tissue, and the results are shown in Figure 2. Subpopulation 8 is a newly discovered chondrocyte subpopulation of human intervertebral disc nucleus pulposus tissue.

[0089] To more precisely identify subpopulation 8, differential genetic analysis was performed on this subpopulation in this example, and the results are shown in Figure 3. As can be seen from the results, CITED4 and METRN are characteristic genes of this newly discovered chondrocyte subpopulation of intervertebral disc nucleus pulposus tissue.

[0090] Example 2 MRI grading of intervertebral disc nucleus pulposus tissue and sample grouping 1. Experimental materials 50 cases of human degenerative disc tissue.

[0091] 2. Experimental method (1) Sample collection Fifty cases of human degenerative disc tissue were collected. The samples were pathological tissue samples of degenerative discs taken from patients with traumatic lumbar burst fractures or degenerative disc herniations who required surgical resection treatment based on actual clinical diagnosis and treatment conditions. This study was approved by the hospital's ethics committee and conducted with informed consent from the subjects or their families (guardians).

[0092] (2) Identifying the degree of degeneration of different intervertebral disc tissues using MRI (magnetic resonance imaging) technology The Pfirrmann MRI (T2WI) grading criteria for disc degeneration were used to grade the degree of disc degeneration in all patients. Grade I: The disc structure was uniform and bright white, the boundary between the nucleus pulposus and the annulus fibrosus was clear, the signal intensity was equal to or greater than that of cerebrospinal fluid, and the disc height was normal. Grade II: The disc structure was heterogeneous, with the horizontal ligament present or absent, the boundary between the nucleus pulposus and the annulus fibrosus was clear, the signal intensity was equal to or greater than that of cerebrospinal fluid, and the disc height was normal. Grade III: The disc structure was heterogeneous and gray, the boundary between the nucleus pulposus and the annulus fibrosus was unclear, the signal intensity was moderate, and the disc height was normal or slightly reduced. Grade IV: The disc structure was heterogeneous and gray or black, the boundary between the nucleus pulposus and the annulus fibrosus was lost, the signal intensity was moderate or low, and the disc height was normal or moderately reduced. Grade V: The disc structure is uneven and black, the boundary between the nucleus pulposus and the annulus fibrosus is lost, the signal intensity is low, and the disc space is collapsed.

[0093] (3) Sample grouping Pfirrmann grading was performed on intervertebral disc tissue based on MRI results, and the results were classified as follows: Grades I-II are mild degeneration group (L), Grade III is moderate degeneration group (M), and Grades IV-V are severe degeneration group (S). To distinguish the expression differences of the biomarkers according to the degree of degeneration and to better evaluate the diagnostic efficacy of CITED4 and METRN as biomarkers, Grades I-II and III are further combined into the mild-moderate degeneration group (LM), and Grades III and IV-V are classified into the moderate-severe degeneration group (MS).

[0094] 3. Experimental results The Pfirrmann degeneration grading results for 50 human disc tissue samples are shown in Table 1. As can be seen from the results, there were 17 samples with Pfirrmann degeneration grades I-II (mild, L), 18 samples with grade III (moderate, M), and 15 samples with grades IV-V (severe, S). After further classification, there were a total of 35 samples with grades I-III (mild-moderate, LM) and 33 samples with grades III-V (moderate-severe, MS).

[0095] Table 1. Pfirrmann degeneration grading of human intervertebral disc tissue [Table 1]

[0096] Example 3: ACAN chondrocyte identification experiment 1. Experimental materials Fifty cases of human degenerative disc tissue, Anti-Aggrecan Antibody (ACAN) (Boster Biological Technology Co., Ltd., BA2967-1, Lot No: D-blj2-08F183).

[0097] 2. Experimental method (1) Sample collection In Example 2, 50 cases of human degenerative disc tissue identified by MRI were collected. The samples were pathological tissue samples of degenerative discs collected from patients with traumatic lumbar burst fractures or degenerative disc herniation who required surgical resection treatment based on actual clinical diagnosis and treatment conditions. This study was approved by the hospital's ethics committee and conducted with informed consent from the subjects or their families (guardians).

