Method for establishing skin injury model of ICR mice and application thereof

Abstract

The application provides a method for establishing a skin injury model of ICR mice and application thereof; ICR mice are used as experimental animals, and a local skin bump is formed by injecting a mucor toxin protein on the back of the near-tail region to form a skin injury model; the model can be applied in mucor infection research, and research related to mucor toxin protein in mucormycosis can be quickly and conveniently carried out, and a new method is provided for basic research and clinical application related to mucormycosis infection and mucor toxin protein.

Description

TECHNICAL FIELD

[0001] The application relates to a method for establishing a model of skin injury of ICR mice and application, and belongs to the technical field of microbiology and experimental zoology. BACKGROUND

[0002] Ricin toxin (RT) is a widely existing plant toxin, belongs to type II ribosome inactivation protein (RIP), is composed of A and B two chains, the A chain (RTA) has the ability of ribosome inactivation and is a toxic chain; the B chain (RTB) is a binding chain and is a Galectin binding protein, can bind to target cells and induce cell damage. Ricin toxin-like protein genes exist in various pathogenic microorganisms, and the RLT proteins found in Rhizophagus, Rhizopus delemar, Streptomyces and Paenibacillus have homology and all have biological virulence.

[0003] The applicant has carried out in-depth research on the genomics of the pathogenicity of Mucorales for many years, and found that the MI supernatant can cause HUVECs injury, which indicates that MI may secrete related virulence factors to damage vascular endothelium. In order to determine the composition of the exogenous factors of MI, the applicant constructs a model of skin injury of ICR mice induced by Mucor toxin protein, so as to carry out basic research and clinical application related to Mucor toxin protein in the infection mechanism of Mucormycosis. SUMMARY

[0004] In view of the above-mentioned technical problems, the purpose of the present application is to provide a method for establishing a model of skin injury of ICR mice.

[0005] The technical solution of the present application is realized as follows: a method for establishing a model of skin injury of ICR mice, comprising the following steps:

[0006] ①6-8 weeks old male ICR mice are used as experimental animals, and after the skin is prepared on the back near the tail region, 25 micrograms of Mucor toxin protein in 1ml is injected intradermally to form a local skin bump;

[0007] ②The mice treated in step ① are fed for 7 days.

[0008] Preferably, the Mucor toxin protein is formed by isolation and purification of Mucorales B50a.

[0009] Preferably, the amino acid sequence of the Mucor toxin protein is as follows:

[0010] MSLYFIRSQYNGRVLDVNDGSLEDGAEIIVWSQKGSDDCLNQLWRIEDGYIVNAKSAKALDISGGEMEPESAIIQYRQKSIEEAANQRWGIDSEGYIYSEARPDLVLDIQGREDEDGTPIILYNKREGEVASNQRWTLEEFQG.

[0011] Preferably, the mucorales include mucor and rhizopus arrhizus.

[0012] The above-constructed mouse skin damage model can be applied in the study of mucor infection.

[0013] Preferably, the mucor infection includes infection caused by mucor and rhizopus arrhizus.

[0014] Compared with the prior art, the application has the following advantages:

[0015] The application uses ICR mice as experimental animals, injects mucor toxin protein in the back of the near-tail region to form a local skin bump, and forms a skin damage model; the model can be applied in the study of mucor infection, and can quickly and conveniently carry out research on mucormycosis related to mucor toxin protein, thereby providing a new way for the basic research and clinical application of mucormycosis infection and mucor toxin protein. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application, the following will briefly introduce the drawings needed to be used in the description of the embodiments of the application. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can be obtained according to the contents of the embodiments of the application and the drawings without creative labor.

