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WO2024183369A1 - Utilisation de cited4 et/ou de metrn dans le diagnostic differentiel du degré de dégénérescence des disques intervertébraux - Google Patents

Utilisation de cited4 et/ou de metrn dans le diagnostic differentiel du degré de dégénérescence des disques intervertébraux Download PDF

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Publication number
WO2024183369A1
WO2024183369A1 PCT/CN2023/136554 CN2023136554W WO2024183369A1 WO 2024183369 A1 WO2024183369 A1 WO 2024183369A1 CN 2023136554 W CN2023136554 W CN 2023136554W WO 2024183369 A1 WO2024183369 A1 WO 2024183369A1
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WIPO (PCT)
Prior art keywords
metrn
cited4
intervertebral disc
degeneration
disc degeneration
Prior art date
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PCT/CN2023/136554
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English (en)
Chinese (zh)
Inventor
朱立国
张平
王源
张静茹
展嘉文
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wang Jing Hospital Of China Academy Of Chinese Medical Sciences Institute Of Orthopedics And Traumatology Of China Academy Of Chinese Medical Sciences
Original Assignee
Wang Jing Hospital Of China Academy Of Chinese Medical Sciences Institute Of Orthopedics And Traumatology Of China Academy Of Chinese Medical Sciences
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Publication of WO2024183369A1 publication Critical patent/WO2024183369A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention belongs to the field of biomedicine technology, and in particular, the present invention relates to the application of CITED4 and/or METRN in the differential diagnosis of the degree of intervertebral disc degeneration.
  • Intervertebral disc degeneration is a degenerative change that occurs due to aging, decreased motor function, reduced stability of the lumbar spine, and increased stress on the intervertebral disc. It is also called disc degeneration and is common in middle-aged and elderly people. When the load on the intervertebral disc exceeds the maximum limit, the annulus fibrosus ruptures, and eventually the nucleus pulposus protrudes from it, causing low back pain and leg pain. Intervertebral disc degeneration is one of the main causes of low back pain. According to statistics, about 84% of adults will experience low back pain at least once in their lifetime, of which low back pain caused by intervertebral disc degeneration accounts for the vast majority.
  • Severe intervertebral disc degeneration can even cause lower limb sensory impairment, incontinence, etc.
  • Intervertebral disc degeneration has become an important factor in global disability, and with the aging of the world's population, the problem of intervertebral disc degeneration threatening health is becoming increasingly serious. Therefore, early diagnosis of disc degeneration and quantification of the degree of disc degeneration have very important clinical value for timely intervention and treatment of disc degeneration and delaying the further development of disc degeneration.
  • the diagnosis of intervertebral disc degeneration is still mainly based on imaging lesions, that is, using imaging techniques such as nuclear magnetic resonance and X-ray to judge the degree of disc tissue degeneration by observing abnormal changes in the disc.
  • imaging techniques such as nuclear magnetic resonance and X-ray to judge the degree of disc tissue degeneration by observing abnormal changes in the disc.
  • imaging diagnosis has better specificity and is non-invasive and safe, the imaging characteristics of the tissue change slowly.
  • imaging characteristics as a sign of intervertebral disc degeneration still has certain limitations in the early basic research of exploring treatment plans and the optimization of clinical diagnosis and treatment plans in the later stage.
  • Chondrocytes account for a large proportion of the nucleus pulposus in adults, bear a large load, and are an important component for 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.
  • the present invention provides the application of CITED4 and/or METRN in the differential diagnosis of the degree of intervertebral disc degeneration.
  • the present invention utilizes single-cell transcriptome sequencing technology and bioinformatics analysis technology, and by analyzing the single-cell gene expression profile of human intervertebral disc nucleus pulposus tissue clinically collected by the inventor of the present invention, separates, characterizes, and identifies a chondrocyte subpopulation that can reflect the intervertebral disc degeneration process, and through further research, determines the new characteristic genes CITED4 and METRN expressed by the cell subpopulation.
  • the characteristic genes have a high diagnostic value for the differential diagnosis of the degree of intervertebral disc nucleus pulposus tissue degeneration, and have a marking significance for the occurrence and development of pathological degeneration of intervertebral disc nucleus pulposus tissue.
  • the 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 preparation of a product for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
  • the reagent includes:
  • Reagents 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
  • a reagent for detecting the number of CITED4 and/or METRN positively expressed cells in a sample is provided.
  • the reagent for detecting the mRNA expression level of CITED4 and/or METRN in the sample includes:
  • Probes that specifically recognize CITED4 and/or METRN are specifically recognize CITED4 and/or METRN.
  • the reagent for detecting the protein expression level of CITED4 and/or METRN in the sample includes:
  • Affinity proteins that specifically bind to proteins encoded by CITED4 and/or METRN.
  • the reagent for detecting the number of CITED4 and/or METRN positively expressed cells in a sample includes a reagent for detecting the number of CITED4 and/or METRN positively expressed cells by immunohistochemistry.
  • the information of the genes CITED4 and METRN are as follows: the Gene ID of gene CITED4 (Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4) is 163732, and the Gene ID of gene METRN (meteorin, glial cell differentiation regulator) is 79006.
  • the detailed information of the genes can be obtained at https://www.ncbi.nlm.nih.gov/gene/.
  • the genes CITED4 and METRN include polynucleotides of human CITED4 gene, human METRN gene and any functional equivalents of human CITED4 gene and human METRN gene. They can be natural or artificially synthesized, or obtained by transfecting cells with vectors that can express DNA fragments of CITED4 and METRN.
  • the vectors include viral vectors and eukaryotic expression vectors.
  • the viral vectors can be any appropriate vectors, including but not limited to retroviral vectors, adenoviral vectors, adenovirus-associated viral vectors, herpes virus (e.g., herpes simplex virus, vaccinia virus and Epstein-Barr virus) vectors, and alphavirus vectors.
