Medicine for treating osteoarthritis

Abstract

The invention discloses a medicine for treating osteoarthritis, and belongs to the field of medicine, the medicine comprises a reagent capable of stimulating chondrocyte development, and the medicine L-arginine can promote expression of chondrocyte anabolic factors and reduce expression of cartilage degradation markers, so that chondrocytes are protected to a certain extent; in addition, the medicine provided by the invention has an excellent cartilage repair function, can inhibit the progress of degenerative osteoarthritis, has good biocompatibility, and has no cytotoxicity to cartilage cells.

Description

Technical Field

[0001] The invention belongs to the field of medicine and specifically relates to a medicine for treating osteoarthritis. Background Art

[0002] In-depth exploration of the molecular mechanism of OA is of great clinical significance for finding accurate and efficient prevention and treatment measures and therapeutic targets, and for finding drugs to improve OA. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention provides a medicine for treating osteoarthritis.

[0004] The first aspect of the present invention relates to an in vitro cell experiment method, comprising the following steps:

[0005] (1) Male rats were selected, killed by cervical dislocation, and then immersed for disinfection; the tibia and femur were separated, the muscle tissue was removed and washed, the distal end of the femur of the rat was selected, the cartilage tissue was removed from the tibiofemoral joint surface, and the tissue blocks were transferred to a culture bottle, rinsed, and the tissue blocks were allowed to stand, the upper liquid and suspended tissue were discarded, trypsin was added, digested, the supernatant was discarded, collagenase was added, digested, DMEM / F-12 medium was added to terminate the digestion, filtered, centrifuged and the supernatant was discarded, the cells were resuspended in cartilage culture medium and inoculated into a tissue culture bottle, incubated, and proliferated;

[0006] (2) The chondrocytes are separated and treated with EDTA and then resuspended, L-arginine is weighed to prepare a cell culture solution, and the mixed solution with the chondrocytes is inoculated into a cell culture incubator for culture, CCK-8 solution and culture solution are added and culture is continued, and the OD value of the solution is measured;

[0007] (3) Chondrocytes were seeded on a well plate, treated with interleukin to induce chondrocyte degeneration, cells were cultured with L-arginine medium, the treated cells were used as a positive control group, and the untreated cells were used as a negative control group. The cell dye solution was diluted in serum-free culture medium, the cell mixture in each well was removed, the diluent was added to each well to cover the cells, incubated, and then the cells were washed with serum-free cell culture medium, and the fluorescence images were taken;

[0008] (4) Evaluate the repair effect of L-arginine on chondrocytes.

[0009] Optionally, the method for evaluating the repair effect of L-arginine on chondrocytes comprises: one or more of real-time quantitative polymerase chain reaction analysis, immunoglobulin analysis, cell immunofluorescence detection and RNA sequencing analysis.

[0010] Optionally, the interleukin comprises 1β (IL-1β).

[0011] Optionally, the cell stain includes dichlorodihydrofluorescein-acetoacetate.

[0012] Optionally, the DMEM / F-12 culture medium includes FPS, penicillin, streptomycin and amphotericin B.

[0013] Optionally, the dilution ratio is 1:1000.

[0014] Optionally, the incubation temperature in step (1) and step (3) is 37°C.

[0015] Optionally, the immersion disinfection time is 15 minutes.

[0016] The second aspect of the present invention relates to the use of the above-mentioned L-arginine in the preparation of a drug for repairing chondrocytes.

[0017] The beneficial effects of the present invention include:

[0018] The drug L-arginine of the present invention can promote the expression of chondrocyte anabolic factors and reduce the expression of cartilage degradation markers, thereby protecting chondrocytes to a certain extent; in addition, the drug of the present invention has excellent cartilage repair function, can inhibit the progression of degenerative osteoarthritis in a rat model, has good biocompatibility, and has no cytotoxicity to chondrocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 , L-arginine cytotoxicity test results;

[0021] Figure 2 , L-arginine maintains the metabolic balance of chondrocytes in vitro;

[0022] Figure 3 , L-arginine in vivo treatment of osteoarthritis effect diagram;

[0023] Figure 4 , the protein blot detection results of this application are shown in FIG. Figure 1 When 0, 5, 10, 15, and 20 ml of L-arginine were added to chondrocytes, the expression of aggrecan was significantly increased, and aggrecan is a special immune marker of chondrocytes. This result also shows that L-arginine can promote the formation of human chondrocytes.

