Application of fragile X intellectual disorder No.1 gene in preparation of reagent for diagnosing osteoarthritis

Abstract

The invention belongs to the field of biological medicine, and relates to a new application of a fragile X intellectual disturbance No.1 gene, namely an application of the fragile X intellectual disturbance No.1 gene in preparation of a reagent for diagnosing osteoarthritis, and the reagent for diagnosing osteoarthritis comprises but is not limited to substances for detecting the fragile X intellectual disturbance No.1 gene or a gene product of the fragile X intellectual disturbance No.1 gene. The research shows that the osteoarthritis is accompanied by the increase of the concentration of the fragile X chromosome intellectual retardation protein in serum and the increase of the synovial inflammatory macrophages, and the fragile X chromosome intellectual retardation protein in the synovial inflammatory macrophages aggravates the progress of the osteoarthritis. The macrophage FMR1 gene for targeted inhibition of articular cavity synovitis can be used as a diagnostic marker of osteoarthritis, a new idea is provided for prevention and treatment of osteoarthritis, and a support is provided for clinical transformation.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to application of fragile X mental retardation 1 gene (FMRP) in preparation of a reagent for diagnosing osteoarthritis. BACKGROUND

[0002] The structure of fragile X mental retardation 1 gene (FMRP, Accession No. Gene ID:2332 of NCBI) includes an RGG-Box-RNA binding domain, a ribonucleoprotein K homology domain (KH binding domain), a nuclear localization signal (NLS) and a nuclear export signal (NES), wherein the KH binding domain and the RGG-Box are combined with U-rich RNA or G-quadruplex in RNA; the mRNA subcellular localization can be affected by the interaction with Motor protein, thereby mediating the regulation of translation, and FMRP is recognized as an RNA binding protein. FMRP is involved in the processing of RNA, including the regulation of subcellular localization of RNA, mediation of the translation process and the like by promoting the shuttle between cytoplasm / nucleus and interaction with actin. Meanwhile, FMRP has a mutual relationship with miRNA and Ago2, and activates or inhibits translation in the form of a regulatory factor. The miRNAs reported to have a mutual relationship with FMRP include miR-124a, miR-323, miR-302 and the like.

[0003] Osteoarthritis is a chronic joint disease characterized by degenerative changes in articular cartilage and secondary hyperostosis, which can be divided into primary osteoarthritis, secondary osteoarthritis, traumatic osteoarthritis, congenital or developmental osteoarthritis, metabolic osteoarthritis, infectious osteoarthritis and the like.

[0004] Articular cartilage is mainly composed of chondrocytes and extracellular matrix of chondrocytes. Chondrocytes are differentiated from bone marrow stromal stem cells (MSCs), and need to go through six stages to mature chondrocytes: stromal cells (chondroblast precursors), aggregated stromal cells, chondrocytes, proliferating chondrocytes, hypertrophic pre-chondrocytes and hypertrophic chondrocytes. The extracellular matrix of chondrocytes mainly contains collagen type II (COLII), aggrecan and water. Collagen type II constitutes the basic scaffold of articular cartilage tissue, provides a fixed semi-circular grid structure for chondrocytes and other extracellular matrix, resists mechanical stress and buffers pressure.

[0005] In the pathological process of OA, on the one hand, chondrocytes appear abnormal proliferation, hypertrophic differentiation and apoptosis, and the transparent layer of cartilage begins to gradually calcify; on the other hand, the metabolism of the extracellular matrix of chondrocytes appears abnormal, the dynamic balance of synthesis and degradation is broken, the degradation level of the extracellular matrix increases, and the extracellular matrix appears degradation and loss. The extracellular proteolytic enzymes responsible for the degradation of the extracellular matrix mainly include two categories, namely: the degradation of proteoglycan of aggrecanase (ADAMTS) and the degradation of collagen of matrix metalloproteinase (MMPs). The former is mainly ADAMTS-4, 5; the latter mainly includes MMP-1, 3, 9, 13 (which has the strongest degradation effect), and the expression of these two types of extracellular proteolytic enzymes increases with the progress of OA (Yang C Y, Chanalaris A, Troeberg L. ADAMTS and ADAM metalloproteinases in osteoarthritis - looking beyond the 'usual suspects'. Osteoarthritis Cartilage, 2017, 25 (7): 1000-1009. Glasson S S, Askew R, Sheppard B, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature, 2005, 434 (7033): 644-8).

[0006] The diagnosis of osteoarthritis needs to be comprehensively judged in combination with the symptoms, signs, imaging examination and laboratory examination of the patient. Imaging examination, such as X-ray examination, CT or nuclear magnetic resonance. Blood routine and inflammation indicators can be used for auxiliary judgment, such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are normal or slightly elevated, which can be used to exclude other inflammatory arthritis (such as rheumatoid arthritis, infectious arthritis). The diagnostic criteria in different countries and regions are slightly different. For example, the American College of Rheumatology standard is: ① knee pain + morning stiffness <30 minutes + bone crepitus during activity + age >40 years + no joint swelling, excluding other diseases. ② knee pain + X-ray showing osteophytes + joint space narrowing + morning stiffness <30 minutes + bone crepitus during activity, excluding other diseases. Therefore, the osteoarthritis marker is still a hot spot and difficulty in the art. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a new use of fragile X mental retardation 1 gene.

