Biomarker for knee osteoarthritis diagnosis and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a biomarker for clinical diagnosis of knee osteoarthritis and application of the biomarker. The biomarker for diagnosing the knee osteoarthritis, disclosed by the invention, is a neuronal glial antigen 2, namely NG2. The inventor finds that the soluble NG2 (sNG2) level is remarkably increased in joint fluid of a knee osteoarthritis patient (OA) and is remarkably positively correlated with the minimum joint gap width of the OA and an inflammatory factor TNF-alpha; compared with a healthy control, the sNG2 level in OA serum is remarkably increased; in addition, the TNF-alpha promotes the expression up-regulation of NG2 in OA cartilage cells and the shedding generation of sNG2. Therefore, by detecting sNG2 in serum and synovial fluid of a patient with knee osteoarthritis, the kit can be used for diagnosing knee osteoarthritis, and a new strategy is provided for clinical diagnosis of knee osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the application of NG2 in the field of biomedical technology, and in particular to a biomarker for diagnosing knee osteoarthritis and an application thereof. Background Art

[0002] Osteoarthritis (OA) is the most common joint disease in the world. Studies in North America and Europe have shown that approximately 30% of people aged ≥65 years suffer from knee OA, and the characteristic signs of OA include pain, transient morning stiffness, and cramping (grating sound or sensation in the joint) during movement. Joint pain and loss of function cause a huge economic burden, often accounting for 1-2.5% of GDP in developed countries. Osteoarthritis affects the entire joint, including articular cartilage, subchondral bone, and synovium. Although the pathological process can affect a single tissue, the entire joint is ultimately involved due to its intimate structural and functional relationship. Substantial changes occur in the articular cartilage matrix, which is caused and affected by chondrocytes. When chondrocytes fail to repair the damaged cartilage matrix, these cells begin to secrete matrix-degrading enzymes, reactive oxygen species, cytokines, and chemokines, etc. Then, synovial inflammation ensues, leading to cartilage degradation. Obesity and increased levels of associated adipokines also increase the inflammatory state. Remodeling of the subchondral plate reflects and causes changes in the mechanical load of cartilage and subchondral bone.

[0003] NG2 (neuronal glial antigen 2), also known as CSPG4 (chondroitin sulfate proteoglycan 4), is a multifunctional transmembrane proteoglycan involved in a variety of cellular processes such as migration, survival, apoptosis and differentiation. NG2 is frequently expressed in a variety of solid organ cancers. NG2 is involved in cancer cell adhesion, invasion, proliferation, angiogenesis, complement inhibition and signal transduction, and is closely related to malignant tumors such as liver cancer, pancreatic cancer, lung cancer and kidney cancer. In bone homeostasis, NG2 is involved in the initiation of bone metastasis and bone remodeling. NG2 expression is upregulated during intrachondral and intramembranous ossification and downregulated with the completion of ossification. Overexpression of lysyl oxidase-like protein 2 (LOXL2) in chondrocytes increases the level of chondroitin sulfate proteoglycan (NG2) mRNA. In addition, the molecule has multiple protein binding domains, including ERK1 / 2 and PKC-α, MUPP-1 and integrin, among which NG2 plays a key role in regulating the ERK1 / 2 signal cascade, and ERK1 / 2 also regulates chondrocyte differentiation, hypertrophy, and osteoblast differentiation. Existing studies have shown that metalloproteinases such as ADAM10, MMP-9, MMP-13 and MMP-14 have various relationships with the proliferation, migration, synovial inflammation, cartilage destruction and other pathological occurrences of synovial fibroblasts during the pathogenesis of osteoarthritis. At the same time, it has been found that the above-mentioned proteases can hydrolyze NG2, forming soluble NG2 (sNG2) that is released outside the cell to exert its effect.