[0098] (2) Immunohistochemical staining and interpretation Tissues were generally fixed, dried, and embedded before paraffin sectioning. Sections were baked at 65°C for 1 hour, dewaxed, hydrated, and antigen retrieval was performed using an enzymatic retrieval method. Cell samples were thoroughly fixed with 95% ethanol, incubated with 3% hydrogen peroxide for 10 minutes, washed twice with PBS, and incubated with primary antibody for 30 minutes. Anti-Aggrecan antibody was diluted 1:50, washed twice with PBS, incubated with secondary antibody for 20 minutes, washed twice with PBS, and then stained with DAB. After thorough washing with tap water, the sections were counterstained, dried, cleared, and mounted.

[0099] Two pathologists independently reviewed the pathological results of the sections, and the consensus interpretation was used as the final result. Discrepant results were adjudicated by a third pathologist.

[0100] 3. Experimental results Aggrecan (ACAN) is an important component of the extracellular matrix of chondrocytes and is commonly used as a marker for identifying chondrocytes. The results of this study are shown in Figure 4. Using an antibody against Aggrecan (ACAN) by immunohistochemistry, positively expressing cells in human intervertebral disc nucleus pulposus tissue were identified as chondrocytes.

[0101] Example 4 CITED4, METRN immunohistochemistry experiments and ROC curve analysis 1. Experimental materials 50 cases of human degenerative disc tissue, Anti-CITED4 antibody (abcam, USA, ab134072, YJ082412CS), Meteorin Polyclonal Antibody (Invitrogen, USA, PA5-62335, XF3619498A).

[0102] 2. Experimental method (1) Sample collection In Example 2, 50 cases of human degenerative disc tissue identified by MRI were collected. The samples were pathological tissue samples of degenerative discs collected from patients with traumatic lumbar burst fractures or degenerative disc herniation who required surgical resection treatment based on actual clinical diagnosis and treatment conditions. This study was approved by the hospital's ethics committee and conducted with informed consent from the subjects or their families (guardians).

[0103] (2) Immunohistochemical staining and interpretation Tissues were fixed, dried, embedded, and then paraffin sections were prepared. The sections were baked at 65°C for 1 hour, dewaxed, hydrated, and antigen retrieval was performed using an enzymatic retrieval method. Cell samples were thoroughly fixed in 95% ethanol, incubated with 3% hydrogen peroxide for 10 minutes, washed twice with PBS, and incubated with primary antibodies for 30 minutes. Anti-CITED4 antibody and Meteorin polyclonal antibody were diluted 1:100 and 1:50, respectively, washed twice with PBS, incubated with secondary antibodies for 20 minutes, washed twice with PBS, and then stained with DAB. After thorough washing with tap water, the sections were counterstained, dried, cleared, and mounted.

[0104] (3)ROC curve analysis For each immunohistochemical section, three fields were randomly selected and photographed under a 10x microscope. The number of CITED4+ positive cells in each of the three randomly selected fields was analyzed using Image Pro Plus software, and the total number of cells was statistically analyzed. The integrated optical density of METRN+ positive expression was calculated as the sum of the three randomly selected fields. Two pathologists independently reviewed the pathological results, and the consensus interpretation was used as the final result. Discrepant results were adjudicated by a third pathologist. Using SPSS software, receiver operating characteristic curves were constructed for mild degeneration and moderate / severe degeneration. The area under the AUC curve and threshold were calculated to evaluate the diagnostic value of the newly discovered chondrocyte subpopulation signature genes CITED4, METRN, and the combination of CITED4 and METRN for the degree of intervertebral disc degeneration.

[0105] In this example, 50 cases of human degenerative disc tissue collected according to the present invention were randomly divided into two groups (training set and validation set), each accounting for 50% of the total, using the createDataPartition function in the caret package in R. That is, the training set included 25 cases of human degenerative disc tissue, and the validation set included 25 cases of human degenerative disc tissue. Further analysis and validation were performed using the training set and validation set.

[0106] 3. Experimental results In the training set, the differential expression results of CITED4 and METRN between each group are shown in Figure 6. As can be seen from the results, there is significant differential expression of CITED4 and METRN between patients with different degrees of disc degeneration.

