A method for constructing a mouse model simulating senile osteoarthritis and application thereof

Abstract

The application belongs to the technical field of biomedicine, and discloses a construction method of a mouse model simulating senile osteoarthritis and application thereof, wherein mouse bone marrow cells are induced to differentiate into bone marrow-derived macrophages in vitro, the macrophages are treated with an induction medium containing 100nM doxorubicin for 2 days, and then cultured in a conventional medium for 5 days to obtain senescent macrophages with a stable senescence phenotype; 1x10 6 The senescent macrophages are injected into the knee joint cavity of a mouse once a week for a total of 4 times to induce chronic low-grade inflammation and characteristic osteoarthritis lesions in the joint. The model constructed by the application has high pathological simulation degree, good repeatability, and a modeling cycle of only 4 weeks, thereby reducing the interference of acute injury factors, and can be widely applied to the research on pathogenesis of senile osteoarthritis, screening of therapeutic targets, and evaluation of the efficacy of candidate drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for constructing a mouse model simulating senile osteoarthritis and its application. Background Technology

[0002] Osteoarthritis is a group of degenerative joint diseases characterized by articular cartilage degeneration, subchondral bone remodeling, osteophyte formation, synovial inflammation, and chronic pain. With the increasing aging of the population, the incidence of age-related osteoarthritis is rising. Its pathological process involves not only chondrocyte degeneration but is also closely related to cellular senescence, chronic low-grade inflammation, and immune homeostasis disorders. Therefore, establishing animal models that can simulate the clinicopathological features of age-related osteoarthritis is crucial for elucidating disease mechanisms and evaluating intervention strategies.

[0003] Currently, animal models of osteoarthritis are mainly divided into: (1) Surgically induced models: including medial meniscus instability models, anterior cruciate ligament transection models, etc. These models induce osteoarthritis-like lesions by destroying the joint stabilizing structure and changing the mechanical environment. They have the advantages of short modeling cycle and good repeatability. However, their pathological starting point is acute trauma, which is significantly different from the slow progression of senile osteoarthritis due to multiple factors. Moreover, the acute inflammatory response caused by the surgical operation itself will interfere with the accuracy of the experimental results. (2) Chemically induced models: including sodium iodoacetate, collagenase induced models, etc. These models directly damage chondrocytes or destroy joint structures through chemical toxicity. The lesions occur quickly and the pain phenotype is obvious. However, the essence is acute toxic injury, which cannot simulate the chronic low-grade inflammatory characteristics of senile osteoarthritis. (3) Mechanical load models: including knee joint axial compression, excessive treadmill exercise models, etc. These models can reflect the role of mechanical factors in osteoarthritis. However, they have high requirements for equipment and experimental parameters, large individual differences, and are difficult to promote. (4) Spontaneous models: including STR / ort mice, naturally aged mice, etc. Although they do not require artificial damage and are closer to the natural process of primary osteoarthritis, they have drawbacks such as long modeling cycle, naturally aged mice need to be fed for more than 18 months, high feeding cost, large individual differences, low experimental efficiency, and some models have strain specificity, which limits their application.

[0004] It is evident that existing models primarily focus on traumatic joint instability, chemically induced cartilage damage, or spontaneous degeneration under specific genetic backgrounds. While these models can induce osteoarthritis-like lesions to some extent, they fail to adequately simulate the pathological processes of immunosenescence, chronic low-grade inflammation, accumulation of senescent macrophages, decline in joint microenvironment homeostasis, and gradual cartilage degeneration in senile osteoarthritis. Therefore, there is a need to develop an animal model construction method that offers relatively controllable cycles, is relatively simple to operate, highlights the effects of aging, and simulates key pathological features of senile osteoarthritis. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for constructing and applying a mouse model simulating senile osteoarthritis. The method involves inducing bone marrow cells to differentiate into bone marrow-derived macrophages in vitro using macrophage colony-stimulating factor (MSF), and then inducing these macrophages to form senescent macrophages with stable cellular senescence phenotypes and senescence-related secretory phenotypes using doxorubicin. These senescent macrophages are then repeatedly reinfused locally into the knee joint cavity of recipient mice once a week for four consecutive weeks. This simulates the unique immunosenescence and chronic low-grade inflammation pathological microenvironment characteristic of senile osteoarthritis in the joint region, thus successfully constructing an animal model whose pathological process closely matches clinical senile osteoarthritis, exhibiting strong reproducibility and a controllable modeling cycle.

