Molecular marker for detecting osteoarthritis, detection kit and antibody chip

Abstract

The invention relates to a molecular marker for detecting osteoarthritis, a detection kit and an antibody chip, and belongs to the technical field of molecular biology. The invention provides a molecular marker for detecting osteoarthritis. The molecular marker comprises a tissue metalloproteinase inhibiting factor 4, a chemotactic factor beta8-1, an interleukin 13 receptor 2, a killer cell activated receptor molecule 1, a tumor necrosis factor related apoptosis inducing ligand 2, a bone forming protein receptor 1A, a nerve nutrient, a B-cell activating factor, a plasminogen activation inhibitor 1 and serine protease 8. Researches show that the molecular marker is very significant in correlation with osteoarthritis, and osteoarthritis patients and healthy people can be well distinguished. When being used for detecting osteoarthritis, the molecular marker has the advantages of high flux, high sensitivity, high specificity and low cost.

Description

Technical Field

[0001] The invention relates to a molecular marker, a detection kit and an antibody chip for detecting osteoarthritis, and belongs to the technical field of molecular biology. Background Art

[0002] Osteoarthritis (OA) is the most common joint disease characterized by articular cartilage damage and involvement of the entire joint tissue. In the later stages of osteoarthritis, articular cartilage degeneration, fibrosis, fracture, defect and damage to the entire joint surface will occur. It is one of the main causes of pain, chronic physical disability and permanent disability. Osteoarthritis is characterized by progressive articular cartilage degeneration, subchondral bone remodeling and failure of synovial joint organs. Although the occurrence and development of osteoarthritis is mainly related to aging, other key factors including inflammation, obesity, joint trauma, joint infection, genetics, genetic metabolic disorders and various bone turnover disorders can also promote the occurrence and progression of osteoarthritis.

[0003] Currently, osteoarthritis is considered an inflammatory disease. Increasing evidence shows that symptoms such as joint pain, swelling and stiffness in osteoarthritis patients are related to inflammation. For example, proinflammatory cytokines IL-1 and TNF-α can play a role in promoting the progression of osteoarthritis in cartilage. The level of TNF-α in serum is correlated with the joint space narrowing (JSN) grade and the severity of knee cartilage loss in osteoarthritis patients. The level of TNF receptor in serum is correlated with the pain level, joint stiffness and radiographic severity of osteoarthritis patients. The level of IL-6 in serum is correlated with the JSN grade, pain level and the probability of early osteoarthritis in osteoarthritis patients. The levels of IL-18 and VEGF in serum are positively correlated with the severity of osteoarthritis in osteoarthritis patients. The level of CRP in serum is correlated with the joint count, imaging score, muscle strength, pain level and Kellgren-Lawrence (KL) grade of osteoarthritis patients. Adipokines play an important role in the occurrence and progression of knee osteoarthritis. However, subsequent studies have shown that these molecular markers in serum are not sufficient for accurate detection of osteoarthritis.

[0004] At present, accurate diagnosis of osteoarthritis still needs to be made based on clinical symptoms and radiography. Among them, radiography is mainly used to measure the typical pathological characteristics of osteoarthritis, including JSN grade, degree of bone sclerosis and degree of subchondral sclerosis. However, radiography is insensitive and can only be seen when symptoms are obvious. Magnetic resonance imaging (MRI) and ultrasound can make up for the shortcomings of radiography. However, due to its high cost, MRI will bring a great financial burden to patients. The penetrating ability of ultrasound has certain limitations. The high density of bones will cause ultrasound to be almost completely reflected, resulting in acoustic shadows, which makes it difficult to see the structure behind the bones. It is only suitable for observing the entire joint. Therefore, it is necessary to find reliable molecular markers that can accurately detect osteoarthritis to monitor the progression of osteoarthritis.