[0008] The research of the present application shows that the level of FMRP in serum exosomes is positively correlated with the pathological progression of KOA, when the level of FMRP in serum exosomes of chondrocytes is significantly higher than that of normal tissues, the bone joint has inflammation, i.e. osteoarthritis occurs. Therefore, FMRP can be used as a diagnostic marker for osteoarthritis.

[0009] The present application provides a new use of fragile X mental retardation 1 gene, which is the use in preparing a reagent for diagnosing osteoarthritis.

[0010] Preferably, the fragile X mental retardation 1 gene or its gene product is a diagnostic marker for osteoarthritis, and the expression level of the fragile X mental retardation 1 gene or its gene product in the osteoarthritis sample is significantly higher than that in the normal sample.

[0011] The reagent for diagnosing osteoarthritis includes but is not limited to a substance for detecting the fragile X mental retardation 1 gene or its gene product, preferably, the reagent for diagnosing osteoarthritis includes but is not limited to a substance specifically recognizing or binding to the fragile X mental retardation 1 gene or its gene product. More preferably, the reagent for diagnosing osteoarthritis includes but is not limited to a nucleic acid, a protein, a small molecule compound, a composition or a compound containing a nucleic acid, a protein or a small molecule compound, and a derivative of the above-mentioned substance. In a preferred embodiment of the present application, the substance for detecting the fragile X mental retardation 1 gene or its gene product used is an antibody of the fragile X mental retardation 1 protein. In another preferred embodiment of the present application, the substance (specifically) recognizing the fragile X mental retardation 1 gene or its gene product used is a probe or a primer thereof.

[0012] Preferably, the reagent for diagnosing osteoarthritis is a reagent for diagnosing synovial osteoarthritis.

[0013] Preferably, the reagent for diagnosing osteoarthritis is a reagent for diagnosing whether the bone joint is damaged or autophagy.

[0014] Preferably, the use includes:

[0015] S1, obtaining a sample to be detected; and / or

[0016] S2, detecting the expression level of the fragile X mental retardation 1 gene or its gene product in the sample.

[0017] Preferably, the use further includes:

[0018] S3, comparing the expression level of the fragile X mental retardation 1 gene or its gene product in the sample with that in normal tissues;

[0019] S4, when the expression level of fragile X mental retardation 1 gene or its gene product in the sample is significantly higher than that in normal tissue, it is judged that the sample has bone joint damage or autophagy;

[0020] On the contrary, it is judged that the sample has no osteoarthritis.

[0021] Preferably, the sample of S1 comprises bone cells; more preferably, the sample of S1 is a chondrocyte.

[0022] The detection in S2 uses a substance capable of specifically recognizing fragile X mental retardation 1 gene or its gene product;

[0023] S4 significantly high means that the expression level of fragile X mental retardation 1 gene or its gene product in the sample is more than 50% higher than that in normal tissue. More preferably, S4 significantly high means that the expression level of fragile X mental retardation 1 gene or its gene product in the sample is more than 2 times that in normal tissue. Inhibition or reduction of the expression level of fragile X mental retardation 1 gene or its gene product can delay or improve the condition of osteoarthritis, bone joint damage or autophagy. The use of substances that inhibit or reduce the expression level of fragile X mental retardation 1 gene or its gene product can include the use in the preparation of drugs for improving or treating bone joint damage or autophagy, especially osteoarthritis and the like.

[0024] Preferably, the sample includes but is not limited to tissues or samples suspected of synovitis, osteoarthritis, and gouty arthritis.

[0025] Osteoarthritis is accompanied by an increase in the concentration of fragile X chromosome mental retardation protein in serum, and fragile X chromosome mental retardation protein in synovial inflammatory macrophages exacerbates the progression of osteoarthritis; the mechanism is that osteoarthritis progresses, accompanied by an increase in inflammatory macrophages, and both inflammatory macrophages and secreted exosomes have significantly increased FMRP. The present application provides a new diagnostic marker for osteoarthritis, and provides a new method for the diagnosis of osteoarthritis and chondrocyte damage. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the present application, the drawings needed in the embodiments will be briefly introduced below. Obviously, each drawing in the following description is directed to some embodiments of the present application, and those skilled in the art can also obtain other drawings based on these drawings without creative labor.

[0027] Figure 1 is an animal experiment model diagram.

[0028] Figure 2This is a histological analysis of osteoarthritis progression. From top to bottom, the same specimen is stained with Safranin O-Fast Green. Scale bars are 500 μm (top) and 100 μm (bottom).

[0029] Figure 3 It is the OARSI score in the progression of osteoarthritis.

[0030] Figure 4 Shows the changes in FMRP protein content in serum exosomes during the development of osteoarthritis (WB detection).

[0031] Figure 5 yes Figure 4 Statistical chart of .

[0032] Figure 6 It shows the FMRP protein content in serum exosomes of clinical normal and KOA patients (WB detection)

[0033] Figure 7 yes Figure 6 Statistical chart of .