[0024] Figure 5 , Schematic diagram of the protein blot detection structure of the present application Figure 2 , adding 0 (blank control group), 0, 5, 10, 20 ml of L-arginine to chondrocytes, it can be seen that the expression of MMP-13, COX-2, and inos is significantly reduced, among which MMP-13, COX-2, and inos are immune markers of osteoarthritis, which shows that L-arginine can effectively reduce the expression of osteoarthritis. . DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] (1) Isolation and culture of rat chondrocytes

[0028] One-month-old male rats were selected, killed by cervical dislocation, and then soaked in 75% ethanol for 15 minutes for disinfection. The tibia and femur were separated under sterile conditions, the muscle tissue was removed, and washed twice with phosphate buffered saline (PBS). The distal end of the rat femur was selected, and the cartilage tissue was removed from the tibiofemoral joint surface and continued to be cut into 0.3-0.5mm tissue blocks, transferred to culture bottles, and rinsed twice with PBS solution containing 2% double antibody. After the tissue block was allowed to stand for 5 minutes, the upper liquid and suspended tissue were discarded, and 0.25% trypsin 10 times the volume of the cartilage was added, and digested at 37°C for 2 hours. The supernatant was discarded again, and 0.2% type II collagenase was added, and digested overnight at 37°C. After a single cell was observed to be free under an inverted microscope, DMEM / F-12 medium (containing 10% FPS, 1% penicillin / streptomycin and amphotericin B) was added to terminate digestion. After blowing the cell mass, the filtrate was filtered with a 70μm cell filter and collected. After centrifugation at 1500r / min for 5min, the supernatant was discarded. The cells were resuspended in complete cartilage medium and inoculated into tissue culture bottles, incubated in a 37°C, 5% CO2 cell culture incubator, and the medium was changed every 3 days. After proliferation to 80% confluence, the cells were separated with 0.25% EDTA and set aside.

[0029] (2) L-arginine cytotoxicity assay

[0030] The cytotoxicity of L-arginine was analyzed using Cell Counting Kit-8 (CCK-8): the previously cultured chondrocytes were separated by EDTA and resuspended in new culture medium. L-arginine was weighed and prepared into 5 / 10 / 20mM, mixed with chondrocytes in cell culture medium, inoculated into 24-well plates (200μL / well, 2.5×104cells) and cultured in a 37°C, 5% CO2 cell culture incubator. After adding CCK-8 solution (50μL) and culture medium (500μL) to each well at 1d, 3d, 5d, and 7d, the 24-well plate was cultured in a dark environment at 37°C for h. The operation was repeated 3 times for each sample group. Finally, the mixed solution was transferred to a 96-well plate in the dark, and the OD value of the solution was measured at a wavelength of 450nm using a microplate reader.

[0031] (3) Evaluation of the anti-ROS effect of L-arginine

[0032] Reactive Oxygen Species Assay Kit (ROS Assay Kit) was used to detect the scavenging ability of L-arginine on intracellular reactive oxygen species (ROS): 1×10 4 / mL chondrocytes were seeded on a 24-well plate and treated with 10 ng / ml interleukin 1β (IL-1β) for 12 h to induce chondrocyte degeneration. The cells were cultured in a medium containing 5 / 10 / 20 mM L-arginine for 12 h. Cells treated with IL-1β served as the positive control group, and cells without treatment served as the negative control group. Dichlorodihydrofluorescein-acetoacetate (DCFH-DA) cell stain was diluted in serum-free culture medium (ratio 1:1000) to a concentration of 10 μmol / L. The cell mixture was removed from each well, and about 500 μL of DCFH-DA dilution was added to each well to fully cover the cells, and then incubated in a cell culture incubator at 37°C and 5% CO2 for 20 min. The cells were then washed three times with serum-free cell culture medium to fully wash away the DCFH-DA dilution solution that did not enter the cells to prevent interference with the photography under the fluorescence microscope due to the bright field of view. Finally, the fluorescence image was observed and photographed under a fluorescence microscope.

