A method for constructing a mechanical stress-induced skin fibrosis mouse model and application thereof

Abstract

The present application relates to the biomedical field, specifically relates to a kind of mechanical stress induced skin fibrosis mouse model construction method and its application.The construction method includes making incision on the back of immunocompetent mouse, bluntly separating subcutaneous loose connective tissue, to form subcutaneous interstitial space;Approximately hemispherical hydrogel with plane diameter of 1~2.2cm and height of 0.6~1.2cm is implanted into the subcutaneous interstitial space, the plane of hydrogel is downward, the curved surface is upward and contacts the skin of mouse back, after suture incision, the mouse is routinely bred, and the hydrogel implantation time is 2~3 weeks, to obtain the mouse model.The construction method of mouse model in the present application is simple and has high repeatability;The mouse model obtained using the method of the present application can be widely used in skin fibrosis mechanism research and anti-skin fibrosis drug screening work.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for constructing a mouse model of mechanical stress-induced skin fibrosis and its application. Background Technology

[0002] In current research on skin diseases, there is a lack of mouse models that can stably and controllably simulate continuous mechanical stress in vivo. In particular, in studies on the formation of skin fibrosis induced by mechanical stress and related pathological mechanisms, traditional fibrosis mouse models cannot truly reflect the effects of mechanical force on dermal tissue structure and extracellular matrix deposition.

[0003] Currently, mouse models for studying skin fibrosis or related pathological processes mainly include: 1) chemically induced models, such as the bleomycin subcutaneous injection-induced skin fibrosis model, which induces inflammation and collagen deposition through chemical damage. 2) genetically modified models, specifically through transgenic or gene-knockout mice, inducing abnormalities in fibrosis-related signaling pathways. 3) trauma or suture traction models: applying a certain tension to the skin through skin incision, suturing, or mechanical traction devices to study skin fibrosis. However, the above-mentioned existing technologies generally have the following shortcomings: chemically induced models mainly focus on inflammatory damage and are difficult to reflect mechanical stress factors alone; genetically modified models are costly and time-consuming, and are not suitable for simulating acquired mechanical stress; existing traction devices in trauma or suture traction models are complex to operate and highly invasive, making it difficult to achieve long-term, stable, and uniform mechanical stress loading in vivo. In addition, most existing models lack clear control of physical structural parameters, resulting in poor repeatability and comparability. Therefore, there is currently a lack of experimental models and methods that can generate continuous mechanical stress in mice to induce skin fibrosis. Summary of the Invention

[0004] Therefore, this invention aims to solve the following technical problems: 1. A mouse model of skin fibrosis that can be continuously subjected to mechanical stress in vivo; 2. Its construction method is simple to operate, highly reproducible, and has good stability; 3. A mouse experimental platform suitable for studying the mechanism of skin fibrosis and screening anti-fibrotic drugs.

[0005] Accordingly, the present invention provides a method for constructing a mouse model of mechanical stress-induced skin fibrosis. The method includes making an incision in the back of an immune-healthy mouse, bluntly dissecting the loose subcutaneous connective tissue to form a subcutaneous cavity; implanting an approximately hemispherical hydrogel with a planar diameter of 1-2.2 cm and a height of 0.6-1.2 cm into the subcutaneous cavity, with the plane of the hydrogel facing down and the curved surface facing up in contact with the back skin of the mouse; suturing the incision and feeding the mouse normally; the hydrogel implantation time is 2-3 weeks to obtain the mouse model.

[0006] In one specific implementation, the mice are BALB / c female mice, and the mice are 6-7 weeks old when the hydrogel is implanted.

[0007] In one specific implementation, after the hydrogel implantation reaches a predetermined time and the mouse is euthanized, the skin tissue on the back of the mouse covering the hydrogel site is taken for analysis.

[0008] In one specific embodiment, the hydrogel is a GelMA hydrogel, namely methacrylamide gelatin hydrogel.

[0009] In one specific embodiment, the planar diameter of the approximately hemispherical hydrogel is 1.4~1.6cm, and the height of the hydrogel is 0.8~1.1cm.

[0010] The present invention also provides the application of a mouse model obtained by the construction method described above in the screening of drugs for anti-dermal fibrosis.