[0004] At present, the pathogenesis of OA is still unclear, and there is no unified gold standard for its diagnosis, staging and treatment. The current clinical diagnosis of OA is based on the patient's clinical symptoms and imaging examinations. The clinical symptoms are mainly pain, limited knee joint movement and narrow joint space. Imaging X-rays and MRI examinations are the main diagnostic methods for OA at this stage, and there is currently a lack of more specific laboratory test indicators for OA. The sNG2 protein we found was significantly increased in the serum and synovial fluid of OA, and was significantly correlated with the severity of OA, suggesting that sNG2 protein can be used as an auxiliary indicator for clinical diagnosis of OA and judgment of severity. Summary of the invention

[0005] In view of the lack of specific laboratory diagnostic indicators for OA, in order to fill the gap in laboratory examinations for OA, the present invention proposes a sNG2 protein from knee joint synovial fluid and serum that can be used as a biomarker for the onset of knee osteoarthritis. By detecting the level of sNG2 protein in the knee joint synovial fluid and serum of patients, early diagnosis of knee osteoarthritis patients and monitoring of the occurrence and development of the disease can be achieved. In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0006] In a first aspect, the present invention provides a biomarker for clinical diagnosis and treatment prognosis of knee osteoarthritis, wherein the marker is neuronal glial antigen 2, namely NG2.

[0007] The present invention detects the content of sNG2 in human serum and knee joint fluid and identifies it as a biomarker for knee osteoarthritis. The expression level of the marker sNG2 in the serum and joint fluid of patients with knee osteoarthritis is significantly increased, and it can be used for the diagnosis of knee osteoarthritis and monitoring of the course of the disease. It has strong specificity and high sensitivity, and provides a new strategy for the clinical diagnosis of knee osteoarthritis.

[0008] Furthermore, as a preferred embodiment of the marker of the present invention, the marker exists in the serum and joint fluid of OA patients.

[0009] In a second aspect, the present invention provides use of a reagent for measuring NG2 expression in preparing a tool for diagnosing knee osteoarthritis.

[0010] As a preferred embodiment of the use described in the present invention, the detection of NG2 expression includes: one or more of ELISA method, Western blot method, flow cytometry method, gene chip detection method, and real-time fluorescence quantitative method.

[0011] As a preferred embodiment of the use described in the present invention, the sNG2 level in the synovial fluid and blood of OA patients is detected to determine whether it is knee osteoarthritis, and the expression change level of sNG2 in the peripheral blood serum of OA patients before and after treatment is used to predict the therapeutic efficacy of knee osteoarthritis.

[0012] Furthermore, the expression of sNG2 is upregulated in the serum and synovial fluid of OA patients, and the sNG2 level of OA patients is significantly positively correlated with the severity of knee arthritis and the inflammatory factor TNF-α.

[0013] As a preferred embodiment of the use of the present invention, the diagnosis of knee osteoarthritis comprises the following steps:

[0014] (1) Collecting samples from test subjects and collecting control samples;

[0015] (2) detecting and comparing the expression levels of NG2 in the test subject samples and control samples;

[0016] If the expression level of NG2 in the sample of the test subject is increased compared with the expression level of NG2 in the control sample, the test subject is diagnosed as having knee osteoarthritis or being at risk of having knee osteoarthritis.

[0017] As a preferred embodiment of the use described in the present invention, the control sample is derived from a healthy population or a healthy tissue of a test subject, and the sample of the test subject is one or both of serum and synovial fluid.

[0018] As a preferred embodiment of the detection kit of the present invention, the method for preparing the serum comprises any of the following steps:

[0019] (a) Centrifuge peripheral blood to obtain; or

[0020] (b) peripheral blood is collected and subjected to density gradient centrifugation, magnetic bead separation, and immunoseparation technology to prepare the product.

[0021] As a preferred embodiment of the detection kit of the present invention, the method for preparing joint fluid comprises any of the following steps:

[0022] (a) Extract the joint fluid using a sterile syringe and centrifuge it; or

[0023] (b) The synovial fluid is obtained by density gradient centrifugation, magnetic bead separation, and immunoseparation technology.

[0024] In a third aspect, the present invention provides a kit for diagnosing knee osteoarthritis.

[0025] Furthermore, the kit comprises a reagent for detecting NG2 expression.