[0006] To achieve the above objectives, the present invention provides a method for constructing a mouse model simulating senile osteoarthritis, comprising the following steps: S1. Differentiate and culture bone marrow cells in a differentiation medium to obtain bone marrow-derived macrophages; S2. Bone marrow-derived macrophages were induced and cultured in senescence-inducing medium, and then cultured in conventional medium to obtain senescent macrophages; S3. The induced senescent macrophages were injected into the knee joint cavity of the recipient animal to construct an senile osteoarthritis model.

[0007] Preferably, in S1, the differentiation medium includes macrophage colony-stimulating factor M-CSF at a concentration of 20 ng / mL; the differentiation culture time is 5 days.

[0008] Preferably, in S2, the aging induction medium includes α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, 20 ng / mL M-CSF, and 100 nM doxorubicin; the induction culture time is 2 days; the conventional medium includes α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, and 20 ng / mL M-CSF, and is cultured for 5 days.

[0009] Preferably, in step S3, the specific steps for injecting senescent macrophages into the knee joint cavity of the recipient animal are as follows: injection once every 7 days, with knee joint cavity injections performed on days 0, 7, 14, and 21, for a total of 4 injections. The modeling period is 28 days, and the number of senescent macrophages injected each time is 1×10⁻⁶. 6 ~1.5×10 6 Each injection volume is 5-10 μL.

[0010] Preferably, in S3, the process also includes acclimatization feeding before injecting senescent macrophages into recipient animals. The acclimatization feeding conditions are a 12-hour light / 12-hour dark cycle, a temperature of 25±1℃, a relative humidity of 75±5%, and a feeding period of 7 days.

[0011] Preferably, in S3, the recipient animal is a C57BL / 6 mouse.

[0012] A mouse model of senile osteoarthritis constructed according to the method is also provided.

[0013] The study also provides the application of the simulated senile osteoarthritis mouse model in the study of the pathogenesis of senile osteoarthritis, the screening of therapeutic targets, or the evaluation of the efficacy of candidate drugs.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention uses senescent macrophages obtained in vitro as the starting point for modeling and allows them to directly participate in the regulation of the local microenvironment of the joint through intra-articular reinfusion. This can better simulate the pathological features of senile osteoarthritis, such as immune senescence, chronic low-grade inflammation and the pathogenic effects of senescent immune cells. At the same time, the construction method can reduce the interference of acute strong injury factors on the experimental results and help to clarify the causal role of senescent macrophages in the occurrence and development of osteoarthritis.

[0015] (2) The present invention stabilizes the modeling cycle at 4 weeks, which greatly shortens the time required for modeling and significantly reduces the animal mortality rate, cost consumption and experimental cycle uncertainty caused by long-term feeding. At the same time, the modeling method is relatively simple to operate, which enables the model to be quickly established under ordinary laboratory conditions, meeting the needs of high-throughput drug screening and batch mechanism verification.

[0016] (3) The model constructed in this invention is not only applicable to the study of the pathogenesis of osteoarthritis in the elderly, but can also be used to screen and evaluate candidate drugs and treatment strategies that have anti-aging, anti-inflammatory, macrophage function regulation, lipid metabolism regulation, mitochondrial function improvement or inhibition of aging-related secretory phenotypes. It provides a new animal model tool for the study of the pathogenesis of osteoarthritis in the elderly, the screening of therapeutic targets and the evaluation of drug efficacy. At the same time, macrophages from different sources (such as peritoneal macrophages) can be replaced to further expand the application scope of the model and provide a new technical solution for the study of age-related degenerative diseases.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the culture and senescence induction process of bone marrow-derived macrophages according to the present invention; Figure 2The figure shows the identification results of the differentiation effect of bone marrow-derived macrophages in this invention. In the figure, A is the flow cytometry detection of F4 / 80 expression in bone marrow cells of the uninduced control group and bone marrow-derived macrophages induced by M-CSF. B is the statistical result of the proportion of F4 / 80 positive cells. **** represents P<0.0001.

