CN114959012B - Product for detecting tendon injury - Google Patents

Product for detecting tendon injury Download PDF

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CN114959012B
CN114959012B CN202210595523.XA CN202210595523A CN114959012B CN 114959012 B CN114959012 B CN 114959012B CN 202210595523 A CN202210595523 A CN 202210595523A CN 114959012 B CN114959012 B CN 114959012B
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tendon injury
kit
rasl10b
tendon
group
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CN114959012A (en
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张耀南
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Beijing Hospital
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Beijing Hospital
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Abstract

The invention discloses a product for detecting tendon injury. Specifically, the product comprises a reagent for detecting the expression level of the RASL10B gene. Based on the expression level of RASL10B gene, tendon injury can be diagnosed or prognosis of tendon injury patients using non-steroidal anti-inflammatory drugs can be predicted. The invention also provides application of RASL10B in screening of candidate drugs for preventing or treating tendon injury.

Description

Product for detecting tendon injury
Technical Field
The invention relates to the field of biomedicine, in particular to a product for detecting tendon injury.
Background
Tendons are uniaxial dense fibrous connective tissues connecting muscles and bones, transmit forces generated by the muscles to drive the bones to move, and play an important role in movement of the organism. However, improper exercise, training or repeated pulling can often cause tendon injury and even rupture, which can have serious effects on human health and quality of life.
Repair of a damaged tendon is a lengthy process that mainly consists of three overlapping stages. First, during the inflammatory phase, erythrocytes and inflammatory cells, especially neutrophils, enter the site of injury, and during the first 24 hours, monocytes and macrophages dominate and phagocytose the necrotic material. As vascular permeability increases, factors acting on vascular activity and chemotaxis are released, thereby stimulating tenocyte proliferation and recruiting more inflammatory factors. After a few days the proliferation phase begins and the fibrous tissue proliferates considerably and begins to synthesize collagen fibers. During the proliferation phase, the water content and mucopolysaccharide concentration are kept high. After about 6 weeks, tendon repair enters the remodeling stage and cell number, collagen and mucopolysaccharide synthesis gradually decreases. The reconstitution period can be divided into consolidation period and maturation period. The consolidation phase begins approximately at week 6 and continues through week 10, during which time tissue repair changes from cellular to fibrous, tenocyte metabolism remains high, and tenocytes and collagen fibers line up in the direction of stress. A high proportion of collagen I synthesis occurs during the consolidation phase. After 10 weeks, a maturation period occurred in which the fibrous tissue gradually changed into scar-like tendon tissue for a period lasting up to one year. In the latter half of the maturation period, the rate of tenocyte metabolism is reduced and vascularity is reduced.
Currently, the treatment of injured tendons is generally treated with either conservative treatment or surgical repair, or a combination of both. Conservative treatments, such as rest, analgesia, corticoid injection, physiotherapy and the like, are usually used for treating chronic tendon injuries, are long in time consumption and low in curative effect, can only reduce pain mostly, and can form scar tissues to influence tendon functions. For acute tendon rupture, surgical treatment such as autografting, allotransplantation, xenotransplantation or surgical suture is often adopted. However, the surgically treated tendons are easily broken again and scar-like tissue is also formed, and there are risks of autograft donor area lesions, immunological rejection of xenotransplantation/xenotransplantation, and disease transmission. In conclusion, the therapeutic effect of tendon injury is not ideal, and finding a more effective method to promote the repair of injured tendon and improve the therapeutic effect is still an important clinical challenge at present. Therefore, there is a need in the art to find biomarkers associated with tendon damage that provide new methods for diagnosing or treating tendon damage.
Disclosure of Invention
In order to remedy the deficiencies of the prior art, the object of the present invention is to provide a product for diagnosing or treating tendon injuries.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a detection reagent of a biomarker in preparing a product for diagnosing tendon injury or predicting the prognosis of a patient with tendon injury by using a non-steroidal anti-inflammatory drug, wherein the biomarker is RASL10B.
The RASL10B refers to a Gene with the Gene ID of 91608.
Further, the non-steroidal anti-inflammatory drugs (NSAID's) include propionic acid derivative NSAID's, acetic acid derivative NSAID's, fenamic acid derivative NSAID's, biphenylcarboxylic acid derivative NSAID's, oxicams, 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.
Further, the detection reagent comprises a probe, a primer or a protein binding agent.