[0034] Figure 8 This is the strategy for constructing FMR1 conditional knockout mice.

[0035] Figure 9 This is a histological analysis of osteoarthritis progression. From top to bottom, the same specimen is stained with Safranin O-Fast Green. Scale bars are 200 μm (top) and 50 μm (bottom).

[0036] Figure 10 The effect of exosomes from M0 / M1 macrophages (THP-1) with FMRP overexpression and knockdown on C28 / I2 cartilage function (WB detection).

[0037] Figure 11 Effects of FMRP overexpression and knockdown on M0 / M1 THP-1 exosomes on C28 / I2 cartilage function (immunofluorescence). Scale bar: 20 μm.

[0038] Figure 12 yes Figure 11 Statistical chart of .

[0039] Figure 13 The effects of FMRP overexpression and knockdown M0 / M1 THP-1 exosomes on C28 / I2 autophagy function were demonstrated (WB detection).

[0040] Figure 14 Immunofluorescence analysis shows the effects of exosomes from M0 / M1 THP-1 cells expressing FMRP overexpression and knockdown on chondrocyte autophagy. Scale bar, 10 μm. Statistical analysis of chondrocyte autophagic flux (number of autophagosomes and autolysosomes). Data are mean ± SD. DETAILED DESCRIPTION

[0041] The application provides an action mechanism of fragile X mental retardation protein in preventing and antagonizing osteoarthritis and application thereof. Osteoarthritis is accompanied by an increase in the concentration of fragile X mental retardation protein in serum, and the fragile X mental retardation protein in synovial inflammatory macrophages aggravates the progression of osteoarthritis; the mechanism is that: osteoarthritis progresses, accompanied by an increase in inflammatory macrophages, and the FMRP in inflammatory macrophages and secreted exosomes is significantly increased, conducts downstream signals through specific sorting of inflammatory molecules miR-155-5p, and causes synovial inflammation and cartilage degeneration. Targeted inhibition of FMR1 gene of joint cavity synovial inflammatory macrophages reverses the osteoarthritis-related phenotype caused by fragile X mental retardation protein. Therefore, the application discloses a new osteoarthritis mechanism, provides a new idea for the prevention and treatment of osteoarthritis, and provides support for clinical transformation.

[0042] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0043] 1. Experimental method

[0044] 1.1 Rat osteoarthritis (OA) model

[0045] SPF level SD male wild (WT) rats (160-180g) of 6 weeks old were purchased and routinely fed. When the rats were 8 weeks old, a knee medial meniscus instability operation (DMM) + anterior cruciate ligament resection (ACLT) was performed to prepare the rat OA model. The specific operation is as follows:

[0046] Before the operation, the rats were fasted and deprived of water for 12 hours, and then anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at a dose of 10ul / g. After anesthesia, the knee joint was extracted around the rat using an electric shaver, and the rat was fixed on the operation board after spraying 75% alcohol.

[0047] The joint capsule was incised along the medial edge of the patellar ligament to expose the knee joint, and the medial collateral ligament and the medial meniscus tibial ligament were cut off; then the anterior foot of the medial meniscus was disconnected, and the anterior cruciate ligament was cut off inward, and attention was paid to protect the integrity of the articular surface to avoid damage to the articular cartilage. The skin and joint capsule of the knee joint in the sham operation group were only incised, and the other tissues remained intact. After the operation, hemostasis was performed, and the opened joint capsule was sutured with absorbable needle and thread, and the skin incision was sutured with nylon thread. After the operation, antibiotics (penicillin, etc.) were injected intraperitoneally for 3 consecutive days.

[0048] 1.2. Rat grouping

[0049] 80 eight-week-old SD male wild (WT) rats were randomly divided into two groups, 40 in each group, respectively: sham operation group (Sham group), OA group.

[0050] Sham operation group (Sham group): 40 eight-week-old SD male wild (WT) rats were subjected to sham operation, and knee joint samples were collected at 8 weeks and 16 weeks, respectively.

[0051] OA group: 40 eight-week-old SD male wild (WT) rats were subjected to bilateral knee joint DMM+ACLT operation, and knee joint samples were collected at 8 weeks and 16 weeks, respectively. Figure 1 ) after operation.

[0052] 1.3 Preparation of plasma samples

[0053] The blood collection time is usually in the morning. Whole blood is collected in an anticoagulant tube and gently inverted up and down 3-5 times. Within 1 hour (at room temperature) or 2 hours (at 4°C), the following operations are performed: 4°C, 1900g (1568rpm) centrifugation for 10 minutes; transfer the supernatant to a clean 1.5mL centrifuge tube, 16,000g (13,200rpm), 4°C, centrifugation for 10 minutes, carefully pipette the supernatant into a new centrifuge tube; store in a -80°C refrigerator.