[0005] In addition, due to the lack of specific molecular markers that can accurately detect osteoarthritis, the treatment options for osteoarthritis are limited. Traditional osteoarthritis treatments include physical therapy measures, drug therapy, and surgery. Among them, physical therapy measures refer to moderate exercise, which may delay the development of osteoarthritis, but the progress is slow and the effect is not obvious. Drug treatments such as acetaminophen and opioids show effective analgesic effects, but cannot reverse the cartilage damage of osteoarthritis and have side effects. Surgeries such as arthroscopic irrigation and debridement are only suitable for severe cases, which is not conducive to long-term treatment and has the risk of aggravating trauma. If reliable molecular markers that can accurately detect osteoarthritis can be found to monitor the progression of osteoarthritis, it may guide the choice of osteoarthritis treatment options. Summary of the invention

[0006] To solve the above problems, the present invention provides a molecular marker for detecting osteoarthritis, wherein the molecular markers include tissue inhibitor of metalloproteinase 4 (TIMP-4), chemokine β8-1 (Ckbeta8-1 / CCL23), interleukin 13 receptor 2 (IL-13R2), killer cell activating receptor molecule 1 (DNAM-1), tumor necrosis factor-related apoptosis-inducing ligand 2 (TRAIL R2), bone morphogenetic protein receptor 1A (BMPR-1A), neurotrophin (NT-4), B-cell activating factor (BAFF / TNFSF13B), plasminogen activator inhibitor-1 (PAI-1) and serine protease 8 (Prostasin / PRSS8).

[0007] In one embodiment of the present invention, the molecular markers are composed of tissue inhibitor of metalloproteinase 4 (TIMP-4), chemokine β8-1 (Ckbeta 8-1 / CCL23), interleukin 13 receptor 2 (IL-13R2), killer cell activating receptor molecule 1 (DNAM-1), tumor necrosis factor-related apoptosis-inducing ligand 2 (TRAIL R2), bone morphogenetic protein receptor 1A (BMPR-1A), neurotrophin (NT-4), B-cell activating factor (BAFF / TNFSF13B), plasminogen activator inhibitor-1 (PAI-1) and serine protease 8 (Prostasin / PRSS8).

[0008] In one embodiment of the present invention, the B-cell activating factor is a truncated protein of the B-cell activating factor; the amino acid sequence of the truncated protein of the B-cell activating factor is shown in SEQ ID NO:1.

[0009] The present invention also provides the use of a reagent for detecting the above molecular marker in preparing a product for detecting osteoarthritis.

[0010] In one embodiment of the present invention, the product comprises a detection kit and / or an antibody chip.

[0011] The present invention also provides an antibody chip for detecting osteoarthritis, the antibody chip comprising a chip body and a core capture antibody coated on the surface of the chip body; the core capture antibody is arranged on the chip body to form a microarray; the core capture antibody comprises a specific antibody targeting the above-mentioned molecular marker.

[0012] In one embodiment of the present invention, the antibody chip further comprises auxiliary capture antibodies coated on the surface of the chip body; the auxiliary capture antibodies are arranged on the chip body to form a microarray; the auxiliary capture antibodies comprise specific antibodies targeting TSP-1, CXCL14, Aggrecan, GITR L, Cadherin-11, Eotaxin, Osteoactivin / GPNMB, WIF-1, RANK, Follistatin, LYVE-1, Galectin-7, Axl, CEACAM-1, FSH, TGFb2, Fas, Cystatin EM, Furin, IL-1F8, Cathepsin S, CTLA4, Angiogenin, EG-VEGF, IL-20, ActivinA, IGFBP-2, FGF-4, CD40, IL-1F7, IL-1F10, IL-18, IL-1R5, PIGF, GROa, MIP-3b and / or MCP-2.

[0013] In one embodiment of the present invention, the chip body is a solid phase carrier; the capture antibody is coated on the surface of the solid phase carrier to form a plurality of independent recognition sites.

[0014] In one embodiment of the present invention, the method for preparing the antibody chip includes: mixing the capture antibody with a sample diluent to obtain an antibody diluent; and spotting the sample in the spotting wells of the solid phase carrier using the antibody diluent to fix the capture antibody in the spotting wells of the solid phase carrier.

[0015] In one embodiment of the present invention, the sample diluent is a PBS buffer solution containing 0.01 to 0.1 vt% Tween-20 at 0.01 to 0.02 mol / L and a pH of 7.1 to 7.5.

[0016] In one embodiment of the present invention, the solid phase carrier is a glass slide.