[0011] Compared with existing technologies, this invention has at least the following beneficial effects: 1) It stably and continuously simulates mechanical stress through in vivo structural implantation; 2) It does not rely on chemical stimulation, reducing non-specific inflammatory interference; 3) The mouse model construction method is simple and highly reproducible; 4) Experimental results show that HE staining confirms a significant increase in the thickness of the mouse dermis; Masson staining confirms a significant increase in collagen deposition; and Western blot analysis shows a significant increase in the expression of fibrosis-related proteins such as COL1A1, Fibronectin (FN), and α-SMA; 5) The mouse model obtained using the method described in this invention can be widely used in research on the mechanism of skin fibrosis and in the screening of anti-skin fibrosis drugs. Attached Figure Description

[0012] Figure 1 Photograph of a hemispherical hydrogel of GelMA.

[0013] Figure 2 This is a schematic diagram of hydrogel implantation under the skin of a mouse.

[0014] Figure 3 This is a schematic diagram showing the effect of hydrogel implantation under the skin of mice for 2 weeks.

[0015] Figure 4 This is a schematic diagram of Western blot analysis for the mouse model group and the control group.

[0016] Figure 5 This diagram illustrates HE staining and Masson staining detection in the mouse model group and control group.

[0017] Figure 6 The image shows the dermal thickness data of the mouse model group and the control group. Detailed Implementation

[0018] This invention induces skin fibrosis in mice by implanting a hydrogel material with a specific morphology and size to create continuous mechanical stress. The invention provides a method for constructing a mouse model of mechanical stress-induced skin fibrosis, comprising: first, preparing a hemispherical GelMA hydrogel of predetermined size and morphology in vitro; forming a subcutaneous cavity in the right side of the mouse's back; implanting the hemispherical GelMA hydrogel into the subcutaneous cavity; generating continuous mechanical stress on the mouse's skin tissue through the volume and morphology of the hydrogel; and after a predetermined treatment time, i.e., 2-3 weeks, collecting and analyzing mouse skin tissue to obtain a stable mouse skin fibrosis phenotype. Hydrogel material: GelMA hydrogel, i.e., methacrylamide gelatin hydrogel, commercially available, purchased from EFL; hydrogel morphology: approximately hemispherical structure; hydrogel size: width approximately 1.5 cm; height approximately 1.0 cm; experimental animals: female BALB / c mice, 6-7 weeks old; implantation site: subcutaneous on the back of the mouse; treatment period: approximately 2 weeks. Control group: Mice that were not implanted with hydrogel.

[0019] Example 1

[0020] Six- to seven-week-old female BALB / c mice were selected as experimental animals and randomly divided into experimental and control groups. In the experimental group, under anesthesia, a subcutaneous cavity was created on the right side of the back skin using blunt dissection. A pre-fabricated hemispherical GelMA hydrogel (approximately 1.5 cm wide, i.e., a plane diameter of 1.5 cm and a height of approximately 1.0 cm, EFL) was implanted into the subcutaneous cavity and routinely sutured. Postoperative adjuvant antibiotic treatment was administered. Mice in the control group did not receive hydrogel implantation. After two weeks of normal feeding postoperatively, the mice were sacrificed, and skin tissue from the implantation area (i.e., the skin tissue covering the hydrogel) was collected for analysis. HE staining results showed a significant increase in dermal thickness in the model group mice; Masson staining showed a significant increase in collagen deposition in the model group mice; Western blot analysis results showed significantly increased expression levels of fibrosis-related proteins such as COL1A1, Fibronectin (FN protein), and α-SMA, indicating that this invention successfully constructed a mechanical stress-induced skin fibrosis mouse model.

[0021] Figure 1 Photograph of a hemispherical hydrogel of GelMA. Figure 1 The image shows the shape and size characteristics of the commercially available GelMA hemispherical hydrogels. Each square on the scale measures 1cm x 1cm. The smaller blue-green transparent hydrogels have a plane diameter of approximately 1.5cm, while the larger purple transparent hydrogels have a plane diameter of approximately 2cm. All hydrogels are approximately 1cm high.