[0054] 1.4 Preparation of exosome samples

[0055] 4°C centrifuge, 500g centrifugation for 10min, remove the suspended cells. 2000g centrifugation for 30min, remove the cell debris. 10,000g centrifugation for 60min, remove the large vesicles. Transfer the supernatant to an ultracentrifuge tube, strictly balance (accurate to 2 decimal places), 4°C ultracentrifuge, 120,000g centrifugation for 70min. After centrifugation, slowly discard the supernatant in a clean bench, resuspend the exosomes with sterile pre-cooled PBS, stand for 2min, again 4°C ultracentrifuge, 120,000g centrifugation for 70min to precipitate the exosomes. After centrifugation, resuspend in sterile pre-cooled PBS in a clean bench. Take 5μl of each sample to determine the protein concentration of the exosomes by BCA technique. Exosome samples can be stored at 4°C for 2 days, and if not used for a long time, they can be stored in a -80°C refrigerator for subsequent experiments.

[0056] 1.5 Western blotting

[0057] 1.5.1 Preparation of PAGE gel

[0058] The glass plate is washed and dried, and the glass plate is loaded with the double distilled water to check if the glue leaks. 3.5 ml of the lower glue solution and buffer, 70 μl of the modified coagulant are taken in a small beaker, mixed well, and injected into the glass plate. A suitable amount of 75% ethanol is added to cover the lower glue, and the glue is sealed with liquid. After the lower glue is completely solidified (about 15-30 min), the ethanol is poured out and dried. 1 ml of the lower glue solution and buffer, 20 μl of the modified coagulant are taken in a small beaker, mixed well, and injected into the glass plate with the lower glue, and the comb is inserted immediately. After 20 min, when the upper glue is completely solidified, the polyacrylamide gel glass plate is clamped into the electrophoresis frame, and the comb is pulled out after the electrophoresis buffer is added.

[0059] 1.5.2 Electrophoresis

[0060] Loading: The loading amount of the protein sample is 20-30 μg. The loading amount of the protein marker is 2.5-5 μl.

[0061] Electrophoresis: 70 V electrophoresis for about 30 min through the concentrated glue; then according to the size of the detected protein, constant voltage 100 V electrophoresis for about 40-90 min. After the electrophoresis is completed, the glass plate is taken out and washed with double distilled water. The glass plate is pried open, and the concentrated glue and unused lanes are cut off. The separation glue is soaked in the pre-cooled wet transfer buffer at 4°C.

[0062] 1.5.3 Transfer

[0063] a) Transfer: according to the size of the separation glue, the same size of PVDF (polyvinylidene fluoride) membrane is cut, and a small part of the upper right corner is cut off for marking. Soak in methanol for about 20 seconds for activation, and soak in the wet transfer buffer. The filter paper and sponge are assembled in the wet transfer "sandwich" structure in the order of negative electrode→positive electrode: wet transfer clamp black side-sponge-filter paper-glue-membrane-filter paper-sponge-wet transfer clamp transparent side. Insert into the wet transfer frame in the correct positive and negative direction. Pay attention to assemble the sandwich structure without bubbles. Put the ice box in the transfer membrane tank, pour in the wet transfer buffer, and put it into the wet transfer frame. The transfer membrane process is carried out in an ice bath. According to the size of the detected protein, constant current 0.22-0.30 A transfer for 1-2 h.

[0064] b) Washing: after the transfer is completed, take out the protein blotting membrane and put it into a plastic box, add an appropriate amount of TBST, and shake on the shaker (speed 20) for 10 min.

[0065] c) Blocking: absorb the TBST, add an appropriate amount of blocking solution, and shake on the shaker (speed 5) for 1 hour at room temperature. After the blocking is completed, according to the protein standard and the molecular weight of the protein to be tested, the protein blotting membrane is cut. And cut a small part of the upper right corner of the cut membrane for marking.

[0066] d) Primary antibody staining: Dilute the primary antibody in blocking solution according to the recommended dilution ratio on the datasheet. Place the cut protein blot membrane into the primary antibody dilution using forceps and incubate at 4°C overnight on a shaker (speed 5).

[0067] e) Washing: Remove the primary antibody dilution and wash the protein blot membrane with sufficient TBST for 10 min per wash on a shaker (speed 40) for 3 times.

[0068] f) HRP secondary antibody staining: Dilute the secondary antibody conjugated with horseradish peroxidase in blocking solution according to the recommended dilution ratio on the datasheet. Transfer the protein blot membrane into the secondary antibody dilution and incubate at room temperature for 1 h on a shaker (speed 5).

[0069] g) Washing: Remove the secondary antibody dilution and wash the protein blot membrane with sufficient TBST for 10 min per wash on a shaker (speed 40) for 3 times.

[0070] h) Exposure: Prepare the ECL luminescence solution: Take equal amounts of A and B solution and mix them together. Keep the solution in the dark. Place the protein blot membrane on a flat piece of plastic wrap using forceps and wipe off the excess TBST with a paper towel. Add the ECL luminescence solution to the membrane evenly and make sure that the protein blot membrane is fully covered. Immediately place the protein blot membrane with the ECL luminescence solution into the chemiluminescence instrument and take a picture using the automatic exposure mode.

