Modeling method for mouse oral lichen planus

Abstract

The invention discloses a mouse oral lichen planus modeling method, and relates to the technical field of animal modeling, and the specific method comprises the following steps: removing mouse abdominal hair, smearing oxazolone on a hair removal part, 5 days later, smearing oxazolone on mouse oral mucosa again, continuously treating for 3-7 days, and completing induction, the problems that an existing animal modeling method cannot effectively reflect the core pathological process of the oral lichen planus, and an animal model well simulating the oral lichen planus is difficult to obtain are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of animal modeling, and in particular to a method for establishing a mouse oral lichen planus model. Background Art

[0002] Oral lichen planus is a common chronic inflammatory oral mucosal disease that mainly affects the oral mucosa and has a potential risk of canceration. It is characterized by unknown etiology and complex pathogenesis. Patients experience a chronic course characterized by alternating non-erosive and erosive lesions. Currently, the research on the etiology and pathogenesis of oral lichen planus is still insufficient, and there is a lack of effective treatment methods clinically. Therefore, in diseases such as oral lichen planus, due to the intricate and elusive pathogenesis, the use of animal models is essential for hypothesis testing and research to clarify the disease mechanism.

[0003] Existing animal models have certain limitations in simulating the pathological characteristics of human oral lichen planus. Existing methods include xenogeneic T cell transfer, exposure to amalgam, or Escherichia coli infection under zinc deficiency conditions, etc. These methods are used to induce oral lichenoid reactions with certain oral lichen planus characteristics. However, these methods have inconsistencies in the stability of lesion development, and the modeling process is complex. Some require the use of rats for modeling, which limits their application value. They cannot effectively reflect the core pathological process of oral lichen planus. Therefore, there is an urgent need for an animal model that can better simulate oral lichen planus to facilitate in-depth research on its pathological mechanism and evaluation of the effectiveness of treatment strategies. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for establishing a mouse oral lichen planus model, which is simple, convenient and effectively establishes a mouse model with the pathological characteristics of oral lichen planus.

[0005] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] A method for establishing a mouse oral lichen planus model, the modeling method is as follows:

[0007] Remove the hair on the abdomen of the mouse, and then apply oxazolone to the depilated area. After 5 days, apply oxazolone again on the oral mucosa of the mouse. After continuous treatment for 3 - 7 days, the induction is completed.

[0008] The modeling mice selected by the present invention are preferably mice aged 10 - 14 weeks and specific pathogen-free, with no gender limitation, and the strain is preferably B57 or Balb / c. During breeding, the environment where the mice are located maintains a circadian rhythm of 12 hours of daylight and 12 hours of darkness per day, and the feed and drinking water are both carried out according to the specific pathogen-free environment to prevent other pathogens from affecting the modeling results.

[0009] When making the model, it is necessary to first remove the long hair on the abdomen of the mouse, and then apply oxazolone to the depilated area for sensitization, and the sensitization process only needs to be carried out once. Then, oxazolone is applied to the oral mucosa of the mouse, preferably on the inner mucosa of the mouse's lips, and continuously applied for 5 days to induce a local lichenoid immune response and simulate the core pathological process of oral lichen planus.

[0010] Furthermore, the oxazolone applied to the depilated area is an oxazolone solution with a mass concentration of 2-4%, and the oxazolone applied to the oral mucosa is an oxazolone solution with a mass concentration of 0.5-1.5%.

[0011] Preferably, when the oxazolone solution is applied to the depilated area, the mass concentration is 3%, and when it is applied to the oral mucosa, the mass concentration is 1%.

[0012] The present invention preferably dissolves oxazolone in absolute ethanol to make an oxazolone absolute ethanol solution with a corresponding concentration. In addition, oxazolone can also be dissolved in other common organic solvents, such as 80% acetone + 20% olive oil, which is also applicable.

[0013] Furthermore, when the oxazolone solution is applied to the depilated area, the dosage is 80-150 μL, preferably 100 μL, and when the oxazolone solution is applied to the oral mucosa, the dosage is 10-20 μL, preferably 15 μL.