[0017] In one embodiment of the present invention, the method for preparing the antibody chip includes: mixing the capture antibody with the sample diluent to obtain the antibody diluent; spotting 2×8 identical antibody microarrays on the surface of a glass slide using a non-contact spotter in the form of a microarray using the antibody diluent; dividing the standard tissue glass slide spotted with the 2×8 antibody microarrays into 16 non-interfering small areas using a detachable 2×8-hole frame structure, each small area containing an antibody microarray; and clamping the glass slide and the frame with frame buckles on both sides to form an antibody chip.

[0018] In one embodiment of the present invention, the surface of the solid phase carrier is further provided with a positive control well and a negative control well; the positive control well is fixed with a biotinylated IgG antibody; the negative control well is fixed with a control antibody; the control antibody refers to an antibody that cannot bind to the above-mentioned molecular marker.

[0019] The present invention also provides a detection kit for detecting osteoarthritis, wherein the detection kit comprises a reagent for detecting the above-mentioned molecular marker and / or the above-mentioned antibody chip.

[0020] In one embodiment of the present invention, the detection kit comprises the above-mentioned antibody chip and a core detection antibody; the core detection antibody comprises a specific antibody targeting the above-mentioned molecular marker, and the core detection antibody is labeled with a molecular marker.

[0021] In one embodiment of the present invention, the detection kit further comprises an auxiliary detection antibody; the auxiliary detection antibody comprises a targeted specific antibody, and the auxiliary detection antibody is labeled with a molecular marker.

[0022] In one embodiment of the present invention, the molecular marker is biotin.

[0023] In one embodiment of the present invention, the detection kit further comprises streptavidin.

[0024] In one embodiment of the present invention, the streptavidin is streptavidin coupled with fluorescein.

[0025] In one embodiment of the present invention, the detection kit is based on a double antibody sandwich method.

[0026] The present invention also provides a diagnostic model for detecting osteoarthritis, the diagnostic model comprising an information collection module, a data analysis module and an evaluation module;

[0027] The information collection module is used to detect and collect information related to the evaluation object and the above-mentioned molecular markers;

[0028] The data analysis module is used to analyze the information obtained in the information collection step to obtain an analysis result of whether the patient suffers from osteoarthritis;

[0029] The evaluation module is used to classify the test subjects into a group suffering from osteoarthritis and a healthy group according to the analysis results obtained by the data analysis module.

[0030] The technical solution of the present invention has the following advantages:

[0031] 1. The present invention provides a molecular marker for detecting osteoarthritis, wherein the molecular marker includes tissue inhibitor of metalloproteinase 4 (TIMP-4), chemokine β8-1 (Ckbeta 8-1 / CCL23), interleukin 13 receptor 2 (IL-13R2), killer cell activating receptor molecule 1 (DNAM-1), tumor necrosis factor-related apoptosis-inducing ligand 2 (TRAILR2), bone morphogenetic protein receptor 1A (BMPR-1A), neurotrophin (NT-4), B-cell activating factor (BAFF / TNFSF13B), plasminogen activator inhibitor-1 (PAI-1) and serine protease 8 (Prostasin / PRSS8). Studies have shown that the molecular marker has a significant correlation with osteoarthritis and can well distinguish osteoarthritis patients from healthy people. The use of the molecular marker to detect osteoarthritis has the advantages of high throughput, high sensitivity, high specificity and low cost.

[0032] 2. The present invention provides an antibody chip for detecting osteoarthritis, the antibody chip comprising a chip body and a core capture antibody coated on the surface of the chip body; the core capture antibody is arranged on the chip body to form a microarray; the core capture antibody comprises a specific antibody targeting a molecular marker; the molecular markers include tissue inhibitor of metalloproteinase 4 (TIMP-4), chemokine β8-1 (Ck beta 8-1 / CCL23), interleukin 13 receptor 2 (IL-13R2), killer cell activating receptor molecule 1 (DNAM-1), tumor necrosis factor-related apoptosis-inducing ligand 2 (TRAILR2), bone morphogenetic protein receptor 1A (BMPR-1A), neurotrophin (NT-4), B-cell activating factor (BAFF / TNFSF13B), plasminogen activator inhibitor-1 (PAI-1) and serine protease 8 (Prostasin / PRSS8). Studies have shown that the molecular markers used in the antibody chip are significantly correlated with osteoarthritis and can well distinguish osteoarthritis patients from healthy people. The results obtained by using the antibody chip to detect osteoarthritis are reliable and accurate, and at the same time, they save costs. They can overcome the defects of the prior art, such as complicated operation, single detection index, expensive instruments and low sensitivity. They have the advantages of being cheap, convenient, sensitive, accurate, high-throughput, requiring less specimens, and being able to be promoted and scaled up in ordinary laboratories. They are conducive to obtaining information that is instructive for treatment methods and prognosis, thereby increasing the effective treatment rate of osteoarthritis and improving the living standards of patients.