  • the eukaryotic expression vectors can be any appropriate expression vectors, including but not limited to pCMV-Myc expression vectors, pcDNA3.0 expression vectors, pcDNA3.1 expression vectors, pEGFP expression vectors, pEF Bos expression vectors, pTet expression vectors, pTRE expression vectors, or vectors modified on the basis of known expression vectors, such as pBin438, pCAMBIA1301, etc.
  • the primer and amplification primer refer to a nucleic acid fragment containing 5-100 nucleotides.
  • the primer or amplification primer contains 15-30 nucleotides that can initiate an enzymatic reaction (e.g., an enzymatic amplification reaction).
  • the primer refers to a primer that specifically amplifies the genes CITED4 and/or METRN.
  • the probe refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule.
  • the probe refers to a probe that specifically recognizes CITED4 and/or METRN.
  • a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a target polynucleotide) through complementary base pairing.
  • the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe.
  • Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase or in situ hybridization assays.
  • Exemplary probes in the present invention include gene-specific DNA oligonucleotide probes, such as microarray probes fixed on a microarray substrate, quantitative nuclease protection test probes, probes connected to molecular barcodes, and probes fixed on beads.
  • the stringency of hybridization reaction can be easily determined by those of ordinary skill in the art, and is usually calculated empirically based on probe length, washing temperature and salt concentration. Generally speaking, longer probes require higher temperatures to anneal correctly, while shorter probes require lower temperatures. Hybridization usually depends on the ability of denatured DNA to reanneal when complementary chains are present in an environment below their melting temperature. The higher the degree of expected homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is inferred that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are also less stringent.
  • the reagents that specifically bind to proteins encoded by CITED4 and/or METRN include, but are not limited to, antibodies, affinity proteins, and also include peptides, aptamers and/or compounds that specifically bind to proteins encoded by CITED4 and/or METRN.
  • the antibody is well known in the art and refers to a specific immunoglobulin for an antigenic site.
  • the antibody described in the present invention refers to an antibody that specifically binds to the protein encoded by CITED4 and/or METRN described in the present invention, and the antibody can be manufactured according to conventional methods in the art.
  • the form of the antibody includes polyclonal antibodies or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen binding site of the antibody, and any other modified immunoglobulin molecules comprising the antigen binding site, as long as the antibody exhibits the desired biological binding activity.
  • antibody fragments such as Fab, Fab', F(ab')2 and Fv fragments
  • scFv single-chain Fv
  • multispecific antibodies such as bispecific antibodies
  • monospecific antibodies monovalent antibodies
  • chimeric antibodies humanized antibodies
  • human antibodies fusion proteins comprising the antigen binding site of the antibody
  • any other modified immunoglobulin molecules comprising the antigen binding site, as long as the antibody exhibits the desired biological binding activity.
  • the peptide has the ability to highly bind to the target substance (protein encoded by CITED4 and/or METRN of the present invention) and will not be denatured during heat treatment or chemical treatment. Moreover, due to its small size, it can be used as a fusion protein by attaching it to other proteins. Specifically, because it can be specifically attached to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.
  • the aptamer refers to a polynucleotide composed of a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid), which has a stable tertiary structure and has the property of being able to bind to the target molecule (the protein encoded by CITED4 and/or METRN described in the present invention) with high affinity and specificity.
  • the target molecule the protein encoded by CITED4 and/or METRN described in the present invention
  • the aptamer can specifically bind to antigenic substances like an antibody, but is more stable than a protein, has a simpler structure, and is composed of a polynucleotide that is easy to synthesize, it can be used instead of an antibody.
  • the reagents for detecting the number of CITED4 and/or METRN positively expressing cells by immunohistochemistry experiments include but are not limited to any reagents required for detecting the number of CITED4 and/or METRN positively expressing cells by immunohistochemistry experiments, for example, fixatives, buffers, color developing solutions, adhesives, mounting agents, enzyme digestion solutions, and sucrose solutions.
  • the fixing agent includes but is not limited to: formaldehyde, glutaraldehyde, paraformaldehyde, ethanol, HneFIX, acetone
  • the buffer includes but is not limited to: PBS buffer, citrate buffer, EDTA buffer, TBS buffer
  • the color developing solution includes but is not limited to: DAB color developing solution, 4-chloro-1-naphthol (4-Cl-1-Naphthol) color developing solution, 3-amino-9-ethylcarbozole (3-amino-9-ethylcarbozole, AEC) color developing solution, TMB color developing solution, NBT color developing solution
  • the adhesive includes but is not limited to: gelatin, resin glue, polylysine, commercial adhesive
  • the sealing agent includes but is not limited to: skimmed milk powder, BSA, serum and Fab fragment single-chain secondary antibody
  • the enzyme digestion solution includes but is not limited to: trypsin digestion solution, pepsin digestion solution.
  • the differential diagnosis of the degree of intervertebral disc degeneration refers to the differential diagnosis of the degree of intervertebral disc degeneration of the subject.
  • the differential diagnosis of the degree of intervertebral disc degeneration especially refers to the differential diagnosis of moderate and severe degenerative tissues, mild and severe degenerative tissues, and mild-moderate and severe degenerative tissues by CITED4 and/or METRN, and experimental verification has found that the AUC values of CITED4 and/or METRN in the differential diagnosis of the above-mentioned tissues with different degrees of degeneration are all greater than 0.9, with high sensitivity and specificity, and have excellent diagnostic efficacy.
  • the evaluation of the therapeutic efficacy of intervertebral disc degeneration refers to evaluating the efficacy of a subject's treatment of a certain treatment method or therapeutic drug.
  • the evaluation of the therapeutic efficacy of intervertebral disc degeneration especially refers to CITED4 and/or METRN performing differential diagnosis on moderate and severe degenerative tissues, mild and severe degenerative tissues, and mild-moderate and severe degenerative tissues of subjects with intervertebral disc degeneration, and further evaluating the efficacy of the subject's treatment of a certain treatment method or therapeutic drug, which has good clinical application prospects.
  • the expression level refers to the absolute amount or relative amount of CITED4 and/or METRN described in the present invention.
