Construction method of tophus mouse model

Abstract

The invention relates to a construction method of a tophus mouse model, relates to the technical field of animal disease model construction, and is used for solving one of the problems of long modeling period, limited maintenance time, tedious operation, harsh conditions and the like of a tophus animal model in the prior art. The construction method of the tophus mouse model comprises the following steps: step 1, preparing a urate crystal-matrigel suspension; and 2, injecting the urate crystal-matrigel suspension prepared in the step 1 into the subcutaneous tissue of the back of a mouse. According to the construction method of the gouty stone mouse model, the experiment threshold and operation cost are low, repeatability is good, stability is high, the core pathological process of the gouty stone can be stably represented, the nodule and crystal morphology, the deposition state and the inflammatory characteristics can stably exist for at least four weeks after modeling, and the model has good application prospects. The requirements of long-term lesion observation, pathogenesis research and long-acting drug screening of the tophus are met.

Description

Technical Field

[0001] This invention relates to the field of animal disease model construction technology, and in particular to a method for constructing a mouse model of gouty tophi. Background Technology

[0002] Gout is a disease caused by excessively high levels of uric acid in the blood, leading to the deposition of uric acid crystals in the joints. Generally, 30% of gout patients who do not receive regular or regular treatment will develop tophi within 5 years of diagnosis, and tophi usually appear within 10 years of diagnosis if uric acid-lowering treatment is not initiated. The formation of tophi is a result of gout inflammation entering a chronic stage and failing to completely resolve. In patients with chronic gout, uric acid accumulates in the subcutaneous tissue of various parts of the body, forming urate crystals that eventually form swollen, nodular lumps—these are tophi. Once tophi appear, without proper treatment, they will not only not disappear on their own but will also gradually increase in size as the disease progresses, causing pain and functional impairment, severely impacting the patient's quality of life.

[0003] Constructing disease-related animal models has become an important tool for studying disease pathogenesis and developing therapeutic drugs. Therefore, constructing animal models of gouty tophi is particularly crucial for conducting research on tophi. However, due to the current limited understanding of gouty tophi, research on animal models related to gouty tophi is still in its early stages.

[0004] It has been reported that only two animal models of gouty tophi exist in current technology. One model, reported in the literature, involves injecting urate crystals to induce gouty tophi; however, this method requires four injections per week for three consecutive weeks, resulting in a long modeling time and short maintenance period. CN114366744A discloses an improved modeling method involving subcutaneous injection of urate crystals combined with gavage of potassium oxonate and a high-yeast diet. While this model can be maintained for approximately 28 days, the procedure is cumbersome and involves many steps, requiring strict aseptic conditions, repeated insufflation, and special dietary treatments, which greatly limits the promotion and application of this animal model.

[0005] Therefore, there is an urgent need for an animal model of gouty tophi that is simpler to operate, has a high modeling rate, a long maintenance time, and requires no special equipment or complex treatment. Summary of the Invention

[0006] Based on the above analysis, the present invention aims to provide a method for constructing a mouse model of gouty tophi, in order to solve one of the problems of existing animal models of gouty tophi, such as long modeling cycle, limited maintenance time, cumbersome operation and harsh conditions.

[0007] The objective of this invention is mainly achieved through the following technical solutions:

[0008] A method for constructing a mouse model of gouty tophi includes the following steps: Step 1: Preparation of urate crystal-matrix suspension; Step 2: Inject the urate crystal-matrix suspension prepared in Step 1 into the subcutaneous tissue on the back of the mouse.

[0009] Furthermore, after the subcutaneous injection in step two, nodules are formed in the subcutaneous tissue of the mice.

[0010] Furthermore, the nodules formed in step two can persist in the subcutaneous tissue of mice for 4 weeks.

[0011] Furthermore, the concentration of urate crystals in the urate crystal-matrix suspension is 10–40 mg / mL.

[0012] Furthermore, in step two, 50–150 μL of urate crystal-matrix suspension is subcutaneously injected into the subcutaneous tissue on the back of the mouse.