[0019] Figure 3 The images show the SA-β-gal staining results of bone marrow-derived macrophages in the conventional culture group and after senescence induction. In the images, A is the staining result of the control group and B is the staining result of the experimental group after senescence induction. The scale bar is 50 μm. Figure 4 The figure shows the expression of p16, p21, and p53 mRNA in bone marrow-derived macrophages in the conventional culture group and after senescence induction. In the figure, A represents p16, B represents p21, and C represents p53. Conventional culture represents bone marrow-derived macrophages cultured in the conventional culture group, and Dox senescence induction represents bone marrow-derived macrophages after senescence induction. ** represents P<0.01. Figure 5 The figure shows the mRNA expression of inflammatory factors IL-6, TNF-α, and IL-1β in bone marrow-derived macrophages after conventional culture and senescence induction. In the figure, A represents IL-6, B represents TNF-α, and C represents IL-1β. Conventional culture represents bone marrow-derived macrophages after conventional culture, and Dox senescence induction represents bone marrow-derived macrophages after senescence induction. ** represents P<0.01, and *** represents P<0.005. Figure 6 The figures represent the ATP production levels of bone marrow-derived macrophages in the conventional culture group and after senescence induction, with ** indicating P < 0.01. Figure 7 This is a schematic diagram of the process for constructing an animal model of senile osteoarthritis using senescent macrophages according to the present invention; Figure 8 The images show the bone and joint conditions of the control and model groups in this invention via Micro-CT scans. In the figure, A is a reconstruction of the microstructure of the mouse tibia, and B is a cross-sectional reconstruction of the femoral-tibial knee joint region. Figure 9 The images show the results of safranin and fast green staining of the knee joints of mice in the control and model groups of this invention. Figure A shows the staining results of the knee joints of mice in the control and model groups, with a scale bar of 200 μm. Figure B shows the OARSI score, and **** represents P < 0.0001. Figure 10 The figures show the gait detection results of mice in the control and model groups of this invention. In the figure, A is the gait trajectory of the mice, B is the statistical graph of the relative maximum contact intensity of the mice, and C is the statistical graph of the stride span of the mice. ** represents P<0.01, and *** represents P<0.005. Figure 11 The images show the COL2A1 / II type collagen staining results of the knee joints of mice in the control and model groups of this invention. In the image, A is the COL2A1 / II type collagen staining result of the knee joints of mice in the control group, and B is the COL2A1 / II type collagen staining result of mice in the model group. The scale bar is 200 μm. Figure 12 The images show the results of p16 and F4 / 80 immunofluorescence staining in the subchondral bone region of mice in the control and model groups of this invention. In the figure, A is a representative image of p16 and F4 / 80 immunofluorescence staining, with a scale bar of 50 μm. B is a statistical result of the proportion of p16 and F4 / 80 co-localized positive cells. **** represents P<0.001. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0023] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0024] Example 1 The process of macrophage culture and senescence induction is as follows: Figure 1 As shown, the specific steps include the following: (1) Acquisition and culture of macrophages: The femur and tibia of mice were isolated, and the muscle and connective tissue attached to the bone surface were removed. The bone marrow cavity of the femur and tibia was flushed with a sterile syringe containing α-MEM basal medium, and the bone marrow cell suspension was collected. After centrifugation, the obtained bone marrow cell suspension was treated with erythrocyte lysis buffer to remove erythrocytes. After lysis, the cells were collected by centrifugation again and resuspended in α-MEM basal medium. Subsequently, the bone marrow cells were seeded in differentiation medium containing macrophage colony-stimulating factor (M-CSF) to differentiate into bone marrow-derived macrophages.

[0025] In a preferred embodiment, the mice are 6-8 week old C57BL / 6 mice, and the differentiation medium is α-MEM basal medium containing M-CSF. The α-MEM basal medium includes α-MEM basal medium, fetal bovine serum, and penicillin-streptomycin antibiotics. Preferably, the α-MEM basal medium includes α-MEM basal medium, 10% fetal bovine serum, and 1% penicillin-streptomycin antibiotics; the concentration of M-CSF is 10-50 ng / mL, more preferably 20 ng / mL. The cultured bone marrow-derived macrophages can be identified using F4 / 80 macrophage markers.

