Construction method and application of PM2.5 induced rheumatoid arthritis cell model

Abstract

The invention discloses a construction method of a PM2.5-induced rheumatoid arthritis cell model, after fibroblast-like synovial cells are subjected to exposure treatment for a period of time through PM2.5 with a specific concentration, when the treated cells present an obvious polygon and rheumatoid arthritis cell form, the expression quantity of inflammatory factors in the cells is significantly increased, and the expression quantity of inflammatory factors in the cells is significantly increased. And when the cell model has the ability to invade and degrade cartilage, completing the construction to obtain the rheumatoid arthritis cell model. The change of the pathological process of inflammation of the synovial tissue of the human body under long-term PM2.5 exposure is simulated, the problem of lack of rheumatoid arthritis cell models caused by specific exposure factors is solved, and a cytology foundation is laid for further discussing mechanism research and treatment schemes of rheumatoid arthritis induced by environmental particulate matter exposure. The construction method provided by the invention is verified for many times and has good repeatability, the operation process is simple and convenient, and experimental conditions are easy to control.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and more specifically, to a method for constructing a PM2.5-induced rheumatoid arthritis cell model and its application. Background Art

[0002] Particulate matter 2.5 (PM2.5) is a type of inhalable lung particle with a particle diameter of ≤2.5μm. Its small particle size allows it to remain suspended in the air for a long time and be transported over long distances, and it can enrich harmful substances in the air. At present, studies have shown that PM2.5 is associated with respiratory system-related rhinitis, lung tumors, interstitial lung disease, and cardiovascular system-related atherosclerosis, coagulation dysfunction and other diseases. However, there is little in-depth research on the relationship between atmospheric particulate matter and other systemic diseases of the body, among which research on autoimmune diseases is necessary and urgent. Autoimmune diseases (ADs) are chronic and complex diseases with unknown etiology, including systemic lupus erythematosus, osteoarthritis, rheumatoid arthritis (RA), etc.

[0003] Rheumatoid arthritis is an autoimmune disease with aggressive symmetrical polyarthritis as the main clinical manifestation. The pathogenesis of RA has not been clarified so far. Although some large-scale epidemiological studies have found that the incidence of rheumatoid arthritis is relatively high in people who are exposed to high-concentration PM2.5 environments for a long time, and their risk of developing rheumatoid arthritis is a certain percentage higher than that of residents living in areas with better air quality. However, these studies only observed correlation and cannot prove causality. Because in real life, people exposed to high-concentration PM2.5 environments may also be affected by many other factors, such as lifestyle (smoking, drinking, etc.), occupational exposure, genetic factors, etc. Previous experimental studies have shown that PM2.5 can activate the MAPK signaling pathway through oxidation, thereby inhibiting the expression of specific miRNAs, inducing inflammatory responses and exacerbating the deterioration of rheumatoid arthritis. However, most of these studies focus on exploring the mechanism of PM2.5 leading to acute exacerbation of rheumatoid arthritis. Whether PM2.5 can induce RA and its molecular mechanism need to be further clarified. Therefore, attempts to construct a PM2.5-induced rheumatoid arthritis transformation cell model and to find the early key molecules of rheumatoid arthritis under PM2.5 exposure are of great significance for the study of rheumatoid arthritis caused by PM2.5. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a method for constructing a rheumatoid arthritis cell model.

[0005] The second object of the present invention is to provide an application of the above cell model.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention provides a method for constructing a rheumatoid arthritis cell model induced by particulate matter 2.5. The construction method comprises treating normal fibroblast-like synoviocytes with particulate matter 2.5 (PM2.5) at a concentration of 18 to 22 μg / mL for 45 to 50 h, subculturing the cells to the 8th to 12th generation, and completing the construction when the treated cells exhibit obvious polygonal and rheumatoid arthritis cell morphology, the expression of inflammatory factors in the cells is significantly increased, and the cells have the ability to invade and degrade cartilage, thereby obtaining a PM2.5-induced rheumatoid arthritis cell model.

[0007] Synovial fibroblasts (SF) are the main effector cells in the progression of rheumatoid arthritis (RA), but it is still unknown whether a rheumatoid arthritis cell model can be obtained by direct induction with PM2.5. The present invention uses a specific concentration of PM2.5 to treat normal fibroblast-like synoviocytes (HFLS) to simulate the long-term exposure environment of the human body to PM2.5, successfully constructs a new RA cell model, and also lays a certain foundation for further searching for key molecules between PM2.5 and RA.

[0008] Specifically, the present invention uses different concentrations (0, 10, 20, 40 μg / mL) of PM2.5 to treat normal fibroblast-like synoviocytes for 24 hours, and then uses flow cytometry and immunofluorescence experiments to detect the entry of PM2.5 into cells. It is found that when the cells are treated with PM2.5 concentrations of 20 μg / mL and above, the lateral angle parameter reflecting the cell granularity increases significantly, indicating that PM2.5 can enter the cells. The CCK-8 experiment found that when PM2.5 was 20 μg / mL, it did not cause cell death, but it could cause increased secretion of inflammatory factors. When the PM2.5 concentration was 40, 80, and 160 μg / mL, some cell death occurred, and when the concentration reached 320 μg / mL, the cells were basically dead. Further investigation was conducted on the transformed cells after 10 generations of passage treated with 20 μg / mL PM2.5 for 48 hours. The results of EdU cell proliferation and Transwell migration and invasion experiments showed that PM2.5 exposure treatment would significantly enhance cell proliferation, migration and invasion ability; the results of PCR and ELISA experiments showed that the expression and secretion of inflammatory factor IL-6 and rheumatoid arthritis specific inflammatory factor IL-32 in cells exposed to PM2.5 were significantly upregulated; HE staining, Masson trichrome staining and Alcian blue staining experiments showed that the PM2.5 exposure-treated cells obtained by the present invention have the ability to invade and degrade mouse cartilage tissue, reaching the level of RA-HFLS cells. In summary, the construction method of the present invention can obtain a rheumatoid arthritis fibroblast synoviocyte model induced by PM2.5 exposure. The method has been verified many times and has good repeatability, and the operation process is simple, the experimental conditions are easy to control, and it can stably produce rheumatoid arthritis fibroblast-like synoviocytes.

[0009] Furthermore, the inflammatory factors are pro-inflammatory cytokines and rheumatoid arthritis-specific inflammatory factors.

[0010] Furthermore, the concentration of PM2.5 treatment is 20 μg / mL, which can increase the inflammatory degree of HFLS cells and does not affect the viability of the cells.