  • the expression level of CITED4 and/or METRN described in the present invention can be determined by a variety of techniques well known to those skilled in the art.
  • the absolute amount or relative amount of CITED4 and/or METRN described in the present invention can be detected by using immunohistochemical detection methods well known to those skilled in the art.
  • the sample refers to a composition obtained from or derived from a target subject, which contains cell entities and/or other molecular entities to be characterized and/or identified, for example, based on physical, biochemical, chemical and/or physiological characteristics.
  • the sample can be obtained from the subject's blood and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom.
  • the source of the tissue sample can be solid tissue, such as from fresh, frozen and/or preserved organ or tissue samples, biopsy tissues or aspirates; blood or any blood component; body fluids; cells from any time of individual pregnancy or development; or plasma.
  • sample includes biological samples that have been processed in any way after they are obtained, such as reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes.
  • the sample includes, but is not limited to, tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, bladder washing fluid, secretions (e.g., breast secretions), oral washing fluid, swabs (e.g., oral swabs), touch preparations, fine needle aspirations, cell extracts, and combinations thereof.
  • the sample is preferably a tissue sample derived from a subject, more preferably a degenerative intervertebral disc tissue sample derived from a subject.
  • the differential diagnosis refers to the discovery, judgment, identification or recognition of the health status or condition of an individual based on one or more symptoms, data or other information related to the individual.
  • the differential diagnosis of the degree of intervertebral disc degeneration refers to the differentiation of individuals with different degrees of intervertebral disc degeneration, especially the differentiation of individuals with moderate and severe intervertebral disc degeneration, mild and severe intervertebral disc degeneration, mild-moderate and severe degenerative intervertebral disc degeneration.
  • the reagent is used to detect the expression level of CITED4 and/or METRN in the test sample of the subject through sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, and immunohistochemistry technology.
  • the sequencing technology is a nucleic acid sequencing technology, including chain terminator (Sanger) sequencing technology and dye terminator sequencing technology. Ordinary technicians in this field will recognize that since RNA is less stable in cells and is more susceptible to nuclease attack in experiments, RNA is usually reverse transcribed into DNA before sequencing.
  • the sequencing technology also includes next-generation sequencing technology (i.e., deep sequencing/high-throughput sequencing technology).
  • nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blotting.
  • nucleic acid amplification technology includes but is 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).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • TMA transcription-mediated amplification
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • the protein immunoassay technique includes sandwich immunoassay, such as sandwich ELISA, in which two antibodies recognizing different epitopes on CITED4 and/or METRN are used to detect the marker; radioimmunoassay (RIA), direct, indirect or comparative enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), Western blotting, immunoprecipitation, and any particle-based immunoassay (eg, using gold, silver or latex particles, magnetic particles or quantum dots).
  • sandwich immunoassay such as sandwich ELISA, in which two antibodies recognizing different epitopes on CITED4 and/or METRN are used to detect the marker
  • RIA radioimmunoassay
  • ELISA direct, indirect or comparative enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • FIA fluorescent immunoassay
  • Western blotting immunoprecipitation
  • any particle-based immunoassay eg
  • a second aspect of the present invention provides a product for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
  • the product includes a reagent for detecting the expression level of CITED4 and/or METRN in a sample.
  • the products include detection kits and biochips.
  • the detection kit includes 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 capable of specifically recognizing CITED4 and/or METRN.
  • the detection kit further comprises one or more substances selected from the following group: a container, instructions for use, a positive control substance, a negative control substance, a buffer, an auxiliary agent or a solvent.
  • 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.
  • the detection kit may further include the elements necessary for reverse transcription polymerase chain reaction.
  • the RT-PCR detection kit includes a pair of primers specific for the gene encoding the marker protein.
  • Each primer is a nucleotide with a nucleic acid sequence specific for the gene, and its length may be about 7 to 50bp, more particularly about 10-39bp.
  • the kit may further include primers specific for the nucleic acid sequence of the control gene; preferably, the RT-PCR detection kit may also include a test tube or a suitable vessel, a reaction buffer (different pH values and magnesium concentrations), a deoxynucleotide (dNTP), an enzyme (such as Taq polymerase and reverse transcriptase), a deoxyribonuclease inhibitor, a ribonuclease inhibitor, DEPC-water, and sterile water.
  • a reaction buffer different pH values and magnesium concentrations
  • dNTP deoxynucleotide
  • an enzyme such as Taq polymerase and reverse transcriptase
  • a deoxyribonuclease inhibitor a ribonuclease inhibitor
  • DEPC-water DEPC-water
  • sterile water sterile water
  • the detection kit may include elements necessary for operating a DNA chip.
  • the DNA chip kit may include a substrate bound to a gene or cDNA or an oligonucleotide equivalent to a fragment thereof, and reagents, agents and enzymes for constructing a fluorescently labeled probe.
  • the substrate may include a control gene or cDNA or an oligonucleotide equivalent to a fragment thereof.
  • the detection kit disclosed in the present invention may include elements necessary for performing ELISA.
  • the ELISA detection kit may include antibodies specific for proteins (proteins encoded by CITED4 and/or METRN described in the present invention).
  • the antibody has high selectivity and affinity for marker proteins, no cross-reactivity with other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody.
  • the ELISA detection kit may include antibodies specific for control proteins.
  • the ELISA detection kit may further include reagents capable of detecting the bound antibody, for example, a labeled second antibody, a chromophore, an enzyme (e.g., conjugated to an antibody), and a substrate thereof or a substance capable of binding the antibody.
  • reagents capable of detecting the bound antibody for example, a labeled second antibody, a chromophore, an enzyme (e.g., conjugated to an antibody), and a substrate thereof or a substance capable of binding the antibody.
  • the biochip is also referred to as an array, which refers to a solid support comprising connected nucleic acid or peptide probes.
  • the array typically comprises a plurality of different nucleic acid or peptide probes connected to the substrate surface according to different known positions.
