A method for constructing an animal inflammatory model of psoriasis with different lesion severity

Abstract

The present invention discloses a method for constructing an animal inflammatory model of psoriasis with different severities of skin lesions, comprising the following steps: raising 30 Balb / C mice according to a standard feeding method; weighing and calculating the body surface area of ​​each mouse at baseline D0, and removing the hair of the mice; randomly dividing the 30 Balb / C mice into three groups according to the lesion area, namely a mild group, a moderate group and a severe group; using 5 mg / cm 2 Imiquimod was evenly applied to the lesion area of ​​each mouse; the mice were killed after 7 days of intervention, and the skin tissue and spleen of the mice were taken for subsequent examination. The changes in the skin lesions of the mice were recorded by taking photos with a camera. After 7 days of treatment with imiquimod cream, typical psoriasis-like lesions appeared. The constructed psoriasis animal model has very similar clinical symptoms to psoriasis patients, can show psoriasis-related histological changes and immune system changes, and can be used to screen psoriasis treatment drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and in particular to a method for constructing animal inflammation models of psoriasis with different skin lesion severity. Background Art

[0002] The incidence of psoriasis accounts for about 2-3% of the world's population, while the incidence in China is 0.47%. Psoriasis is an immune-mediated systemic inflammatory disease, which is associated with metabolic diseases, coronary atherosclerotic heart disease, depression and anxiety. According to the severity of its skin lesions, it is divided into mild, moderate and severe. It is very important to assess the severity of psoriasis in patients with psoriasis. The severity of skin lesions is closely related to the recurrence, prognosis and occurrence and development of comorbidities of the disease. And doctors can choose treatment methods according to the severity of psoriasis lesions, such as local treatment, systemic medication, or combined treatment; currently, the treatment of mild patients mostly adopts local treatment, while patients with "moderate to severe psoriasis" are mostly recommended to adopt systemic treatment; the treatment effect is evaluated according to the changes in the severity of skin lesions. Psoriasis severity assessment also enables researchers to compare different treatments for similar patients. Therefore, in order to further clarify the correlation between the severity of psoriasis lesions and the prognosis of the disease, better and more precise treatment, and compare the advantages and disadvantages of different intervention methods, but because human bodies cannot be directly used for experiments, it is urgent to construct animal models of psoriasis with different severities.

[0003] At present, psoriasis models are mostly focused on the construction of acute or chronic psoriasis inflammation models, or the construction of acute and chronic models of mice lacking related genes. These models are animal models with consistent severity, and cannot fully simulate the clinical reality that the severity of psoriasis lesions varies among different patients in the population or in different stages of the same patient. Therefore, the current methods for constructing psoriasis animal models are insufficient and difficult to meet relevant needs. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing an animal inflammatory model of psoriasis with different degrees of skin lesion severity, and a camera is used to record the changes in mouse skin lesions, and typical psoriasis-like lesions appear; the constructed psoriasis animal model has very similar clinical symptoms to psoriasis patients, can show psoriasis-related histological changes and immune system changes, and can be used for screening psoriasis therapeutic drugs.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A method for constructing an animal inflammatory model of psoriasis with different skin lesion severity comprises the following steps:

[0007] (1) Thirty Balb / C mice were raised according to standard feeding methods for one week;

[0008] (2) At baseline D0, weigh and calculate the body surface area of ​​each mouse, and remove the hair of the mice;

[0009] (3) Thirty Balb / C mice were randomly divided into three groups according to the lesion area: mild group, moderate group, and severe group;

[0010] (4) Using 5mg / cm 2 Imiquimod was evenly applied on the lesion area of ​​each mouse;

[0011] (5) Seven days after intervention, the mice were killed and their skin tissues and spleens were obtained for subsequent testing.

[0012] Further, in step S3, the administration area is calculated according to the BSA standard: 2 cm for the mild group 2 , moderate group 7cm 2 , severe group: 15.75cm 2 .