[0013] Furthermore, the mice were 8–10 week old male C57BL / 6 mice.

[0014] Further, step one includes step 1.1, preparing a urate crystal dispersion.

[0015] Furthermore, step one also includes step 1.2, which involves mixing the urate crystal dispersion prepared in step 1.1 with the matrix gel on ice.

[0016] A second aspect of the present invention also provides a mouse model of gouty tophi, which is constructed using the above-described construction method.

[0017] The third aspect of this invention also provides the application of the above-mentioned tophi mouse model in the study of tophi formation mechanism and the screening of drugs for the treatment of tophi.

[0018] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: (1) The method for constructing the tophi mouse model of the present invention greatly simplifies the model construction process: there is no need to construct subcutaneous air sacs, reducing complex aseptic operations; there is no need to combine potassium oxonate gavage or high yeast diet to induce hyperuricemia; the model can be formed simply by subcutaneous injection of urate crystals encapsulated in matrix gel; there are fewer steps and no special equipment dependence, reducing the experimental threshold and operating costs.

[0019] (2) The method for constructing the tophi mouse model of the present invention has a wide range of applications and strong versatility. No genetic modification of mice or special dietary intervention is required; ordinary experimental mice can be successfully used to establish the model.

[0020] (3) The tophi mouse model constructed using the method of the present invention has a high modeling rate and strong stability. The three-dimensional structure of the matrix gel can fix urate crystals, reduce the probability of urate crystals being rapidly degraded by uricase, and achieve continuous crystal deposition. Small sample experiments have verified that the modeling rate is 100% and the model has good repeatability, and can stably reproduce the core pathological process of tophi formation.

[0021] (4) The tophi mouse model constructed using the method of the present invention has a long maintenance time and is suitable for long-term research: the nodules remain stable for at least 4 weeks after modeling, and the crystal morphology, deposition state and inflammatory characteristics are continuously preserved, breaking through the limitation of the short maintenance time of existing models, and can meet the needs of long-term observation of tophi lesions, research on pathogenesis and screening of long-acting drugs.

[0022] (5) The tophi mouse model constructed using the method of the present invention has typical pathological features and high clinical simulation. Macroscopically, it forms stable and hard local nodules, and microscopically, a large number of urate crystals with typical birefringence characteristics are visible. It is accompanied by chronic inflammatory response with continuous recruitment of macrophages, which is highly consistent with the crystal deposition and chronic inflammatory pathological pattern of clinical tophi. Attached Figure Description

[0023] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0024] Figure 1 This is a flowchart of the method for constructing a mouse model of gouty tophi according to Embodiment 1 of the present invention; Figure 2 The images show the body appearance of the mouse model of tophi in Example 1 of the present invention from week 1 to week 4 after its construction, and the anatomical images at week 4. Figure 3 This is a comparative pathological section image of hematoxylin-eosin staining of gouty tophi-related tissues in Example 2 of the present invention; Figure 3 -A and B are staining images of the monosodium urate control group; Figure 3 -C and D are staining images of the experimental group (urate crystal-matrix suspension); Figure 4 This is a birefringence image of urate crystals under a polarizing microscope in Embodiment 2 of the present invention; Figure 5 This is a bar chart comparing the number of F4 / 80 positive macrophages in different treatment groups in Example 2 of the present invention. Figure 6 This is a bar chart showing the statistical comparison of α-smooth muscle actin-positive cells in different treatment groups in Example 2 of the present invention. Figure 7This is a bar chart comparing the relative mRNA expression levels of interleukin-1β in different treatment groups in Example 2 of the present invention. Figure 8 This is a bar chart comparing the relative mRNA expression levels of tumor necrosis factor-α in different treatment groups in Example 2 of the present invention. Figure 9 This is a bar chart comparing the relative mRNA expression levels of interleukin-6 in different treatment groups in Example 2 of the present invention. Figure 10 This is a comparison of immunohistochemical staining between the experimental animal group (urate crystal-matrix suspension) and the urate crystal control group in Example 4 of the present invention. Detailed Implementation

[0025] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0026] Example 1 This embodiment is mainly used to solve one of the following technical problems in the existing technical solutions: solving one of the problems of long modeling cycle, limited maintenance time, cumbersome operation and strict conditions in the existing animal models of gouty tophi.

