CN114807358A - Biomarker related to tendon injury - Google Patents
Biomarker related to tendon injury Download PDFInfo
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- CN114807358A CN114807358A CN202210595517.4A CN202210595517A CN114807358A CN 114807358 A CN114807358 A CN 114807358A CN 202210595517 A CN202210595517 A CN 202210595517A CN 114807358 A CN114807358 A CN 114807358A
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- 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
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- 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
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- 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
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- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The invention discloses a biomarker related to tendon injury. The invention firstly researches the relation between GPRC5C and tendon injury, and discovers that after a tendon injury model mouse is treated by using a non-steroidal anti-inflammatory drug, the expression level of GPRC5C in achilles tendon is up-regulated, which indicates that GPRC5C can be used for predicting prognosis of tendon injury patients by using the non-steroidal anti-inflammatory drug. Meanwhile, the invention verifies the relevance of GPRC5C and tendon injury in GEO data set. The invention also discloses a method for screening candidate drugs for preventing or treating tendon injury.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a biomarker related to tendon injury.
Background
Tendon is composed of two parts, tenocyte and extracellular matrix. Tendon cells are fibroblasts with a special morphology as parenchymal cells of tendons, and account for the most abundant cell types in tendons. The extracellular matrix of the tendon is mainly composed of collagen fibers arranged in rows along the long axis of the tendon. Due to the sustained mechanical load, tendons are often affected by a variety of pathologies, collectively referred to as tendinopathies. Tendon injury has become a common clinical condition due to overuse or age-related degeneration. The pathogenesis of tendinopathy is still not well understood, but many points of view are receiving increasing attention, such as the oxidative stress theory. Ischemia occurs when the tendon is subjected to maximal tension, and ischemia reperfusion occurs after the tendon is restored to a relaxed state, generating oxidative free radicals. The enzyme peroxiredoxin 5 is an antioxidant enzyme that protects cells against active oxygen-induced damage and has been found by researchers to be expressed more in tendinopathies in humans. In addition, tendon requires oxidative energy metabolism to maintain cellular ATP levels, and hypoxia in large amounts triggers tenocyte cell death, so it is speculated that the hypoxic environment in tendon may be a major cause of tendon degeneration.
Injured tendons tend to heal slowly and it is difficult to restore the structural integrity and mechanical strength of intact tendons, which often places a burden on the clinic and the patient. The clinical treatment of tendon injury is mainly injection of non-steroidal anti-inflammatory drugs, physical therapy or surgery. At present, no literature reports how to predict the prognosis of tendon injury patients using non-steroidal anti-inflammatory drugs so as to select individual treatment schemes according to differences among patients and improve treatment effects.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a product for predicting the prognosis of a patient with tendon injury by using a non-steroidal anti-inflammatory drug.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a reagent that is capable of specifically detecting GPRC5C in a sample.
The "GPRC 5C" refers to a Gene with Gene ID 55890.
The terms "sample" and "sample" are used interchangeably herein to refer to a composition obtained or derived from a subject (e.g., an individual of interest) that comprises cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cells and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluids, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.
Further, the reagent comprises a reagent for detecting GPRC5C by a sequencing technology, a nucleic acid hybridization technology, a nucleic acid amplification technology and a protein immunization technology.
Nucleic acid sequencing methods of the invention include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.
The nucleic acid sequencing method also comprises next generation sequencing (deep sequencing/high-throughput sequencing), and the high-throughput sequencing technology is a unimolecular cluster-based sequencing-by-synthesis technology and is based on a special reversible termination chemical reaction principle. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.
Methods of nucleic acid hybridization in the present invention include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNAISH can be used to determine the structure of chromosomes. Rnash is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.
Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.
The nucleic acid amplification method of the present invention is selected from the group consisting of 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). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.