  • These arrays also referred to as microarrays, can typically be produced using mechanical synthesis methods or light-guided synthesis methods, which incorporate a combination of photolithography and solid phase synthesis methods.
  • the array can comprise a flat surface, or can be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, glass or any other suitable substrate.
  • the array can be packaged in a certain manner to allow for diagnosis of a fully functional device or manipulation of other methods.
  • Microarrays are hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), arranged in an orderly manner on a substrate.
  • the substrate can be a solid substrate, for example, a glass or silica slide, a bead, a fiber optic binder, or a semi-solid substrate, such as a nitrocellulose membrane.
  • the nucleotide sequence can be DNA, RNA, or any arrangement thereof.
  • the biochip includes a gene chip and a protein chip; the gene chip includes a solid phase carrier; and oligonucleotide probes fixed in order on the solid phase carrier, and the oligonucleotide probes specifically correspond to part or all of the sequences shown by CITED4 and/or METRN.
  • the protein chip includes a solid phase carrier, and specific antibodies or ligands of proteins encoded by CITED4 and/or METRN fixed on the solid phase carrier.
  • the antibodies specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity.
  • chimeric antibodies immunoglobulins in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity.
  • the ligand may comprise a peptide, an antibody or a fragment thereof, or an aptamer or an oligonucleotide that can specifically bind to CITED4 and/or METRN.
  • the antibodies used in the present invention against proteins encoded by CITED4 and/or METRN are used in the broadest sense, specifically covering, for example, monoclonal antibodies, polyclonal antibodies, antibodies with multi-epitope specificity, multispecific antibodies and antibody fragments. Such antibodies may be chimeric, humanized, human and synthetic. As long as the fragment can retain The ability to bind to the protein encoded by CITED4 and/or METRN is sufficient.
  • the 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 preparation of a system and/or device for differentially diagnosing the degree of intervertebral disc degeneration or evaluating the therapeutic efficacy of intervertebral disc degeneration.
  • a fourth aspect of the present invention provides a system and/or device for differentially diagnosing the degree of intervertebral disc degeneration or evaluating the efficacy of treatment for intervertebral disc degeneration.
  • system/device includes a processor, an input module, and an output module;
  • the processor is used to perform 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, and contains a computer-readable medium of instructions, which, when executed by the processor, executes an algorithm on the input expression level of CITED4 and/or METRN;
  • the output module is used to output the degree of intervertebral disc degeneration of the subject or the therapeutic efficacy of intervertebral disc degeneration.
  • non-human animals includes all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc.
  • the subject is preferably a human.
  • the present invention further provides a computer-readable storage medium having a computer program stored thereon, and when the computer program is executed by a processor, the system and/or device according to the fourth aspect of the present invention is implemented.
  • the system/device is a method for distinguishing different components, elements, parts, parts or assemblies at different levels.
  • the words can be replaced by other expressions.
  • the present invention can be implemented as an apparatus, method or computer program product. Therefore, the content disclosed in the present invention can be specifically implemented in the following forms, that is, it can be complete hardware, it can be complete software (including firmware, resident software, microcode, etc.), and it can also be a combination of hardware and software.
  • the present invention can also be implemented in the form of a computer program product in one or more computer-readable media, and the computer-readable medium contains computer-readable program code.
  • 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 electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, device or device, or any combination thereof.
  • the computer-readable storage medium may be any tangible medium containing or storing a program that may be used by or in conjunction with an instruction execution system, device or device.
  • a fifth aspect of the present invention provides a biomarker for differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
  • biomarkers are CITED4 and/or METRN.
  • the sixth aspect of the present invention provides the use of the biomarkers CITED4 and/or METRN in differential diagnosis of the degree of intervertebral disc degeneration or evaluation of the therapeutic efficacy of intervertebral disc degeneration.
  • biomarkers CITED4 and/or METRN described in the present invention are used to differentially diagnose the degree of intervertebral disc degeneration or evaluate the therapeutic efficacy of intervertebral disc degeneration, such methods fall within the protection scope of the present invention and are not limited to the specific methods of using the biomarkers CITED4 and/or METRN.
  • the purpose of differentially diagnosing the degree of intervertebral disc degeneration or evaluating the therapeutic efficacy of intervertebral disc degeneration can be achieved or basically achieved, it falls within the protection scope of the present invention.
  • a seventh aspect of the present invention provides a method for differentially diagnosing the degree of intervertebral disc degeneration or evaluating the therapeutic efficacy of intervertebral disc degeneration.
  • the method comprises the following steps:
  • the 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.
  • the present invention has the following advantages and beneficial effects:
  • the present invention first discovered that CITED4 and METRN were significantly differentially expressed in degenerative disc tissues of different degrees of degeneration, and that they were highly correlated with the degree of disc degeneration. In the clinical samples collected by the present invention, they showed a high differential diagnostic efficacy for the degree of disc degeneration, with high accuracy, sensitivity and specificity.
  • the AUC values of CITED4 for 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 of METRN for differential diagnosis of moderate vs. severe, mild vs. severe, and mild-moderate vs.
  • CITED4 and METRN can be used as biomarkers for differential diagnosis of the degree of intervertebral disc degeneration, and can be effectively used in the differential diagnosis of the degree of intervertebral disc degeneration, thereby allowing for timely and effective intervention and treatment in the early stages.
  • the CITED4 and/or METRN provided by the present invention have high diagnostic efficacy for the differential diagnosis of the degree of intervertebral disc degeneration and have good prospects for development as diagnostic methods.
  • the present invention provides a new solution strategy to solve the technical problems in the field that imaging diagnosis in clinical practice depends on the imaging characteristics of tissues, and the imaging characteristics of tissues change slowly, resulting in delayed diagnostic results, and the changes in disease tissue morphology are often later than the abnormal changes in molecular biology, resulting in low sensitivity of related diagnostic methods based on tissue morphology.
  • the characteristic genes identified at the molecular level by the present invention can provide a certain theoretical basis for the discovery and promotion of biomarkers of intervertebral disc degeneration, and have important application significance for the differential diagnosis of intervertebral disc degeneration, the effectiveness evaluation of related intervention measures, and the understanding of the pathogenesis and progression of the disease.