[0107] The ROC curve analysis results for each group corresponding to CITED4+ in the training set are shown in Figure 7 and Table 2 below. Except for mild and moderate (L / M), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.875, with a corresponding specificity of 77.8% and a sensitivity of 87.5%. For the comparison between mild and severe (L / S), the AUC value was 0.931, with a corresponding specificity of 88.9% and a sensitivity of 87.5%. For the comparison between mild, moderate, and severe (LM / S), the AUC value was 0.903, with a corresponding specificity of 83.3% and a sensitivity of 87.5%. As can be seen from the results, CITED4 is a biomarker that can indicate the degree of degeneration of intervertebral disc nucleus pulposus tissue, and is particularly valuable in distinguishing between moderate and severe, mild and severe, and mild, moderate, and severe degenerative tissue.

[0108] Table 2. ROC curve parameters for each CITED4+ cell group in the training set [Table 2]

[0109] The ROC curve results for each group corresponding to METRN+ in the training set are shown in Figure 8 and Table 3 below. Except for mild vs. moderate / severe (L / M) and mild vs. moderate / severe (L / MS), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.806, with a corresponding specificity of 66.7% and a sensitivity of 100%. For the comparison between mild and severe (L / S), the AUC value was 0.875, with a corresponding specificity of 77.8% and a sensitivity of 100%. For the comparison between mild / moderate and severe (LM / S), the AUC value was 0.840, with a corresponding specificity of 72.2% and a sensitivity of 100%. As can be seen from the results, METRN is a biomarker that can indicate the degree of degeneration of intervertebral disc nucleus pulposus tissue, and is particularly valuable in distinguishing between moderate and severe, mild and severe, and mild, moderate, and severe degenerative tissue.

[0110] Table 3. ROC curve parameters for each METRN+ cell group in the training set [Table 3]

[0111] The ROC curve results for the CITED4+ and METRN+ groups in the training set are shown in Figure 9 and Table 4 below. Except for mild vs. moderate (L / M) and mild vs. moderate-severe (L / MS), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.875, with a corresponding specificity of 77.8% and a sensitivity of 87.5%. For the comparison between mild and severe (L / S), the AUC value was 0.875, with a corresponding specificity of 100% and a sensitivity of 75.0%. For the comparison between mild-moderate and severe (LM / S), the AUC value was 0.917, with a corresponding specificity of 88.9% and a sensitivity of 87.5%. The results show that the combination of CITED4 and METRN can label the degree of degeneration of the intervertebral disc nucleus pulposus tissue, and is particularly useful in distinguishing between moderate and severe, mild and severe, and mild-moderate and severe degenerative tissues.

[0112] Table 4. ROC curve parameters for each CITED4+ and METRN+ cell group in the training set. [Table 4]

[0113] In the validation set, the differential expression results of CITED4 and METRN between each group are shown in Figure 10. As can be seen from the results, CITED4 and METRN have significant differential expression between patients with different degrees of disc degeneration.

[0114] The ROC curve analysis results for each group corresponding to CITED4+ in the validation set are shown in Figure 11 and Table 5 below. Except for mild and moderate (L / M), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.968, with a corresponding specificity of 100% and sensitivity of 85.7%. For the comparison between mild and severe (L / S), the AUC value was 1.000, with a corresponding specificity of 100% and sensitivity of 100%. For the comparison between mild, moderate, and severe (LM / S), the AUC value was 1.000, with a corresponding specificity of 100% and sensitivity of 100%. As can be seen from the results, CITED4 is a biomarker that can indicate the degree of degeneration of intervertebral disc nucleus pulposus tissue, and is particularly valuable in distinguishing between moderate and severe, mild and severe, and mild, moderate, and severe degenerative tissue.

[0115] Table 5. ROC curve parameters for each CITED4+ cell group in the validation set [Table 5]

[0116] The ROC curve results for each group corresponding to METRN+ in the validation set are shown in Figure 12 and Table 6 below. Except for mild and moderate (L / M), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.984, with a corresponding specificity of 88.9% and a sensitivity of 100%. For the comparison between mild and severe (L / S), the AUC value was 0.982, with a corresponding specificity of 87.5% and a sensitivity of 100%. For the comparison between mild, moderate, and severe (LM / S), the AUC value was 0.983, with a corresponding specificity of 88.2% and a sensitivity of 100%. As can be seen from the results, METRN is a biomarker that can indicate the degree of degeneration of intervertebral disc nucleus pulposus tissue, and is particularly valuable in distinguishing between moderate and severe, mild and severe, and mild, moderate, and severe degenerative tissue.