[0026] As a preferred embodiment of the kit of the present invention, the sample to be detected is serum and / or joint fluid.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention proposes for the first time that the expression level of sNG2 in the serum and knee synovial fluid of OA patients is significantly increased (high expression). This molecular marker can be used for the diagnosis of OA, has strong specificity and high sensitivity, can conveniently and quickly obtain test samples, and economically and effectively obtain test results, which can not only reduce the test cost, but also shorten the test time, improve the test efficiency and accuracy, reduce the patient's pain and economic costs, and shorten the cycle of predicting the therapeutic effect; in addition, the knee osteoarthritis biomarker of the present invention can be repeatedly tested, which is convenient for re-examination and can improve the accuracy of the test results; the present invention found that the severity of knee osteoarthritis is significantly positively correlated with the level of sNG2 protein in the synovial fluid, which can provide a new strategy for the diagnosis and staging of knee osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This figure shows the difference in expression of NG2 in articular cartilage between healthy subjects and OA patients.

[0030] Figure 2 This is a graph showing the results of sNG2 content in the synovial fluid of osteoarthritis patients with different KL levels.

[0031] Figure 3 This is a graph showing the correlation between the minimum joint space width and the sNG2 level in the synovial fluid of OA patients.

[0032] Figure 4 This is a graph showing the correlation between sNG2 levels in the synovial fluid of OA patients and the inflammatory factor TNF-α.

[0033] Figure 5 This figure shows the difference in expression of sNG2 in the serum of healthy people and osteoarthritis patients.

[0034] Figure 6 NG2 is upregulated in the cartilage tissue of DMM osteoarthritis model mice.

[0035] Figure 7 TNF-α promotes the upregulation of NG2 expression and increased sNG2 shedding in chondrocytes.

[0036] Figure 8 TNF-α induces EGR1-mediated upregulation of NG2 expression. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The following examples are convenient for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used can all be obtained from commercial sources.

[0038] Example 1

[0039] This example introduces the expression changes of neuroglial antigen 2 (NG2) in the knee articular cartilage of normal subjects and OA patients.

[0040] 1. Sample collection

[0041] Three patients with normal knee joints (amputation patients) and 3 patients with OA were admitted to the First Affiliated Hospital of Anhui Medical University (North District). All patients were diagnosed with OA by clinical and related knee bone examinations, and their ages ranged from (23 to 79) years old. The diagnosis of all patients in the case group was based on the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Osteoarthritis" of the Chinese Medical Association. The research subjects were all unrelated Han people. The results of H&E staining and NG2 fluorescence staining of the two groups were as follows: Figure 1 As shown, Figure 1 A is the hematoxylin-eosin (H&E) pathological staining and NG2 fluorescent staining of articular cartilage of healthy people and OA patients; Figure 1B is the quantitative statistical result of NG2 fluorescence staining in Figure A. It can be seen from the figure that the chondrocytes of OA patients are hypertrophic, and the expression of NG2 in the hypertrophic chondrocytes of OA patients is significantly increased.

[0042] Example 2

[0043] This example introduces the expression of sNG2 in the synovial fluid of healthy subjects and OA patients at different clinical stages.

[0044] 1. Sample collection

[0045] Ten healthy subjects and 40 patients with knee osteoarthritis admitted to the First Affiliated Hospital of Anhui Medical University (North District) were collected. All enrolled cases were diagnosed with OA by clinical manifestations and related bone and joint imaging examinations, and their ages ranged from (50 to 83) years old. The diagnosis of all patients in the case group was based on the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Osteoarthritis" of the Chinese Medical Association, and the research subjects were all unrelated Han people. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (North District), with the approval number PJ-YX2024-022. All patients and healthy volunteers have obtained informed consent. The basic information and clinical data of the 10 normal subjects and 40 OA patients are summarized in Table 1.

[0046] Table 1

[0047]

[0048] 2. Extraction of joint fluid Joint fluid from 40 OA patients was collected by syringe extraction. This operation was performed before joint replacement surgery in OA patients. After the patients were anesthetized, 2-3 mL of joint fluid was extracted and then injected into a clean blood collection tube. Within 30 minutes after specimen collection, centrifuge at 4°C and 3000g for 20 minutes. The upper layer of light yellow clear liquid after centrifugation is the joint fluid. If the color is reddish, hemolysis may occur. Hemolytic specimens will affect the final test results. Therefore, hemolytic specimens are not suitable for this test. Carefully draw the joint fluid and dispense it into sterile EP tubes and store it at -80°C. Repeated freezing and thawing should be avoided.