[0011] Furthermore, the PM2.5 treatment time is 48 hours.

[0012] Furthermore, the subculture is carried out to generation 10. The present invention shows that compared with HFLS, the proinflammatory cytokine IL-6 and the RA-specific inflammatory factor IL-32 are highly secreted in PM2.5 transformed cells (PM-5th, PM-10th), and the secretion levels of inflammatory factors IL-6 and IL-32 in the 10th generation PM2.5 transformed cells (PM-10th) are not significantly different from those in RA-HFLS cells.

[0013] Furthermore, the cells are cultured in a complete DMEM medium containing non-essential amino acids and fetal bovine serum.

[0014] Furthermore, the pro-inflammatory cytokine is IL-6.

[0015] Furthermore, the rheumatoid arthritis specific inflammatory factor is IL-32.

[0016] Furthermore, the method for identifying the properties of transformed cells is a mouse chimera animal experiment.

[0017] Furthermore, the normal fibroblast-like synoviocytes are human-derived normal fibroblast-like synoviocytes.

[0018] The present invention treats HFLS with PM2.5 to simulate humans being in a PM2.5 exposure environment for a long time, successfully constructs a PM2.5 exposure-induced rheumatoid arthritis transformation cell model, searches for early key molecules of rheumatoid arthritis under PM2.5 exposure, and provides suggestions for rheumatoid arthritis caused by PM2.5.

[0019] Therefore, the present invention also provides the application of the above-mentioned cell model in exploring the mechanism of PM2.5 exposure-induced rheumatoid arthritis.

[0020] The present invention also provides the use of the above cell model in screening and preparing therapeutic drugs for rheumatoid arthritis induced by PM2.5 exposure.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on normal fibroblast synoviocytes and uses a specific concentration of PM2.5 for treatment to establish a PM2.5 exposure-induced normal cell conversion into rheumatoid arthritis fibroblast-like synoviocyte model, which solves the problem of lack of rheumatoid arthritis cell models caused by specific exposure factors, and lays a cytological foundation for further exploration of the mechanism research and treatment of rheumatoid arthritis induced by environmental particulate matter exposure. The construction method provided by the present invention has been verified many times and has good repeatability, and the operation process is simple, the experimental conditions are easy to control, and it can stably produce rheumatoid arthritis fibroblast-like synoviocytes.

[0022] Compared with the existing rheumatoid arthritis cell models, the cell model of the present invention directly induces the transformation of normal fibroblast-like synoviocytes into rheumatoid arthritis characteristic cells through exogenous PM2.5 stimulation, which makes up for the shortcomings of traditional models in simulating environmental pathogenic factors, and provides a new experimental tool for in-depth research on the pathogenesis of rheumatoid arthritis and screening potential therapeutic targets. It can be used to screen anti-inflammatory drugs, antioxidants and other intervention agents, and can also be used for PM2.5 exposure risk assessment, etc., further expanding the application scope of the model and having high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Flow cytometry and immunofluorescence experiments were used to detect the entry of PM2.5 into cells. Figure 1 A in the figure is the flow cytometry test of HFLS cell side angle (SSC) and front angle (FSC) parameters; B is the immunofluorescence experiment to detect the entry of PM2.5 into HFLS cells. Blue: cell nucleus, red arrow: PM2.5.

[0024] Figure 2 To detect the effect of PM2.5 on the proliferation of HFLS cells. Figure 2 A in the figure is the CCK-8 experiment to detect the effect of PM2.5 on the proliferation ability of HFLS cells; B is the PCR experiment to detect the relative expression levels of IL-6, IL-1β, MMP3, and CCL2 in HFLS cells after PM2.5 treatment; C is the ELISA experiment to detect the level of IL-6, an inflammation-related molecule, secreted by HFLS cells after different concentrations of PM2.5. * P <0.05,** P <0.01,*** P <0.001.

[0025] Figure 3 EdU is used to detect newly synthesized DNA in HFLS, PM-HFLS, and RA-HFLS cells. Blue: cell nucleus, red: newly synthesized DNA.

[0026] Figure 4 PCR was used to detect the relative expression levels of IL6 and IL32 mRNA in HFLS cells after PM2.5 treatment at different stages.

[0027] Figure 5 ELISA experiment was used to detect the levels of inflammatory-related factors IL-6 (A) and IL-32 (B) secreted by HFLS cells of different generations treated with PM2.5 (PM-HFLS).

[0028] Figure 6The results show the effect of PM2.5 on the invasion and migration of FLS cells. Figure 6 A in the figure is the Transwell migration assay to detect the migration ability of HFLS, PM-HFLS, and RA-HFLS cells; B is the Transwell invasion assay to compare the invasion ability of HFLS, PM-HFLS, and RA-HFLS cells. * P <0.05, ** P <0.01, *** P <0.001.

[0029] Figure 7 This is a flow chart of the SCID mouse chimera animal experiment. Figure 7 A is the flow chart of the SCID mouse chimera animal experiment; B is the weight change of the three groups of mice (HFLS cell group, PM-HFLS group, RA-HFLS cell group) after cartilage implantation; C is the cartilage attachment of the three groups of mice 60 days after subcutaneous implantation; D is the expression levels of inflammatory factors IL-6 and IL-32 in the serum of the three groups of mice 60 days after cartilage implantation.

[0030] Figure 8 HE staining results of implanted cartilage tissues in three groups of mice. Black arrows: the site of cartilage tissue invasion.

[0031] Fig. 9 Results of Alcian blue staining of implanted cartilage tissues in three groups of mice. Red arrows: Degraded cartilage tissue sites.

[0032] Fig.10 Masson trichrome staining results of implanted cartilage tissues in three groups of mice. Red arrows: damaged cartilage tissues. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] 1. Cell source: Normal fibroblast-like synoviocytes (HFLS): The human normal fibroblast-like synoviocytes (HFLS) used in the present invention are derived from the Cell Applications cell bank.

[0036] Rheumatoid arthritis fibroblast-like synoviocytes (RA-HFLS): The rheumatoid arthritis fibroblast-like synoviocytes (RA-HFLS) used in the present invention are derived from the Cell Applications cell bank.

[0037] 2. Cell culture method: Cell Recovery: (1) Preparation before cell recovery: Place the prepared DMEM complete medium containing 1% non-essential amino acids and 10% fetal bovine serum at room temperature for 30 min. Heat the water bath to 37°C. Light an alcohol lamp in the cell clean bench. Take 10 mL sterile centrifuge tubes, label them and place them on a test tube rack. Add 6 mL of DMEM complete medium to each centrifuge tube for later use.