[0033] Furthermore, the antibody chip also includes auxiliary capture antibodies coated on the surface of the chip body; the auxiliary capture antibodies are arranged on the chip body to form a microarray; the auxiliary capture antibodies include targeting TSP-1, CXCL14, Aggrecan, GITR L, Cadherin-11, Eotaxin, Osteoactivin / GPNMB, WIF-1, RANK, Follistatin, LYVE-1, Galectin-7, Axl, CEACAM-1, FSH, TGFb2, Fas, Cystatin EM, Furin, IL-1F8, Cathepsin S, CTLA4, Angiogenin, EG-VEGF, IL-20, ActivinA, IGFBP-2, FGF-4, CD40, IL-1F7, IL-1F10, IL-18, IL-1R5, PIGF, GROa, MIP-3b, MMP-1, IGFBP-3, SCF / KITLG and / or MCP-2 specific antibodies. Studies have shown that the 50 molecular markers detected by the antibody chip are closely related to osteoarthritis, and can effectively distinguish osteoarthritis patients from healthy people by their expression differences. The simultaneous determination of these 50 molecular markers by the antibody chip helps to fully and deeply understand the mechanism of the occurrence and progression of osteoarthritis from the perspective of the cytokine signaling network, so that the occurrence and course of osteoarthritis can be more accurately and comprehensively reflected, and the level of accurate diagnosis, course monitoring and prognosis evaluation of osteoarthritis can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 :Statistical results of differentially expressed proteins obtained from 48 samples.

[0035] Figure 2 : Results of principal component analysis of the difference results obtained from 48 samples.

[0036] Figure 3 : Volcano plot of the difference results obtained from 80 samples.

[0037] Figure 4 : ROC curve of a single molecular marker (down) in distinguishing healthy subjects from patients with osteoarthritis.

[0038] Figure 5 : ROC curve of a single molecular marker (up) in distinguishing healthy subjects from patients with osteoarthritis. DETAILED DESCRIPTION

[0039] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0040] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0041] Experimental Example 1: Screening of Molecular Markers

[0042] The experimental process is as follows:

[0043] 1. Screening of molecular markers

[0044] Protein chip technology was used to detect the expression levels of 440 cytokines in synovial fluid samples from 24 osteoarthritis patients (all from the General Hospital of Ningxia Medical University) and 24 healthy volunteers to identify osteoarthritis-related biomarkers. The specific steps are as follows:

[0045] 100 μL synovial fluid was obtained from the joints of 24 osteoarthritis patients (OA group) and 24 healthy volunteers (Control group) and added to the human cytokine antibody array pool (RayBiotech, Norcross, GA, USA). Then, 440 primary antibodies were added and incubated overnight at 4°C. Unbound proteins were washed away, and then the biotin-conjugated detection antibody mixture was added to the array pool. Then, streptavidin conjugated with cyanine dye (Cy3) was added and incubated in the chip for 2 hours. The fluorescence signal in the chip was converted into a signal value by an InnoScan 300 scanner (Innopsys, France), and 50 differential proteins (cytokines) were obtained. The results are shown in Figure 2. Figure 1 As shown in the figure, OA-related biomarkers identified in the antibody array were analyzed using moderated t-statistics test, with a corrected p-value less than 0.05 and a fold change >1.2 or <0.83, which were considered to be significantly associated with the pathogenesis of OA.