[0049] 3. Index detection

[0050] In this example, the conventional double antibody sandwich method (sandwich ELISA) was used to detect the sNG2 content in the sample.

[0051] (a) On the first day, the capture antibody (NG2 monoclonal antibody) was diluted to 2.0 μg / mL in PBS. 100 μL of the diluted capture antibody was immediately added to each well of the ELISA plate, the plate was sealed with a sealing film, and incubated at 4°C overnight.

[0052] (b) Perform the test on the next day. Discard the liquid, pat dry on absorbent paper, add 300 μL of washing solution to each well and let stand for 1 min, discard the liquid, pat dry on absorbent paper, and repeat the above operation three times.

[0053] (c) Add 300 μL of blocking solution to each well and incubate at room temperature for 1 hour.

[0054] (d) Repeat the wash in step (b) and prepare to add the sample when completed.

[0055] (e) 100 μL of joint fluid sample diluted 1:5 with PBS and serially diluted standards were added to each well, the plate was sealed with a sealing film, and incubated at room temperature for 2 hours.

[0056] (f) Repeat the wash in (b).

[0057] (g) 100 μL of 0.5 μg / mL horseradish peroxidase (HRP)-conjugated NG2 polyclonal antibody was added to each well, the plate was sealed with a sealing film, and incubated at room temperature for 1 hour.

[0058] (h) Repeat the wash in (b).

[0059] (j) Add 150 μL of TMB substrate solution to each well and incubate at room temperature in the dark for 20 minutes. Avoid placing the plate in direct sunlight.

[0060] (k) Add 50 μL of stop solution (2M H2SO4) to each well and tap gently to ensure thorough mixing.

[0061] (l) Immediately use an ELISA reader to measure the optical density of the well plate at 450 nm. The obtained value needs to be further analyzed and processed.

[0062] 4. Concentration calculation

[0063] Calculate the average absorbance of each group of standards, controls, and samples. Construct a standard curve by plotting the absorbance of each standard on the y-axis and the concentration on the x-axis, and draw a best fit curve through these points on the graph. To determine the concentration of the sample, find the absorbance value of the sample on the y-axis and draw a horizontal line to the standard curve. Draw a vertical line to the x-axis at the intersection and read the concentration. The read concentration multiplied by 5 (dilution factor) is the actual concentration of the sample. In addition to the above method, computer-based curve fitting statistical software can also be used to calculate the concentration of the sample. The expression difference results of sNG2 content in the synovial fluid of patients with osteoarthritis of different KL levels are shown in Figure 2. Figure 2 As shown, it can be seen that the sNG2 content in the synovial fluid of patients with knee osteoarthritis detected by us increased significantly with the progression of the disease.

[0064] In summary, the sNG2 content in the synovial fluid of patients with knee osteoarthritis is significantly increased.

[0065] Example 3

[0066] This example introduces the correlation analysis between the above-mentioned knee osteoarthritis biomarker (sNG2) and the clinical staging index (mJSW).

[0067] In order to find more evidence to prove that sNG2 is a potential marker for the diagnosis of knee osteoarthritis, we conducted a correlation analysis between the sNG2 content in the patient's synovial fluid and the common clinical diagnostic indicators of the patient in clinical diagnosis. This example is based on computer software (GraphPad Prism 8) to calculate the correlation between the sNG2 content and the clinical indicators, and the steps are as follows:

[0068] (a) Import data: Create a new XY table in GraphPad Prism 8 and input the clinical data of each patient into the table one by one.

[0069] (b) Normality test: Click the Analysis button on the toolbar to enter the Analyze Data interface. Select the Normality and Lognormality Tests option in Column analyses, keep the default parameter settings, and click OK.