[0017] Figure 1 is a diagram of the damage of HUVECs induced by mucor toxin protein according to the application; Figure 1 Figure 2 is a diagram of the general changes of three groups of ICR mice according to the application;

[0018] Figure 2 Figure 3 is a model of skin damage of ICR mice induced by mucor toxin protein according to the application;

[0019] Figure 4 is a negative control group of ICR mice induced by subcutaneous injection of PBS according to the application; Figure 3 Figure 5 is a diagram of the damage of HUVECs induced by mucor toxin protein according to the application;

[0020] Figure 4 Figure 6 is a diagram of the general changes of three groups of ICR mice according to the application;

[0021] Figure 7 is a model of skin damage of ICR mice induced by mucor toxin protein according to the application; Figure 5 ​​The positive control group of ICR mice is subcutaneously injected with the Mucor strain CAMS-CCPM-D B50a. DETAILED DESCRIPTION

[0022] To make the technical problems solved by the present application, the technical solutions adopted and the technical effects achieved more clear, the technical solutions of the embodiments of the present application will be further described in detail below with reference to the drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person skilled in the art without creative work fall within the protection scope of the present application.

[0023] The present application provides a method for establishing a model of skin injury in ICR mice, comprising the following steps:

[0024] ① 6-8 weeks old male ICR mice are used as experimental animals, and after the skin is prepared on the back near the tail region, 25 μg of Mucor toxin protein in 1 ml is injected intradermally to form a local skin bump; the Mucor toxin protein is formed by isolation and purification of Mucor B50a; the amino acid sequence of the Mucor toxin protein is as follows:

[0025] MSLYFIRSQYNGRVLDVNDGSLEDGAEIIVWSQKGSDDCLNQLWRIEDGYIVNAKSAKALDISGGEMEPESAIIQYRQKSIEEAANQRWGIDSEGYIYSEARPDLVLDIQGREDEDGTPIILYNKREGEVASNQRWTLEEFQG.

[0026] The Mucor includes but is not limited to Mucor, Rhizopus arrhizus, etc.

[0027] ② The mice treated in step ① are raised for 7 days.

[0028] The model can be applied in the research of Mucor infection, which includes but is not limited to the infection caused by Mucor, Rhizopus arrhizus, etc.

[0029] The main process is: first, the damage of Mucor toxin protein (50 μg / ml) to HUVECs cells is preliminarily verified by in vitro experiment, and this concentration can cause 47.58% cell damage. Second, intradermal injection is performed on the back skin of mice, and 1×10 6 (±0.1×10 6) Spore quantity, negative control group of mice injected with Mucor toxin protein solvent (0.1 ml), toxin protein model group of mice injected with Mucor toxin protein (25 μg, 0.1 ml), clear Mucor toxin protein induced ICR mouse skin damage. At the same time, observe the general changes of infected mice, general inflammatory response (HE staining) and inflammatory cell infiltration (CD68 and LY6G immunohistochemical staining), prove that Mucor toxin protein (25 μg, 0.1 ml) can induce ICR mice with neutrophil reaction as the main skin damage.

[0030] The specific steps are as follows:

[0031] ① 6-8 weeks old male ICR mice were used as experimental animals, and the back of the near tail region was prepared for skin, and then Mucor toxin protein (25 μg, 0.1 ml) was injected intradermally to form a local skin bump, and the treated mice were raised for 7 days.

[0032] ② Set 6-8 weeks old male ICR mice as positive control group, inject Mucor strain CAMS-CCPM-D B50a bacterial suspension (1×10 6 Spore quantity, 0.1 ml) to form a local skin bump in the same way, and raise the treated mice for 7 days.

[0033] ③ Set 6-8 weeks old male ICR mice as negative control group, inject protein solvent to form a local skin bump in the same way, and raise the treated mice for 7 days.

[0034] ④ After raising the mice for 7 days, the local skin lesions were taken, 4% paraformaldehyde was used for immersion fixation, and HE staining and inflammation-related factor histochemical staining were performed.

[0035] Results:

[0036] I. In vitro experiment of vascular endothelial damage

[0037] After Mucor toxin protein induced human umbilical vein endothelial cells (HUVECs) damage, CCK8 was used to detect the damage degree, and the experiment proved that 50 μg / ml concentration of Mucor toxin protein could induce 47.58% cell damage (as shown in Figure 1 ), proving that Mucor toxin protein can induce in vitro damage of HUVECs, and the concentration of 50 μg / ml is used.