[0013] Further, in step S4, the mild group was topically treated with 10 mg of imiquimod, the moderate group was topically treated with 35 mg of imiquimod, and the severe group was topically treated with 78.75 mg of imiquimod.

[0014] Furthermore, the method further comprises step S6, wherein the severity of the skin lesions of mice is evaluated using a modified psoriasis lesion area and severity index mPASI scoring standard, and an mPASI score is given.

[0015] Furthermore, the mice were housed in a ventilated environment with a constant temperature of 22°C and a constant humidity of 50%-70%, with a photoperiod of 12 h dark and 12 h light cycle, and were fed water and standard animal feed ad libitum.

[0016] Furthermore, the mice are fed for more than 8 weeks.

[0017] The beneficial effect of the present invention is that the changes of skin lesions of mice were recorded by a camera photographing method, and typical psoriasis-like skin lesions appeared after 7 days of treatment with imiquimod cream, such as increased skin thickness, obvious erythema, and silvery white scales on the erythema. As the dose of imiquimod increases, the characteristics of psoriasis-like skin lesions become more obvious.

[0018] The constructed psoriasis animal model has clinical symptoms that are very similar to those of psoriasis patients and can demonstrate psoriasis-related histological changes and immune system changes. It can not only be used to screen psoriasis therapeutic drugs, but also provide a good platform for studying the occurrence and development mechanism of psoriasis. The modeling method is simple, convenient and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 A technical route for preparing a severity-grading inflammatory model for psoriasis;

[0021] Figure 2 Schematic diagram of psoriasis-like lesions of different severity;

[0022] Figure 3 This is a schematic diagram of PASI scoring for psoriasis mice;

[0023] Figure 4 This is a schematic diagram of the weight changes in psoriasis mice;

[0024] Figure 5 Schematic diagram of psoriasis-like mouse pathology and epidermal thickness assessment;

[0025] Figure 6 This is a schematic diagram of the changes in the spleen and spleen index. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0027] In the description of the present invention, it is necessary to understand that the terms "upper", "lower", "front", "back", "left", "right", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0028] See attached Figure 1-Figure 6 , according to the method for constructing psoriasis animal inflammation models with different skin lesion severity of the embodiment of the present invention, comprising the following steps:

[0029] (1) Thirty Balb / C mice were raised according to standard feeding methods for one week;

[0030] (2) At baseline D0, weigh and calculate the body surface area of ​​each mouse, and remove the hair of the mice;

[0031] (3) Thirty Balb / C mice were randomly divided into three groups according to the lesion area: mild group, moderate group, and severe group;

[0032] (4) Using 5mg / cm 2 Imiquimod was evenly applied on the lesion area of ​​each mouse;

[0033] (5) Seven days after intervention, the mice were killed and their skin tissues and spleens were obtained for subsequent testing.

[0034] The mice were weighed at baseline D0, and the body surface area of ​​each mouse was calculated. The hair of the mice was removed, and the administration area was calculated according to the BSA score standard: the control group was not treated, and the mild group was 2 cm 2 , moderate group: 7cm 2 , severe group: 15.75cm 2 . And 5 mg of imiquimod cream was administered per square centimeter. Specifically, the control group was not treated, the mild group was treated with 10 mg of imiquimod cream, the moderate group was treated with 35 mg of imiquimod cream, and the severe group was treated with 78.75 mg of imiquimod cream.

[0035] Among them, the classification standards of mild, moderate and severe refer to the BSA score of psoriasis: the percentage of psoriasis lesions to body surface area (less than 3% is mild, 3-10% is moderate, and greater than 10% is severe). The body surface area of ​​mice is calculated according to the MeeHRubner formula: S=K*2W / 3. S: body surface area of ​​mice, in cm2, K: is a constant, the value of mice is 9.1, and W: weight of mice, in g.

[0036] Experimental Example 1, Model Evaluation - Skin Lesions

[0037] like Figure 2 As shown in the figure, the changes of skin lesions in mice were recorded by camera photography. After 7 days of treatment with imiquimod cream, typical psoriasis-like lesions appeared, such as increased skin thickness, obvious erythema, and silvery white scales on the erythema. With the increase of imiquimod dose, the characteristics of psoriasis-like lesions became more obvious.