[0070] (c) Correlation analysis: Click the Analysis button on the toolbar to enter the Analyze Data interface. Select the Correlation option in XY analyses. According to the normality test results in step (b), if both variables follow a normal distribution, the Pearson correlation analysis in the parameters is used. If one or both of the two variables are rank variables or do not meet the normal distribution, the Spearman correlation analysis is required. Keep the default options for other parameter settings and click OK.

[0071] (d) The results of the correlation analysis can be viewed in Results: If the P value is less than 0.05, there is a significant difference in the correlation.

[0072] The results of the correlation analysis between the minimum joint space width (mJSW) and the sNG2 content in the synovial fluid of patients with osteoarthritis are as follows Figure 3As shown in the figure, the sNG2 content in the synovial fluid of patients with knee osteoarthritis is significantly correlated with the mJSW index. In clinical diagnosis, mJSW is an important index for determining the clinical stage of OA. Its calculation is based on the weight-bearing X-ray of the patient's knee joint. By measuring the width of the narrowest part of the patient's key gap, this index can reflect the thickness of the patient's articular cartilage and determine the severity of the lesion. This index can help determine the clinical stage of knee arthritis.

[0073] Example 4

[0074] This example detected the content of TNF-α in the synovial fluid of OA patients and analyzed its correlation with sNG2.

[0075] TNF-α (tumor necrosis factor) is an important indicator for determining knee osteoarthritis inflammation. It is associated with the progression and severity of the disease and can reflect the level of joint inflammation. Existing literature shows that the level of TNF-α in the synovial fluid of patients with knee osteoarthritis increases significantly during the course of the disease. The TNF-α-induced mouse arthritis model exhibits all symptoms of inflammatory arthritis, including synovitis, pannus formation, and bone erosion. Therefore, we detected the TNF-α content in the synovial fluid of OA patients with different clinical stages and conducted a correlation analysis with sNG2. The correlation between the sNG2 level in the synovial fluid of osteoarthritis patients and the tumor necrosis factor-α (TNF-α) content is as follows: Figure 4 As shown, it can be seen that the content of sNG2 in synovial fluid is significantly correlated with TNF-α.

[0076] Example 5

[0077] This example introduces the expression of sNG2 in the serum of healthy subjects and OA patients.

[0078] 1. Sample collection

[0079] Ten healthy subjects admitted to the First Affiliated Hospital of Anhui Medical University (North District) and 10 randomly selected from 40 patients with knee osteoarthritis were collected. All enrolled cases were diagnosed with OA by clinical manifestations and related bone and joint imaging examinations, and their ages ranged from (50 to 83) years old. The diagnosis of all case groups was based on the diagnostic criteria of the Chinese Medical Association's "Guidelines for the Diagnosis and Treatment of Osteoarthritis", and the research subjects were all unrelated Han people.

[0080] The basic and clinical data of the 10 normal subjects and 40 OA patients are summarized in Table 1.

[0081] 2. Serum extraction

[0082] 4 mL of peripheral blood was collected from 10 healthy people and 10 OA patients by venous blood sampling. The blood collection tube used in this example was a blood collection tube with a blood anticoagulant. The specimen was centrifuged at 4°C and 1000g for 20 minutes within 30 minutes after collection. The upper pale yellow liquid after centrifugation was serum. If the color was reddish, hemolysis may occur. Hemolyzed specimens will affect the final test results. Therefore, hemolyzed specimens are not suitable for this test. The serum was carefully aspirated and dispensed into sterile EP tubes and stored at -80°C. Repeated freezing and thawing should be avoided.

[0083] 3. Index detection

[0084] In this example, the conventional double antibody sandwich method (sandwich ELISA) was used to detect the sNG2 content in the sample (refer to Example 2 for the specific steps). The expression difference of sNG2 in the serum of healthy people and osteoarthritis patients is shown in the following figure: Figure 5 As shown, it can be seen that the sNG2 content in the serum of patients with knee osteoarthritis detected by us increased significantly with the progression of the disease.

[0085] In summary, the serum sNG2 level in patients with knee osteoarthritis is significantly increased.