[0033] (4) Evaluation of the repair effect of L-arginine microspheres on chondrocytes

[0034] ① Real-time quantitative polymerase chain reaction (qRT-PCR) analysis

[0035] The cells in each group were treated as above. The total RNA of chondrocytes was extracted using TRIZOL reagent, and the purity and concentration of the RNA solution were obtained by measuring the absorbance at 260nm and 280nm. The first-strand cDNA synthesis kit was used to react with 1μg RNA solution to synthesize the corresponding cDNA. The qRT-PCR-mediated cDNA amplification process was then completed using the ABI 7500 sequencing detection system and the Perfect Real time dye real-time fluorescence quantitative kit. The relative mRNA expression levels of COL2, Aggrecan, MMP13, and ADAMTS 5 were calculated using various specific primers.

[0036] ② Western blot analysis (WB)

[0037] The cells in each group were treated as above, lysed on ice with RIPA lysis buffer, centrifuged and the supernatant was collected. After BCA quantification, the protein was boiled and denatured and stored at -80°C. Using SDS-PAGE gel electrophoresis technology, after electrophoresis, transfer, blocking, primary and secondary antibody incubation, etc., the expression of COL2, Aggrecan, MMP13 and ADAMTS 5 proteins in chondrocytes after treatment was observed. Figure 4 , Figure 5 shown.

[0038] ③Cell immunofluorescence detection

[0039] The cells in each group were treated as above. The chondrocytes were fixed with 4% paraformaldehyde for 10 minutes, then permeated with 0.1% triton X-100 for 15 minutes at 25°C, and blocked with 3% bovine serum albumin / phosphate buffered saline (BSA / PBS) for 30 minutes. After washing with PBS, the cells were stained with rat primary antibodies specific for COL2, Aggrecan, MMP13, and ADAMTS 5 and incubated overnight at 4°C. After washing, the secondary antibodies were incubated at room temperature for 1 hour. After sealing, the fluorescence images were observed and captured using a fluorescence microscope.

[0040] ④RNA sequencing analysis

[0041] Each group of cells was treated as above, lysed with Trizol reagent and immediately immersed in liquid nitrogen. RNA was extracted and its purity was measured using a NanoDrop spectrophotometer. The concentration of RNA samples was measured using the Qubit RNA BR Assay Kit, and the integrity of the extracted RNA was evaluated by the RNA screening band system. Library preparation for high-throughput sequencing was completed and sequenced on the NovaSeq 6000 platform using the NovaSeq S4 reagent kit, generating 150 base pairs (Bp) of paired-end sequence data. The initial processing of raw reads in FASTQ format was to trim adapters and filter out low-quality sequences using FAST software to obtain clean reads. These CLEAN sequences were then aligned to the reference genome using HISAT2, and the subsequent gene expression levels were quantified using HTSeq-Count in units of transcript fragments per million mapped reads (FPKM). The reproducibility of different biological replicates was evaluated using principal component analysis (PCA) software (version 3.2.0). Differential expression analysis was performed using the DESeq2 package, with stringent thresholds for identification of significantly differentially expressed genes (Degs) with a Q value less than 0.05 and an absolute fold change greater than 2 or less than 0.5. Hierarchical clustering of DEGs was performed using R software (version 3.2.0) to elucidate the expression patterns between different experimental conditions and samples. Volcano plots constructed using the ggplot2 package in R visually represented the expression differences of up-regulated or down-regulated DEGs. Gene Ontology (GO) terms and pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases were enriched in DEGs using the hypergeometric test in the R environment (version 3.2.0). Then, the significantly enriched terms were clustered using R software.

[0042] The expression data were visualized in various formats, including bar charts, chord charts, and bubble charts. Finally, the gene expression data were further evaluated using Gene Set Enrichment Analysis (GSEA), allowing the identification of relevant biological pathways or processes using stand-alone GSEA software.

[0043] (5) In vitro anti-inflammatory function detection

[0044] ① Culture of rat bone marrow-derived macrophages

[0045] Rat bone marrow-derived macrophages were purchased from Pronosai Biotechnology Co., Ltd. (Cat NO.: CP-R141). The cells were resuspended in complete chondrogenic medium and inoculated into 150 cm2 tissue culture flasks, incubated in a 37°C, 5% CO2 cell culture incubator, and the medium was changed every 3 days. After proliferation to 80% confluence, 1 mL of lidocaine (12 mM) digestion solution was added to the culture flask, and the culture flask was gently rotated until the digestion solution covered the entire bottom of the culture flask, and then incubated at 37°C for 3 minutes (not more than 5 minutes); observed under an inverted microscope, after the cells shrank and became round, 5 ml of complete culture medium was added to dilute the digestion solution; gently blow to mix and disperse the cells, aspirate the cell suspension, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant; resuspend the cells in complete culture medium, count and inoculate in the culture flask, and start the experiment after the cells are completely attached to the wall. The treatment of each group of cells is the same as above, so that the chondrocytes of the same number of cells are inoculated on a 6-well plate and treated with LPS (50 ng / ml) +