Generally, PCR uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; RT-PCR Reverse Transcriptase (RT) is used to prepare complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of the DNA; TMA autocatalytically synthesizes multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength, and pH, wherein the multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally includes the use of blocking, partial, terminating moieties, and other modifying moieties to improve the sensitivity and accuracy of the TMA process; LCR with target nucleic acid adjacent region hybridization of two sets of complementary DNA oligonucleotides. The DNA oligonucleotides are covalently linked by DNA ligase in repeated cycles of heat denaturation, hybridization, and ligation to produce a detectable double-stranded ligated oligonucleotide product; the SDA uses multiple cycles of the following steps: primer sequence pairs anneal to opposite strands of the target sequence, primer extension in the presence of dNTP α S to produce double-stranded hemiphosphorothioated (phosphorothioated) primer extension products, endonuclease-mediated nicking of the hemimodified restriction enzyme recognition site, and polymerase-mediated extension from the 3' end of the nick to displace the existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, thereby causing geometric amplification of the products.
The protein immunization methods of the invention include sandwich immunoassays, such as sandwich ELISA, in which the detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.
Further, the agent is selected from:
a probe that specifically recognizes GPRC5C gene; or
Primers for specifically amplifying GPRC5C gene; or
A binding agent that specifically binds GPRC5C protein.
The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modes include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.
The term "primer" refers to 7 to 50 nucleic acid sequences capable of forming a base pair (bas e pair) complementary to a template strand and serving as a starting point for replication of the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.
The term "hybridization" refers to the annealing of two complementary nucleic acid strands to one another under conditions of appropriate stringency. Hybridization is generally carried out using nucleic acid molecules of probe length. Nucleic acid hybridization techniques are well known in the art. Those skilled in the art know how to estimate and adjust the stringency of hybridization conditions such that sequences with at least the desired degree of complementarity will stably hybridize, while sequences with lower complementarity will not stably hybridize.
Examples of binding agents of the invention are peptides, peptidomimetics, aptamers, spiegelmers, dappin, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies and monovalent antibody fragments.
In a second aspect, the invention provides a kit comprising the reagents of the first aspect of the invention.
In the present invention, the kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an auxiliary agent or a solvent, and instructions for use with the kit, wherein the instructions describe how to use the kit for detection, and how to use the detection results to determine the development of a disease and select a treatment regimen.
The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and is included in the scope of the present invention as long as the reagents are used for diagnosing tendon injury or predicting the prognosis of tendon injury patients using nsaids based on the detection of GPRC5C gene.
In a third aspect, the invention provides a chip comprising a reagent according to the first aspect of the invention.
The term "chip," also referred to as an "array," as used herein, refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.
In a fourth aspect, the present invention provides a composition comprising an agent that promotes the expression of GPRC 5C.
Furthermore, the composition also comprises other medicines which are compatible with the reagent and pharmaceutically acceptable carriers and/or auxiliary materials.
In a fifth aspect, the present invention provides a method for screening a candidate drug for preventing or treating tendon injury, the method comprising the steps of:
treating a system expressing or containing GPRC5C with a substance to be screened; detecting the expression level of GPRC5C in the system; wherein, if the substance can promote the expression of GPRC5C, the substance is a candidate drug for preventing or treating tendon injury.
The term "treatment" as used herein generally relates to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect may be achieved, for example, inhibiting the development of a condition (including reducing the rate of development, halting development), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".
In a sixth aspect, the present invention provides a method for identifying and assessing the effect of an agent and/or physical therapy in the prevention or treatment of tendon injury, said method comprising:
(1) collecting a first sample provided by a subject having a tendon injury;
(2) obtaining a GPRC5C expression profile from said first sample;
(3) administering to or on said subject one or more drug candidates and/or one or more physical treatments;
(4) providing a second sample from the subject in step (3);
(5) obtaining a GPRC5C expression profile from said second sample;
(6) comparing the expression profile of GPRC5C obtained in steps (2) and (5) to a reference GPRC5C expression profile;
(7) assessing whether said one or more drug candidates and/or physical treatments are effective in preventing or treating tendon damage based on said comparison in step (6);
further, the pharmaceutical agents are non-steroidal anti-inflammatory drugs (NSAID's).