[0038] (2) Quickly remove the cell cryopreservation tube from the liquid nitrogen tank or -80℃ refrigerator, place it in sterile film gloves, and shake it continuously in 37℃ warm water to thaw it quickly for about 1~2 minutes. During this period, pay attention to observe the melting state.

[0039] (3) Take out the cryotube from the gloves, spray it with 75% ethanol for disinfection, and place it on the cell ultra-clean operating table. Aspirate the cell suspension and transfer it to a 10 mL sterile centrifuge tube that has been added with complete culture medium. Carefully blow and resuspend the cells, and centrifuge at 800 rpm for 3 min.

[0040] (4) Carefully discard the supernatant, resuspend the cell pellet in 1 mL of fresh culture medium, and inoculate it into a 6 cm sterile cell culture dish. Then add 4 mL of culture medium to the dish, shake the cells and place them in a conditioned incubator at 5% CO2 and 37°C for incubation. Replace the culture medium with fresh medium the next day.

[0041] Cell passaging: (1) When the cell confluence in the culture dish reaches 80-90%, subculture the cells.

[0042] (2) Discard the old culture medium and add 2 mL of sterile PBS to wash the cells twice to remove the remaining old culture medium and dead cells. After discarding the PBS, add 800 μL of 0.25% trypsin, gently shake the culture dish so that all cells in the dish can come into contact with trypsin, and place the culture dish in a conditioned incubator for digestion.

[0043] (3) After 2-3 minutes of cell digestion, place the cells under a microscope for observation. When the cells shrink, become round, and the intercellular spaces become wider, add 1 mL of fresh culture medium to the culture dish to terminate cell digestion.

[0044] (4) Tilt the culture dish and gently blow the cells on the bottom of the dish repeatedly to make the cells completely fall off the bottom of the dish to form a cell suspension. Then transfer the cells to a 5 mL centrifuge tube and centrifuge at 1300 rpm for 3 min.

[0045] (5) Carefully discard the supernatant, gently resuspend the cells in 1 mL of fresh culture medium, inoculate the cells into a new culture dish at an appropriate ratio, gently shake the cells, and place the cells in a conditioned incubator for incubation.

[0046] Cryopreservation of cells: (1) Take cells that are in the logarithmic growth phase and in good condition, discard the old culture medium, wash the cells twice with PBS, and then add 0.25% trypsin to digest the cells. After sufficient digestion, transfer the cell suspension to a centrifuge tube and centrifuge at 1300 rpm for 3 minutes.

[0047] (2) Carefully discard the supernatant, aspirate 1 mL of serum-free freezing solution, gently resuspend the cells, blow evenly and transfer the cell suspension to a 2 mL sterile cell freezing tube, seal with sterile sealing film, and label the cell name and freezing date.

[0048] (3) Store the cell cryovials in a -80°C refrigerator or liquid nitrogen tank.

[0049] Example 1 Using PM2.5 to transform cells 1. Experimental Methods 1. PM2.5 induces normal HFLS cells (1) Select normal HFLS cells with good condition and appropriate cell density for PM2.5 treatment to induce rheumatoid arthritis transformed cells (taking a 6 cm culture dish as an example).

[0050] (2) Discard the old culture medium, wash once with PBS, discard the PBS, take a 5 mL sterile centrifuge tube, and add 4 mL of 10% DMEM fresh culture medium for later use.

[0051] (3) Take 12.5 mg / mL PM2.5 working solution, aspirate 6.4 μL PM2.5 working solution in a dark place and add it to the previously prepared centrifuge tube containing 4 mL culture medium. The final concentration of PM2.5 is 20 μg / mL. After repeated pipetting and mixing, add the culture medium containing PM2.5 to the 60 mm 2 Gently shake the culture dish to evenly distribute the PM2.5 in the dish, place the dish in a conditioned incubator and continue incubating. After 48 hours of exposure, the cells will be fully grown and then be subcultured.

[0052] The PM2.5 reagent preparation shown is: Mother solution: Weigh 50 mg of PM2.5 particle standard (ERMCA-110), first dissolve it in 200 μL of DSMO, then add 200 μL of sterile PBS solution to further dilute, mix thoroughly to make the mother solution concentration of the particle suspension 125 mg / mL, a total of 400 μL, and place in a -20°C refrigerator away from light for later use.

[0053] Working solution: Place the PM2.5 mother suspension in an ultrasonic oscillator ice bath for at least 20 minutes, and vortex for 1 minute to disperse the particles to the greatest extent and reduce their aggregation. Take 100 μL of PM2.5 mother suspension, add 900 μL of PBS to dilute it to a PM2.5 suspension with a concentration of 12.5 mg / mL, a total of 1 mL. Use a pipette to repeatedly blow it evenly and store it in a refrigerator at 4°C.

[0054] 2. Flow Cytometry Observation of PM2.5 uptake by cells: The present invention uses a BD Accuri™ C6Plu flow cytometer (BD, USA) to analyze PM2.5 uptake by cells.

[0055] (1) Take cells in the logarithmic growth phase with a cell density of 90%, digest them with 0.25% trypsin, and centrifuge at 1300 rpm for 3 min. Carefully discard the supernatant, resuspend the cells in fresh culture medium and count the cells. Inoculate 170,000 cells per dish into a 3.5 cm culture dish, shake well, and incubate in a conditioned incubator.

[0056] (2) After the cells were attached for 24 h, different concentrations of PM2.5 (0, 10, 20, 40 μg / mL) were added to treat the cells for 24 h in the dark.

[0057] (3) Discard the supernatant, add 2 mL of PBS and wash the cells 3 to 4 times to wash away residual PM2.5, digest the cells with 0.25% trypsin without EDTA for 5 to 6 minutes, observe under a microscope that the cells are completely digested, add fresh culture medium to terminate digestion, and centrifuge at 1500 rpm for 3 minutes.

[0058] (4) Carefully discard the supernatant, add 1 mL of PBS and gently blow the cell pellet to resuspend the cells, transfer them to a 1.5 mL sterile centrifuge tube, and centrifuge at 1300 rpm for 3 min.

[0059] (5) The changes in the forward angle parameters and lateral angle parameters of the cells after being treated with different concentrations of PM2.5 were measured using BDACCuRIC6Plus software, and then further analyzed using FlowJoV10 software.

[0060] 3. Immunofluorescence Experiment Observation of PM2.5 entering cells: The present invention uses an Olympus FV1000 Confocal laser scanning microscope (Tokyo, Japan) to analyze PM2.5 uptake by cells.