  • FIG1 is a diagram showing the quality control results of the preparation of a single cell suspension of human intervertebral disc nucleus pulposus tissue
  • FIG2 is a diagram showing the results of single cell subpopulations in human intervertebral disc nucleus pulposus tissue
  • FIG3 is a violin plot showing the mRNA expression of CITED4 and METRN, marker molecules of the 8th subpopulation of chondrocytes in the nucleus pulposus of human intervertebral disc;
  • FIG4 is a diagram showing the result of Aggrecan (ACAN)-positive cells in human intervertebral disc nucleus pulposus tissue being identified as chondrocytes, wherein the left image is 100X, and the right image is 400X;
  • ACAN Aggrecan
  • Figure 5 is a graph showing differences in CITED4+-expressing chondrocytes in nucleus pulposus tissues of human intervertebral discs at different Pfirrmann grades after immunohistochemical staining, 100X;
  • Figure 6 is a statistical diagram showing the difference in the number of CITED4+ cells and the integrated optical density of METRN+ cells in the nucleus pulposus tissue of intervertebral discs of different groups in the training set, wherein Figure A: CITED4+ cells, Figure B: METRN+ cells;
  • Figure 7 is a ROC curve obtained by plotting CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the training set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • Figure 8 is a ROC curve diagram obtained by plotting METRN+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the training set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • Figure 9 is a ROC curve obtained by plotting METRN+CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the training set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • Figure 10 is a graph showing the statistical results of the difference in the number of CITED4+ cells and the integrated optical density of METRN+ cells in the nucleus pulposus tissue of intervertebral discs of different groups in the validation set, wherein Figure A: CITED4+ cells, Figure B: METRN+ cells;
  • Figure 11 is a ROC curve obtained by plotting CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues in the validation set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • Figure 12 is a ROC curve diagram obtained by plotting METRN+ cells against different degenerative human intervertebral disc nucleus pulposus pathological tissues in the validation set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • Figure 13 is a ROC curve obtained by plotting METRN+CITED4+ cells against different degenerative human intervertebral disc nucleus pulposus pathological tissues in the validation set, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • Figure 15 is a ROC curve diagram obtained by plotting CITED4+ cells on different degenerative human intervertebral disc nucleus pulposus pathological tissues, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild-moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • FIG. 16 is a graph showing the difference in the expression of METRN+ chondrocytes in the nucleus pulposus tissue of the intervertebral disc with different Pfirrmann grades after immunohistochemical staining, 100X;
  • FIG18 is a ROC curve diagram obtained by plotting METRN+ cells for different degenerative nucleus pulposus pathological tissues, wherein, FIGA: L/M (comparison between mild degeneration and moderate degeneration), FIGB: M/S (comparison between moderate degeneration and severe degeneration), FIGC: L/S (comparison between mild degeneration and severe degeneration), FIGD: LM/S (comparison between mild-moderate degeneration and severe degeneration), FIGE: L/MS (comparison between mild degeneration and moderate-severe degeneration);
  • Figure 19 is a ROC curve diagram obtained by plotting METRN+CITED4+ cells on different degenerated nucleus pulposus pathological tissues, wherein, Figure A: L/M (comparison between mild degeneration and moderate degeneration), Figure B: M/S (comparison between moderate degeneration and severe degeneration), Figure C: L/S (comparison between mild degeneration and severe degeneration), Figure D: LM/S (comparison between mild to moderate degeneration and severe degeneration), Figure E: L/MS (comparison between mild degeneration and moderate to severe degeneration).
  • CITED4 and METRN were significantly differentially expressed in degenerative disc tissues of different degrees of degeneration, and that they were highly correlated with the degree of disc degeneration.
  • they showed a high differential diagnostic efficacy for the degree of disc degeneration, with high accuracy, sensitivity and specificity, and can be used in clinical practice as biomarkers for differential diagnosis of the degree of disc degeneration.
  • biomarker used in this article refers to an indicator of the phenotype of a patient (the patient in the present invention specifically refers to a patient with degenerative disc disease), such as an indicator of a pathological state or possible responsiveness to a therapeutic agent, which can be detected in a biological sample derived from the patient.
  • the biomarker includes but is not limited to: DNA, RNA, protein, small molecule metabolites, carbohydrates, glycolipid-based molecules, etc.; in a specific embodiment of the present invention, the biomarker is CITED4 and/or METRN.
  • expression level used in this article is the same as “level”, which refers to the absolute amount or relative amount of the biomarkers CITED4 and/or METRN described in the present invention.
  • level refers to the absolute amount or relative amount of the biomarkers CITED4 and/or METRN described in the present invention.
  • the expression level of the biomarkers CITED4 and/or METRN described in the present invention can be determined by a variety of techniques.
  • the absolute amount or relative amount of the biomarkers CITED4 and METRN described in the present invention can be detected by using methods well known to those skilled in the art.
  • the expression level of a gene can be measured by a variety of techniques well known in the art.
  • the expression level of a gene can be determined by measuring the amount of mRNA. Methods for measuring the amount of mRNA are well known in the art. For example, nucleic acids contained in a sample (e.g., blood, cell or tissue sample extracted from a patient) are first extracted according to a standardized method, such as using a cell enzyme or chemical solution or extracted by a nucleic acid binding resin according to the manufacturer's instructions.
  • the extracted mRNA is then detected by hybridization (e.g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • 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).
  • the term "significant difference” as used herein refers to the difference in the content or concentration of one or more biomarkers of the present invention in one sample, compared to the expression level of the same one or more biomarkers of the present invention in a second sample, after measuring the content or concentration of the biomarkers.
  • the "significant difference gene” used herein can be measured by the ratio of the level of a given biomarker (gene) relative to the average level of a given biomarker in a control, wherein the ratio is not equal to 1.0.
  • the difference can also be measured using a p-value. When using a p-value, a biomarker is identified as showing a difference between the first and second populations when the p-value is less than 0.1.