[0086] Example 6

[0087] This example detects the expression of NG2 gene and protein, and the expression of MMP13 protein in mouse chondrocytes under an inflammatory environment.

[0088] 1. Sample collection

[0089] From six 6-8 week old C57BJ / 6 male mice purchased from the SPF animal room of Anhui Medical University, three were randomly selected for DMM surgery modeling, and the other three underwent sham surgery. Six mice were killed one month later, and the cartilage of both legs of the mice was collected.

[0090] 2. Cartilage protein extraction

[0091] Take the cartilage of each mouse and put it in a grinding tube. Add 1 mL of RIPA lysis buffer, grind it thoroughly and pour it into a 1.5 mL EP tube. After 40% power ultrasound (10 s working, 20 s pause, 3 cycles), let it stand on ice for 20 minutes, centrifuge it at 4°C, 12000 rpm for 20 minutes, and collect the supernatant in a new EP tube.

[0092] 3. Cartilage RNA Extraction

[0093] Take the cartilage of each mouse and put it in a grinding tube, add 1mL Trizol lysis buffer, grind it thoroughly and pour it into an enzyme-free 1.5mL EP tube, add 200μL of chloroform to each tube, shake it thoroughly, centrifuge it at 4℃, 12000g for 15 minutes, take about 400μL of the supernatant and put it in a new enzyme-free EP tube, add 1μL glycogen to each tube, 700μL 70% ethanol and mix it by inverting, put it in -80℃ for 3 minutes, then centrifuge it at 4℃, 12000rpm for 15 minutes, discard the supernatant, add 1mL 70% ethanol to wash, then centrifuge it at 4℃, 12000rpm for 5 minutes, discard the supernatant, blow dry the remaining ethanol in the clean bench, and dissolve the RNA with 20μL RNAfree water.

[0094] 4. Index detection

[0095] In this example, the Western blot method was used to detect the expression of NG2 and MMP13 in mouse cartilage.

[0096] (a) Prepare the separation gel and stacking gel according to the purchased Western blot kit.

[0097] (b) Assemble the electrophoresis mold, pour in the electrophoresis solution and load the sample, first at 80V for 30 minutes, wait for the sample to run out of the concentrated gel, then at 120V for 1 hour to end the electrophoresis.

[0098] (c) Assemble the mold in the order of black gel and white film, pour in the transfer solution, transfer the film at 300mA and ice bath for 2 hours.

[0099] (d) After the transfer, the NC membrane was removed and placed in 5% skim milk powder (1 g skim milk powder + 20 mL 1×TBST) and blocked at room temperature for 1 h.

[0100] (e) Antibodies were diluted according to the recommended ratio and incubated at 4°C overnight (antibody dilution ratio: NG2, 1:1000; MMP13, 1:1000; Actin, 1:5000).

[0101] (f) The next day, the NC membrane was removed and washed three times with 1× TBST, 5 min each time.

[0102] (g) Incubate with HRP-conjugated secondary antibodies of different species at room temperature for 1 hour.

[0103] (h) The membrane was washed three times with 1× TBST, 5 min each time, and then developed and counted.

[0104] In this example, the Q-PCR method was used to detect the expression of NG2 in mouse cartilage.

[0105] (a) The extracted cartilage RNA was converted to cDNA and diluted 5-fold.

[0106] (b) Configure the qPCR system according to the following scheme.

[0107]

[0108] (c) Detection was performed using a qPCR instrument (program: 95°C, 2 min; (95°C, 10 sec; 60°C, 30 sec) × 40 cycles).

[0109] 5. Results Analysis

[0110] The grayscale value of each target protein and internal reference protein band was obtained by ImageJ software analysis. A ratio of target protein / internal reference protein was obtained, and the obtained ratios were substituted into the GraphPad Prism 8 table in turn. The normality test was used for analysis. The specific scheme refers to Example 3.

[0111] In the Excel spreadsheet, calculate the CT value of each target gene minus the internal reference gene to obtain the ΔCT value, calculate the mean ΔCT value of the Control group, and then use the calculated CT value of each group to subtract the mean ΔCT value of the Control group to obtain the ΔΔCT value of each group. Calculate 2 (-ΔΔCT) Obtain the difference in gene expression, substitute the obtained values ​​into the GraphPadPrism 8 table in turn, and use the normality test for analysis. The specific scheme refers to Example 3.