[0027] A specific embodiment of the present invention discloses a method for constructing a mouse model of gouty tophi, such as... Figure 1 As shown, it includes the following steps: Step 1: Preparation of urate crystal-matrix suspension; Step 2: Inject the urate crystal-matrix suspension prepared in Step 1 into the subcutaneous tissue on the back of the mouse.

[0028] The method for constructing a tophi mouse model described in this embodiment significantly simplifies the modeling process by preparing a suspension of urate crystals encapsulated in matrix gel and administering it subcutaneously to the back. It eliminates the need for subcutaneous airbags, combined with potassium oxonate gavage or a high-yeast diet to induce hyperuricemia, thus simplifying existing model procedures and resolving the technical problem of redundant steps. Simultaneously, the three-dimensional structure of matrix gel fixes the crystals, reducing the rate of crystal degradation by uricase and enabling continuous crystal deposition. This method efficiently constructs a tophi mouse model, lowering the experimental threshold and operational costs, and improving the convenience and reliability of model construction.

[0029] Furthermore, step one specifically includes the following steps: Step 1.1 Preparation of crystal dispersion: Sodium urate crystals (MSU) were added to a sterile 0.5% (w / v) sodium carboxymethyl cellulose (CMC-Na) solution and pre-dispersed by vortexing and ice bath sonication to form a uniform primary crystal dispersion.

[0030] Step 1.2 Prepare the matrix gel: Place the matrix gel (Corning® Matrigel® GFR) on ice to allow it to completely melt and keep it at a low temperature.

[0031] Step 1.3 Mixing and resuspension: Mix the crystal dispersion obtained in step 1.1 with the melted matrix gel in step 1.2 on ice and gently blow it to make it initially mixed.

[0032] Step 1.4 Homogenization treatment: The mixture obtained in step 1.3 was subjected to brief, intermittent, and mild ultrasonic treatment under ice bath conditions to finally obtain a urate crystal-matrix suspension with a uniform urate crystal concentration of 25 mg / mL, which was then placed on ice for later use.

[0033] Furthermore, in step 1.1, the concentration of urate crystals is 10–40 mg / mL.

[0034] The urate crystal concentration is set within an adjustable range of 10–40 mg / mL, which can be flexibly optimized according to experimental objectives: low concentrations can be used to explore the initiation conditions of tophi formation, while high concentrations can simulate the crystal deposition state in patients with severe gout, adapting to different research scenarios (such as mechanism exploration, drug dosage screening, etc.), significantly improving the model's versatility and the flexibility of experimental design. For example, urate crystal concentrations of 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, or 40 mg / mL can be used. When the urate crystal concentration is 10 mg / mL, it simulates the state of small-scale urate crystal deposition in patients with early-stage clinical gout. This allows observation of the threshold effect of crystal deposition and the initiation process of the body's inflammatory response to low-dose crystals, providing model support for elucidating the early molecular mechanisms of tophi formation. For example, a urate crystal concentration of 25 mg / mL achieves a high modeling rate and can stably form typical tophi nodules, balancing crystal deposition volume with animal tolerance. This is suitable for routine studies of tophi formation mechanisms and initial screening of universal therapeutic drugs. At a urate crystal concentration of 30 mg / mL, it can simulate the crystal deposition state of patients with moderate to severe gout, forming larger and denser tophi nodules. This concentration significantly increases crystal deposition and intensifies the chronic inflammatory response, making it suitable for evaluating the efficacy of potent crystal-dissolving drugs or exploring the effects of high-dose crystals on tissue fibrosis and functional damage. For example, a urate crystal concentration of 40 mg / mL is suitable for constructing a severe tophi model, simulating the pathological state of severely ill patients with long-term untreated and massive accumulation of urate crystals. This concentration can form persistent, stable, and large nodules, making it suitable for studying the pathological progression of severe tophi (such as nodule invasion of surrounding tissues, high expression of inflammatory factors, etc.) or screening for specific drugs for severe gout.