  • the p-value is less than 0.05. Even more preferably, the p-value is less than 0.01. Still more preferably, the p-value is less than 0.005. Most preferably, the p-value is less than 0.001.
  • the difference is determined based on a ratio, if the ratio of the levels in the first and second samples is greater than or less than 1.0, the biomarker is different. For example, greater than 1.2, 1.5, A ratio of 1.7, 2, 3, 4, 10, 20, or a ratio less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1, 0.05.
  • diagnosis refers to the discovery, judgment or recognition of an individual's health status or condition based on one or more symptoms, data or other information related to the individual.
  • the individual's health status can be diagnosed as healthy/normal (i.e., the absence of disease or illness), or can be diagnosed as unhealthy/abnormal (i.e., the presence of disease or illness), or can be diagnosed as a specific severity of illness (e.g., mild, mild-moderate, moderate, moderate-severe, severe).
  • diagnosis, early diagnosis, diagnosis and variations of these terms include early discovery of diseases/disorders associated with specific diseases or illnesses (specifically, patients with degenerative disc disease in the present invention); characteristics or classification of the disease; discovery of the progression, cure or recurrence of the disease; discovery of the individual's response to the disease after treatment or treatment.
  • the detection probe provided by the present invention may also include one or more detectable markers, and specific examples of the detectable markers include fluorescent molecules (or fluorescent dyes), fluorescent nanoparticles, radioactive isotopes, detectable enzymes, etc.
  • the fluorescent molecules can be selected from the User Manual - Fluorescent Probes and Labeling Technology Guide, and special fluorescent groups that can be bound to nucleic acid molecules include but are not limited to: 4-acetylamino-4'-isothiocyanate stilbene-2,2'-disulfonic acid, acridine, isothiocyanate acridine, 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid, 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate, N-(4-anilino-1-naphthyl)maleimide, o-aminobenz
  • the fluorescent nanoparticles include but are not limited to: semiconductor nanocrystals, quantum dots, etc.
  • the radioactive isotopes include but are not limited to: radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt, etc.
  • the detectable enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, ⁇ -galactosidase, ⁇ -glucuronidase, ⁇ -lactamase, and the like.
  • area under the curve refers to the area under the curve of the receiver operating characteristic (ROC) curve, both of which are well known in the art.
  • the AUC measurement is useful for comparing the accuracy of classifiers across the entire data range. A classifiers with higher AUC have a higher ability to correctly classify unknowns between two or more target groups (e.g., intervertebral disc nucleus pulposus tissue samples of patients with intervertebral disc degeneration of different degrees of degeneration).
  • the ROC curve is useful for describing the performance of a specific feature (e.g., any biomarker described herein and/or any item of additional biomedical information) when distinguishing between two populations.
  • ROC curves can be generated with respect to individual features, and can be generated with respect to other individual outputs, for example, a combination of two or more features can be combined mathematically (e.g., added, subtracted, multiplied, etc.) to provide a separate sum value, and the separate sum value can be plotted in the ROC curve.
  • any combination of multiple features in which the combination is derived from a separate output value can be plotted in the ROC curve.
  • These combinations of features can include tests.
  • the ROC curve is a plot of the true positive rate (sensitivity) of a test against the false positive rate (1-specificity) of the test, which can be used to analyze the accuracy of diagnosis.
  • sample refers to a composition obtained from or derived from a target subject, which contains cell entities and/or other molecular entities to be characterized and/or identified, for example, based on physical, biochemical, chemical and/or physiological characteristics.
  • the sample can be obtained from the subject's blood and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom.
  • the source of the tissue sample can be a solid tissue, such as an organ or tissue sample, a biopsy tissue or an aspirate from a fresh, frozen and/or preserved organ; blood or any blood component; body fluid; cells from any time of pregnancy or development of an individual; or plasma.
  • sample includes biological samples that have been processed in any way after they are obtained, such as reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes.
  • the sample or sample described in the present invention includes, but is not limited to: a tissue sample, a blood sample, a blood-derived cell sample, a serum sample, a plasma sample, a lymph sample, a synovial fluid sample, a cell extract sample, or any combination thereof.
  • the nucleus pulposus tissue was cleaned with PBS and minced with ophthalmic scissors.
  • the minced tissue was transferred to a 5 mL centrifuge tube containing tissue dissociation solution, digested with trypsin at 37°C for 0.5 h, and digested with type II collagenase at 37°C for 2.5-3 h, and passed through a 70 ⁇ m cell sieve membrane.
  • the red blood cells were lysed, and the dead cells were removed by passing through a 40 ⁇ m cell sieve membrane, and gradient centrifuged at 4°C, and the cells were resuspended in PBS to an appropriate volume.
  • the Chromium TM Single Cell 3'Solution based on the 10X Genomics platform is used to isolate and label all single cells in the range of 500-10,000 at one time, and perform gene expression detection at the single-cell level.
  • the specific process is that the Chromium TM Single Cell 3'Solution is a microfluidic platform based on the GemCode technology, which encapsulates gel beads with barcodes and primers and single cells in oil droplets; then in each oil droplet, the gel beads are dissolved, the cells are lysed to release mRNA, and reverse transcription is used to produce barcoded cDNA for sequencing.
  • the cDNA is subsequently used to construct a library, and then the library is sequenced and detected using the Illumina sequencing platform, obtaining a large amount of single-cell gene expression data at one time, thereby realizing expression sequencing at the single-cell level.
  • the prepared single-cell suspension was quality controlled. Taking a 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 agglomeration rate was 26.09%
  • the live cell concentration was 730 cells/ ⁇ L
  • the average cell diameter was 12.26 ⁇ m
  • the live cell particle ratio was 77.05%.
  • the Chromium TM Single Cell 3'Solution based on the 10X Genomics platform was used to isolate single cells from human intervertebral disc nucleus pulposus tissue, and gene expression was detected at the single cell level.