[0071] 1.6 Collection of clinical samples

[0072] This study recruited 20 patients admitted for total hip arthroplasty (THA) (osteoarthritis patients = 13) from the Department of Orthopedics, the Sixth People's Hospital of Shanghai Jiao Tong University School of Medicine. Control samples were derived from patients with femoral neck fractures without a history of arthritis (n = 7). All subjects signed the informed consent for the use of clinical research data. Samples were immediately stored in tissue preservation solution (Miltenyi Biotec) and transported immediately after surgery. This study was approved by the Ethics Committee of the Sixth People's Hospital of Shanghai Jiao Tong University School of Medicine (Approval No.: 2023-KY-033(K) and 2024-KY-270(K)).

[0073] 1.7 Collection of cellular proteins

[0074] Example 6: Cell lysis and protein extraction 6-well plate as an example, pre-cooled PBS wash 2 times, then add 500 μl 0.25% Trypsin-EDTA, 37°C incubator for 3 min or so, visible to the naked eye adherent cells fine sand, add 500 μl complete medium, collection of digested cells in 1.5 ml EP tube, 1000 rpm centrifugation 5 min, discard the supernatant, get the cell pellet, according to 106 cells / 80 μl RIPA lysis buffer (in advance to add protease inhibitors and phosphatase inhibitors), ultrasonic, ice for 15 min, 12000 rpm centrifugation 15 min, take the supernatant. Each tube 5 μl supernatant, using BCA technique to measure the protein concentration. Add 5x loading buffer, and use 1x loading buffer to balance the protein, 100°C boiling 10 min, -80°C.

[0075] Western blot: same as 1.5.

[0076] 1.8 Immunofluorescence experiment (IF-IC) of cells

[0077] 1.8.1 Fixation

[0078] Note: All subsequent incubations should be carried out at room temperature (20-25°C), unless otherwise stated.

[0079] Wash the cells plated on 24-well plate glass with pre-cooled 1x PBS three times for 5 min each time.

[0080] Cover the sample to a depth of 2-3 mm with 4% formaldehyde (let the specimen fix at room temperature for 15-20 min) or -20°C pre-cooled methanol solution (-20°C fixation for 5 min). Rinse with 1x PBS three times for 5 min each time.

[0081] 1.8.2 Immunostaining

[0082] a) Block the specimen in blocking buffer for 60 min.

[0083] b) While blocking, prepare the primary antibody in antibody dilution buffer (preliminary experiment to find the appropriate dilution range).

[0084] c) Aspirate the blocking solution, then add the diluted primary antibody. Incubate overnight at 4°C.

[0085] d) Rinse with 1x PBS three times for 5 min each time.

[0086] e) Dilute the fluorescein-conjugated secondary antibody in antibody dilution buffer, and incubate the specimen in the dark for 1-2 h.

[0087] f) Rinse with 1x PBS three times for 5 min each time, keeping it in the dark.

[0088] g) Appropriate counterstaining.

[0089] h) Mounting with Mounting medium, store the sample at 4°C in the dark.

[0090] 1.9 Autophagy double-label lentivirus infection of C28 / I2 cells

[0091] 1.9.1 Pre-experiment of autophagy double-label lentivirus infection of C28 / I2 cells (MOI exploration)

[0092] a) Cell preparation (Day 1):

[0093] C28 / I2 cells in good growth state were inoculated into 96-well plates, so that the cell concentration was 1 x 10 5 / ml, 100 μl / well, and the number of cells in each well was about 1 x 10 4 , so as to ensure that the cell confluence rate was 30-50% when the virus was infected, and the cells were cultured overnight in a 5% CO2 incubator at 37°C.

[0094] b) Virus infection (Day 2):

[0095] 1 / 2 small volume infection technique, that is, when the virus is infected, 1 / 2 volume of fresh culture solution is added, and the culture volume is supplemented after 4 hours of lentivirus infection.

[0096] Before infection, the virus was taken out and slowly thawed on ice, the original culture medium of the cells was aspirated, 1 / 2 volume of fresh culture medium was added, 50 μl of culture medium was added to the 96-well plate, the value MOI = 3, 10, 30, 100, the virus stock solution was added to the culture medium, and polybrene was added to make the final concentration 10 μg / ml.

[0097] Taking the virus titer of 1 x 10 8 TU / ml as an example, the amount of virus added at different MOI values is shown in the following table.

[0098] Amount of virus added per well (μl) = MOI x cell number / virus titer (TU / ml) x 1000

[0099] Table 1 Virus volume added per well of 96-well plate at different MOI values

[0100]

[0101] c) Medium change (Day 3):

[0102] The old one was aspirated and fresh complete culture medium was added the day after infection (about 24 h), and the culture was continued.

[0103] d) Observation of fluorescence (Day 4-5):

[0104] 48 hours after infection, fluorescence expression can be initially observed using a fluorescence microscope. 72 hours after infection, if the infection efficiency is around 80% and the cells are in good condition, the corresponding infection conditions and MOI can be used as the reference MOI for subsequent infection experiments.

[0105] 1.9.2 Infection of C28 / I2 Cells with Autophagy Double-labeled Lentivirus

[0106] a) Cell preparation (Day 1):

[0107] C28 / I2 cells in good condition were seeded into 24-well plates at 500 μl / well, with the number of cells per well being approximately 1×10 5 The cell concentration was 2 × 10 5 / ml to ensure that the cell confluence rate is 30-50% during virus infection. Place in a 37°C, 5% CO2 incubator and culture overnight.