[0035] Furthermore, step two includes the following steps: Step 2.1 Shave the fur on the back of the mouse.

[0036] Step 2.2 The mice were anesthetized with a gas anesthesia machine.

[0037] Step 2.3 Using an insulin injection needle, inject 50 μL–150 μL of urate crystal-matrix suspension subcutaneously into the subcutaneous tissue on the back of the mouse.

[0038] Furthermore, after the subcutaneous injection in step two, nodules are formed in the subcutaneous tissue of the mice.

[0039] Furthermore, the nodules formed in step two can persist in the subcutaneous tissue of mice for 4 weeks, such as... Figure 2 The image shows the external appearance of the mouse model of gouty tophi from week one to week four, as well as the actual image of the mice after dissection in week four.

[0040] Example 2 This embodiment 1 uses the construction method described in embodiment 1 to construct a mouse model of tophi.

[0041] 1. Animals and main materials Laboratory animals: Male C57BL / 6J mice were provided by the Laboratory Animal Department of Peking University Health Science Center. The mice were 8 to 10 weeks old and weighed 20 to 23g. All animals were acclimatized to their environment for at least one week to ensure good adaptation before any procedures were performed. All animal husbandry and experimental projects were approved by the Animal Protection and Ethics Committee of Peking University Health Science Center and strictly followed the Peking University Biomedical Ethics Guidelines.

[0042] Control animals: C57BL / 6j mice of the same age (8–10 weeks) and sex (male) as the experimental group (source: Department of Laboratory Animal Science, Peking University School of Medicine). The rearing conditions, ethical review, and adaptation feeding process were exactly the same as the experimental group.

[0043] Crystal source: Sodium urate crystals (Uricacidsodium (Monosodiumurate)) were purchased from Shanghai Haoyuan Pharmaceutical Technology Co., Ltd. (MCE), product code HY-B2130A. The standard was analyzed by high performance liquid chromatography (HPLC), and its chemical purity was 99.58%.

[0044] Corning® Matrigel® GFR, part number: REF354230, purchased from Corning Corporation, USA, and stored at -20°C.

[0045] 2. The experimental group of animals was used to construct a mouse model of tophi. The specific steps are as follows: Step 1: Preparation of urate crystal-matrix suspension; Step 1.1 Preparation of crystal dispersion: Sodium urate crystals were added to a sterile 0.5% (w / v) sodium carboxymethyl cellulose (CMC-Na) (molecular weight 40,000 Da to 1,000,000 Da) solution and pre-dispersed by vortexing and ice bath sonication. The sonication power was 100W, 2000-3000 rpm, under ice bath conditions, each sonication lasted 5 seconds, with a 5-second interval, and was repeated for 10 cycles to ensure that the crystals were fully and uniformly dispersed to form a homogeneous primary crystal dispersion.

[0046] Step 1.2 Prepare the base adhesive: Place the base adhesive on ice to allow it to completely melt and keep it at a low temperature.

[0047] Step 1.3 Mixing and resuspension: Mix the primary dispersion of crystals obtained in step 1.1 with the melted matrix gel in step 1.2 on ice and gently blow it to make it initially mixed.

[0048] Step 1.4 Homogenization treatment: The mixture obtained in step 1.3 was subjected to brief, intermittent, and mild ultrasonic treatment under ice bath conditions to finally obtain a urate crystal-matrix suspension with a uniform urate crystal concentration of 25 mg / mL, which was then placed on ice for later use.

[0049] Furthermore, in step 1.1, the concentration of urate crystals is 10–40 mg / mL.

[0050] Step 2: Select male C57BL / 6 mice aged 8–10 weeks and shave their backs; Take 100 μL of the urate crystal-matrix suspension prepared in step one and inject it subcutaneously into the subcutaneous tissue of the mouse back using a 29G insulin needle to a depth of 2-3 mm to form local nodules.