[0112] Expression of NG2 gene and protein in mouse chondrocytes under inflammatory conditions; Expression of MMP13 protein Figure 6 As shown, Figure 6 A is the results of Western blot detection of NG2 expression and quantification in articular cartilage of healthy mice and DMM (a mouse osteoarthritis model); Figure 6 B is the expression and quantitative results of MMP13 in articular cartilage of healthy mice and DMM mice detected by Western blot; Figure 6 C is the quantitative statistical result of Q-PCR detection of NG2 mRNA expression level in articular cartilage of healthy mice and DMM mice. It can be seen that in the cartilage tissue of the DMM surgery-induced mouse osteoarthritis model, both the protein and mRNA levels of NG2 increased with the increase of MMP13.

[0113] Example 7

[0114] This example describes the expression of MMP13 gene, NG2 gene and protein, and sNG2 shedding in C28 chondrocytes under the stimulation of TNF-α.

[0115] 1. Sample collection

[0116] C28 cells were plated at 40% between cells (12-well plate), and TNF-α was added after 24 hours for different purposes.

[0117] (a, b) RNA samples were collected after stimulation with different concentrations of TNF-α (0, 20, 40 ng / mL) for 24 h.

[0118] (c) The protein-like structure was detected after 0, 6, 12, and 24 h of TNF-α stimulation.

[0119] (d) The supernatants were collected after TNF-α stimulation for 0, 4, 8, 12, 16, and 24 h.

[0120] 2. C28 chondrocyte protein extraction

[0121] For details, please see Example 6.

[0122] 3. RNA extraction from C28 chondrocytes

[0123] For details, please see Example 6.

[0124] 4. Index detection

[0125] In this example, the Western blot method was used to detect the expression of NG2 in C28 cartilage.

[0126] For details, please see Example 6.

[0127] In this example, the qPCR method was used to detect the expression of NG2 in chondrocytes.

[0128] For details, please refer to Example 6

[0129] In this example, the conventional double antibody sandwich method (sandwich ELISA) was used to detect the sNG2 content in the sample.

[0130] The specific scheme is detailed in Example 2.

[0131] The results of MMP13 gene expression, NG2 gene expression, protein expression and shedding in C28 chondrocytes under inflammatory environment are as follows Figure 7 As shown, Figure 7 A is the quantitative statistical result of qPCR detection of NG2 mRNA expression level in C28 cells after TNF-α stimulation for 12 hours; Figure 7 B is the quantitative statistical result of the mRNA expression level of MMP13 in C28 cells detected by qPCR after 12h of TNF-α stimulation; Figure 7 C is the expression and quantitative results of NG2 in C28 (human chondrocyte cell line) cells detected by Western blot after different time stimulation with TNF-α; Figure 7D is the ELISA test of sNG2 content in the supernatant of C28 cells after different time of TNF-α stimulation. It can be seen that after C28 chondrocytes were stimulated by TNF-α, the expression of MMP13 gene increased significantly, the protein level and gene level of NG2 were significantly induced, and TNF-α also significantly induced the production of sNG2 in the supernatant of C28 chondrocytes.

[0132] Example 8

[0133] This example describes the expression of NG2 gene and the shedding of sNG2 in C28 chondrocytes after treatment with TNF-α and EGR1 inhibitor (ML264).

[0134] 1. Sample collection

[0135] (a) C28 cells were plated at 40% intercellular plating (12-well plate). After 24 hours, different concentrations of TNF-α (10, 10, 20, 40 ng / mL) were added for stimulation and RNA samples were collected after 12 hours.

[0136] (b) C28 cells were plated at 40% between cells (12-well plate), ML264 was added 24 hours later for 6 hours, and then TNF-α was added for 24 hours before protein absorption.

[0137] (c) C28 cells were plated at 40% between cells (12-well plate), ML264 was added 24 hours later for 6 hours, and then TNF-α was added for 24 hours before RNA samples were collected.