[0026] The identification process involved collecting cultured bone marrow-derived macrophages, discarding the culture medium, washing with PBS, and gently digesting or collecting adherent cells by pipetting to prepare a single-cell suspension. After centrifugation, the supernatant was discarded, and the cells were resuspended in a buffer containing protein blocking components to reduce non-specific antibody binding. Subsequently, an appropriate amount of the cell suspension was incubated with fluorescently labeled F4 / 80 antibody. After incubation, the cells were washed with PBS to remove unbound antibodies. The cells were then resuspended in PBS containing 1% BSA, and the proportion of F4 / 80 positive cells was detected by flow cytometry. The identification results are as follows: Figure 2 As shown, the bone marrow-derived macrophages successfully differentiated.

[0027] (2) Obtaining senescent macrophages: Doxorubicin (Dox) was used to induce senescent phenotypes in macrophages. Specifically, the obtained bone marrow-derived macrophages were treated in induction medium (which included α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, 20 ng / mL M-CSF and 100 nM Dox) for 2 days, and then cultured in conventional medium (which included α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics and 20 ng / mL M-CSF) for 5 days to induce senescent phenotypes and enhanced inflammatory expression in macrophages, thus obtaining Dox-induced senescent macrophages.

[0028] Set up a routine culture group: Place the bone marrow-derived macrophages obtained in step (1) in a routine culture medium (the routine culture medium includes α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics and 20 ng / mL M-CSF) and culture for 7 days.

[0029] Phenotypic identification was performed on the conventional culture group and the senescent macrophages induced by senescence.

[0030] β-gal staining of cells: Macrophages treated with in vitro induction were collected, the culture medium was discarded, and the cells were washed with PBS and fixed with cell fixation solution. After fixation, the fixative was removed, and the cells were washed again with PBS. Then, β-gal staining working solution was added for incubation to allow senescence-associated β-galactosidase-positive cells to develop staining. After staining, the staining working solution was removed, and the cells were washed with PBS. Cell images were observed and acquired using an optical microscope. Cells showing blue-green positive staining were identified as β-gal positive cells.

[0031] Real-time quantitative PCR (q-PCR): Bone marrow-derived macrophages were collected and washed with PBS. Total RNA was extracted from the cells using a total RNA extraction reagent. The total RNA extraction process was performed according to the instructions of the RNA extraction kit. The extracted RNA was tested for concentration and purity and then used for subsequent reverse transcription. The obtained total RNA was reverse transcribed into cDNA using a reverse transcription kit. Preferably, Evo M-MLV RT Premix for qRT-PCR reverse transcription reagent was used, and the reverse transcription reaction was performed according to the reagent instructions to obtain the corresponding cDNA sample. Subsequently, using the cDNA as a template, the expression level of the target gene was detected by real-time quantitative PCR. The target genes include, but are not limited to, genes related to cell senescence, genes related to inflammatory factors, genes related to macrophage function, and genes related to cartilage degeneration. The target genes include at least three of p16, p21, p53, IL-6, TNF-α, and IL-1β. The real-time quantitative PCR reaction was performed according to the instructions of the quantitative PCR reagent. Gapdh was used as an internal control gene, and the expression level of the target gene was normalized. -ΔΔCt The method calculates the relative expression level of the target gene in each group of samples.

[0032] ATP level detection: Bone marrow-derived macrophages from the conventional culture group and those induced by senescence were collected. After discarding the culture medium, the cells were washed with PBS to remove residual culture medium components. Cell lysis buffer was then added according to the ATP assay kit instructions to fully lyse the cells. The cell lysis buffer was collected, centrifuged, and the supernatant was used as the test sample. The ATP assay working solution was prepared according to the ATP assay kit instructions, and ATP standards were set up to establish a standard curve. The test sample and the ATP assay working solution were added to the assay plate, allowing the ATP in the sample to react with the assay system to generate a chemiluminescent signal. The luminescence value of each well was detected using a multi-mode microplate reader, and the ATP content in the sample was calculated based on the ATP standard curve. Preferably, the ATP content was corrected using BCA protein quantification results.