[0108] b) Viral infection (Day 2):

[0109] The culture volume of a 24-well plate is 500 μl, and the 1 / 2 culture volume is 250 μl.

[0110] Aspirate the original cell culture medium and add 1 / 2 volume of fresh culture medium. Based on the calculated MOI, add the appropriate volume of virus for infection. Add polybrene to the virus solution to a final concentration of 10 μg / ml. 4 hours after infection, top up to the full culture volume.

[0111] c) Medium change (Day 3):

[0112] 24 hours after infection, remove the old culture medium, replace it with fresh complete culture medium, and continue culturing.

[0113] d) Observe fluorescence (Day 4-5):

[0114] GFP and RFP expression can be initially observed using a fluorescence microscope 48 hours after infection, with peak expression typically reaching 72 hours after infection. For viruses carrying the puromycin-resistant gene, wait until cells stabilize after infection and replace with fresh complete culture medium containing puromycin to screen for stably transduced C28 / I2 cells. These cells can then be used in the next step of the experiment.

[0115] 1.10 Transgenic knockout mice

[0116] 1.10.1 Mouse osteoarthritis OA model

[0117] When the transgenic mice were 8 weeks old, medial meniscus destabilization (DMM) was performed to establish an OA model. The specific procedures were as follows:

[0118] Before operation, mice were fasted and watered for 12 hours. After weighing, 1% sodium pentobarbital solution was injected intraperitoneally at a dose of 10 ul / g for anesthesia. After anesthesia, the hair around the knee joint was extracted with an electric shaver, and the mouse was fixed on the operation board in a supine position after spraying 75% alcohol.

[0119] The joint capsule was incised along the medial margin of the patellar ligament to expose the knee joint, and the medial collateral ligament and the medial meniscus tibial ligament were cut off. Then the front foot of the medial meniscus was disconnected, and the integrity of the articular surface was protected to avoid damage to the articular cartilage. The skin and joint capsule at the knee joint site were only incised in the sham operation group, and the other tissues remained intact. After operation, hemostasis was performed, and the opened joint capsule was sutured with absorbable needle, and the skin incision was sutured with nylon thread. After operation, antibiotics (penicillin, etc.) were injected intraperitoneally for 3 consecutive days.

[0120] 1.10.2 Mouse grouping:

[0121] All experimental mice were C57BL / 6J background, LysM-Cre mice, FMR1 flox / flox Mice were purchased from SJAIE Biotechnology Co., Ltd. FMR1 flox / flox Mice (Control group) and myeloid macrophage conditional knockout transgenic mice: FMR1 flox / flox LysM-Cre positive mice (CKO group), 10 in each group (half male and half female), unilateral knee DMM operation was performed, and knee joint samples were collected at 10 weeks.

[0122] 2. Experimental materials

[0123] The primary antibody in the WB experiment was derived from: FMRP-antibody (1:1000, CST, 4317), β-actin-antibody (1:1000, CST, 8457), Beclin1-antibody (1:1000, CST, 3495), ATG3-antibody (1:1000, CST, 3415), ATG7-antibody (1:1000, CST, 8558), LAMP1-antibody (1:1000, Abeam, ab62562), GAPDH-antibody (1:1000, CST, 5174), COL2A1 (1:1000, Invitrogen, MA5-12789), MMP13-antibody (1:1000, Invitrogen, MA5-14238), Sox9-antibody (1:1000, Abeam, ab185966).

[0124] Primary antibodies used for immunofluorescence of cells: COL2A1 (1:1000, Invitrogen, MA5-12789), MMP13-antibody (1:1000, Invitrogen, MA5-14238).

[0125] Unless otherwise specified, the experimental materials of the present application can use commercially available conventional materials.

[0126] Example 1

[0127] At 8 weeks after modeling, the wild rats showed obvious joint degeneration, and at 16 weeks after modeling, the joint degeneration became more serious. The joint degeneration gradually increased over time. At 8 weeks after modeling, the tibial plateau cartilage surface of the rat knee was thin, and the tide line moved up. The articular surface was not complete, and the cartilage surface was partially broken and worn. The local cartilage surface was stained lighter, and the chondrocyte was empty. The cartilage matrix was partially lost. In summary, the proteoglycan in the joint cartilage was lost, the cartilage was partially lost and significantly degenerated, and the OA medium pathological symptoms were obvious.

[0128] At 16 weeks after modeling, the rat knee joint safranin O-fast green staining showed that the joint space was severely worn, the articular surface was not complete, and cracks appeared. The medial tibial plateau had almost no cartilage tissue left, the matrix was severely lost, and the subchondral bone was exposed. Figure 2 In summary, the articular cartilage surface was severely worn and lost, the cartilage was lost and significantly degenerated, and the OA late pathological symptoms were more serious. According to the results of safranin O-fast green staining of the rat knee joint, the OARSI score was as shown in Figure 3 Regardless of whether it was 8 weeks after DMM surgery or 16 weeks after DMM surgery, the OA group of rats had significantly higher scores than the Sham group, and the 16-week post-surgery rats had significantly higher scores than the 8-week post-surgery rats. This shows that the degree of OA in rats after DMM modeling gradually developed.