[0138] (d) C28 cells were plated at 40% between cells (12-well plate), ML264 was added 24 hours later for 6 hours, and then TNF-α was added for stimulation for 24 hours before collecting the cell supernatant.

[0139] 2. RNA extraction from C28 cells

[0140] For details, please see Example 6.

[0141] 3. C28 chondrocyte protein extraction

[0142] For details, please see Example 6.

[0143] 4. Index detection

[0144] This example uses the qPCR method to detect the expression of the NG2 gene in C28 cartilage.

[0145] For details, please see Example 6.

[0146] In this example, the Western blot method was used to detect the expression of NG2 protein in C28 cartilage.

[0147] For details, please see Example 6.

[0148] In this example, the conventional double antibody sandwich method (sandwich ELISA) was used to detect the sNG2 content in the sample.

[0149] The specific scheme is detailed in Example 2.

[0150] After C28 chondrocytes were treated with EGR1 inhibitors under inflammatory conditions, EGR1 gene expression; NG2 gene and protein expression were as follows Figure 8 As shown, Figure 8 A is the mRNA expression and quantitative results of EGR1 in C28 chondrocytes after treatment with different concentrations of TNF-α detected by Q-PCR; Figure 8 B is the protein expression and quantitative results of NG2 in C28 chondrocytes after treatment with TNF-α and EGR1 inhibitor (ML264) detected by Western blot; Figure 8 C is the Q-PCR detection of the mRNA expression and quantitative results of NG2 in C28 chondrocytes after treatment with TNF-α and EGR1 inhibitor (ML264). It can be seen that the presence of TNF-α promoted the expression of EGR1 in C28 chondrocytes. At the same time, the inhibition of EGR1 by ML264 also reduced the upregulation of NG2 in chondrocytes and the shedding of sNG2 induced by TNF-α.

[0151] In summary, the present invention proposes for the first time that the expression level of sNG2 in the serum and synovial fluid of patients with knee osteoarthritis is significantly increased. This molecular marker can be used for the diagnosis of knee osteoarthritis, provide a reference for the clinical staging of knee osteoarthritis, and provide a new direction for the treatment of knee osteoarthritis.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A biomarker for the diagnosis of knee osteoarthritis, It is characterized in that The biomarker is neuronal glial antigen 2, namely NG2; including soluble NG2 in OA joint fluid and serum, namely sNG2, and NG2 in chondrocytes.

2. The biomarker according to claim 1, It is characterized in that The marker is present in the synovial fluid and serum, as well as in articular chondrocytes of patients with knee osteoarthritis.

3. A kit for diagnosing knee osteoarthritis, It is characterized in that The kit comprises reagents for detecting NG2 expression.

4. The kit according to claim 3, It is characterized in that The sample to be tested is selected from at least one of OA patient's joint fluid, serum, and articular chondrocytes.

5. Use of a reagent for detecting NG2 expression in the preparation of a tool for diagnosing knee osteoarthritis.

6. The use according to claim 5, It is characterized in that Whether the patient has knee osteoarthritis is determined by detecting the level of NG2 in the patient's articular chondrocytes and the level of sNG2 in the synovial fluid and serum.

7. The use according to claim 5, It is characterized in that The detection of NG2 expression includes: one or more of ELISA method, Western blot method, flow cytometry method, gene chip detection method, and real-time fluorescence quantitative method.

8. The use according to claim 5, It is characterized in that The knee osteoarthritis diagnosis comprises the following steps: (1) Collecting samples from test subjects and collecting control samples; (2) detecting and comparing the expression levels of NG2 in the test subject samples and control samples; If the expression level of NG2 in the sample of the test subject is increased compared with the expression level of NG2 in the control sample, the test subject is diagnosed as having knee osteoarthritis or being at risk of having knee osteoarthritis.

9. The use according to claim 8, It is characterized in that In step (1), samples of the test subjects and control samples are collected, and the specific operations include: (a) centrifuging peripheral blood to obtain the sample; or (b) extracting joint fluid before surgery and obtaining articular cartilage during knee replacement surgery in patients with knee osteoarthritis.

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