[0046] Depend on Figure 1It can be seen that there are significant differences in the expression of 50 cytokines in the synovial fluid of the OA group and the Control group. These 50 cytokines are considered to be related to the pathogenesis of osteoarthritis (OA). Among these differentially expressed cytokines, the expression of 20 cytokines in the synovial fluid of the OA group is lower than that of the Control group, while the expression of 30 cytokines in the synovial fluid of the OA group is higher than that of the Control group.

[0047] In order to prove that the expression of these cytokines is different between osteoarthritis patients and healthy people, the array data of differential cytokines in the OA group and the Control group were analyzed by principal component analysis (PCA), and the distribution areas of the OA group and the Control group were plotted. The results are shown in Figure 2. Figure 2 As shown in the figure, red represents the OA group and blue represents the Control group. Figure 2 It can be seen that the 50 cytokines screened out in this example can indeed distinguish osteoarthritis patients from healthy people and can be used as markers for osteoarthritis detection.

[0048] 2. Confirmation of molecular markers

[0049] In order to reduce false positives and expand the sample size for analysis, 24 cytokines with relatively large differential folds (difference fold FC>2.4AND FC<0.53) were selected from the 50 cytokines. Forty-nine osteoarthritis patients (OA group) and 31 healthy volunteers (Control group) were used as samples, and the array data of differential cytokines in the OA group and the Control group were analyzed.

[0050] On this basis, a customized antibody chip was used to detect the expression levels of these 24 cytokines in synovial fluid samples from 49 osteoarthritis patients (all from the General Hospital of Ningxia Medical University) and 31 healthy volunteers to further identify osteoarthritis-related molecular markers. The specific steps are as follows:

[0051] 1. Preparation of antibody chip

[0052] The capture antibody was diluted 100 times with the sample diluent to obtain the antibody diluent; a glass slide was taken, and the antibody diluent was spotted on the surface of a glass slide with a non-contact spotter at a spotting amount of 0.1 ng / spotting well, and 2×8 identical antibody microarrays (4 repeated wells were set for each antibody) were spotted through a microarray method; the standard tissue glass slide spotted with 2×8 antibody microarrays was divided into 16 non-interfering small areas with a detachable 2×8-well frame structure, and each small area contained an antibody microarray; two The side frame is clamped to form an antibody chip; wherein, the sample diluent is a PBS buffer solution of 0.02 mol / L and pH=7.4 containing 0.05 vt% Tween-20; among the capture antibodies used, except for the specific antibody of the truncated protein of B-cell activating factor (BAFF / TNFSF13B) (the specific antibody targeting the truncated protein of B-cell activating factor is shown in the patent disclosure with publication number CN117756918A), the other capture antibodies are commercially available specific antibodies.

[0053] 2. Detection of molecular markers

[0054] Ultrapure water and synovial fluid samples were mixed in a volume ratio of 1:1 to obtain a sample diluent; the antibody chip was equilibrated at room temperature (25°C) for 30 minutes and then dried in a vacuum dryer for 1 hour; after drying, the sample diluent was added to the antibody chip at a volume of 50 μL / spot well and incubated at room temperature for 2 hours; after incubation, the liquid in each well was drawn out and the antibody chip was washed 5 times with a washing solution, each washing for 5 minutes; after washing, the biotinylated detection antibody was diluted 100 times with a sample diluent to obtain an antibody diluent; the antibody diluent was added at a volume of 80 μL / spot well. After adding the sample to the antibody chip, incubate at room temperature for 2 hours; after the incubation, remove the liquid in each well, use the washing solution to wash the antibody chip 5 times, each washing for 5 minutes; after the washing, use the sample diluent to prepare Cy3-streptavidin to a concentration of 1μg / mL to obtain Cy3-streptavidin solution; add the Cy3-streptavidin solution to the antibody chip at an addition amount of 80μL / spotting well, and incubate at room temperature in the dark for 1 hour; after the incubation, remove the liquid in each well, use the washing solution to wash the antibody chip 5 times, each washing for 5 minutes; after the washing, use Axon GenePix laser scanner scanned the signal, and during scanning, Cy3 channel (excitation frequency 532nm) was used; after scanning, GenePix software was used to read the fluorescence value of the biochip, and the value selected after reading was the median reading (F532 Median-Local Background) after removing the local background; based on the reading results, principal component analysis was performed on the expression levels of 24 cytokines in osteoarthritis patients and healthy people, and the results of principal component analysis are shown in Table 3; based on the mean and standard deviation data of the expression levels of the 24 cytokines that can be used as molecular markers in the OA group and the Control group measured by the antibody chip, the p-value between the OA group and the Control group was calculated, and the p-value was corrected for multiple hypothesis testing using the FDR error control method, and the FDR values ​​of the 24 cytokines that can be used as molecular markers in distinguishing the OA group and the Control group were obtained, and the FDR values ​​are shown in Table 1; wherein, the washing solution is a 1 mol / L PBS buffer solution containing 0.1vt% Tween-20, pH=7.4.