Further, the non-steroidal anti-inflammatory drugs include propionic acid derivative NSAID's, acetic acid derivative NSAID's, fenamic acid derivative NSAID's, biphenylcarboxylic acid derivative NSAID's, oxicam NSAID's, cyclooxygenase-2 (COX-2) selective NSAID's, or pharmaceutically acceptable salts of the foregoing.
Further, the non-steroidal anti-inflammatory drugs are cyclooxygenase-2 (COX-2) selective NSAID's.
Further, the cyclooxygenase-2 (COX-2) selective NSAID's include celecoxib.
The term "subject" means any animal, also human and non-human animals. The term "non-human animal" includes all vertebrates, e.g., 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 domestic or pet animal; and non-mammals, such as chickens, amphibians, reptiles, and the like.
A seventh aspect of the invention provides the use of any one of:
(1) the use of an agent according to the first aspect of the invention in the manufacture of a product for predicting prognosis of a patient with tendon injury using a non-steroidal anti-inflammatory drug;
(2) use of an agent according to the first aspect of the invention in the manufacture of a product for diagnosing tendon injury;
(3) use of a kit according to the second aspect of the invention in the manufacture of a product for diagnosing tendon injury;
(4) the kit of the second aspect of the invention is used for preparing a product for predicting the prognosis of tendon injury patients using non-steroidal anti-inflammatory drugs;
(5) the chip of the third aspect of the invention is applied to the preparation of products for diagnosing tendon injury;
(6) the chip of the third aspect of the invention is applied to the preparation of products for predicting the prognosis of tendon injury patients using non-steroidal anti-inflammatory drugs;
(7) use of a composition according to the fourth aspect of the invention in the manufacture of a medicament for the prevention or treatment of tendon injury;
(8) use of a method according to the fifth aspect of the invention for screening a candidate for preventing or treating tendon injury;
(9) use of a method according to the sixth aspect of the invention for identifying and assessing the effect of an agent and/or physical therapy in the prevention or treatment of tendon injury.
Further, the non-steroidal anti-inflammatory drugs include propionic acid derivative NSAID's, acetic acid derivative NSAID's, fenamic acid derivative NSAID's, biphenylcarboxylic acid derivative NSAID's, oxicam NSAID's, cyclooxygenase-2 (COX-2) selective NSAID's, or pharmaceutically acceptable salts of the foregoing.
Further, the non-steroidal anti-inflammatory drugs are cyclooxygenase-2 (COX-2) selective NSAID's.
Further, the cyclooxygenase-2 (COX-2) selective NSAID's include celecoxib. The invention has the advantages and beneficial effects that:
the invention firstly discovers that the differential expression of GPRC5C is related to tendon injury, and the detection of the expression level of GPRC5C can diagnose tendon injury or predict the prognosis of tendon injury patients using non-steroidal anti-inflammatory drugs.
The invention also discloses a method for screening candidate drugs for preventing or treating tendon injury.