[0046] IFN-γ (20 ng / ml) was treated for 12 h to induce macrophage M1 polarization. The cells were cultured in medium containing 5 / 10 / 20 mM L-arginine for 12 h. Cells treated with LPS + IFN-γ served as the positive control group, and cells without treatment served as the negative control group.

[0047] ② Real-time quantitative polymerase chain reaction (qRT-PCR) analysis

[0048] The cells in each group were treated as above. The total RNA of chondrocytes was extracted using TRIZOL reagent, and the purity and concentration of the RNA solution were obtained by measuring the absorbance at 260nm and 280nm. The first-strand cDNA synthesis kit was used to react with 1μg R solution to synthesize the corresponding cDNA. The qRT-PCR-mediated cDNA amplification process was then completed using the ABI 7500 sequencing detection system and the PerfectReal time dye real-time fluorescence quantitative kit. The relative mRNA expression levels of iNOS and CD206 were calculated using various characteristic primers.

[0049] ③ Western blot analysis (WB)

[0050] The cells in each group were treated as above, RIPA lysis buffer was added, lysed on ice, centrifuged and the supernatant was taken, the protein was boiled and denatured after BCA quantification, and stored at -80°C. SDS-PAGE gel electrophoresis technology was used to observe the expression of iNOS and CD206 proteins in chondrocytes after treatment, after electrophoresis, transfer, blocking, primary and secondary antibody incubation and other processes.

[0051] ④Cell immunofluorescence detection

[0052] The cells in each group were treated as above. The chondrocytes were fixed with 4% paraformaldehyde for 10 minutes, then permeated with 0.1% triton X-100 for 15 minutes at 25°C, and blocked with 3% bovine serum albumin / phosphate buffered saline (BSA / PBS) for 30 minutes. After washing with PBS, the cells were stained with anti-iNOS and CD206 specific rat primary antibodies and incubated overnight at 4°C. After washing, the secondary antibodies were incubated at room temperature for 1 hour. After sealing, the fluorescence images were observed and photographed using a fluorescence microscope.

[0053] Comparative Example 2

[0054] The anterior cruciate ligament was removed and the meniscus was surgically modeled, and no L-arginine was given for intervention. Other aspects were the same as in Example 2.

[0055] Example 2

[0056] (1) Experimental animal grouping

[0057] Thirty-six healthy male SD rats aged 2 months were selected and randomly divided into 6 groups, with 6 rats in each group. The specific grouping is as follows:

[0058] Blank control group: The animals were fed normally. Sham operation group: The animals were simply operated without removing the anterior cruciate ligament and meniscus, and were fed normally. Experimental group 1: The anterior cruciate ligament and meniscus were removed for surgical modeling, and 0.25g / kg L-arginine was given orally every day. Experimental group 2: The anterior cruciate ligament and meniscus were removed for surgical modeling, and 0.5g / kg L-arginine was given orally every day. Experimental group 3: The anterior cruciate ligament and meniscus were removed for surgical modeling, and 1g / kg L-arginine was given orally every day. The rats were killed 2 months later and the samples were taken for the next experiment.

[0059] (2) Preparation of OA model in SD rats by ACLT+DMM method

[0060] Preparation of rat OA model: The rat osteoarthritis model was designed by ACLT+DMM surgery. First, the rats were injected with sodium pentobarbital (30 mg / kg body weight) for intraperitoneal anesthesia. After disinfection, the knee joint of the right lower limb was exposed and a medical lateral incision was set up. A skin incision was made from the distal end of the patella to the proximal end of the tibial plateau. Subsequently, the patellar tendon in the knee joint capsule was opened with scissors. The patellar tendon was retracted, and the fat pad was bluntly dissected to expose the medial meniscus ligament. The MMTL was incised with a microscalpel, the medial meniscus was removed, and the anterior cruciate ligament was cut and the incision was sutured. In addition, the sham operation used the same method without removing the medial meniscus and cutting the anterior cruciate ligament. The mice were provided with sufficient food and water and allowed to move without any restrictions.