In a second aspect, the invention provides a product for diagnosing tendon injury or predicting tendon injury prognosis in a patient with tendon injury, the product comprising a reagent for detecting RASL10B gene expression level in a sample.
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.
Further, the product comprises a kit, a chip or a test strip.
Further, the kit comprises a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry kit, an immunohistochemical detection kit, an ELISA kit and an electrochemiluminescence detection kit.
Further, the kit further comprises instructions for assessing whether the subject is suffering from or susceptible to tendon injury.
Further, the product also comprises reagents for processing the sample.
In a third aspect, the invention provides a pharmaceutical composition comprising an agent that promotes expression of RASL10B.
Further, the pharmaceutical composition may further comprise a stabilizer, an antiseptic, a buffer, an isotonic agent, a chelating agent, a PH controlling agent or a surfactant.
In a fourth aspect, the present invention provides a use of the pharmaceutical composition of the third aspect of the present invention in the preparation of a medicament for preventing or treating tendon injury.
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:
(1) Treating the system expressing or containing the RASL10B gene with a test substance;
(2) Detecting expression of the RASL10B gene in the system;
(3) Selecting as the drug candidate an agent that increases the expression level of the RASL10B gene.
In a sixth aspect, the invention provides the use of RASL10B in screening for a candidate for the prevention or treatment of tendon injury.
The invention has the advantages and beneficial effects that:
the invention firstly discovers that the differential expression of RASL10B is related to tendon injury, and can diagnose the tendon injury or predict the prognosis of tendon injury patients using non-steroidal anti-inflammatory drugs by detecting the expression level of RASL10B.
The invention provides a product for diagnosing tendon injury or predicting 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, the model group and the celecoxib drug 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 masson trichrome staining result graph (× 100), panel D is a blank group masson 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 masson trichrome staining result graph (× 100), panel H is a model group masson trichrome staining result graph (× 400), panel I is a drug group HE staining result graph (× 100) for celecoxib, panel J is a drug group HE staining result graph (× 400) for celecoxib, panel K is a drug group masson trichrome staining result graph (× 100) for celecoxib, and panel L is a drug group masson trichrome staining result graph (× 400) for celecoxib; panel M is a graph of results of immunohistochemical staining for the blank group RECA-1 (x 400), panel N is a graph of results of immunohistochemical staining for the model group RECA-1 (x 400), and panel O is a graph of results of immunohistochemical staining for the drug group RECA-1 of celecoxib (x 400);
FIG. 4 is a line graph showing differential expression of RASL10B gene in the experimental group;
FIG. 5 is a plot of RASL10B gene differential expression profiles in a GSE26051 dataset;
FIG. 6 is a ROC plot of RASL10B in diagnosis of tendon injury.
Detailed Description
The invention aims to screen a biomarker for predicting the prognosis of a patient with tendon injury using a non-steroidal anti-inflammatory drug, construct a tendon injury model mouse, comprehensively analyze gene expression profiles before and after drug administration, screen genes with significant difference in expression level, and further verify the correlation between the genes and the tendon injury in a GEO data set, thereby finding the biomarker suitable for diagnosing the tendon injury or predicting the prognosis of the patient with tendon injury using the non-steroidal anti-inflammatory drug.
In the present invention, the term "biomarker" means a compound, preferably a gene, which is differentially present (i.e. increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g. having a disease) compared to a biological sample from a subject or a group of subjects having a second phenotype (e.g. no disease). The term "biomarker" generally refers to the presence/concentration/amount of one gene or the presence/concentration/amount of two or more genes.
Biomarkers can be differentially present at any level, but are typically present at levels that are increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more; or generally at a level that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (i.e., absent).
Preferably, the biomarkers are differentially present at levels of statistical significance (i.e., p-value less than 0.05 and/or q-value less than 0.10, as determined using the Welch's T-Test or the Wilcoxon rank-sum Test).
In a specific embodiment of the invention, the biomarker is RASL10B.
The RASL10B refers to a Gene with the Gene ID of 91608.
The reagent for detecting RASL10B and/or its expression product described in the present invention includes a probe, a primer or a protein-binding agent.
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 formats 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 (base pair) complementary to a template strand and serving as a starting point for replication of the template strand. The primer is generally synthesized, but a naturally occurring nucleic acid may 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.