Drawings
FIG. 1 is a technical roadmap;
FIG. 2 is a graph of the results of comparative experiments on day 1 for the blank group, model group and celecoxib group; wherein, panel a is a blank group HE staining result graph (× 100), panel B is a blank group HE staining result graph (× 400), panel C is a blank group massson trichrome staining result graph (× 100), panel D is a blank group massson trichrome staining result graph (× 400), panel E is a model group HE staining result graph (× 100), panel F is a model group HE staining result graph (× 400), panel G is a model group massson trichrome staining result graph (× 100), panel H is a model group massson trichrome staining result graph (× 400), panel I is a celecoxib medicinal group HE staining result graph (× 100), panel J is a celecoxib medicinal group HE staining result graph (× 400), panel K is a celecoxib medicinal group massson trichrome staining result graph (× 100), and panel L is a celecoxib medicinal group HE staining result graph (× 400); FIG. M is a graph of the results of the blank group RECA-1 immunohistochemical staining (X400), FIG. N is a graph of the results of the model group RECA-1 immunohistochemical staining (X400), and FIG. O is a graph of the results of the celecoxib drug group RECA-1 immunohistochemical staining (X400);
FIG. 3 is a graph of comparative experimental results for the blank group, model group, and celecoxib group on day 14; wherein, panel a is a blank group HE staining result graph (× 100), panel B is a blank group HE staining result graph (× 400), panel C is a blank group massson trichrome staining result graph (× 100), panel D is a blank group massson trichrome staining result graph (× 400), panel E is a model group HE staining result graph (× 100), panel F is a model group HE staining result graph (× 400), panel G is a model group massson trichrome staining result graph (× 100), panel H is a model group massson trichrome staining result graph (× 400), panel I is a celecoxib medicinal group HE staining result graph (× 100), panel J is a celecoxib medicinal group HE staining result graph (× 400), panel K is a celecoxib medicinal group massson trichrome staining result graph (× 100), and panel L is a celecoxib medicinal group HE staining result graph (× 400); FIG. M is a graph of the results of the blank group RECA-1 immunohistochemical staining (X400), FIG. N is a graph of the results of the model group RECA-1 immunohistochemical staining (X400), and FIG. O is a graph of the results of the celecoxib drug group RECA-1 immunohistochemical staining (X400);
FIG. 4 is a line graph of differential expression cassettes of the GPRC5C gene in the experimental group;
FIG. 5 is a line graph of the differential expression cassette of the GPRC5C gene in the GSE26051 dataset;
figure 6 is a ROC graph of GPRC5C diagnosis of tendon injury.
Detailed Description
The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration and explanation only, not limitation.
Example 1 evaluation of the Effect of celecoxib on tendon injury treatment
Firstly, experimental materials:
1. experimental animals: SD rats (sbefu);
2. experimental equipment: a full-automatic dehydrator (Leica, ASP200S), a paraffin slicer (Leica, RM2235), a baking sheet table (Leica, HI1220), a bathtub (Leica, HI1220), a heating paraffin embedding system (Leica, G1150H) and a microscope (Leica, DM 3000);
3. experimental reagent: xylene (national medicine), absolute alcohol (national medicine), Citrate buffer (pH6.0) (China fir Jinqiao, ZLI-9064), PBS phosphate buffer (pH7.2-7.4) (China fir Jinqiao, ZLI-9061), hematoxylin dye (China fir Jinqiao, ZLI-9609), DAB kit (China fir Jinqiao, ZLI-9017), SPkit of general type (China fir Jinqiao, SP-9000), neutral gum (China fir Jinqiao, ZLI-9555), sealant (AR 0150)
II, an experimental method:
1. grouping animals
SD rats were purchased from Sibefu (Beijing) Biotechnology Ltd, 6 weeks old, 30, sex randomized, 200-. The rats were randomized into three groups of 10 animals each: normal control group, tendon injury model group, tendon injury + celecoxib treatment group. The technical route is shown in figure 1.
2. Rat modeling and drug administration
(1) After 1 week of acclimation, 10% chloral hydrate was anesthetized by intraperitoneal injection, and the right achilles tendon was severed and immediately sutured. The normal control group did not sever Achilles tendon, and the other operations were the same as those of the experimental group.
(2) The celecoxib group was injected daily with celecoxib (10mg/Kg) after surgery.
(3) The rats in each group were fed normally.
3. Drawing materials from rat
(1) Rats were sacrificed on days 1 and 14 after molding, 3 rats per group were sacrificed, and achilles tendons at the injured site were taken.
(2) After quick freezing, a part of the sample is stored in a refrigerator at minus 80 ℃ and embedded in paraffin.
4. Dyeing process
(1) Embedding and slicing the sample
1) Fixing: the tissue is fixed in 4% paraformaldehyde for more than 24 h. Taking out the tissue from the fixing solution, flattening the tissue of the target part in a fume hood by using a scalpel, and placing the trimmed tissue and the corresponding label in a dehydration box.