[0061] (1) Select normal HFLS cells with good condition and appropriate cell density for PM2.5 treatment to induce rheumatoid arthritis transformed cells. Collect samples 24 hours after treatment. The 10th generation cells are transformed rheumatoid arthritis cells, and select appropriate cell amounts to plate in confocal dishes.

[0062] (2) The infected cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were permeabilized with 100% methanol for 10 minutes at -20°C and then blocked with 3% BSA in PBS for 0.5 hours.

[0063] (3) Cell nuclei were stained with Hoechst reagent for 5 min at room temperature. Images were captured using an Olympus FV1000 Confocal laser scanning microscope (Tokyo, Japan).

[0064] 2. Experimental Results Flow cytometry and immunofluorescence experiments were used to detect whether PM2.5 could be absorbed into HFLS cells. The results of PM2.5-induced morphological changes in normal HFLS cells are shown in Figure 2. Figure 1 As shown in Figure B, untreated normal HFLS cells showed typical fiber-like morphology, with long spindle-shaped cells and good cell adhesion. As the PM2.5 concentration gradient increased, the cell polygonal shape increased, and obvious PM2.5 black particles appeared. The flow cytometry test results are shown in Figure 2. Figure 1 As shown in A, after treating cells with PM2.5 at different concentrations (0, 10, 20, 40 μg / mL) for 24 hours, the lateral angle (SSC) parameter reflecting the cell granularity increased, and increased significantly when treated with PM2.5 concentrations of 20 μg / mL and above, while the forward angle (FSC) parameter reflecting the cell size did not change significantly, indicating that PM2.5 can enter the cells. Among them, the forward angle (FSC) can reflect the size of the cell; the lateral angle (SSC) is proportional to the cell granularity, that is, the number of organelles, nuclei, etc. contained in the cell. At the same time, the results of immunofluorescence experiments are shown in Figure 1 As shown in B, the entry of PM2.5 into the cells was further observed under a microscope, and the results also showed that PM2.5 could enter the HFLS cells. The results show that PM2.5 exposure can directly induce cell damage.

[0065] Example 2 Cytological trait detection 1. Experimental Methods 1.CCK-8 Experiment The CCK-8 kit of the present invention comes from Beijing Lanjieke Technology Co., Ltd. The present invention uses CCK-8 experiment to detect the effect of PM2.5 of different concentrations (0, 10, 20, 40, 80, 160, 320 μg / mL) on cell viability.

[0066] (1) Select cells in the logarithmic growth phase with a cell density of 80%, and change the medium one day in advance. After 0.25% trypsin digestion, centrifuge at 1300rpm for 3 minutes, carefully discard the supernatant, resuspend the cells in fresh culture medium and count the cells. Dilute and prepare the cell suspension based on the assumption that 100μL of cell suspension contains 3000 cells per well. Inoculate 3000 cells per well in a 96-well plate, try to ensure uniform inoculation, set up a blank control for each group, and set up 5 replica wells for each group.

[0067] (2) After 24 hours, the cells adhered to the wall, the medium was changed, and different concentrations of PM2.5 were added for treatment.

[0068] (3) According to the instructions for the CCK-8 reagent, prepare a mixture of the corresponding volume of cell complete culture and CCK-8 solution at a ratio of 1:10, mix thoroughly, add 100 μL of the mixture to each well, shake well, and continue culturing for 3 h.

[0069] (4) After 3 hours, use an ELISA reader to measure the absorbance of each well at 450 nm, i.e., the OD value, and record the OD value of each well.

[0070] 2. RNA Extraction (1) RNA extraction from cells or tissues A. Cell lysis: Take the adherent cells in a 3.5 cm culture dish as an example. After the cells are treated, remove the old culture medium, wash them three times with pre-cooled PBS on ice, add 600 μL of Trizol to the culture dish, and let it stand on ice for 10 minutes. Puff the cells to fully lyse them, transfer Trizol to a 1.5 mL enzyme-free centrifuge tube, mark it and store it in a -80°C refrigerator or extract RNA immediately.

[0071] B. Add appropriate amount of chloroform to the tube in a ratio of 5:1 Trizol:chloroform, shake well, and let stand for 10 minutes. Centrifuge at 12000g for 15 minutes at 4℃. The solution will be divided into three layers: the colorless upper solution contains RNA; the white middle solution is the DNA and protein layer; the pink lower solution is the organic layer.

[0072] C. Pipette at least 2 times the chloroform supernatant into a new enzyme-free EP tube, without touching the white DNA, protein layer, and pink organic layer. Add isopropanol equal to the supernatant, shake well, and let stand for 10 minutes. Centrifuge at 4℃, 12000g for 10 minutes, at which point the RNA precipitate is at the bottom.

[0073] D. Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water), and shake well. Centrifuge at 7500g for 5 min at 4°C, discard the supernatant; repeat the wash with 1 mL of 75% ethanol, discard the supernatant after centrifugation, invert on filter paper, and dry thoroughly. Add 10 μL~20 μL of PM2.5 according to the amount of sediment at the bottom of the tube to test whether different concentrations (0, 10, 20, 40, 80 μg / mL) of PM2.5 can promote HFLS cells to secrete inflammatory factors and matrix metalloproteinases, etc.

[0074] (2) Measuring RNA concentration A. Take 1 μL of RNA solution and use Nanodrop2000 micro-nucleic acid protein detector to detect RNA concentration, repeat twice. OD260 / 280 less than 1.8 indicates protein residue; 1.8~2.0 indicates high-purity RNA; greater than 2.0 indicates partial RNA degradation. OD260 / 230 less than 2.0 indicates salt ion residue. Calculate the volume of RNA solution X μL to be added according to the formula V=1000 / c.

[0075] (3) RNA reverse transcription A. Removal of genomic DNA: (Takara reverse transcription kit is used as an example. For specific steps, refer to the instruction manual of the reverse transcription kit.) Prepare the reaction premix on ice. The system is as shown in Table 1. Perform the reaction in a PCR instrument: 42°C, 2min; 4°C.

[0076] Table 1 Reaction premix system

[0077] B. Reverse transcription reaction (SYBR® Green qPCR method): Prepare the reaction premix on ice. Perform the reaction in a PCR instrument: 37°C, 15 min; 85°C, 5 s; 4°C. The reverse transcription product cDNA is stored at -20°C.

[0078] 3. Conventional PCR The present invention uses a common PCR instrument.

[0079] (1) Primer sequences are shown in Table 2.

[0080] Table 2 Primer sequences

[0081] (2) After diluting the cDNA 5-fold, prepare the PCR reaction solution on ice. The system is as shown in Table 3. Perform the reaction in a PCR instrument: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 60°C for 30 s.