[0055] The array data analysis results are as follows Figure 3 As shown in ((Volcano plot), blue represents the OA group and red represents the Control group). Figure 3As shown in the results of Table 1, among the 24 cytokines, the FDR values ​​of 15 cytokines that can be used as potential molecular markers, including TIMP-4, Ckbeta 8-1 / CCL23, IL-13R2, DNAM-1, BMPR-IA, GITR L, GROa, IL-1R5, IL-18, MIP-3b, TRAIL R2, NT-4, BAFF / TNFSF13B, PAI-1 and Prostasin / PRSS8, were all less than 1 in distinguishing the OA group from the Control group. Among them, the FDR values ​​of some cytokines were even close to 0, which verified the possibility of these 15 cytokines as molecular markers of osteoarthritis.

[0056] Table 1. Expression and FDR of 24 cytokines in the Control group and OA group

[0057] proteinID FC p FDR sig Aggrecan 0.881678378 0.286086086 0.312093912 NotSig BAFF / TNFSF13B 1.859285705 2.13E-11 5.12E-10 Up BMPR-IA 0.654040435 1.04E-06 5.40E-06 Down Cadherin-11 0.763720548 0.016883262 0.025324892 NotSig Ckbeta8-1 0.657086615 0.000362719 0.000967249 Down CXCL14 0.797761664 0.033013392 0.044017856 NotSig DNAM-1 0.602323012 0.000171881 0.000515644 Down Eotaxin 0.752114557 0.024403231 0.03445162 NotSig GITRL 0.603921219 0.001712526 0.003161586 Down GRO 1.460930523 0.005261324 0.008418118 Up IL-1F10 0.996929609 0.982508891 0.982508891 NotSig IL-1R5 1.578714512 4.53E-07 3.62E-06 Up IL-13R2 0.645150195 0.004378441 0.007505899 Down IL-18 1.867390174 1.13E-06 5.40E-06 Up MCP-2 1.009800204 0.936445428 0.977160446 NotSig MIP-3b 2.397780245 2.61E-09 3.13E-08 Up NT-4 1.515845404 0.00050228 0.001205472 Up Osteoactivin 0.785132432 0.055225898 0.069759029 NotSig PAI-1 1.436377157 0.000734201 0.001468403 Up PIGF 1.156421344 0.165215288 0.188817472 NotSig Prostasin 1.821155623 2.38E-06 9.52E-06 Up TIMP-4 0.625106089 0.000564258 0.001231109 Down TRAILR2 0.600130058 1.98E-05 6.80E-05 Down TSP-1 0.841698551 0.059306966 0.071168359 NotSig

[0058] 3. Combination of molecular markers

[0059] according to Figure 1 to Figure 3 Based on the results in Table 1, a single molecular marker or a combination of molecular markers that can be used to detect osteoarthritis was obtained. The synovial fluid of 49 osteoarthritis patients (all cases were from the General Hospital of Ningxia Medical University) and 31 healthy volunteers were used as samples. Binomial Logistic regression was used to fit the test results and then the receiver operating characteristic curve (ROC curve, which represents the detection ability of a specific diagnostic method to distinguish specific patient group from non-patient group samples, is a curve drawn with true positive rate, i.e. sensitivity, as the ordinate and false positive rate, i.e. 1-specificity, as the abscissa, and the point closest to the upper left corner is the optimal critical point) was established to verify the effectiveness of these molecular marker combinations. The molecular marker combinations and effectiveness verification results are shown in Tables 2 to 4 and Figure 4-5 .