2) And (3) dehydrating: and (5) sequentially carrying out gradient alcohol dehydration. The method comprises the following steps of 1h of 75% alcohol, 4h of 85% alcohol, 2h of 90% alcohol, 2h of 95% alcohol, 1h of absolute ethanol I, 30min of absolute ethanol II, 5min to 10min of alcohol benzene, 5min to 10min of xylene I, 5min to 10min of xylene II, 1h of paraffin and 1h of paraffin.
3) Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, molten wax is put into an embedding frame, tissues are taken out from a dehydration box and put into the embedding frame according to the requirements of an embedding surface before the wax is solidified, and corresponding labels are attached. And (4) freezing and cooling at-20 ℃, taking out the wax block from the embedding frame after the wax is solidified, and trimming the wax block.
4) Slicing: the trimmed wax block was sliced on a paraffin slicer to a thickness of 4 μm. The slices float on a spreading machine at 40 ℃ warm water to flatten the tissues, the tissues are taken out by a glass slide, and the slices are baked in a 60 ℃ oven. Taking out after water baking and wax baking and roasting for standby at normal temperature.
(2) HE staining
1) Placing the slices into xylene I10min, xylene II10min, absolute ethyl alcohol I5 min, absolute ethyl alcohol II 5min, 95% alcohol 5min, 90% alcohol 5min, 80% alcohol 5min, 70% alcohol 5min, and distilled water washing in sequence.
2) The sections were stained with hematoxylin for 3-8 min, washed with tap water, 1% HCl-alcohol differentiated for several seconds, and washed with tap water.
3) The sections were stained with eosin stain for 1-3 min.
4) And (3) putting the slices into 95% alcohol I for 5min to 95% alcohol II for 5min to absolute ethyl alcohol I for 5min to absolute ethyl alcohol II for 5min to xylene I for 5min to xylene II for 5min to dehydrate and transparent, taking the slices out of the xylene, slightly drying the slices, and sealing the slices with neutral gum.
5) And (6) microscopic observation.
(3) Masson staining (Masson trichrome staining kit, Solarbio, G1340)
1) The slices were dewaxed conventionally to water.
2) And dyeing with a prepared Weigart hematoxylin staining solution for 5min to 10 min.
3) And (5) differentiating the acidic ethanol differentiation solution for 5-15s, and washing with water.
4) Returning the Masson bluing solution to blue for 3-5min, and washing with water.
5) Washing with distilled water for 1 min.
6) And dyeing with ponceau fuchsin dyeing solution for 5-10 min.
7) During the operation, according to the weight ratio of distilled water: weak acid working solution is prepared according to the ratio of 2:1, and the solution is washed for 1min by the weak acid working solution.
8) Washing with phosphomolybdic acid solution for 1-2min
9) Washing with prepared weak acid working solution for 1 min.
10) Directly placing into aniline blue staining solution for dyeing for 1-2 min.
11) Washing with prepared weak acid working solution for 1 min.
12) 95% ethanol dehydrates rapidly.
13) Dehydrating with anhydrous ethanol for 3 times, each for 5-10 s.
14) The xylene is transparent for 3 times, each for 1-2 min.
15) And (5) sealing by using neutral gum.
16) And (6) microscopic observation.
(4) Immunohistochemistry (RECA1)
1) Paraffin wax slices were baked at 60 deg.C for 1 hr, and dewaxed with xylene (xylene I10min, xylene II10min), gradient alcohol (100% 3min, 100% 3min, 95% 3min, 90% 3min, 85% 3min, 75% 3min, distilled water 3min, PBS 3 min).
2) Antigen retrieval: placing the slices in 0.1mol/L citric acid solution with ph of 6.0, boiling with microwave oven 100 fire for three minutes to slightly boil, maintaining 50 fire for 7 minutes, stopping heating, and naturally cooling for 20-30 minutes. Rinse 5min × 3/time with PBS.