[0051] like Figure 3 As shown, after 1–4 weeks of observation, white or clump-like material was observed at the injection site, and crystal deposition areas were visible upon dissection. H&E staining revealed numerous needle-like / rod-like crystals, inflammatory cell infiltration, and macrophage-encapsulated structures, similar to the structure of clinical tophi.

[0052] 2.1 Detection structure of experimental group animals Local white or pale yellow nodules can be observed 1–4 weeks after injection; the nodules become denser over time.

[0053] Dissection of local tissues, H&E revealed: numerous needle-like crystal deposits; significant inflammatory cell infiltration in the mouse subcutaneous tissue, such as... Figure 3 As shown.

[0054] Observation under a polarized light microscope reveals distinct needle-like or rod-like crystals exhibiting typical yellow-blue birefringence; such as Figure 4 As shown, the crystal morphology and birefringence characteristics remained relatively stable 4 weeks after injection, indicating that the model can continuously retain MSU crystal deposition during the observation period and has a certain long-term stability.

[0055] 3. The treatment process for the sodium urate control group animals is as follows: Preparation of crystal dispersion: An equal amount (same mass as the experimental group) of monosodium urate (MSU) crystals was directly added to the same volume of sterile 0.5% CMC-Na solution and subjected to vortexing and ice bath sonication under the same conditions to form a crystal dispersion (without matrix gel).

[0056] Male C57BL / 6 mice aged 8–10 weeks were selected, their backs were shaved, and 100 μL of the above-mentioned matrix gel-free crystal dispersion was subcutaneously injected into the same location on the back of the mice using a 29G insulin needle.

[0057] 3.1 Results of animal tests in the control group like Figure 3 As shown, H&E staining analysis was performed on the control group tissue 28 days after injection. The results showed: Crystal distribution: Only a small number of scattered needle-like crystals are visible in the field of view.

[0058] Inflammatory response: mainly acute and transient inflammatory cell infiltration, without the formation of typical tophi-like pathological changes.

[0059] Structural integrity: The tissue lacked the persistent, well-defined crystal deposition cores and surrounding fibrous encapsulation tendencies observed in the experimental group. Figure 3 -A and B are staining images of the monosodium urate control group, showing only a small number of scattered needle-like crystals and acute inflammatory cell infiltration; Figure 3 - C and D are staining images of the experimental group (urate crystal-matrix suspension), showing a large amount of crystal deposition, macrophage encapsulation structure, typical pathological features of tophi; The control group animals were treated with 4.0.5% sodium carboxymethyl cellulose (CMC-Na) according to the following steps: Take an equal volume of sterile 0.5% CMC-Na solution from the same batch as the above crystal dispersion, place it in an identical container, and subject it to vortexing and ice bath sonication under identical conditions (i.e., the same vortex parameters, sonicator, sonic power, number of pulse cycles, and ice bath conditions) to prepare a blank control dispersion free of urate crystals.

[0060] Male C57BL / 6 mice aged 8–10 weeks were selected. After shaving the back of the mice, 100 μL of the above-mentioned blank control dispersion was subcutaneously injected into the back of the mice at the same location as the sodium urate control group using a 29G insulin needle.

[0061] 5. Conclusion The experimental group animals successfully induced persistent structures with typical tophi pathological features; while the control group animals only induced transient acute inflammatory responses, the crystals were quickly cleared, and a stable lesion model could not be formed.