[0129] KOA experiments were conducted on rats, and the peripheral blood of rats 8 weeks and 16 weeks after surgery was collected Figure 1 , and then the exosomes in the serum were separated, and the level of FMRP in the serum exosomes of each group was detected by WB technology. The results showed that the OA group was significantly higher than the Sham group, and the 16-week post-surgery group of the OA group was significantly higher than the 8-week post-surgery group. This shows that as the OA condition worsens, the level of FMRP in the serum exosomes gradually increases Figures 4-5 .

[0130] Example 2 Clinical osteoarthritis samples

[0131] Clinical KOA patients and normal patients were collected, serum was collected and exosomes were isolated, and the FMRP level in serum exosomes was detected by WB technology. The results show that the FMRP level in serum exosomes of OA patients is significantly higher than that of normal patients Figure 6 and Figure 7 ). This shows that as the OA condition worsens, the FMRP level in serum exosomes gradually increases.

[0132] Example 3

[0133] 10 weeks after FMRP knockdown mouse modeling Figure 8 ), the knee joint of the mouse was stained with safranin O-fast green. Compared with the control group, the cartilage matrix of the CKO group increased, the chondrocyte empty staining decreased, the area of the chondral surface with broken and worn regions decreased, and the chondral surface integrity increased Figure 9 ).

[0134] In summary, it is shown that myeloid macrophage knockout of FMRP protein significantly alleviates the progression of osteoarthritis.

[0135] Example 4 Effect of FMRP overexpression and knockdown macrophage-derived exosomes on cartilage cell matrix metabolism

[0136] COL2A1 is the main component of the extracellular matrix of chondrocytes and is a marker of articular cartilage tissue. Sox9 is an important transcription factor in cells and is an important molecule for regulating metabolism in the extracellular matrix of articular chondrocytes, which can promote the transcription of COL2A1, proteoglycan and type IX collagen, while inhibiting the expression of ADAMTS and MMPs, thereby maintaining the phenotype of articular chondrocytes. The present application selects COL2A1 and Sox9 as indicators for measuring anabolism in cartilage tissue homeostasis. During the development of OA, matrix metalloproteinases MMPs that degrade collagen, the most important component of the extracellular matrix, gradually increase, among which MMP-13 has the strongest degradation effect. Therefore, the present application selects MMP-13 as an indicator for measuring catabolism in cartilage tissue homeostasis.

[0137] Western blot technology was used to detect the effect of FMRP overexpression and knockdown M0 / M1 type macrophages (THP-1) on C28 / I2 cartilage function-related proteins: COL2A1, Sox9 and MMP-13. The results show that Vector / FMRP OE (FMRP overexpression) and SCR KD / FMRP KDThe exosomes secreted by the THP-1 cell lines of the four groups (FMRP knockdown) induced the differentiation of M0 and M1 macrophages. The latter significantly reduced the levels of COL2A1 and Sox9 proteins in C28 / I2 cells and significantly increased the level of MMP-13 protein. KD Compared with SCR KD The group could significantly increase the levels of COL2A1 and Sox9 proteins and significantly reduce the level of MMP-13 protein in C28 / I2 cells; however, FMRP OE Compared with the Vector group, the COL2A1 and Sox9 protein levels in C28 / I2 cells were significantly reduced, and the MMP-13 protein level was significantly increased. KD Compared with FMRP OE The Vector / FMRP group significantly increased the levels of COL2A1 and Sox9 proteins in C28 / I2 cells and significantly decreased the level of MMP-13 protein. OE and SCR KD / FMRP KD The effects of exosomes secreted by these four cell lines on the protein levels of COL2A1, Sox9 and MMP-13 in C28 / I2 cells were not significant (see Figure 10 ).

[0138] Immunofluorescence was used to detect the effects of exosomes secreted by FMRP-overexpressing and knockdown M0 / M1 THP-1 on C28 / I2 cartilage function-related proteins: COL2A1, Sox9 and MMP-13 (see Figure 11 and 12 ), and the results were consistent with the WB test results above.

[0139] Example 5 Effects of Macrophage-derived Exosomes with FMRP Overexpression and Knockdown on Chondrocyte Autophagy

[0140] ① Detection of autophagy-related proteins

[0141] Beclin1, Atg3, Atg7 and Lamp1 proteins were selected as markers of autophagy. Western blot was used to detect the effects of exosomes secreted by FMRP-overexpressing and knockdown M0 / M1 THP-1 on C28 / I2 autophagy-related proteins. OE and SCR KD / FMRP KDThe exosomes secreted by the four groups of THP-1 cell lines, which induced differentiation of M0 and M1 type macrophages, can significantly reduce the content of autophagy-related proteins in C28 / I2 cells.