The invention provides a product for diagnosing tendon injury or predicting the prognosis of a patient with tendon injury by using non-steroidal anti-inflammatory drugs, which comprises a reagent for detecting the expression level of RASL10B in a sample; and instructions for using the kit to assess whether a subject is suffering from or susceptible to tendon injury can be included.
The most reliable results are possible when processing samples in a laboratory environment. For example, a sample may be taken from a subject in a doctor's office and then sent to a hospital or commercial medical laboratory for further testing. However, in many cases, it may be desirable to provide immediate results at the clinician's office or to allow the subject to conduct the test at home. In some cases, the need for testing that is portable, prepackaged, disposable, ready to use by the subject without assistance or guidance, etc., is more important than a high degree of accuracy. In many cases, especially in the presence of a physician's follow-up, it may be sufficient to perform a preliminary test, even a test with reduced sensitivity and/or specificity. Thus, assays provided in product form can involve detecting and measuring relatively small amounts of biomarkers to reduce the complexity and cost of the assay.
Any form of sample assay capable of detecting a sample biomarker described herein may be used. Typically, the assay will quantify the biomarkers in the sample to an extent, for example whether their concentration or amount is above or below a predetermined threshold. Such kits may take the form of test strips, dipsticks, cartridges, chip-based or bead-based arrays, multi-well plates, or a series of containers, and the like. One or more reagents are provided to detect the presence and/or concentration and/or amount of a biomarker in a selected sample. The sample from the subject may be dispensed directly into the assay or indirectly from a stored or previously obtained sample. The presence or absence of a biomarker above or below a predetermined threshold may be indicated, for example, by chromogenic, fluorogenic, electrochemiluminescent or other output (e.g., in an Enzyme Immunoassay (EIA), such as an enzyme-linked immunoassay (ELISA)).
In one embodiment, the product may comprise a solid substrate such as a chip, slide, array, or the like, having reagents capable of detecting and/or quantifying one or more sample biomarkers immobilized at predetermined locations on the substrate. As an illustrative example, the chip may be provided with reagents immobilized at discrete predetermined locations for detecting and quantifying the presence and/or concentration and/or amount of a biomarker in a sample. As described above, a reduced or increased level of the biomarker is found in a sample of a subject suffering from tendon injury. The chip may be configured such that a detectable output (e.g. a color change) is provided only when the concentration of the biomarker exceeds a threshold value selected or differentiated between the concentration and/or amount of the biomarker indicative of a control subject and the concentration and/or amount of the biomarker indicative of a patient suffering from or susceptible to tendon injury. Thus, the presence of a detectable output (such as a color change) immediately indicates that a significantly reduced level of the biomarker is included in the sample, indicating that the subject is suffering from or susceptible to tendon injury.
The invention also provides a pharmaceutical composition comprising an agent that promotes RASL10B expression.
Further, the pharmaceutical compositions comprise the active ingredient in combination with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as necessary to aid in the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration, and the agents which may be used in such compositions may be in the form of their original compounds themselves, or optionally in the form of their pharmaceutically acceptable salts. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired. When using a pharmaceutical composition, a safe and effective amount of a drug of the present invention is administered to a human.
The appropriate dose of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, diet, time of administration, route of administration, excretion rate and reaction sensitivity of the patient, and a skilled physician can easily determine the prescription and the dose of administration effective for the desired treatment or prevention.
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 present application, are given by way of illustration and explanation only, and are not intended to limit the present application.
Example 1 evaluation of the Effect of celecoxib on the treatment of tendon injury
1. Experimental materials:
1. experimental animals: SD rats (sbefu);
2. experimental equipment: a full-automatic dehydrator (Leica, ASP 200S), a paraffin slicer (Leica, RM 2235), a baking sheet table (Leica, HI 1220), a bathtub (Leica, HI 1220), a heating paraffin embedding system (Leica, G1150H) and a microscope (Leica, DM 3000);
3. experimental reagent: xylene (national medicine), anhydrous 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 (China fir Jinqiao, SP-9000), neutral gum (China fir Jinqiao, ZLI-9555), sealant (AR 0150)
2. The experimental method comprises the following steps:
1. grouping animals
SD rats were purchased from Sibefu (Beijing) Biotechnology, inc., 6 weeks old, 30, random gender, 200-300g, normal light cycle, normal diet. The rats were randomized into three groups of 10 animals each: a normal control group, a tendon injury model group, and a 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 (10 mg/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) Putting the slices into dimethylbenzene I for 10min, dimethylbenzene II for 10min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, 95% alcohol for 5min, 90% alcohol for 5min, 80% alcohol for 5min, 70% alcohol for 5min, and washing with distilled water.