[0062] like Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 The bar chart shown here is a multidimensional comparison of animals in the 0.5% carboxymethyl cellulose sodium (CMC-Na) control group, the monosodium urate control group, and the experimental group (monosodium urate and matrix gel), including cell markers ( Figure 4 and Figure 5 As shown), inflammatory factor mRNA ( Figure 6 , Figure 7 and Figure 8As shown in the figure): Among the cell markers, the number of F4 / 80 positive macrophages and α-smooth muscle actin positive fibrotic cells in the experimental group were significantly higher than those in the 0.5% carboxymethyl cellulose sodium (CMC-Na) and sodium urate control groups, demonstrating the recruitment effect of matrix gel encapsulation on cells related to chronic inflammation. In the inflammatory factor mRNA module, the expression of acute inflammatory factors (IL-1β, TNF-α, IL-6) mRNA was significantly increased in the sodium urate control group, while the expression in the experimental group decreased to a level not significantly different from that in the 0.5% carboxymethyl cellulose sodium (CMC-Na) control group, indicating that matrix gel encapsulation transforms "acute transient inflammation" into "chronic inflammation". Overall, this figure, through the comparison of cell composition and inflammation type, directly corroborates that matrix gel-encapsulated sodium urate can simulate the pathological features of chronic inflammation with fibrosis in clinical tophi.

[0063] Example 3 To further verify the success rate of constructing a tophi mouse model using the method described in Example 1, this example uses the method described in Example 1 to construct a tophi mouse model from 5 male C57BL / 6J mice. The following procedures were performed on each mouse: Step 1: Preparation of urate crystal-matrix suspension; Step 1.1 Preparation of crystal dispersion: Sodium urate crystals were added to a sterile 0.5% (w / v) sodium carboxymethyl cellulose (CMC-Na) solution and pre-dispersed by vortexing and ice bath sonication. The sonication power was 100W. Under ice bath conditions, each sonication lasted 5 seconds, with a 5-second interval, and was repeated for 10 cycles to ensure that the crystals were fully and uniformly dispersed to form a uniform primary crystal dispersion.

[0064] Step 1.2 Prepare the base adhesive: Place the base adhesive on ice to allow it to completely melt and keep it at a low temperature.

[0065] Step 1.3 Mixing and resuspension: Mix the primary dispersion of crystals obtained in step 1.1 with the melted matrix gel in step 1.2 on ice and gently blow it to make it initially mixed.

[0066] Step 1.4 Homogenization treatment: The mixture obtained in step 1.3 was subjected to brief, intermittent, and mild ultrasonic treatment under ice bath conditions to finally obtain a urate crystal-matrix suspension with a uniform urate crystal concentration of 25 mg / mL, which was then placed on ice for later use.

[0067] Furthermore, in step 1.1, the concentration of urate crystals is 10–40 mg / mL.

[0068] Step 2: Shave the fur on the back of the mouse; Take 100 μL of the urate crystal-matrix suspension prepared in step one and inject it subcutaneously into the subcutaneous tissue on the back of a mouse using a 29G insulin needle to form local nodules.

[0069] On day 28 post-injection, samples were taken from all mice. The initial criterion for successful modeling was the formation of stable, palpable nodules in the subcutaneous tissue of the back, with typical white / off-white crystalline deposits visible upon incision. Results showed that in the model of this invention, 5 mice met the above criteria, resulting in a 100% success rate.

[0070] Currently, most published literature and patents focus on acute gouty arthritis models, lacking standardized animal models for stable tophi-like nodules, and there is also a lack of systematic statistical analysis of the "nodule formation rate." This invention, with clearly defined model formation criteria, achieves a small-sample model formation rate of approximately 100%, achieving a high success rate while maintaining simple modeling procedures. It also features reproducibility and long-term maintenance, making it suitable as a reproducible mouse model of tophi that can be easily constructed in routine laboratory settings.

[0071] Example 4: Histopathological Analysis A mouse model of tophi was constructed using the construction method described in Example 2, and the following operations were performed.

[0072] 1. Sample preparation Sampling time points: Mice were sacrificed on days 7, 14, 21 and 28 after modeling. After sacrifice, the tophi and surrounding tissues were completely removed and embedded in paraffin. Experimental design: n=3 biological replicates were set up for each group at each time point.

[0073] 2. Perform H&E staining on each sample. 2.1 Reagents: Hematoxylin staining solution (Beijing Zhongshan Jinqiao Company), eosin staining solution (Beijing Zhongshan Jinqiao Company), xylene, gradient ethanol, etc.