[0082] Table 3 PCR reaction system

[0083] (3) Agarose gel electrophoresis: Accurately weigh 1.6g of agarose powder and place it in a clean conical flask, add 80mL of 0.5×TBE electrophoresis working solution, shake well and place in a microwave oven to heat until boiling. After boiling, take it out and shake well. Repeat three times to observe whether the agarose powder is fully dissolved. After dissolving, cool it to room temperature. When the temperature drops to about 50℃, aspirate 8μL of fluorescent dye EB and add it to the solution. After gently shaking, pour the liquid into the prepared horizontal plate of the electrophoresis tank and solidify it at room temperature for 1h. After the agarose gel solidifies, carefully pull out the comb. Add an appropriate amount of 0.5×TBE electrophoresis working solution to the electrophoresis tank and place the agarose gel in it. The liquid level should not exceed the gel. Aspirate 5μL of DNA sample and 2μL of DNAMaker into the gel loading hole respectively, turn on the power, adjust the voltage to 140v, start electrophoresis, observe the position of bromophenol blue after 15min, and stop electrophoresis when it is about 2cm away from the spotting hole. Gels were scanned using a Bio-Rad imaging system, and images were acquired and analyzed using Image Lab 4.0 software.

[0084] 4.BeyoClick™ EdU-594 cell proliferation assay The EdU-594 cell proliferation detection kit of the present invention comes from Bio-Tech Biotech Co., Ltd. It is used to compare the changes in the labeled DNA replication activity in the transformed cells and HFLS cells, further indicating that the proliferation activity of PM-HFLS cells is stronger than that of HFLS cells.

[0085] (1) Select cells in the logarithmic growth phase with a cell density of 80%, inoculate 50,000 cells per dish into a 3.5 cm laser confocal culture dish, and culture it in a conditioned incubator at 37°C and 5% CO2 for 24 h.

[0086] (2) Preparation: pre-cooled PBS, 4% paraformaldehyde fixative, washing solution (PBS containing 3% BSA), permeabilization solution (PBS containing 0.3% TritonX-100), and deionized water.

[0087] (3) EdU labeling: discard the old culture medium and dilute EdU (10 mM) with cell culture medium at a ratio of 1:1000 according to the instructions to obtain 1× EdU working solution (10 μM). Add 100 μL of 10 μM EdU solution into the groove of the glass dish for DNA labeling and continue to incubate the cells for 2.5 h.

[0088] (4) Cell fixation: After EdU labeling, discard the culture medium, add 1 mL PBS to wash three times, add fixative (4% paraformaldehyde) to fix at room temperature for 15 min, remove the fixative, and wash the cells three times with 1 mL washing solution (PBS containing 3% BSA) per well, each time for 3 to 5 min. Remove the washing solution, add 1 mL permeabilization solution (PBS containing 0.3% TritonX-100) to each well, and incubate at room temperature for 10 to 15 min. Remove the permeabilization solution, and wash the cells 1 to 2 times with 1 mL washing solution per well, each time for 3 to 5 min.

[0089] (5) Click reaction: Add 0.5 mL of Click reaction solution to the groove at the bottom of the glass dish, gently shake the dish to ensure that the reaction mixture can evenly cover the sample, incubate at room temperature in the dark for 30 min, remove the Click reaction solution, and wash three times with washing solution, each time for 3 to 5 min.

[0090] (6) Nuclear staining: Add 100 μL of 1× Hoechst 33342 working solution to the bottom of the glass dish, incubate at room temperature in the dark for 10 min, remove the reaction solution, wash three times with washing solution, each time for 3-5 min, and seal the slide with glycerol.

[0091] (7) Use confocal fluorescence microscopy to observe the newly synthesized DNA in cells.

[0092] 2. Experimental Results 1. Treat HFLS cells with PM2.5 at different concentrations (0, 10, 20, 40, 80, 160, 320 μg / mL) for 48 h, and detect cell viability using CCK-8 assay. The results are as follows Figure 2 As shown in A, when the concentration of PM2.5 is 40μg / mL and above, the cell viability gradually decreases with the action concentration, some cells die, and when the concentration reaches 320μg / mL, the cells are basically dead; in addition, when the cells are treated with 20μg / mL PM2.5 for 0, 12, 24, and 48h, there is no obvious change in the cell proliferation activity.

[0093] 2. HFLS cells were treated with PM2.5 at different concentrations (0, 10, 20, 40, 80 μg / mL) for 24 h, and the levels of inflammation-related molecules were detected by PCR. The results are as follows Figure 2 As shown in B, the relative expression levels of IL-6, IL-1β, MMP3, and CCL2 in HFLS cells treated with 20, 40, and 80 μg / mL PM2.5 were higher than those in the control group; Figure 2C in the figure shows that when the concentration of PM2.5 is 20μg / mL or above, the level of IL-6 secreted by HFLS cells for 24 hours after treatment with PM2.5 significantly increases, indicating that PM2.5 at a concentration of 20μg / mL can increase the degree of inflammation in HFLS cells without affecting the viability of the cells.

[0094] HFLS cells were treated with 3.20 μg / mL PM2.5 until the 10th generation, and the replication activity of cell DNA was detected by EdU experiment. Figure 3 As shown, compared with the HFLS cell group, the EdU-labeled DNA replication activity in the PM-HFLS cell group (i.e., the 10th generation cells treated with 20μg / mL PM2.5, each generation of cells was exposed for 48h) and the RA-HFLS cell group was significantly increased, indicating that the proliferation activity of PM-HFLS cells was higher than that of HFLS cells. This shows that after the HFLS cells were treated with 20μg / mL PM2.5 for the 10th generation, the inflammatory degree and cell viability of the HFLS cells were affected.

[0095] Example 3 Analysis of the expression and secretion of inflammatory factors and RA markers 1. Experimental Methods 1.ELISA experiment The human IL-6 ELISA detection kit of the present invention comes from Jiangsu Enzyme Immunity Industrial Co., Ltd.; the human IL-32 ELISA detection kit comes from Excellent Biotechnology Co., Ltd.; the mouse IL-6 ELISA detection kit comes from Excellent Biotechnology Co., Ltd.; the mouse IL-32 ELISA detection kit comes from Jiangsu Enzyme Immunity Industrial Co., Ltd., and is used to compare the expression of the above-mentioned inflammatory factors and markers in HFLS cells and transformed cells.