[0014] Beneficial effects:

[0015] The modeling method of the present invention can induce a local lichenoid immune response, simulate the core pathological process of oral lichen planus, and can effectively establish a mouse model with the pathological characteristics of oral lichen planus, providing a reliable tool for further studying its pathological mechanism and evaluating the effectiveness of treatment strategies. At the same time, this method is simple and easy to operate, with high repeatability. On the one hand, it is suitable for the study of the pathogenesis, and on the other hand, this model can be used to evaluate the effectiveness of new drugs in the treatment of oral lichen planus, providing an experimental basis for the clinical treatment of oral lichen planus. Brief description of the drawings

[0016] Figure 1 : Schematic diagram of the modeling process;

[0017] Figure 2 : Dermoscopic manifestations of the mouse lips before and after induction;

[0018] Figure 3 : Histological characteristics of the oral lichen planus-like mouse model. Detailed implementation manners

[0019] The present invention will be described in detail below in combination with specific embodiments and drawings:

[0020] Example 1: Mice with Oral Lichen Planus Model

[0021] 1. Preparation of the modeling agent: Oxazolone (OXA) was purchased from MedChemExpress and dissolved in absolute ethanol (Vehicle) to prepare Oxazolone absolute ethanol solutions with mass concentrations of 3.0% and 1.0%.

[0022] 2. Selection and feeding of mice: Female mice of 12 weeks old and specific pathogen - free (SPF) with the strain of B57 were selected for modeling. During feeding, the environment where the mice were located maintained a circadian rhythm of 12 - hour day and 12 - hour night, and the feed and drinking water were both carried out according to the SPF environment.

[0023] 3. Experimental grouping and treatment:

[0024] The mice were randomly divided into four groups, namely: OXA - sensitization (Sensitization)+OXA - challenge group, OXA - sensitization+Vehicle (solvent) - challenge group, OXA - non - sensitization (No sensitization)+OXA - challenge group, OXA - non - sensitization+Vehicle - challenge group. The specific treatment methods for each group are as follows:

[0025] OXA - sensitization+OXA - challenge group: The long hair on the abdomen of the mice was removed, and 100 μL of Oxazolone absolute ethanol solution with a mass concentration of 3% was applied once on the shaved abdominal surface for sensitization. Five days later, 15 μL of Oxazolone absolute ethanol solution with a mass concentration of 1% was used to treat the oral mucosa of the lip, and the treatment was continued for five consecutive days.

[0026] OXA - sensitization+Vehicle - challenge group: The long hair on the abdomen of the mice was removed, and 100 μL of Oxazolone absolute ethanol solution with a mass concentration of 3% was applied once on the shaved abdominal surface for sensitization. Five days later, 15 μL of absolute ethanol solution with a mass concentration of 1% was used to treat the oral mucosa of the lip, and the treatment was continued for five consecutive days.

[0027] OXA - non - sensitization+OXA - challenge group: The mice were not sensitized, and directly 15 μL of Oxazolone absolute ethanol solution with a mass concentration of 1% was applied to the oral mucosa of the lip, and the treatment was continued for five consecutive days.

[0028] OXA - non - sensitization+Vehicle - challenge group: The mice were not sensitized, and directly 15 μL of absolute ethanol solution with a mass concentration of 1% was applied to the oral mucosa of the lip, and the treatment was continued for five consecutive days.

[0029] The schematic diagram of the specific modeling process of the above experiment is as Figure 1 shown.

[0030] 4. Experimental results and analysis:

[0031] After the induction ended, the dermoscopic manifestations of the lesions in each group were as follows Figure 2 shown, and the histological features were as follows Figure 3 shown.

[0032] The analysis results showed that:

[0033] (1) As Figure 2 shown, compared with the other three groups, obvious swelling and redness were visible on the lips of mice in the OXA sensitization + OXA challenge group.

[0034] (2) Figure 3 A measured the lip thickness of mice in the OXA sensitization + Vehicle challenge group and the OXA sensitization + OXA challenge group on the 5th day. It was visible that the lip thickness of mice in the OXA sensitization + OXA challenge group increased significantly.

[0035] (3) Figure 3 B showed the representative hematoxylin-eosin staining sections of mice in the OXA sensitization + Vehicle challenge group and the OXA sensitization + OXA challenge group. The results showed that, compared with the solvent (Vehicle) control group, the basal layer cells of the mucosal epidermis in the OXA challenge group showed liquefactive degeneration, there was a band-like lymphocyte infiltration around the basement membrane, and the blood vessels in the lamina propria mucosa were dilated.