[0060] From Table 2 to Table 4 and Figure 4-5It can be seen that when using a single molecular marker to detect osteoarthritis, TIMP-4, Ckbeta 8-1 / CCL23, IL-13R2, DNAM-1, TRAIL R2, BMPR-IA, NT-4, BAFF / TNFSF13B, PAI-1, MIP-3b and Prostasin / PRSS8 are all effective, and the AUC can reach above 0.7; when using a combination of molecular markers to detect osteoarthritis, TIMP-4+Ck beta8-1 / CCL23+IL-13R2+DNAM-1+TRAIL R2+BMPR-IA+NT-4+BAFF / TNFSF13B+PAI-1+Prostasin / PRSS8 has the best effect, with an AUC of 0.962, a sensitivity of 0.878, and a specificity of 0.903.

[0061] Table 2. ROC curve data of single molecular markers (down) in distinguishing healthy people from osteoarthritis patients

[0062]

[0063] Table 3. ROC curve data of single molecular markers (up) in distinguishing healthy people from osteoarthritis patients

[0064]

[0065]

[0066] Table 4. ROC curve data of molecular marker combination in distinguishing healthy people from osteoarthritis patients

[0067]

[0068] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A molecular marker for detecting osteoarthritis, characterized in that: The molecular markers include tissue inhibitor of metalloproteinase 4, chemokine β8-1, interleukin 13 receptor 2, killer cell activating receptor molecule 1, tumor necrosis factor-related apoptosis-inducing ligand 2, bone morphogenetic protein receptor 1A, neurotrophin, B-cell activating factor, plasminogen activator inhibitor-1 and serine protease 8.

2. The molecular marker according to claim 1, characterized in that The molecular markers consist of tissue metalloproteinase inhibitor 4, chemokine β8-1, interleukin 13 receptor 2, killer cell activating receptor molecule 1, tumor necrosis factor-related apoptosis-inducing ligand 2, bone morphogenetic protein receptor 1A, neurotrophin, B-cell activating factor, plasminogen activator inhibitor-1 and serine protease 8.

3. The molecular marker according to claim 1 or 2, characterized in that The B-cell activating factor is a truncated protein of the B-cell activating factor; the amino acid sequence of the truncated protein of the B-cell activating factor is shown in SEQ ID NO:

1.

4. Use of a reagent for detecting the molecular marker according to any one of claims 1 to 3 in the preparation of a product for detecting osteoarthritis.

5. An antibody chip for detecting osteoarthritis, characterized in that: The antibody chip comprises a chip body and core capture antibodies coated on the surface of the chip body; the core capture antibodies are arranged on the chip body to form a microarray; the core capture antibodies comprise specific antibodies targeting the molecular markers described in any one of claims 1 to 3.

6. The antibody chip according to claim 5, characterized in that The chip body is a solid phase carrier; the capture antibody is coated on the surface of the solid phase carrier to form a plurality of independent recognition sites.

7. A detection kit for detecting osteoarthritis, characterized in that: The detection kit comprises a reagent for detecting the molecular marker according to any one of claims 1 to 3 and / or the antibody chip according to claim 5.

8. The detection kit according to claim 7, characterized in that The detection kit comprises the antibody chip according to claim 5 and a core detection antibody; the core detection antibody comprises a specific antibody targeting the molecular marker according to any one of claims 1 to 3, and the core detection antibody is labeled with a molecular marker.

9. The detection kit according to claim 8, characterized in that The molecular marker is biotin.

10. A diagnostic model for detecting osteoarthritis, characterized in that: The diagnostic model includes an information collection module, a data analysis module and an evaluation module; The information collection module is used to detect and collect information related to the evaluation object and the molecular marker according to any one of claims 1 to 3; The data analysis module is used to analyze the information obtained in the information collection step to obtain an analysis result of whether the patient suffers from osteoarthritis; The evaluation module is used to classify the test subjects into a group suffering from osteoarthritis and a healthy group according to the analysis results obtained by the data analysis module.

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