[0033] The results are as follows Figures 3-6As shown, the proportion of SA-β-gal-positive senescent macrophages after senescence induction was significantly higher than that in the conventional culture group; the expression levels of p16, p21, and p53 mRNA were all higher than those in the conventional culture group; the expression levels of IL-6, TNF-α, and IL-1β mRNA were all higher than those in the conventional culture group; and the intracellular ATP production capacity was lower than that in the conventional culture group. These results indicate that a stable senescence phenotype was successfully induced in macrophages.

[0034] Successful induction of the senescence phenotype requires meeting at least three of the following indicators: increased proportion of SA-β-gal positive cells, increased expression of p16, p21, and p53, increased expression of inflammatory factors such as IL-6, TNF-α, and IL-1β, and decreased ATP production capacity. After washing with serum-free culture medium, successfully induced senescent macrophages are resuspended in sterile saline and stored at 4°C. They should be used on the same day as induction, preferably within 2 hours.

[0035] Example 2 Construction of a mouse model of senile osteoarthritis.

[0036] Healthy 8-12 week old C57BL / 6 mice, weighing 20-25g, were selected and housed in an SPF-grade barrier environment with a 12h light / 12h dark cycle, an ambient temperature of around 25℃, and a relative humidity of 75%. They were allowed free access to food and water and were used to construct a mouse model of senile osteoarthritis after 7 days of acclimatization.

[0037] Before construction, a small amount of senescent macrophages prepared in Example 1 were mixed with 0.4% trypan blue solution at a 1:1 ratio and incubated at room temperature for 1-3 minutes. Unstained live cells and blue-stained dead cells were counted using a hemocytometer or automated cell counter, and cell viability was calculated. The cell viability calculation formula is: Cell viability (%) = Number of unstained live cells / Total number of cells × 100%.

[0038] The viability of senescent macrophages used for subsequent construction should not be less than 80%. Simultaneously, a control group and a model group were set up. The construction steps for the senile osteoarthritis mouse model (model group) are as follows: Figure 7 As shown.

[0039] (1) Construction of a mouse model of senile osteoarthritis (model group).

[0040] Processing and labeling of senescent macrophages: The induced senescent macrophages were collected, washed and resuspended in physiological saline or a suitable cell reinfusion solution to obtain a suspension of senescent macrophages (the reinfused cells can also be fluorescently labeled to track their retention, distribution and interaction with synovial tissue in the knee joint cavity).

[0041] Construction method: The prepared suspension of senescent macrophages was injected into the knee joint cavity of mice once a week (7 days) for 4 consecutive weeks (28 days). The number of senescent macrophages injected each time was 1×102 6 The injection volume is 10 μL (to reduce the risk of increased joint pressure, fluid leakage, mechanical damage, or non-specific inflammatory reactions caused by excessive injection volume). By continuously reinfusing senescent macrophages, they continuously participate in the regulation of the local immune microenvironment of the joint, thereby inducing the formation of age-related osteoarthritis-like lesions.

[0042] (2) Control group: The same volume of physiological saline as the suspension of senescent macrophages in the model group was injected into the knee joint cavity of mice once a week for a total of 4 times.

[0043] One week after the last injection, the mice were sacrificed and knee joint tissue was collected for further evaluation.

[0044] Example 3 Evaluation of a mouse model of senile osteoarthritis.

[0045] (1) Micro-CT detection.

[0046] Experimental Procedure: In a preferred embodiment, a Skyscan 1176 Micro-CT system was used for scanning. Scanning parameters were set as follows: X-ray source voltage 60 kV, X-ray source current 385 μA, pixel size 9.96 μm, and rotation step size 0.450°. After scanning, NRecon software was used to perform 3D reconstruction of the scanned images. Preferably, the reconstruction parameters included: ring artifact correction set to 8, smoothness set to 2, and beam hardening correction set to 30%.