3) Immune reaction: 3% H 2 O 2 Incubate for 10min to eliminate endogenous peroxidase activity. PBS wash, 5min X3 times. Blocking solution (5% BSA) was added dropwise and the mixture was left in a wet box at room temperature for 30 minutes. The blocking solution was wiped off with filter paper without washing. Adding primary antibody with proper concentration dropwise, incubating overnight at 4 ℃ in a wet box, washing the primary antibody with PBS for 5 minutes by 3 times, and wiping the PBS outside the specimen with filter paper.
4) The biotinylated secondary antibody working solution was added dropwise, incubated in a wet box at room temperature for 20 minutes, and the secondary antibody was washed off with PBS 5 minutes × 3 times, and the PBS outside the specimen was wiped off with filter paper. And (3) dripping horseradish enzyme labeled streptavidin working solution, incubating in a wet box for 20 minutes at room temperature, washing with PBS for 5 minutes multiplied by 3 times, and wiping the PBS outside the sample with filter paper.
5) DAB color developing agent color development, and tap water fully washes.
6) Hematoxylin counterstaining, dehydration, transparency, and sealing with neutral gum.
7) Microscopic observation
Thirdly, experimental results:
as shown in fig. 2 and 3, the blank group has no significant change; the model group 14d had severe injury and a strong inflammatory response; the group 14d of celecoxib is in the stage of injury repair.
Example 2 biomarkers associated with the prognosis of celecoxib treatment of tendon injury
Samples were selected for RNA sequencing 14 days after drug administration and the model set was analyzed for differentially expressed genes from the celecoxib drug set by using DESeq2 with a p value < 0.05.
As a result:
in comparison to the model group, the celecoxib-treated group had 949 differentially expressed mrnas (552 mRNA expression up-regulated, 397 mRNA expression down-regulated) and 34 differentially expressed lncrnas (20 lncrnas expression up-regulated, 14 lncrnas expression down-regulated). The GPRC5C gene involved in the invention is up-regulated (as shown in figure 4), and the difference has statistical significance, which suggests that the GPRC5C gene can be used for predicting the prognosis of patients with tendon injury using non-steroidal anti-inflammatory drugs.
Example 3 expression of GPRC5C in tendon injury samples
One, data source
The GSE26051 dataset was downloaded from GEO in 46 samples, 23 tendon injury samples and 23 normal tendon samples.
Second, differential expression analysis
Differential expression analysis was performed using the "limma" package in the R software, with the differential gene screening standard p<0.05，|log 2 FC|>1。
Three, result in
The analysis result shows that the biomarker GPRC5C involved in the invention is expressed down-regulated in a tendon injury sample (as shown in FIG. 5 and Table 1).
TABLE 1 expression of GPRC5C in tendon injury samples
| Gene | fold change | AveExpr | t | P.Value |
| GPRC5C | -1.85 | 5.05 | -3.16 | 0.00 |
Example 4 diagnostic Performance validation
Receiver Operating Curves (ROCs) were plotted using the R package "pROC" (version 1.15.0), AUC values, sensitivity and specificity were analyzed, and the diagnostic efficacy of the markers alone or in combination was judged.
When the diagnostic efficacy of the individual index is judged, the expression level of the gene (log2 expression level) is directly used for analysis, and the level corresponding to the point with the maximum john index is selected as the cutoff value thereof, i.e., the optimal division threshold is determined by the point with the maximum john index.
The diagnostic efficacy of GPRC5C is shown in fig. 6 and table 2, suggesting that GPRC5C may be a biomarker for tendon injury.
TABLE 2 diagnostic efficacy of GPRC5C
The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.
In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.
Claims (10)
1. A reagent that specifically detects GPRC5C in a sample.
2. The reagent of claim 1, wherein the reagent comprises reagents for detecting GPRC5C by sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques, protein immunization techniques.
3. The agent according to claim 1 or 2, wherein said agent is selected from the group consisting of:
a probe that specifically recognizes GPRC5C gene; or
Primers for specifically amplifying GPRC5C gene; or
A binding agent that specifically binds GPRC5C protein.