[0022] Figure 2 This is a schematic diagram of hydrogel implantation under the skin of a mouse. Figure 2 The image shows a schematic diagram of a commercially available hemispherical GelMA hydrogel implanted subcutaneously on the right side of a mouse's back and then sutured. Figure 3 This is a schematic diagram showing the effect of hydrogel implantation under the skin of mice for 2 weeks. Figure 3 The image shows a schematic diagram of a mouse model of skin fibrosis formed after the commercially available GelMA hemispherical hydrogel was implanted subcutaneously on the right side of the mouse back and raised normally for two weeks.

[0023] Figure 4 This is a schematic diagram of Western blot analysis of the mouse model group and the control group. The Western blot results show that, compared with the control group, the expression of Fibronectin (FN), COL1A1, and α-SMA in the model group mice were significantly increased. Figure 4 In the figure, because the four internal references GAPDH in the control group were significantly higher than those in the model group, it can be clearly seen from the figure that the expression of α-SMA in the model group mice, like COL1A1 and FN, was significantly higher than that in the control group.

[0024] Figure 5 This diagram illustrates HE staining and Masson staining detection in the mouse model group and control group. Figure 5 The results showed that, compared with the control group, the dermal thickness and collagen deposition in the model group mice were significantly increased. In Masson staining, the blue area represents the collagen deposition status, and the blue area in the model group was darker, indicating excessive collagen deposition, disordered arrangement, and density.

[0025] Figure 6 The graph shows the dermal thickness data of the mouse model group and the control group. There were 4 mice in each group. The mean dermal thickness of the control group mice was 141.25 μm, while the mean dermal thickness of the model group mice was 384.83 μm.

[0026] In summary, this invention specifically relates to a method for constructing a mouse model of mechanical stress-induced skin fibrosis and its application. The method involves making an incision in the back of an immunocompetent mouse, bluntly dissecting the loose subcutaneous connective tissue to form a subcutaneous cavity; implanting an approximately hemispherical hydrogel with a planar diameter of 1-2.2 cm and a height of 0.6-1.2 cm into the subcutaneous cavity, with the flat surface of the hydrogel facing downwards and the curved surface upwards contacting the mouse's back skin; suturing the incision; and then feeding the mouse as usual. The hydrogel implantation time is 2-3 weeks, resulting in the mouse model. The method for constructing the mouse model in this invention is simple and highly reproducible; the mouse model obtained using the method described in this invention can be widely used in research on the mechanism of skin fibrosis and in the screening of anti-skin fibrosis drugs.

[0027] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of 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 mechanical stress-induced skin fibrosis, characterized in that, The method involves making an incision in the back of an immune-healthy mouse, bluntly dissecting the loose subcutaneous connective tissue to form a subcutaneous cavity; implanting an approximately hemispherical hydrogel with a planar diameter of 1-2.2 cm and a height of 0.6-1.2 cm into the subcutaneous cavity, with the flat side of the hydrogel facing down and the curved side facing up, contacting the mouse's back skin; suturing the incision and then feeding the mouse as usual; the hydrogel implantation time is 2-3 weeks to obtain the mouse model.

2. The method for constructing a mouse model of mechanical stress-induced skin fibrosis according to claim 1, characterized in that, The mice were BALB / c female mice, and were 6-7 weeks old when the hydrogel was implanted.

3. The method for constructing a mouse model of mechanical stress-induced skin fibrosis according to claim 1, characterized in that, After the hydrogel implantation reached the predetermined time and the mice were euthanized, the skin tissue on the back of the mice covering the hydrogel area was taken for analysis.

4. The method for constructing a mouse model of mechanical stress-induced skin fibrosis according to claim 1, characterized in that, The hydrogel is a GelMA hydrogel, i.e., methacrylamide gelatin hydrogel.

5. The method for constructing a mouse model of mechanical stress-induced skin fibrosis according to claim 1, characterized in that, The approximately hemispherical hydrogel has a planar diameter of 1.4~1.6cm and a height of 0.8~1.1cm.

6. The application of a mouse model obtained by the construction method according to any one of claims 1 to 5 in the screening of drugs for anti-dermal fibrosis.

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