[0036] (4) Figure 3 C showed the immunohistochemical staining images of CD8 + T cells of mice in the OXA sensitization + Vehicle challenge group and the OXA sensitization + OXA challenge group. The results showed that under high magnification, the number of CD8 + T cells in the OXA sensitization + OXA challenge group was significantly higher than that in the OXA sensitization + Vehicle challenge group, and the CD8 + T cells were mainly located around the basement membrane (white dotted line), and Figure 3 the quantitative analysis of CD8 + T cells in each high-power field (HPF) in D also confirmed this conclusion. And Figure 3 E was the immunofluorescence staining of CD8 (yellow) and granzyme B (GMZB, the absence of which indicates the degranulation phenomenon of CD8 + T cells) in the treated mice. The results showed that there was co-staining of CD8 + T cells and granzyme B in the mucosa of mice in the OXA sensitization + Vehicle challenge group, while there was no co-staining of CD8 + T cells and granzyme B in the mucosa of mice in the OXA sensitization + OXA challenge group. This indicated that the granzyme B release phenomenon of CD8 + T cells occurred in the OXA sensitization + OXA challenge group. Figure 3 The CD8 of F +T cell statistics also prove this point. In the OXA sensitization + OXA challenge group, nearly 95% of the CD8 + T cells in the mucosa showed degranulation, suggesting that after the establishment of this model, CD8 + T cells locally showed the phenomenon of releasing granzyme B.

[0037] (5) Figure 3 G shows the TUNEL immunofluorescence (for detecting apoptotic signals) staining of mice in the OXA sensitization + vehicle challenge group and the OXA sensitization + OXA challenge group (the white dotted line indicates the epithelial-lamina propria junction in Figure 3 G). The green fluorescence signal is TUNEL-positive cells. It can be seen that TUNEL-positive cells appear in the basal layer and within the epidermis of the mucosa of mice in the OXA sensitization + OXA challenge group, while few positive cells are seen in the OXA sensitization + vehicle challenge group. Figure 3 H shows the quantification of TUNEL-positive cells per HPF. The results show that the number of TUNEL-positive cells in the OXA sensitization + OXA challenge group is significantly increased, suggesting a significant increase in the number of apoptotic cells locally after model establishment.

[0038] In summary, the mouse oral lichen planus model established by the method of the present invention is highly consistent with human oral lichen planus in pathological characteristics, specifically manifested as the death of keratinocytes in the basal layer of the epithelium and the zonal lymphocyte infiltration near the basement membrane zone. In addition, the establishment of this model depends on cytotoxic CD8 + T cells and interferon-γ, which is consistent with the currently recognized pathogenesis of human lichen planus. Therefore, this mouse model can effectively simulate the core pathogenesis of human lichen planus.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. A method for modeling oral lichen planus in mice, characterized in that: The modeling method is as follows: The hair on the mouse abdomen was removed, and then oxazolidinone was applied to the depilated area. After 5 days, oxazolidinone was applied again to the oral mucosa of the mouse. The induction was completed after continuous treatment for 3-7 days.

2. A mouse oral lichen planus modeling method according to claim 1, characterized in that: The oxazolone applied to the hair removal area is an oxazolone solution with a mass concentration of 2-4%, and the oxazolone applied to the oral mucosa is an oxazolone solution with a mass concentration of 0.5-1.5%.

3. A mouse oral lichen planus modeling method according to claim 2, characterized in that: The mass concentration of the oxazolone solution when applied to the hair removal area is 3%, and the mass concentration when applied to the oral mucosa is 1%.

4. A mouse oral lichen planus modeling method according to claim 2, characterized in that: When the oxazolone solution is applied to the hair removal area, the usage amount is 80-150 μL, and when the oxazolone solution is applied to the oral mucosa, the usage amount is 10-20 μL.

5. A mouse oral lichen planus modeling method according to claim 3, characterized in that: When applying the oxazolidinone solution to the hair removal area, the amount used is 100 μL, and when applying it to the oral mucosa, the amount used is 15 μL.

6. A mouse oral lichen planus modeling method according to claim 1, characterized in that: The oral mucosa is specifically the inner lip mucosa.

7. A mouse oral lichen planus modeling method according to claim 1, characterized in that: The mice are selected to be 10-14 weeks old, free of specific pathogens, and of any gender.

8. A method for modeling oral lichen planus in mice according to claim 7, characterized in that: The mouse strain was selected as B57 or Balb / c.

9. A mouse oral lichen planus modeling method according to claim 8, characterized in that: Before modeling, the mice were raised according to a circadian rhythm of 12 hours of daylight and 12 hours of darkness per day, and the feed and drinking water were provided in an environment free of specific pathogens.

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