[0047] After reconstruction, CTAn software was used to analyze the regions of the mouse tibia and femur containing the knee joint and subchondral bone. Preferably, the subchondral bone region of the knee joint was selected as the region of interest, and the microstructural changes of the tibial plateau subchondral bone and the femoral condyle subchondral bone were analyzed separately. A uniform threshold range was used for bone tissue segmentation during the analysis, with a preferred threshold range of 80-255 to ensure consistent analysis conditions across different samples. The thickness of the subchondral bone plate was quantitatively observed using CTAn software.

[0048] The results are as follows Figure 8 As shown, compared with the control group, the tibial bone microstructure of the model group mice was more porous, the trabecular bone arrangement was disordered, the intertrabecular bone gaps were increased, and bone hyperplasia and structural remodeling were observed in the subchondral bone region of the knee joint, suggesting that the model mice had osteoarthritis-related subchondral bone abnormalities.

[0049] (2) Safranin-Fix-Green staining and OARSI score.

[0050] Experimental Procedure: Knee joint tissue from the model animal was collected, fixed, decalcified, dehydrated, and embedded to prepare paraffin sections. The paraffin sections were dewaxed and subjected to gradient rehydration, followed by hematoxylin staining, Fast Green staining, and Safranin O staining to visualize the articular cartilage, cartilage matrix, and subchondral bone structures. After staining, the sections were dehydrated, cleared, and mounted to obtain joint sections for histological observation. Furthermore, the OARSI scoring system was used for semi-quantitative evaluation of the degree of articular cartilage degeneration. The scoring criteria included cartilage surface integrity, the degree of cartilage matrix loss, and subchondral bone exposure. Scoring was performed independently by at least two evaluators unaware of the experimental group, and the average score was taken as the final score. The age-related osteoarthritis animal model was considered successfully established when the joint sections of the model animal showed at least one of the following: decreased cartilage matrix staining, cartilage structure destruction, and an elevated OARSI score.

[0051] The results are as follows Figure 9 As shown, the model group mice exhibited decreased safranin O staining in the knee joint cartilage, thinning of the cartilage layer, and reduced cartilage surface integrity, accompanied by varying degrees of cartilage matrix loss and cartilage structural damage. Further semi-quantitative analysis using the OARSI score revealed that the OARSI score of the OA model group mice was significantly higher than that of the control group, suggesting that the model group mice exhibited significant osteoarthritis-like cartilage degeneration.

[0052] (3) Gait analysis.

[0053] Experimental Procedure: The model animal was placed in a gait analysis device and allowed to walk autonomously without significant external stimuli. Gait parameters such as foot contact trajectory, stride length, cadence, support time, swing time, footprint area, and weight distribution on both hind limbs were collected. Animals could undergo acclimatization training before the experiment to reduce the impact of environmental unfamiliarity on walking behavior. During the testing process, continuous, stable, uninterrupted, and non-turning walking segments were selected as valid analysis segments. The gait analysis system was used to analyze the limb movement trajectories and changes in plantar pressure to obtain behavioral indicators related to motor function and joint pain in the model animal.

[0054] The results are as follows Figure 10 As shown, compared with the control group, the relative maximum contact intensity and stride length of the model group mice were significantly reduced, suggesting that the weight-bearing capacity of the lower limbs and walking stability of the model group mice were decreased.

[0055] (4) Immunohistochemistry / fluorescence staining.

[0056] Experimental Procedure: Knee joint tissue from model animals was collected, fixed, decalcified, dehydrated, and embedded to prepare paraffin sections. The paraffin sections were then dewaxed and subjected to gradient rehydration. Antigen retrieval was performed according to the properties of the antigen to be detected, and non-specific binding sites were blocked using a suitable blocking solution. Subsequently, the sections were incubated with the primary antibody corresponding to the target indicator. For immunohistochemical staining, after incubation with the primary antibody, the corresponding immunohistochemical detection reagent was added, followed by chromogenic reaction using a chromogenic substrate. The sections were then counterstained, dehydrated, cleared, and mounted. Images were observed under a microscope, and the expression level of the target protein was evaluated using methods such as positive staining area, positive cell ratio, average optical density, or comprehensive staining score.