4. A kit comprising the reagent of any one of claims 1 to 3.
5. A chip comprising the reagent according to any one of claims 1 to 3.
6. A composition comprising an agent that promotes the expression of GPRC 5C.
7. The composition of claim 6, further comprising other drugs compatible with the agent and a pharmaceutically acceptable carrier and/or adjuvant.
8. A method for screening a candidate drug for preventing or treating tendon injury, comprising the steps of:
treating a system expressing or containing GPRC5C with a substance to be screened; detecting the expression level of GPRC5C in the system; wherein, if the substance can promote the expression of GPRC5C, the substance is a candidate drug for preventing or treating tendon injury.
9. A method for identifying and evaluating the effect of an agent and/or physical therapy in the prevention or treatment of tendon injury, said method comprising:
(1) collecting a first sample provided by a subject having a tendon injury;
(2) obtaining a GPRC5C expression profile from said first sample;
(3) administering to or on said subject one or more drug candidates and/or one or more physical treatments;
(4) providing a second sample from the subject in step (3);
(5) obtaining a GPRC5C expression profile from said second sample;
(6) comparing the expression profile of GPRC5C obtained in steps (2) and (5) to a reference GPRC5C expression profile;
(7) assessing whether said one or more drug candidates and/or physical treatments are effective in preventing or treating tendon damage based on said comparison in step (6);
preferably, the medicament is a non-steroidal anti-inflammatory drug, preferably, the non-steroidal anti-inflammatory drug comprises a propionic acid derivative non-steroidal anti-inflammatory drug, an acetic acid derivative non-steroidal anti-inflammatory drug, a fenamic acid derivative non-steroidal anti-inflammatory drug, a biphenylcarboxylic acid derivative non-steroidal anti-inflammatory drug, an oxicam non-steroidal anti-inflammatory drug, a cyclooxygenase-2 selective non-steroidal anti-inflammatory drug or a pharmaceutically acceptable salt of the foregoing, preferably, the non-steroidal anti-inflammatory drug is a cyclooxygenase-2 selective non-steroidal anti-inflammatory drug, preferably, the cyclooxygenase-2 selective non-steroidal anti-inflammatory drug comprises celecoxib.
10. Use according to any one of the following:
(1) use of an agent according to any one of claims 1 to 3 for the manufacture of a product for predicting prognosis of a patient with tendon injury using a non-steroidal anti-inflammatory drug;
(2) use of an agent according to any one of claims 1-3 in the manufacture of a product for diagnosing tendon injury;
(3) use of the kit of claim 4 for the manufacture of a product for diagnosing tendon injury;
(4) use of the kit of claim 4 for the manufacture of a product for predicting a prognosis for a patient with tendon injury using a non-steroidal anti-inflammatory drug;
(5) use of the chip of claim 5 in the preparation of a product for diagnosing tendon injury;
(6) the use of the chip of claim 5 in the preparation of a product for predicting the prognosis of a patient with tendon injury using a non-steroidal anti-inflammatory drug;
(7) use of a composition according to claim 6 or 7 for the manufacture of a medicament for the prevention or treatment of tendon injury;
(8) use of the method of claim 8 for screening a candidate drug for preventing or treating tendon injury;
(9) use of the method of claim 9 to identify and assess the effect of an agent and/or physical therapy for preventing or treating tendon injury;
preferably, the non-steroidal anti-inflammatory drug comprises propionic acid derivative non-steroidal anti-inflammatory drug, acetic acid derivative non-steroidal anti-inflammatory drug, fenamic acid derivative non-steroidal anti-inflammatory drug, biphenylcarboxylic acid derivative non-steroidal anti-inflammatory drug, oxicam non-steroidal anti-inflammatory drug, cyclooxygenase-2 selective non-steroidal anti-inflammatory drug or pharmaceutically acceptable salts of the foregoing substances, preferably, the non-steroidal anti-inflammatory drug is cyclooxygenase-2 selective non-steroidal anti-inflammatory drug, preferably, the cyclooxygenase-2 selective non-steroidal anti-inflammatory drug comprises celecoxib.
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