[0096] (1) Sample collection: Serum samples should be collected in test tubes that do not contain pyrogens or endotoxins. Avoid any cell stimulation during the operation. After collecting the blood, centrifuge at 3000 rpm for 10 minutes to quickly and carefully separate the serum and red blood cells. After the cell supernatant is collected, centrifuge at 3000 rpm for 10 minutes to remove particles and polymers. After the sample is collected, proceed to the next step of testing. If the test is not carried out in time, divide it into a single-use amount and freeze it in a -20℃ refrigerator to avoid repeated freezing and thawing. Thaw it at room temperature before use and ensure that the sample is evenly and fully thawed.

[0097] (2) Sample detection: (Taking the mouse IL-32 ELISA detection kit as an example, refer to the kit manual for specific steps) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, seal the remaining strips with a ziplock bag and return them to the 4°C refrigerator for storage, add 50 μL of different concentrations of standard wells to each standard well; first add 10 μL of the sample to be tested to the sample well, and then add 40 μL of sample diluent; do not add to the blank well, and set up two replicate wells; except for the blank well, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and incubate in a constant temperature oven for 60 minutes.

[0098] (3) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times; add 50 μL of substrate A and B to each well, incubate at 37°C in the dark for 15 min; add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0099] (4) Interpretation of results: In an Excel worksheet, use the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis to draw a linear regression curve for the standard, and calculate the concentration value of each sample according to the curve equation.

[0100] 2. Experimental Results HFLS cells were treated with 1.20 μg / mL PM2.5 for 48 h to the 5th and 10th generations, and the mRNA levels were detected by conventional PCR experiments. The results are as follows Figure 4 As shown, the mRNA levels of IL-6 and IL-32 in the PM-5th and PM-10th transformed cell groups were significantly higher than those in the HFLS cell group, and there was no significant difference in the mRNA level of IL-32 in the PM-10th cell group compared with the RA-HFLS cell group.

[0101] HFLS cells were treated with 20 μg / mL PM2.5 for 48 h to the 5th and 10th generations, and the secretion levels of inflammatory factors were detected by ELISA. The results are as follows: Figure 5 As shown, compared with HFLS cells, proinflammatory cytokine IL-6 and RA-specific inflammatory factor IL-32 were highly secreted in PM2.5-transformed cells (PM-5th and PM-10th), and the secretion levels of inflammatory factors IL-6 and IL-32 in the 10th generation PM2.5-transformed cells (PM-10th) were not significantly different from those in RA-HFLS cells. This result shows that the secretion levels of inflammatory factors IL-6 and IL-32 in PM-HFLS group cells were significantly increased, suggesting that PM2.5-transformed cells have the characteristics of high secretion of inflammatory factors similar to rheumatoid arthritis cells.

[0102] Example 4 Analysis of the effect of PM2.5 on cell invasion and migration 1. Experimental Methods 1. Transwell migration and invasion assay Used to evaluate the changes in invasion and migration ability of transformed cells after 10 generations of treatment with 20μg / mL PM2.5 for 48h.

[0103] (1) Take cells in the logarithmic growth phase with a cell density of 90%, digest them with 0.25% trypsin, and centrifuge at 1300 rpm for 3 min. Carefully discard the supernatant, resuspend the cells in fresh culture medium and inoculate them in a 6 cm culture dish, and culture them for 24 h until they adhere to the wall; pour out the old culture medium, wash the cells three times with PBS, and add DMEN basal culture medium for starvation culture for 24 h.

[0104] (2) Plating: Wet the Transwell chamber with DMEM basal medium and discard it. Dilute the matrix gel with DMEM basal medium at a ratio of 1:9. Pipette 50 μL of matrix gel and evenly add it to the chamber. Place it in the incubator for 30 min to solidify (this step is ignored in the Transwell migration experiment). Digest the cells with 0.25% trypsin for 2-3 min, terminate the digestion with basal medium, centrifuge at 1300 rpm for 2 min, carefully discard the medium and wash twice with PBS. Add basal medium to resuspend and count the cells. 4 The cells were inoculated in the upper chamber, and 600 μL of fresh DMEM medium was added to the lower chamber, and the cells were cultured in a 37°C incubator for 48 h.

[0105] (3) Remove the culture plate, discard the culture medium in the chamber, gently wipe the inside of the chamber with a cotton swab, and gently rinse the outside of the chamber with PBS, avoiding contact with the bottom of the chamber.

[0106] (4) Place the chamber in 600 μL of 4% paraformaldehyde and fix it at room temperature for 30 min. Remove the chamber and gently wipe the remaining cells in the chamber with a cotton swab. Place the chamber in 0.1% crystal violet staining solution and stain it at room temperature for 15 min. Wash the chamber twice in deionized water to remove the residual crystal violet staining solution. Air dry the chamber at room temperature or dry it in an oven, then take it out and take pictures under an inverted microscope to count the number of migrated or invaded cells.

[0107] 2. Experimental Results Transwell migration and invasion assay results Figure 6 As shown, Figure 6 The A in the figure indicates that the migration ability of PM-HFLS cells is significantly higher than that of HFLS cells. The Transwell invasion assay also yielded consistent results. Figure 6As shown in B, the invasion ability of PM-HFLS cells was significantly increased compared with HFLS cells.

[0108] Example 5 Analysis of the ability of cells to degrade and destroy cartilage in SCID mice 1. Experimental Methods SCID mouse chimera animal experiment: In the present invention, 15 female severely immunodeficient SCID mice (6 weeks old) were purchased from Guangdong Medical Experimental Animal Center, SPF grade, and the quality inspection certificate number was No. 44007200121092.

[0109] This experiment involves PM-HFLS cells: fibroblast-like synoviocytes that were cultured for 10 generations and treated with PM2.5 at a concentration of 20 μg / mL for 48 hours.

[0110] (1) Animal grouping: 15 female severely immunodeficient SCID mice (6 weeks old) were ordered from the Guangdong Medical Experimental Animal Center. The weight of the SCID mice was weighed and recorded. According to the random number table method, the 15 mice were divided into 3 groups, namely the negative control group (normal human cartilage + HFLS cells), the PM2.5 treatment group (normal human cartilage + PM2.5 transformed cells PM-HFLS cells), and the positive control group (normal human cartilage + RA-HFLS cells), with 5 mice in each group.

[0111] (2) Preparation of materials: Prepare small pieces of absorbable hemostatic gelatin sponge trimmed into 0.8 cm*0.8 cm, absorbable surgical sutures, 1 mL syringe, tribromoethanol, iodine tincture, 75% alcohol, sterile cotton balls, sterile drapes, surgical instruments (ophthalmic curved forceps, surgical scissors, hemostatic forceps, scalpel), etc.