[0061] (3) Experimental animal sampling and gross observation

[0062] Rats were killed by cervical dislocation, and a median incision was made at the knee joint to expose the knee joint structure. Bone forceps were used to cut off the distal 1 / 3 of the femur and the tibial tubercle to remove the complete knee joint. The patella was then removed, and the remaining soft tissue was removed with sterile ophthalmic scissors and surgical blades to fully expose the femoral trochlea and the internal and external condyle structures for observation and video recording.

[0063] (4) Micro-CT scanning:

[0064] A Micro-CT scanner was used to measure the size of knee osteophytes and structural parameters of knee bones, including total bone volume (TV), trabecular volume (BV), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number (Tb.N). The bone volume fraction (BV / TV) and bone surface / bone volume ratio (BS / BV) were calculated.

[0065] (5) Histological staining and immunohistochemical staining

[0066] The removed knee joint and synovium were fixed in 4% paraformaldehyde for 24 hours. The knee joint was decalcified in 10% EDTA for 4 weeks. After the bone tissue softened, the specimen was washed with distilled water to remove the residual decalcification solution, and then placed in ethanol for dehydration for 4 hours. The femoral part of the specimen was then trimmed into two halves in the coronal position, and the parts with obvious structures of the medial and lateral condyles and trochlear groove were selected for paraffin embedding. The synovial tissue of the joint was also embedded in paraffin to prepare continuous paraffin sections (5μm layer thickness). After dewaxing, the articular cartilage sections were alternately stained with hematoxylin-eosin staining (HE) and Safranin O-Fast Green staining (SF) and then sealed. Other sections were processed for immunohistochemical staining: after the sections were dewaxed and hydrated, they were immersed in H2O2 solution at room temperature for 15 minutes. After being treated with digestive fluid for 10 minutes, they were blocked in 20% normal sheep serum at room temperature for 20 minutes. Rat COL2, Aggrecan, MMP13, and ADAMTS5 antibodies were added to cartilage sections, and iNOS and CD206 antibodies were added to synovial sections as primary antibodies, overnight at 4°C. After rewarming at 37°C for 45 minutes and washing with PBS, biotinylated goat anti-mouse IgG (secondary antibody) was added and allowed to stand for 1 hour. Then, diaminobenzidine (DAB) was used for color development for 10 minutes, and the color development was observed under a microscope, then rinsed with PBS and counterstained with hematoxylin for 2 minutes. Finally, the sections were dehydrated in alcohol, transparentized in xylene, and sealed. The specimens were observed under a microscope and photographed. The modified Mankin's score was used to score the HE-stained sections, and the Osteoarthritis Research Society International (OARSI) score was used to score the SF-stained sections.

[0067] Example 3

[0068] To determine the feasibility of L-arginine for OA treatment, we first tested the cytotoxicity of different concentrations of L-arginine by CCK-8 assay. Chondrocytes were co-cultured with L-arginine at concentrations of 5mM, 10mM, and 20mM for 1, 3, and 5 days, respectively. CCK-8 assay showed that on day 1, there was no significant difference in cell proliferation between the groups. On day 3, however, 10mM and 20mM L-arginine promoted chondrocyte proliferation. By day 5, the promoting effect of 10mM and 20mM L-arginine was further increased ( Figure 1 ). Therefore, 5mM, 10mM, and 20mM L-arginine all have good biocompatibility and are non-cytotoxic to chondrocytes, which is a prerequisite for in vivo experiments and clinical applications.

[0069] Example 4

[0070] In order to study the effect of L-arginine on chondrocytes under inflammatory conditions, we used IL-1β (10 ng / mL) to induce chondrocyte inflammation and then co-cultured them with 5 mM, 10 mM, and 20 mM L-arginine for 48 hours. Figure 2 ) were used to detect the expression of COL 2 and MMP13 in chondrocytes. Compared with the control group, the expression level of COL 2 in chondrocytes was significantly decreased after IL-1β induction (PBS group). Compared with the PBS group, the expression level of COL 2 in chondrocytes in the 20mM group was significantly increased, but the effect of the 5mM and 10mM groups was limited. On the other hand, compared with the control group, the expression level of MMP13 was higher after IL-1β induction (PBS group). After the addition of 10mM and 20mM L-arginine, MMP13 was significantly downregulated. The results showed that L-arginine can promote the expression of chondrocyte anabolic factors (COL 2) and reduce the expression of cartilage degradation markers (MMP13), thereby protecting chondrocytes to a certain extent.