[0057] For immunofluorescence staining, after incubation with primary antibody, sections were incubated with corresponding fluorescently labeled secondary antibody and counterstained with nuclear dye. The sections were then mounted and observed under a fluorescence microscope. The co-localization of F4 / 80 and p16 aging markers can be detected as needed to evaluate the distribution of senescent macrophages in the joint and their relationship with osteoarthritis-like lesions. Sections were scanned and joint tissue images were acquired using a slide scanner to evaluate the expression of corresponding markers in subchondral bone or periarticular tissues.

[0058] The results are as follows Figure 11 , Figure 12 As shown, compared with the control group, the expression level of COL2A1 in the articular cartilage tissue of the model group mice was decreased, indicating reduced expression of type II collagen in cartilage and impaired cartilage matrix homeostasis. Simultaneously, the proportion of p16 and F4 / 80 colocalized fluorescence in the subchondral bone tissue of the model group mice was increased, suggesting that F4 / 80 positive macrophages exhibited a more pronounced aging-related phenotype. These results demonstrate that the model described in this invention can simulate the pathological features of senile osteoarthritis, such as increased macrophage aging and abnormal cartilage matrix metabolism.

[0059] In summary, this invention successfully constructed a mouse model capable of mimicking the core pathological features of senile osteoarthritis by inducing macrophage senescence in vitro and repeatedly reinfusing them into the joint cavity. This model has advantages such as short modeling cycle, simple operation, good reproducibility, and high pathological simulation, providing a reliable tool for the study of senile osteoarthritis. Furthermore, the bone marrow-derived macrophages involved in the embodiments can be replaced with peritoneal macrophages, spleen-derived macrophages, macrophages induced by peripheral blood mononuclear cells, etc., and applied to the construction of subsequent models.

[0060] Finally, it should be noted that the senile osteoarthritis mouse model described in this invention refers to a mouse model that simulates the immunosenescent pathological characteristics of senile osteoarthritis through the reinfusion of senile macrophages, and does not limit the recipient mice to naturally aged mice. The above embodiments are only used to illustrate the technical solutions of this invention and not to limit them. Although this invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of this invention, and these modifications or equivalent substitutions should not cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of this invention.

Claims

1. A method for constructing a mouse model simulating senile osteoarthritis, characterized in that, Includes the following steps: S1. Differentiate and culture bone marrow cells in a differentiation medium to obtain bone marrow-derived macrophages; S2. Bone marrow-derived macrophages were induced and cultured in senescence-inducing medium, and then cultured in conventional medium to obtain senescent macrophages; S3. Senescent macrophages were injected into the knee joint cavity of recipient animals to construct a model simulating senile osteoarthritis.

2. The method according to claim 1, characterized in that, In S1, the differentiation medium included macrophage colony-stimulating factor M-CSF at a concentration of 20 ng / mL; the differentiation culture time was 5 days.

3. The method according to claim 1, characterized in that, In S2, the aging induction medium included α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, 20 ng / mL M-CSF, and 100 nM doxorubicin; the induction culture time was 2 days; the conventional medium included α-MEM basal medium, fetal bovine serum, penicillin-streptomycin antibiotics, and 20 ng / mL M-CSF, and the culture time was 5 days.

4. The method according to claim 1, characterized in that, In S3, the specific steps for injecting senescent macrophages into the knee joint cavity of the recipient animals were as follows: injections were given once every 7 days, on days 0, 7, 14, and 21, for a total of 4 injections. The modeling period was 28 days, and the number of senescent macrophages injected each time was 1×10⁻⁶. 6 ~1.5×10 6 Each injection volume is 5-10 μL.

5. The method according to claim 1, characterized in that, S3 also includes acclimatization feeding before injecting senescent macrophages into recipient animals. The acclimatization feeding conditions are a 12h light / 12h dark cycle, a temperature of 25±1℃, a relative humidity of 75±5%, and a feeding period of 7 days.

6. The method according to claim 1, characterized in that, In S3, the recipient animal was the C57BL / 6 mouse.

7. The application of the mouse model of senile osteoarthritis constructed by the method according to any one of claims 1 to 6 in the study of the pathogenesis of senile osteoarthritis, screening of therapeutic targets, or evaluation of the efficacy of candidate drugs.

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