[0117] Table 6. ROC curve parameters for each METRN+ cell group in the validation set [Table 6]

[0118] The ROC curve results for the CITED4+ and METRN+ groups in the validation set are shown in Figure 13 and Table 7 below. Except for mild and moderate (L / M), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 1.000, with a corresponding specificity of 100% and sensitivity of 100%. For the comparison between mild and severe (L / S), the AUC value was 1.000, with a corresponding specificity of 100% and sensitivity of 100%. For the comparison between mild, moderate, and severe (LM / S), the AUC value was 1.000, with a corresponding specificity of 100% and sensitivity of 100%. The results show that the combination of CITED4 and METRN can label the degree of degeneration of the intervertebral disc nucleus pulposus tissue, and is particularly useful in distinguishing between moderate and severe, mild and severe, and mild-moderate and severe degenerative tissues.

[0119] Table 7. ROC curve parameters for each CITED4+ and METRN+ cell group in the validation set [Table 7]

[0120] In the total sample set collected in this study, CITED4 was positively expressed in the nuclei of chondrocytes. As can be seen in Figure 5, the number of CITED4+ positive cells clearly increased with the severity of degeneration. CITED4+ cells accounted for approximately 3% of all cells in mild degeneration, approximately 10% in moderate degeneration, and approximately 60% in severe degeneration. The results of statistical analysis of differences between groups are shown in Figure 14. There was a significant difference between moderate and severe (M / S) (P<0.001), between mild and severe (L / S) (P<0.001), between mild-moderate and severe (LM / S) (P<0.001), and between mild and moderate-severe (L / MS) (P=0.002).

[0121] The ROC curve analysis results for each group corresponding to CITED4+ in the total sample set collected in this study are shown in Figure 15 and Table 8 below. Except for mild and moderate (L / M), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.917, with a corresponding specificity of 77.8% and a sensitivity of 93.3%. For the comparison between mild and severe (L / S), the AUC value was 0.961, with a corresponding specificity of 88.2% and a sensitivity of 93.3%. For the comparison between mild-moderate and severe (LM / S), the AUC value was 0.938, with a corresponding specificity of 82.9% and a sensitivity of 93.3%. As can be seen from the results, CITED4 is a biomarker that can indicate the degree of degeneration of intervertebral disc nucleus pulposus tissue, and is particularly valuable in distinguishing between moderate and severe, mild and severe, and mild, moderate, and severe degenerative tissue.

[0122] Table 8. ROC curve parameters for each CITED4+ cell group [Table 8]

[0123] In the total sample set collected in this study, METRN was positively expressed in the cytoplasm of chondrocytes. As can be seen in Figure 16, the number of METRN+ positive cells clearly increased with the severity of degeneration. METRN+ cells were approximately 1% in mild degeneration, approximately 5% in moderate degeneration, and approximately 50% in severe degeneration. Statistical differences between groups are shown in Figure 17. There was a significant difference between moderate and severe degeneration (M / S) (P<0.001), between mild and severe degeneration (L / S) (P<0.001), between mild-moderate and severe degeneration (LM / S) (P<0.001), and between mild and moderate-severe degeneration (L / MS) (P=0.012).

[0124] The ROC curve results for each group corresponding to METRN+ in the total sample set collected in this study are shown in Figure 18 and Table 9 below. Except for mild and moderate (L / M), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.911, with a corresponding specificity of 77.8% and a sensitivity of 100%. For the comparison between mild and severe (L / S), the AUC value was 0.929, with a corresponding specificity of 82.4% and a sensitivity of 100%. For the comparison between mild, moderate, and severe (LM / S), the AUC value was 0.920, with a corresponding specificity of 80% and a sensitivity of 100%. As can be seen from the results, METRN is a biomarker that can indicate the degree of degeneration of intervertebral disc nucleus pulposus tissue, and is particularly valuable in distinguishing between moderate and severe, mild and severe, and mild, moderate, and severe degenerative tissue.