[0071] Comparative Example 5

[0072] The rat OA model was established by ACTL+MMX surgery. The NS group was given normal saline by gavage, and the effect of L-arginine on osteoarthritis in vivo was evaluated by safranin fast green staining (SF) and immunohistochemical staining of cartilage tissue 4 weeks later ( Figure 3 ).

[0073] Example 5

[0074] The rat OA model was established by ACTL+MMX surgery. The rats in the sham operation group only had their skin incised without any other treatment. Four weeks later, the L-arginine group was given by oral gavage (250 mg / kg / d). Four weeks later, the cartilage tissue was stained with safranin fast green (SF) and immunohistochemical staining to evaluate the effect of L-arginine in treating osteoarthritis in vivo ( Figure 3 ). SF staining showed that after OA modeling, the content of glycosaminoglycan (red) in cartilage was significantly reduced, and after the use of L-arginine, the expression of glycosaminoglycan was significantly increased. Immunohistochemistry results showed that after OA modeling, the content of COL 2 in cartilage was significantly reduced, and the expression of MMP13 was significantly increased. After the use of L-arginine, the expression of COL 2 increased significantly, while the expression of MMP13 decreased significantly. This shows that L-arginine has excellent cartilage repair function and can inhibit the progression of OA in rat models.

[0075] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0076] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. An in vitro cell experiment method, characterized in that: The following steps are involved: (1) Male rats were selected, killed by cervical dislocation, and then immersed for disinfection; the tibia and femur were separated, the muscle tissue was removed and washed, the distal end of the femur of the rat was selected, the cartilage tissue was removed from the tibiofemoral joint surface, and the tissue blocks were transferred to a culture bottle, rinsed, and the tissue blocks were allowed to stand, the upper liquid and suspended tissue were discarded, trypsin was added, digested, the supernatant was discarded, collagenase was added, digested, DMEM / F-12 medium was added to terminate the digestion, filtered, centrifuged and the supernatant was discarded, the cells were resuspended in cartilage culture medium and inoculated into a tissue culture bottle, incubated, and proliferated; (2) The chondrocytes are separated and treated with EDTA and then resuspended, L-arginine is weighed to prepare a cell culture solution, and the mixed solution with the chondrocytes is inoculated into a cell culture incubator for culture, CCK-8 solution and culture solution are added and culture is continued, and the OD value of the solution is measured; (3) Chondrocytes were seeded on a well plate, treated with interleukin to induce chondrocyte degeneration, cells were cultured with L-arginine medium, the treated cells were used as a positive control group, and the untreated cells were used as a negative control group. The cell dye solution was diluted in serum-free culture medium, the cell mixture in each well was removed, the diluent was added to each well to cover the cells, incubated, and then the cells were washed with serum-free cell culture medium, and the fluorescence images were taken; (4) Evaluate the repair effect of L-arginine on chondrocytes.

2. The in vitro cell experiment method according to claim 1, characterized in that: The method for evaluating the repairing effect of L-arginine on chondrocytes comprises: one or more of real-time quantitative polymerase chain reaction analysis, immunoglobulin analysis, cell immunofluorescence detection and RNA sequencing analysis.

3. The in vitro cell experiment method according to claim 1, characterized in that: The interleukins include 1β (IL-1β).

4. The in vitro cell experiment method according to claim 1, characterized in that: The cell stain includes dichlorodihydrofluorescein-acetoacetate.

5. The in vitro cell experiment method according to claim 1, characterized in that: The DMEM / F-12 culture medium includes FPS, penicillin, streptomycin and amphotericin B.

6. The in vitro cell experiment method according to claim 1, characterized in that: The dilution ratio is 1:1000.

7. The in vitro cell experiment method according to claim 1, characterized in that: The incubation temperature in step (1) and step (3) is 37°C.

8. The in vitro cell experiment method according to claim 1, characterized in that: The immersion disinfection time is 15 minutes.

9. Use of the L-arginine according to claim 1 in preparing a drug for repairing chondrocytes.

Images