[0074] 2.2 Operating Steps: Sectioning and baking: Prepare 4-6 μm thick paraffin sections and bake at 60℃ overnight.

[0075] Dewaxing and hydration: Immerse in xylene 3 times, 10 minutes each time; immerse in anhydrous ethanol 2 times, 5 minutes each time; immerse in 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes; rinse with distilled water.

[0076] dyeing: Hematoxylin staining: Add hematoxylin staining solution and stain for 5-8 minutes, then rinse with tap water.

[0077] Differentiation and blueing: Differentiation with 1% hydrochloric acid and ethanol for a few seconds, then rinse with tap water; Blueing with 0.6% ammonia for a few seconds, then rinse with tap water.

[0078] Eosin staining: Add eosin staining solution and stain for 1-3 minutes.

[0079] Dehydration and mounting: The slides were dehydrated sequentially with 95% ethanol and anhydrous ethanol, cleared with xylene, and mounted with neutral resin. The slides were then scanned.

[0080] 3. Perform immunohistochemical staining on each sample. 3.1 Main reagents: Antigen retrieval solution: Sodium citrate buffer (Beijing Solarbio) or EDTA antigen retrieval solution (Beijing Solarbio).

[0081] Blocking solution: 10% goat serum (Beijing Zhongshan Jinqiao Company).

[0082] Primary antibody: Anti-F4 / 80 Rabbit pAb (Servicebio, GB11027-100); Anti-α-SMA RabbitpAb (Servicebio, GB111364-50).

[0083] Secondary antibody and colorimetric system: HRP-labeled goat anti-rabbit / mouse IgG secondary antibody and DAB colorimetric kit (Beijing Zhongshan Jinqiao Company).

[0084] Counterstaining reagent: hematoxylin (Beijing Zhongshan Jinqiao Company).

[0085] 3.2 Operating Steps: 1) Section preparation: Prepare tissue paraffin sections with a thickness of 4-6µm, fix the paraffin sections with glass slides and air dry them, put the sections in a 60℃ oven overnight, and bake them again at 60℃ for 2 hours before use.

[0086] 2) Dewaxing and hydration of sections: Place the paraffin sections sequentially into three reagent jars containing xylene, soaking in each jar for 15 minutes; after removing the sections, place them sequentially into two reagent jars containing anhydrous ethanol, soaking in each jar for 5 minutes, then transfer them to two reagent jars containing 95% ethanol, soaking in each jar for 5 minutes, and finally place them into a reagent jar containing 80% ethanol, soaking for 5 minutes; place the sections that have undergone gradient dehydration into distilled water and soak for 3 minutes; remove the sections and wash them three times with PBS buffer, each time for 4 minutes.

[0087] 3) Antigen retrieval: Place sufficient sodium citrate antigen retrieval solution or EDTA antigen retrieval solution into the antigen retrieval box, add the slides, and tighten the lid. Microwave: 6 minutes on high, then 15 minutes on low. Rinse three times with PBS, 4 minutes each time.

[0088] 4) Blocking: Wipe the liquid around the tissue section dry, and draw a hydrophobic zone similar in size to the tissue block using a histochemical pen. Add goat serum blocking solution to the hydrophobic zone and block at room temperature for 30 minutes.

[0089] 5) Primary antibody incubation: After pouring out the goat serum, add the recommended concentration of primary antibody working solution directly into the hydrophobic zone. Place in a dedicated incubation chamber and incubate overnight at 4°C or for 2 hours at 37°C. Wash three times with PBS, 4 minutes each time.

[0090] 6) Secondary antibody incubation: Add horseradish peroxidase-labeled secondary antibody and incubate at room temperature for 30 minutes. Wash three times with PBS, 4 minutes each time.

[0091] 7) Color development: Add an appropriate amount of DAB color developer, observe under a microscope to determine the color development time, and immediately stop the color development with tap water.

[0092] 8) Counterstaining: stain with hematoxylin for 30 seconds, treat with differentiation solution for 5 seconds, and treat with blue solution for 15 seconds. Rinse twice with tap water between each step.