  • single cell subpopulations of human intervertebral disc nucleus pulposus tissue were identified, and the results are shown in Figure 2.
  • subpopulation 8 is a newly discovered subpopulation of chondrocytes in intervertebral disc nucleus pulposus tissue.
  • Example 2 MRI grading of intervertebral disc nucleus pulposus tissue and sample grouping
  • the degree of disc degeneration in all patients was graded using the Pfirrmann MRI (T2WI) grading standard for disc degeneration.
  • Grade I The disc structure is homogeneous and bright white, the boundary between the nucleus pulposus and the annulus fibrosus is clear, the signal intensity is high or equal to the cerebrospinal fluid, and the disc height is normal.
  • Grade II The disc structure is heterogeneous, with or without horizontal bands, the boundary between the nucleus pulposus and the annulus fibrosus is clear, the signal intensity is high or equal to the cerebrospinal fluid, and the disc height is normal.
  • Grade III The disc structure is heterogeneous and gray, the boundary between the nucleus pulposus and the annulus fibrosus is unclear, the signal intensity is medium, and the disc height is normal or slightly reduced.
  • Grade IV The disc structure is heterogeneous, gray or black, the boundary between the nucleus pulposus and the annulus fibrosus disappears, the signal intensity is medium or low, and the disc height is normal or moderately reduced.
  • Grade V The disc structure is heterogeneous, gray or black, the boundary between the nucleus pulposus and the annulus fibrosus disappears, the signal intensity is medium or low, and the disc height is normal or moderately reduced.
  • the disc structure is heterogeneous and black, the boundary between the nucleus pulposus and the annulus fibrosus disappears, the signal intensity is low, and the intervertebral disc space is collapsed.
  • grades I to II were divided into mild degenerative lesions group (L), III into moderate degenerative lesions group (M), and IV to V into severe degenerative lesions group (S).
  • grades I to II and III were further combined into mild to moderate degenerative lesions group (LM), and grades III and IV to V were divided into moderate to severe degenerative lesions group (MS).
  • the Pfirrmann degeneration grading results of 50 cases of human intervertebral disc degeneration tissues are shown in Table 1 below. The results show that there are 17 samples with Pfirrmann degeneration grading I-II (mild, L), 18 samples with III (moderate, M), and 15 samples with IV-V (severe, S). Further grouping resulted in 35 samples with I-III (mild-moderate, LM) and 33 samples with III-V (moderate-severe, MS).
  • Anti-Aggrecan Antibody (ACAN) (Boster Biotech, BA2967-1, Lot No: D-blj2-08F183).
  • Example 2 50 cases of human degenerative intervertebral disc tissue identified by MRI in Example 2 were collected.
  • the samples were pathological tissues of degenerative intervertebral discs collected from patients with lumbar burst fractures or degenerative lumbar disc herniation caused by trauma, which must be surgically removed according to actual clinical diagnosis and treatment. This study was approved by the hospital's medical ethics committee and the informed consent of the subjects or their family members (guardians).
  • Cartilage proteoglycan Aggrecan is an important component of the extracellular matrix of cartilage cells and is usually used as a marker to identify chondrocytes.
  • the identification results of this example are shown in Figure 4.
  • Cells expressing positive in human intervertebral disc nucleus pulposus tissue identified by immunohistochemistry using Aggrecan (ACAN) antibody are chondrocytes.
  • Example 2 50 cases of human degenerative intervertebral disc tissue identified by MRI in Example 2 were collected.
  • the samples were pathological tissues of diseased intervertebral discs obtained from patients with lumbar burst fractures or degenerative lumbar disc herniation caused by trauma, which must be surgically removed according to actual clinical diagnosis and treatment. This study was approved by the hospital's medical ethics committee and the informed consent of the subjects or their family members (guardians).
  • SPSS software was used to draw ROC curves for mild degenerative lesions and moderate to severe degenerative lesions by using the average value, and the area under the AUC curve and threshold were calculated to evaluate the diagnostic value of the newly discovered chondrocyte subset characteristic genes CITED4, METRN, and the combination of CITED4 and METRN for the degree of intervertebral disc degeneration.
  • the 50 cases of human degenerative intervertebral disc tissues collected by the present invention are randomly divided into two groups (training set and validation set) using the createDataPartition function of the caret package in the R language, each accounting for 50%, that is, the training set contains 25 cases of human degenerative intervertebral disc tissues, and the validation set contains 25 cases of human degenerative intervertebral disc tissues.
  • the training set and the validation set are further used for analysis and verification.
  • the ROC curve analysis results of CITED4+ between the groups are shown in Figure 7 and Table 2 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.875, the corresponding specificity is 77.8%, and the sensitivity is 87.5%; the AUC value of mild compared with severe (L/S) is 0.931, the corresponding specificity is 88.9%, and the sensitivity is 87.5%; the AUC value of mild-moderate compared with severe (LM/S) is 0.903, the corresponding specificity is 83.3%, and the sensitivity is 87.5%.
  • M/S moderate compared with severe
  • L/S mild compared with severe
  • L/S mild-moderate compared with severe
  • CITED4 is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues, which has extremely high application value.
  • the ROC curve results of each group corresponding to METRN+ are shown in Figure 8 and Table 3 below. Except for mild to moderate (L/M) and mild to moderately severe (L/MS), the AUC values of the ROC curves of the remaining groups are all greater than 0.7. Among them, the AUC value of moderate to severe (M/S) is 0.806, the corresponding specificity is 66.7%, and the sensitivity is 100%, the AUC value of mild to severe (L/S) is 0.875, the corresponding specificity is 77.8%, and the sensitivity is 100%, and the AUC value of mild to moderate (LM/S) is 0.840, the corresponding specificity is 72.2%, and the sensitivity is 100%.
  • M/S moderate to severe
  • L/S mild to severe
  • L/MS mild to moderately severe
  • METRN is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild to moderate and severe degenerative tissues, which has extremely high application value.
  • the ROC curve results of CITED4+METRN+ for each group are shown in Figure 9 and Table 4 below.