[0125] Table 9. ROC curve parameters for each METRN+ cell group [Table 9]

[0126] The ROC curve results for the CITED4+ and METRN+ groups in the total sample set collected in this study are shown in Figure 19 and Table 10 below. Except for mild and moderate (L / M), the AUC values ​​for the remaining groups were all greater than 0.7. For the comparison between moderate and severe (M / S), the AUC value was 0.933, with a corresponding specificity of 72.2% and a sensitivity of 100%. For the comparison between mild and severe (L / S), the AUC value was 0.953, with a corresponding specificity of 100% and a sensitivity of 86.7%. For the comparison between mild, moderate, and severe (LM / S), the AUC value was 0.949, with a corresponding specificity of 85.7% and a sensitivity of 93.3%. The results show that the combination of CITED4 and METRN can label the degree of degeneration of the intervertebral disc nucleus pulposus tissue, and is particularly useful in distinguishing between moderate and severe, mild and severe, and mild-moderate and severe degenerative tissues.

[0127] Table 10. ROC curve parameters for each CITED4+ and METRN+ cell group [Table 10]

[0128] The above description of the embodiments is only for understanding the method and core idea of ​​the present invention, and those skilled in the art may make some improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications are also within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting the expression level of CITED4 and / or METRN in a sample in the manufacture of a product for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic effect of intervertebral disc degeneration.

2. The reagent comprises: A reagent for detecting the mRNA expression level of CITED4 and / or METRN in a sample; A reagent for detecting the protein expression level of CITED4 and / or METRN in a sample, or The use according to claim 1, characterized in that it comprises a reagent for detecting the number of CITED4 and / or METRN positive expressing cells in a sample.

3. The reagent for detecting the mRNA expression level of CITED4 and / or METRN in the sample includes: Primers that specifically amplify CITED4 and / or METRN, or The use according to claim 2, characterized in that it comprises a probe that specifically recognizes CITED4 and / or METRN.

4. The reagent for detecting the protein expression level of CITED4 and / or METRN in the sample comprises: an antibody that specifically binds to a protein encoded by CITED4 and / or METRN, or The use according to claim 2, characterized in that it comprises an affinity protein that specifically binds to the protein encoded by CITED4 and / or METRN.

5. The use according to claim 2, characterized in that the reagent for detecting the number of CITED4 and / or METRN-positive expressing cells in the sample comprises a reagent for detecting the number of CITED4 and / or METRN-positive expressing cells by immunohistochemistry.

6. A product for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic effect of intervertebral disc degeneration, comprising a reagent for detecting the expression level of CITED4 and / or METRN in a sample.

7. 10. The article of manufacture of claim 6, wherein the article of manufacture comprises a detection kit and a biochip.

8. The detection kit comprises primers and probes that specifically bind to CITED4 and / or METRN, 8. The product of claim 7, wherein the biochip comprises a solid support and a probe attached to the solid support that specifically recognizes CITED4 and / or METRN.

9. Use of a reagent for detecting the expression level of CITED4 and / or METRN in a sample in the manufacture of a system and / or device for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic effect of intervertebral disc degeneration.

10. a processor, an input module, an output module, and a computer-readable medium; The processor performs logical operations on the input information using bioinformatics methods, The input module is used to input the expression level of CITED4 and / or METRN in the subject's sample; The computer-readable medium includes instructions that, when executed by the processor, execute an algorithm on input CITED4 and / or METRN expression levels; A system and / or device for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the effect of treatment of intervertebral disc degeneration, characterized in that the output module outputs the degree of intervertebral disc degeneration or the effect of treatment of intervertebral disc degeneration of the subject.

11. A biomarker for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic effect of intervertebral disc degeneration, characterized by being CITED4 and / or METRN.

12. Use of the biomarkers CITED4 and / or METRN in the differential diagnosis of the degree of disc degeneration or in assessing the efficacy of treatment of disc degeneration.

13. (1) collecting a sample from a subject; (2) detecting the expression level of CITED4 and / or METRN in a sample derived from a subject; and (3) differentially diagnosing the degree of intervertebral disc degeneration of the subject or evaluating the therapeutic effect of intervertebral disc degeneration of the subject based on the detected expression levels of CITED4 and / or METRN.

14. Use of the biomarkers CITED4 and / or METRN in the treatment and / or prevention of intervertebral disc degeneration.

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