[0112] (3) Preparation of cartilage tissue: Trim the cartilage into small pieces of 0.5 cm*0.5 cm in size and 1~3 mm in thickness, store them in EMEM complete culture medium at low temperature for future use, and embed them in sterile gelatin sponge before implantation.

[0113] (4) Cell preparation: HFLS cells, PM-HFLS cells, and RA-HFLS cells were digested with 0.25% trypsin, centrifuged at 1300 rpm for 3 min, the supernatant was discarded, washed twice with PBS, and centrifuged again. The cell supernatant was removed, and an appropriate amount of DMEM basal medium was added to resuspend the cells. The cells were counted, and 7.5*10 cells were required for each mouse. 5 cells, prepare 6*10 cells in three groups respectively 6 cells and place on ice. Adjust the cell concentration to 1.5*10 4 / μL, and each mouse was injected with 50μL of cell suspension.

[0114] (5) Animal anesthesia: Anesthetize the mouse by intraperitoneal injection. Grasp the mouse with the thumb and index finger, with the head facing down to move the organs upward. Disinfect the injection site with a 75% alcohol cotton ball. The injection site is 1.5 cm on both sides of the intersection of the line connecting the roots of the mouse's hind limbs and the midline of the abdomen. Slowly insert the needle at about 45 degrees, with a depth of less than 1 cm. The feeling of needle insertion is the disappearance of local skin depression and a sense of emptiness. When there is no blood or liquid returning to the needle plug, tribromoethanol can be injected. The final concentration of tribromoethanol is 20 mg / mL, and SCID mice are injected at 0.1 mL / 10 g body weight.

[0115] (6) Subcutaneous embedding: Remove the hair on the right side of the midline of the back of the SCID mouse, disinfect with iodine, and then deiodinate with 75% alcohol. Place the mouse on a sterile drape. Use ophthalmic curved forceps to gently lift the skin about 1 cm to the right of the midline of the back of the mouse, and use ophthalmic scissors to make a longitudinal incision about 1 to 1.5 cm long. Bluntly separate the subcutaneous tissue, gently lift the incision skin on one side, wrap the trimmed cartilage piece with a trimmed sterile gelatin sponge, fully mix the cell suspension, inject 50 μL of the cell suspension into the sponge, and then implant the cell-cartilage-sponge complex subcutaneously along the incision and lay it flat. Suture the incision skin and disinfect it. During the operation, closely observe the mouse's breathing, heartbeat, etc.

[0116] (7) Postoperative observation and treatment: Disinfect the skin incision daily within one week after surgery, observe the wound condition, eating and activity of the mice, whether the limbs are swollen, whether the weight changes, etc., and weigh the mice. If the wound recovers well after one week, observe and weigh the mice every 3 days for a total of 60 days.

[0117] (8) Animal blood collection: After 60 days, the experiment was terminated. After the mice were anesthetized, blood was collected from the eyeballs. The thumb and index finger of the left hand grasped the ears and skin behind the neck of the mouse, the little finger fixed the tail, the middle finger lightly pressed the heart area of ​​the sternum, and the ring finger pressed the abdomen. The thumb was twisted and the skin of the eye on the blood collection side was lightly pressed to make the eyeball congested and protruding. The whiskers of the mice were trimmed with surgical scissors to prevent blood from stagnating in the whiskers and causing hemolysis. The eyeballs were clamped with elbow forceps for blood collection. The whole blood was allowed to stand for 2 hours, centrifuged at 2000 rpm for 10 minutes, and the upper serum was taken and stored at -80°C. IL-6 and IL-32 were subsequently detected by ELISA.

[0118] (9) Collection of implanted cartilage tissue in animals: After blood collection, mice were killed by cervical dislocation. The mice were placed on a bed towel, and the hair near the transplanted cartilage on the back was manually trimmed. The skin above the graft was gently lifted to peel off the cartilage. The skin and tissue near the cartilage were cut off together and fixed in 4% paraformaldehyde for 24 hours. Paraffin sections were then made for HE staining, Masson's trichrome staining, and Alcian blue staining experiments.

[0119] 1. Preparation of paraffin sections: (1) Decalcification of cartilage tissue: After the fixed cartilage is washed with running water for 30 minutes, it is placed in a chronic decalcification solution (EDTA), marked on the tube, and placed in a 37°C constant temperature box. The decalcification solution is changed every other day. The cartilage is decalcified for 3 days, and it is considered good when there is no obstruction when the needle is inserted. The decalcification solution is washed with running water for 30 minutes, and the cartilage tissue blocks are cut into 0.5*0.5cm in size.

[0120] (2) Embedding: The cartilage tissue blocks were dehydrated in a gradient manner (60% alcohol for 1 h, 70% alcohol for 1 h, 80% alcohol for 1 h, 90% alcohol for 1 h, 95% alcohol for 2 h, 100% alcohol for 2 h, 100% alcohol for 2 h), immersed in xylene (xylene for 30 min, xylene for 30 min, xylene for 30 min), immersed in wax (paraffin for 40 min, paraffin for 40 min, paraffin for 40 min), and embedded to make small paraffin blocks.

[0121] (3) Tissue sectioning: Slice the embedded paraffin blocks into 4 μm thick sections, dry them under ventilation, and then stain them after dewaxing.

[0122] 2. HE staining: (1) Place the slices in an oven and bake for 2 hours. Dewax the slices with xylene and wash them with various levels of ethanol and water: xylene (I) 5 minutes → xylene (II) 5 minutes → 100% ethanol 2 minutes → 95% ethanol 1 minute → 80% ethanol 1 minute → 75% ethanol 1 minute → distilled water wash for 2 minutes.

[0123] (2) Hematoxylin staining for 3 min. Differentiation with hydrochloric acid ethanol (1:100) for 10 s (lift and insert up and down 3 to 5 times).

[0124] (3) Rinse with tap water for 15 minutes.

[0125] (4) Eosin staining for 10 minutes.

[0126] (5) Conventional dehydration, transparency, and sealing: 95% ethanol for 30 seconds → 100% ethanol for 30 seconds → neutral resin sealing.

[0127] 3.Masson trichrome staining: A classic method that can be used to identify collagen fibers and muscle fibers. Through staining, collagen fibers appear blue, while muscle fibers appear red. If the cartilage tissue is destroyed, the number of collagen fibers in the tissue decreases, and the degree of blue staining of the tissue decreases.