  • the ROC curve AUC values of the remaining groups were greater than 0.7, except for the groups of moderate to moderate (L/M) and mild to moderately severe (L/MS).
  • the AUC value of moderate to severe (M/S) was 0.875, with a corresponding specificity of 77.8% and a sensitivity of 87.5%
  • the AUC value of mild to severe (L/S) was 0.875, with a corresponding specificity of 100% and a sensitivity of 75.0%
  • the AUC value of mild to moderate (LM/S) was 0.917, with a corresponding specificity of 88.9% and a sensitivity of 87.5%.
  • the results showed that the combination of CITED4 and METRN can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in distinguishing moderate from severe, mild from severe, and mild to moderate from severe degenerative tissues, and has a significantly better application value.
  • the ROC curve analysis results of CITED4+ between the groups are shown in Figure 11 and Table 5 below. Except for mild and moderate (L/M), the ROC curve AUC values of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.968, the corresponding specificity is 100%, and the sensitivity is 85.7%; the AUC value of mild compared with severe (L/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%; the AUC value of mild-moderate compared with severe (LM/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%.
  • CITED4 is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues, which has extremely high application value.
  • the ROC curve results of each group corresponding to METRN+ are shown in Figure 12 and Table 6 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.984, the corresponding specificity is 88.9%, and the sensitivity is 100%, the AUC value of mild compared with severe (L/S) is 0.982, the corresponding specificity is 87.5%, and the sensitivity is 100%, and the AUC value of mild-moderate compared with severe (LM/S) is 0.983, the corresponding specificity is 88.2%, and the sensitivity is 100%.
  • M/S moderate compared with severe
  • L/S mild compared with severe
  • L/S mild-moderate compared with severe
  • L/S mild-moderate compared with severe
  • METRN is a biomarker that can mark the severity of intervertebral disc nucleus pulposus tissue degeneration, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues, which has extremely high application value.
  • the ROC curve results of CITED4+METRN+ for each group are shown in Figure 13 and Table 7 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the other groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%. The AUC value of mild compared with severe (L/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%. The AUC value of mild-moderate compared with severe (LM/S) is 1.000, the corresponding specificity is 100%, and the sensitivity is 100%.
  • CITED4 is positively expressed in the nucleus of chondrocytes.
  • Figure 5 As the severity of degeneration increases, the number of CITED4+ positive cells increases significantly. Mildly degenerated CITED4+ cells account for about 3% of the total cells, moderately degenerated CITED4+ cells account for about 10%, and severely degenerated CITED4+ cells account for about 60%. The results of the statistical difference analysis between the groups are shown in Figure 14.
  • the AUC value of moderate compared with severe is 0.917, the corresponding specificity is 77.8%, and the sensitivity is 93.3%;
  • the AUC value of mild compared with severe (L/S) is 0.961, the corresponding specificity is 88.2%, and the sensitivity is 93.3%;
  • the AUC value of mild to moderate compared with severe (LM/S) is 0.938, the corresponding specificity is 82.9%, and the sensitivity is 93.3%.
  • CITED4 is a biomarker that can mark the severity of degeneration of intervertebral disc nucleus pulposus tissue, especially in the diagnosis of moderate and severe, mild and severe, mild to moderate and severe degenerative tissues, and has extremely high application value.
  • METRN is positively expressed in the cytoplasm of chondrocytes.
  • Figure 16 As the severity of degeneration increases, the number of METRN+ positive cells increases significantly. Mildly degenerated METRN+ cells account for about 1%, moderately degenerated METRN+ cells account for about 5%, and severely degenerated METRN+ cells account for about 50%. The statistical differences between the groups are shown in Figure 17.
  • the ROC curve results of each group corresponding to METRN+ are shown in Figure 18 and Table 9 below. Except for mild and moderate (L/M), the AUC values of the ROC curves of the remaining groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.911, the corresponding specificity is 77.8%, and the sensitivity is 100%. The AUC value of mild compared with severe (L/S) is 0.929, the corresponding specificity is 82.4%, and the sensitivity is 100%. The AUC value of mild-moderate compared with severe (LM/S) is 0.920, the corresponding specificity is 80%, and the sensitivity is 100%.
  • METRN is a biomarker that can mark the severity of degeneration of intervertebral disc nucleus pulposus tissue, especially in the diagnosis of moderate and severe, mild and severe, mild-moderate and severe degenerative tissues. There is a very high application value.
  • the ROC curve results of each group corresponding to CITED4+METRN+ are shown in Figure 19 and Table 10 below. Except for mild and moderate (L/M), the ROC curve AUC values of the remaining groups are all greater than 0.7. Among them, the AUC value of moderate compared with severe (M/S) is 0.933, the corresponding specificity is 72.2%, and the sensitivity is 100%, the AUC value of mild compared with severe (L/S) is 0.953, the corresponding specificity is 100%, and the sensitivity is 86.7%, and the AUC value of mild-moderate compared with severe (LM/S) is 0.949, the corresponding specificity is 85.7%, and the sensitivity is 93.3%.

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Abstract

La présente invention concerne une utilisation de CITED4 et/ou de METRN dans le diagnostic différentiel d'un degré de dégénérescence de disque intervertébral. Le CITED4 et le METRN présentent des différences significatives dans des tissus présentant différents degrés de dégénérescence de disque intervertébral, ont une valeur de diagnostic différentielle élevée pour le degré de dégénérescence de disque intervertébral, et peuvent être utilisés dans le diagnostic différentiel de la dégénérescence de disque intervertébral et l'évaluation d'efficacité de mesures d'intervention.
PCT/CN2023/136554 2023-03-08 2023-12-05 Utilisation de cited4 et/ou de metrn dans le diagnostic differentiel du degré de dégénérescence des disques intervertébraux Pending WO2024183369A1 (fr)

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CN116797597B (zh) * 2023-08-21 2023-11-17 邦世科技(南京)有限公司 一种基于三阶段全网络的脊柱退行性疾病全检测方法及系统

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