[0038] II. Mouse experiment of tissue damage

[0039] Reference to the attached Figures 2-5 .

[0040] Figure 2The ICR mouse gross change situation from left to right is as follows: mold toxin protein induces ICR mouse skin damage model; PBS subcutaneous injection induces ICR mouse negative control group; mold strain CAMS-CCPM-D B50a strain subcutaneous injection induces ICR mouse positive control group,

[0041] Figure 3 The ICR mouse gross change situation from left to right is as follows: mold toxin protein induces ICR mouse skin damage model; PBS subcutaneous injection induces ICR mouse negative control group; mold strain CAMS-CCPM-D B50a strain subcutaneous injection induces ICR mouse positive control group,

[0042] Figure 4 The ICR mouse gross change situation from left to right is as follows: mold toxin protein induces ICR mouse skin damage model; PBS subcutaneous injection induces ICR mouse negative control group; mold strain CAMS-CCPM-D B50a strain subcutaneous injection induces ICR mouse positive control group,

[0043] Figure 5 The ICR mouse gross change situation from left to right is as follows: mold toxin protein induces ICR mouse skin damage model; PBS subcutaneous injection induces ICR mouse negative control group; mold strain CAMS-CCPM-D B50a strain subcutaneous injection induces ICR mouse positive control group,

[0044] Conclusion:

[0045] 1. The mold toxin protein induces ICR mouse skin damage model, and the subcutaneous injection site of the local skin lesion is broken, HE staining shows that inflammatory cells infiltrate subcutaneously, and vascular thrombosis occurs, and immunohistochemical staining results suggest that a large number of neutrophils gather and a small number of macrophages surround.

[0046] 2. The ICR mouse negative control group induced by PBS subcutaneous injection, the subcutaneous injection site is not obviously damaged, and HE staining and immunohistochemical staining are not obviously abnormal.

[0047] 3. The ICR mouse positive control group induced by mold strain CAMS-CCPM-D B50a subcutaneous injection, the subcutaneous injection site of the local skin lesion is broken, and the size of the cyst is not uniform, HE staining shows that inflammatory cells infiltrate subcutaneously, and a large number of spore hyphae are surrounded by inflammatory cells, and immunohistochemical staining results suggest that a large number of neutrophils gather and a small number of macrophages surround.

[0048] The above-mentioned embodiments are only for illustrating the technical concept and characteristics of the present application, the purpose is to enable the person skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application, any equivalent changes or modifications made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A method for establishing a skin lesion model in ICR mice, characterized in that, Includes the following steps: ① Using 6-8 week old male ICR mice as experimental animals, after preparing the skin on the back near the tail area, 25 μg and 1 ml of mycotoxin protein were injected intradermally to form local wheals. ② The mice treated in step ① were fed for 7 days; The aforementioned mucortoxin protein was isolated and purified from Mucor B50a; The amino acid sequence of the mucortoxin protein is as follows: MSLYFIRSQYNGRVLDVNDGSLEDGAEIIVWSQKGSDDCLNQLWRIEDGYIVNAKSAKALDISGGEMEPESAIIQYRQKSIEEAANQRWGIDSEGYIYSEARPDLVLDIQGREDEDGTPIILYNKREGEVASNQRWTLEEFQG.

2. The method for establishing an ICR mouse skin lesion model according to claim 1, characterized in that: The aforementioned *Mucor* fungi include *Mucor* and *Rhizopus*.

3. The application of the method for establishing an ICR mouse skin lesion model as described in claim 1, characterized in that: The constructed mouse skin lesion model was applied to the study of Mucor infection.

4. The application of the method for establishing an ICR mouse skin lesion model according to claim 3, characterized in that: The aforementioned Mucor infection includes infections caused by Mucor and Rhizopus.

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