[0093] 9) Dehydration and Transparency: 2 tanks of 95% ethanol (2 minutes each) → 2 tanks of anhydrous ethanol (2 minutes each) → 2 tanks of xylene (4 minutes each). Place in a fume hood for about 1 minute to air dry.

[0094] 10) Mounting and Scanning: Mount the slides with neutral resin. Scan the slides using the scanner.

[0095] (3) Key parameters: Negative control: A negative control (using PBS instead of the primary antibody) was set up for each group of experiments to eliminate non-specific staining interference.

[0096] (4) Results like Figure 9 The immunohistochemical staining image shows that F4 / 80 was used as a specific marker. F4 / 80 is a classic surface marker of mouse macrophages, used to locate and quantify macrophages. Figure 4 The image shows a comparison of staining between the experimental group and the control group animals.

[0097] Experimental group animals: Staining results showed a large number of F4 / 80 stains in localized areas. + Macrophages aggregated and infiltrated, and were distributed around urate crystals. The inflammatory response showed obvious "chronic inflammatory features," with some areas showing a tendency to fibrosis, which is consistent with the "chronic inflammatory microenvironment" in the clinical process of tophi formation. Control group animals: Only a small number of F4 / 80 were observed. + Macrophages, mainly acute inflammatory cells, do not show a sustained aggregation trend, and the inflammation is "acute and transient," unable to form a stable chronic inflammatory state.

[0098] 4. Statistical Analysis: In this invention, all immunohistochemical experiments were performed independently in triplicate, using the same antibody batch and staining conditions. Percentage of positive immunohistochemical area: Using QuPath software, the percentage of DAB-positive stained area relative to the total tissue area was calculated as a quantitative indicator of the marker's expression level.

[0099] This study used GraphPad Prism 9.0 software for statistical analysis and graphing. Unless otherwise specified, experimental data are expressed as mean ± standard deviation (Mean ± SD). For normally distributed data, two-sided unpaired t-tests were used for comparisons between two groups; one-way ANOVA was used for comparisons among multiple groups. For non-normally distributed data, the Mann-Whitney U test was used for comparisons between two groups. P < 0.05 indicated statistical significance.

[0100] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model of gouty tophi, characterized in that, Includes the following steps: Step 1: Prepare a urate crystal-matrix suspension; Step 2: Inject the urate crystal-matrix suspension prepared in Step 1 into the subcutaneous tissue on the back of the mouse.

2. The method for constructing a mouse model of gouty tophi according to claim 1, characterized in that, In step two, after the subcutaneous injection, nodules form in the subcutaneous tissue of the mice.

3. The method for constructing a mouse model of gouty tophi according to claim 2 and its application, characterized in that, The nodules formed in step two can persist in the subcutaneous tissue of mice for 4 weeks.

4. The method for constructing a mouse model of gouty tophi according to claim 1, characterized in that, The concentration of urate crystals in the urate crystal-matrix suspension is 10–40 mg / mL.

5. The method for constructing a mouse model of gouty tophi according to claim 1, characterized in that, In step two, 50–150 μL of urate crystal-matrix suspension is subcutaneously injected into the subcutaneous tissue on the back of the mouse.

6. The method for constructing a mouse model of gouty tophi according to claim 1, characterized in that, The mice were male C57BL / 6 mice aged 8–10 weeks.

7. The method for constructing a mouse model of gouty tophi according to claim 1, characterized in that, Step one includes step 1.1, preparing a urate crystal dispersion.

8. The method for constructing a mouse model of gouty tophi according to claim 7, characterized in that, Step 1 further includes step 1.2, which involves mixing the urate crystal dispersion prepared in step 1.1 with the matrix gel on ice.

9. A mouse model of gouty tophi, characterized in that, It is constructed using the construction method described in any one of claims 1-8.

10. The application of the tophi mouse model of claim 9 in the study of tophi formation mechanism and the screening of drugs for the treatment of tophi.

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