2) The sections are stained with hematoxylin for 3-8 min, washed with tap water, differentiated by 1% hydrochloric acid alcohol 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 10min.
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 1min.
6) And dyeing with ponceau fuchsin dyeing solution for 5-10min.
7) During the operation, according to the weight ratio of distilled water: weak acid solution =2, 1, preparing weak acid working solution, and washing for 1min with the weak acid working solution.
8) Washing with phosphomolybdic acid solution for 1-2min
9) Washing with prepared weak acid working solution for 1min.
10 Directly placing into aniline blue staining solution for staining for 1-2min.
11 Washed with prepared weak acid working solution for 1min.
12 ) 95% ethanol.
13 ) dehydrating with anhydrous ethanol for 5-10s for 3 times.
14 Xylene clear 3 times, each for 1-2min.
15 Neutral gum blocking.
16 ) microscopic observation.
(4) Immunohistochemistry (RECA 1)
1) Paraffin sections were baked at 60 ℃ for 1 hour, dewaxed with xylene (xylene I10min, xylene II10 min), gradient alcohol (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 repair solution with ph =6.0, boiling slightly with microwave oven 100 firepower for three minutes, maintaining 50 firepower for 7 minutes, stopping heating, and naturally cooling for 20-30 minutes. Rinse 5min × 3/time with PBS.
3) Immune reaction: 3% of H 2 O 2 Incubate for 10min to eliminate endogenous peroxidase activity. PBS wash, 5min X3 times. Blocking solution (5% BSA) was added dropwise thereto, and the mixture was allowed to stand at room temperature for 30 minutes in a wet box. 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 is dripped, the mixture is placed in a wet box at room temperature and incubated for 20 minutes, the secondary antibody is washed away by 5 minutes multiplied by 3 times by PBS, and the PBS outside the sample is wiped away by 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 examination observation
3. The experimental results are as follows:
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 differentially expressed genes from the model group and the celecoxib drug group were analyzed by using DESeq2 with a p-value <0.05.
As a result:
in comparison with the model group, the celecoxib treatment group had 949 differentially expressed mrnas (552 mRNA expression was up-regulated and 397 mRNA expression was down-regulated) and 34 differentially expressed lncrnas (20 lncrnas were up-regulated and 14 lncrnas were down-regulated). The RASL10B gene expression referred to in the present invention was up-regulated (as shown in fig. 4), and the difference was statistically significant, suggesting that RASL10B gene could be used to predict prognosis for patients with tendon injury using nsaids.
Example 3RASL10B expression in tendon injury samples
1. Data source
The GSE26051 dataset was downloaded from GEO in 46 samples, 23 tendon injury samples and 23 normal tendon samples.
2. 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。
3. Results
The analysis result shows that the biomarker RASL10B related to the invention is expressed and down-regulated in tendon injury samples (as shown in figure 5 and table 1).
TABLE 1 RASL10B expression in tendon injury samples
Gene foldchange AveExpr t P.Value
RASL10B -2.12 3.75 -3.81 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 indicators alone or in combination was judged.
When the diagnostic efficacy of the individual index is judged, the expression level (log 2 expression level) of the gene 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 RASL10B is shown in fig. 6 and table 2, suggesting that RASL10B may be a biomarker for tendon injury.
TABLE 2 diagnostic potency of RASL10B
Gene AUC Sensitivity of the composition Specificity of the drug
RASL10B 0.78 0.70 0.78
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 (6)

1. The application of the detection reagent of the biomarker in preparing products for diagnosing tendon injury is characterized in that the biomarker is RASL10B.
2. The use of claim 1, wherein the detection reagent comprises a probe, a primer, or a protein binding agent.
3. The use of claim 2, wherein the product comprises a kit, chip or strip.
4. The use of claim 3, wherein the kit comprises a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometric assay kit, an immunohistochemical detection kit, an ELISA kit, and an electrochemiluminescent detection kit.
5. The use of claim 4, wherein the kit further comprises instructions for assessing whether the subject is suffering from or susceptible to tendon injury.
6. The use according to any one of claims 1 to 5, wherein the product further comprises reagents for processing the sample.
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