[0128] (1) After conventional dewaxing to distilled water, the sections were immersed in mordant solution for 8-12 hours at room temperature, and then rinsed with distilled water three times, each time for 3 minutes.

[0129] (2) Dye with lapis lazuli blue solution for 2 minutes and then rinse with water twice, 10 seconds each time.

[0130] (3) Stain with Mayer's hematoxylin solution for 2 minutes, wash twice with distilled water for 10 seconds each time, differentiate with acidic differentiation solution for 2-3 seconds, wash with water to terminate differentiation, and rinse with distilled water for 10 minutes.

[0131] (4) Dye with Ponceau fuchsin solution for 10 min and wash twice with distilled water, each time for 10-15 s.

[0132] (5) Treat with phosphomolybdic acid solution for about 10 minutes, pour off the supernatant, and directly add aniline blue staining solution for 5 minutes without washing the slices with water. After washing away the aniline blue solution with weak acid solution, continue to add weak acid solution to cover the slices for 2 minutes.

[0133] (6) Dehydrate with 95% ethanol for 30 seconds, dehydrate twice with anhydrous ethanol for 10 seconds each time, and seal with neutral gum.

[0134] 4. Alcian blue staining: Alcian blue staining is a special staining of cartilage tissue. Alcian blue can form a complex by combining with acidic glycosamine secreted by chondrocytes. The depth of Alcian blue staining can be used to judge whether the cartilage tissue is damaged. If the staining color becomes lighter, it means that the acidic glycosamine is reduced, the chondrocytes are damaged, and the cartilage tissue is invaded. Alcian blue staining was performed on the implanted cartilage of the three groups of mice.

[0135] (1) Dewax the sections and hydrate them.

[0136] (2) Soak the slices in Alcian acidified solution for 3 minutes.

[0137] (3) Immerse the sections in Alcian staining solution for 30 minutes and then rinse in running water for 1 minute.

[0138] (4) Immerse the sections in Nuclear Fast Red staining solution for 5 minutes and rinse with running water for 1 minute.

[0139] (5) Dehydrate with gradient ethanol, make transparent with xylene, and seal with neutral gum.

[0140] 2. Experimental Results SCID mouse chimera animal experiment flow chart Figure 7 As shown in A. The weight changes of the three groups of mice (HFLS cell group, PM-HFLS group, and RA-HFLS cell group) after cartilage implantation are shown in Figure 7 As shown in B. The cartilage attachment of the three groups of mice 60 days after subcutaneous implantation Figure 7 As shown in C. The levels of inflammatory factors were detected by ELISA experiment. The expression levels of inflammatory factors IL-6 and IL-32 in the serum of the three groups of mice 60 days after cartilage implantation are shown in Figure 7As shown in D, the expression levels of inflammatory factors IL-6 and IL-32 in PM-HFLS group mice were significantly increased, and there was no significant difference compared with RA-HFLS group mice, indicating that PM-HFLS cells promoted the secretion of inflammatory factors in mice.

[0141] 1. HE experimental results are as follows Figure 8 As shown, compared with the HFLS negative control group, the cartilage tissue in the PM-HFLS group and the RA-HFLS group mice was invaded and the matrix degradation was more serious, indicating that PM-HFLS cells and RA-HFLS cells have the ability to invade and degrade cartilage.

[0142] 2. Alcian blue staining test results are as follows Fig. 9 As shown, compared with the HFLS group mice, the Alcian blue staining color of the implanted cartilage tissue in the PM-HFLS group mice and the RA-HFLS group mice became significantly lighter, indicating that the integrity of the cartilage tissue in these two groups was severely damaged, further indicating that PM-HFLS cells and RA-HFLS cells can invade and degrade cartilage tissue.

[0143] 3. Masson trichrome staining test results are as follows Fig.10 As shown, the implanted cartilage of the three groups of mice was stained with Masson's trichrome, and it was found that the collagen fibers of the implanted cartilage of the PM-HFLS group and the RA-HFLS group were significantly destroyed, indicating that compared with the HFLS group, the integrity of the cartilage tissue of these two groups of mice was severely damaged, and PM-HFLS cells can destroy the collagen fibers in the cartilage tissue.

[0144] Animal experiments have shown that after being treated with 20μg / mL PM2.5 for 48h, the cells after 10 generations of subculture had basically no significant difference from the RA-HFLS group.

[0145] In summary, the present invention used 20 μg / mL concentration of PM2.5 to treat each generation of HFLS cells, and each exposure time was 48 hours. It was finally determined that the transformed cells passed down to the 10th generation reached the RA-HFLS cell level, and successfully established a PM2.5-induced rheumatoid arthritis cell model.

[0146] The construction method of the present invention has been verified many times and has good repeatability, and can stably produce PM2.5-induced rheumatoid arthritis fibroblast-like synoviocytes.

Claims

1. A method for constructing a rheumatoid arthritis cell model induced by particle 2.5, characterized in that: The construction method comprises treating normal fibroblast-like synoviocytes with PM2.5 at a concentration of 18 to 22 μg / mL for 45 to 50 hours, and subculturing the cells to the 8th to 12th generation. When the treated cells exhibit obvious polygonal and rheumatoid arthritis cell morphology, the expression of inflammatory factors in the cells is significantly increased, and the cells have the ability to invade and degrade cartilage, the construction is completed, and a PM2.5-induced rheumatoid arthritis cell model is obtained.

2. The construction method according to claim 1, characterized in that: The inflammatory factors are pro-inflammatory cytokines and rheumatoid arthritis specific inflammatory factors.

3. The construction method according to claim 1, characterized in that: The concentration of the particles 2.5 treated was 20 μg / mL.

4. The construction method according to claim 1, characterized in that: The treatment time of the particles 2.5 is 48 hours.

5. The construction method according to claim 1, characterized in that: The cells were cultured in complete DMEM medium containing nonessential amino acids and fetal bovine serum.

6. The construction method according to claim 2, characterized in that: The pro-inflammatory cytokine is IL-6.

7. The construction method according to claim 2, characterized in that: The rheumatoid arthritis specific inflammatory factor is IL-32.

8. The construction method according to claim 1, characterized in that: The normal fibroblast-like synoviocytes are human-derived normal fibroblast-like synoviocytes.

9. Use of the rheumatoid arthritis cell model obtained by the construction method described in any one of claims 1 to 8 in exploring the mechanism of rheumatoid arthritis induced by particulate matter 2.5 exposure.

10. Use of the rheumatoid arthritis cell model obtained by the construction method of any one of claims 1 to 8 in screening and preparing therapeutic drugs for rheumatoid arthritis induced by particulate matter 2.5 exposure.

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