[0038] According to the modified psoriasis-like lesion area and severity index (mPASI) scoring standard, the erythema, scaling and infiltration thickening of the mouse lesions were scored, and the three scores were added together to obtain the total score. PASI scoring standard: 0 points, none; 1 point, mild; 2 points, moderate; 3 points, severe; 4 points, extremely severe.

[0039] After scoring, the experimental results are as follows Figure 3 As shown in the figure, when the total mPASI score is greater than 3 points, psoriasis-like lesions are considered to have appeared. The higher the dose of imiquimod cream, the more severe the psoriasis-like lesions are and the earlier they enter the plateau phase.

[0040] Experimental Example 2, Model Evaluation-Weight

[0041] Weigh the mice on D0 and D7, and calculate the weight change rate of mice = (W7-W0) / W0. Figure 4 As shown, the results showed that after the application of imiquimod, the body weight C of mice fluctuated, and the overall trend was negatively correlated with the dose of imiquimod applied.

[0042] Experimental Example 3, Model Evaluation - Pathology

[0043] Paraffin sections were made from the skin lesions on the back of mice and HE staining was performed. The thickness of the epidermis was evaluated using ImageJ software P. The results are shown in Figure 2. Figure 5 The results showed that after 7 days of imiquimod application, mice developed some typical pathological features of psoriasis, such as hyperkeratosis, parakeratosis, epidermal thickening, finger-like epidermal protrusions into the dermis, increased and dilated dermal capillaries, and dermal inflammatory cell infiltration; after 7 days of topical application of imiquimod cream, the epidermis of mice was significantly thickened, and the overall trend was positively correlated with the dose of imiquimod applied.

[0044] Experimental Example 4, Model Evaluation - Spleen

[0045] Weigh the weight of mice on day 7 W7 and spleen weight Wspleen, and calculate the small spleen index = Wspleen / W7*10. The spleen is the largest secondary lymphoid organ in the immune system, and splenomegaly represents significant systemic immune activation. Changes in mouse spleen and spleen index are as follows Figure 6 The results showed that in the imiquimod-induced psoriasis mouse model, the spleen and spleen index had no significant correlation with the imiquimod dose.

[0046] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for constructing an animal inflammatory model of psoriasis with different lesion severity, comprising the following steps: (1) Thirty Balb / C mice were raised according to standard feeding methods for one week; (2) At baseline D0, weigh and calculate the body surface area of ​​each mouse, and remove the hair of the mice; (3) Thirty Balb / C mice were randomly divided into three groups according to the lesion area: mild group, moderate group, and severe group; (4) Using 5mg / cm 2 Imiquimod cream was evenly applied on the lesion area of ​​each mouse; (5) Seven days after intervention, the mice were killed and their skin tissues and spleens were taken for subsequent examination; In step S3, the administration area is calculated according to the BSA standard: 2 cm for the mild group 2 , moderate group: 7cm 2 , severe group: 15.75cm 2 ; In step S4, the mild group was topically applied with 10 mg of imiquimod cream, the moderate group was topically applied with 35 mg of imiquimod cream, and the severe group was topically applied with 78.75 mg of imiquimod cream.

2. The method for constructing an animal inflammatory model of psoriasis with different skin lesion severity according to claim 1, characterized in that: The method further comprises step S6, wherein a modified psoriasis lesion area and severity index mPASI scoring standard is used to evaluate the severity of the skin lesions of mice and an mPASI score is given.

3. The method for constructing an animal inflammatory model of psoriasis with different skin lesion severity according to claim 1, characterized in that: The mice were housed in a ventilated environment with a constant temperature of 22°C and a constant humidity of 50%-70%, with a photoperiod of 12 h dark and 12 h light cycle, and were fed water and standard animal chow ad libitum.

4. The method for constructing an animal inflammatory model of psoriasis with different skin lesion severity according to claim 1, characterized in that: The mice were fed for more than 8 weeks.

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