[0142] Among them, when comparing the exosomes of M1 type macrophages, the FMRP KD group can significantly increase the content of autophagy-related proteins in C28 / I2 cells, but the FMRP KD group can significantly reduce the content of autophagy-related proteins in C28 / I2 cells. OE Among them, when comparing the exosomes of M0 type macrophages, the FMRP KD group can significantly increase the content of autophagy-related proteins in C28 / I2 cells. At the same time, when comparing the exosomes of M1 type macrophages, the Vector / FMRP OE and SCR OE / FMRP KD groups can significantly increase the content of autophagy-related proteins in C28 / I2 cells. KD The effects of the exosomes secreted by the four groups of cell lines on the autophagy-related proteins in C28 / I2 cells are not significant (see Figure 13 ).

[0143] ② Detection of autophagy flow

[0144] The effects of the exosomes secreted by the four groups of THP-1 cell lines, which induced differentiation of M0 and M1 type macrophages, on the autophagy flow level of C28 / I2 were detected by LC3 double fluorescence lentivirus. The results show that: the Vector / FMRP OE and SCR KD / FMRP KD groups can significantly reduce the autophagy flow level in C28 / I2 cells.

[0145] Among them, when comparing the exosomes of M1 type macrophages, the FMRP KD group can significantly increase the autophagy flow level in C28 / I2 cells, but the FMRP KD group can significantly reduce the autophagy flow level in C28 / I2 cells. OE Among them, when comparing the exosomes of M0 type macrophages, the FMRP KD group can significantly increase the autophagy flow level in C28 / I2 cells. At the same time, when comparing the exosomes of M1 type macrophages, the Vector / FMRP OE and SCR OE / FMRP KD groups can significantly increase the autophagy flow level in C28 / I2 cells. KDThe effects of the exosomes secreted by the four groups of cell lines on the autophagy flow level in C28 / I2 cells were not significant (see Figure 14 ).

[0146] The above-described embodiments are merely specific implementations of the present application, and the protection scope of the present application is not limited thereto. Any person skilled in the art can conceive of changes or replacements within the technical scope disclosed in the present application without creative labor, and such changes or replacements should be encompassed in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims in the present application.

Claims

1. Application of fragile X intellectual disability gene 1, characterized in that, The application is application in preparing reagents for diagnosing osteoarthritis.

2. The use of the fragile X intellectual disability gene 1 according to claim 1, characterized in that: The fragile X intellectual disability 1 gene or its gene product is a diagnostic marker for osteoarthritis. The expression level of the fragile X intellectual disability 1 gene or its gene product in osteoarthritis samples is significantly higher than that in normal samples. The reagents for diagnosing osteoarthritis include but are not limited to substances for detecting the fragile X intellectual disability 1 gene or its gene product.

3. The use of the fragile X intellectual disability gene 1 according to claim 1, characterized in that: The reagent for diagnosing osteoarthritis is a reagent for diagnosing synovitis; the reagent for diagnosing osteoarthritis includes but is not limited to a substance that specifically recognizes the fragile X intellectual disability 1 gene or its gene product.

4. The use of the fragile X intellectual disability 1 gene according to claim 1, characterized in that: The reagent for diagnosing osteoarthritis is a reagent for diagnosing whether the bone and joint are damaged or autophagic; the reagent for diagnosing osteoarthritis includes but is not limited to nucleic acids, proteins, and small molecule compounds that specifically recognize the fragile X intellectual disability 1 gene or its gene product.

5. The use of the fragile X intellectual disability 1 gene according to claim 1, characterized in that: The applications described include: Step S1, obtaining a sample to be tested; and / or Step S2: detecting the expression level of the fragile X intellectual disability 1 gene or its gene product in the sample.

6. The use of the fragile X intellectual disability 1 gene according to claim 5, characterized in that: The applications described include: Step S3, comparing the expression levels of the fragile X intellectual disability 1 gene or its gene product in the sample and normal tissue; Step S4, when the expression level of the fragile X intellectual disability 1 gene or its gene product in the sample is significantly higher than that in normal tissue, the sample is judged to have bone and joint damage or autophagy; Otherwise, the sample is judged to have no osteoarthritis.

7. The use of the fragile X intellectual disability 1 gene according to claim 6, characterized in that: The sample in step S1 contains bone cells; In step S2, the detection uses a substance that can specifically recognize the fragile X intellectual disability 1 gene or its gene product to determine whether the fragile X intellectual disability 1 gene or its gene product is present in the sample and its expression level; The gene product in step S3 is not limited to a protein, a polypeptide, or a substance containing a fragment of the fragile X intellectual disability protein 1; The "significantly higher" in step S4 means that the expression level of the fragile X intellectual disability 1 gene or its gene product in the sample is higher than that in normal tissue by more than 50%.

8. The use of the fragile X intellectual disability 1 gene according to claim 6, characterized in that: The sample in step S1 is chondrocytes; The "significantly higher" in step S4 means that the expression level of the fragile X intellectual disability 1 gene or its gene product in the sample is more than twice that of normal tissue.

9. The use of the fragile X intellectual disability 1 gene according to claim 6, characterized in that: The samples include but are not limited to samples suspected of synovitis, osteoarthritis, and anserinitis.

10. The use of the fragile X intellectual disability 1 gene according to claim 1, characterized in that: The reagent is an agent for diagnosing arthritis accompanied by inflammatory macrophages.

Images