A method for constructing a vitiligo model and its application

Abstract

The present invention discloses a method for constructing a vitiligo model and its application. The invention belongs to the technical field of vitiligo. The method uses a 40% mass concentration of monobenzone cream to smear mice every day, and at the same time gives restraint stress for 18 hours every other day; the restraint stress time is from 16:00 to 10:00 the next day; the modeling time is 4 weeks. Compared with the prior art, the beneficial effect achieved by the present invention is that the present invention simulates "emotional frustration" through a mouse animal model with restraint stress load, which is closer to the clinical situation and provides a new basis for clinical research. Moreover, this model can be realized in ordinary mice, and has more reference basis.

Description

Technical Field

[0001] The present invention relates to the technical field of vitiligo, and more particularly to a construction method and application of a vitiligo model. Background Art

[0002] Vitiligo is an acquired localized or generalized skin depigmentation disease caused by the loss of melanocyte function or a decrease in the number of melanocytes. It is common in the head, face, front of the trunk, forearms and other parts of the body. It is a common skin disease in clinical practice. Its main feature is the occurrence of white spots on the skin or mucous membranes. It is easy to diagnose but difficult to treat, and has a high recurrence rate. According to the morphology, location, etiology, and development of the white spots, vitiligo can be divided into two types, two categories, and two stages, namely, common type and segmental type, complete white spots and incomplete white spots, progressive stage and stable stage. The common type is divided into localized, scattered, generalized, and acral. Because this disease affects the appearance of the skin, it often brings psychological pressure to patients, seriously affecting their social activities and quality of life.

[0003] At present, the pathogenesis of vitiligo has not been fully clarified. Modern medicine generally believes that the occurrence of vitiligo is mainly related to the body's autoimmune abnormalities, the melanocyte self-destruction hypothesis and the inflammatory theory. Some scholars believe that autoimmune abnormalities are an important factor in the pathogenesis of vitiligo, and the occurrence of vitiligo is mainly related to CD8 + T cells and CD4 + Related studies have shown that pathological sections of skin tissues of patients with vitiligo show that the number of melanocytes in the basal layer of the epidermis in and around the lesions is significantly reduced or disappeared, and CD8 + T cells increased significantly, CD4 + T cell reduction, CD4 + T / CD8 + The data showed that psychological stress increased the original CD8 + T cell levels, decreased CD4 + Therefore, psychological stress promotes CD8 T cells associated with vitiligo. + Increase of T cells.

[0004] The pathogenesis of vitiligo may be related to many factors, such as genetic background, stress, environment, innate immunity, adaptive immunity, and mental nerves, among which psychological stress is one of the more important factors. Psychological stress is extremely harmful. Statistical and clinical studies have shown that psychological stress can lead to CD8 + Cell increase, CD4 + The number of cells decreases, blood viscosity increases, and the immune system is stimulated. This shows that stress leads to CD8 +The increase of melanocytes can specifically kill melanocytes, which can accelerate the development of vitiligo. Other studies have shown that psychological stress events such as the death of family members, work and family economic problems are closely related to the onset of vitiligo, which will increase the levels of catecholamines, neuropeptides and cortisol in patients with vitiligo and accelerate the development of vitiligo.

[0005] In patients with vitiligo, multiple immunoregulatory cytokines, including macrophage inhibitory factor (MIF) and CD27, are increased. MIF is one of the multiple immunoregulatory cytokines secreted by T lymphocytes, macrophages and pituitary glands, and has been shown to play a key role in several types of immune and autoimmune diseases. MIF is stored in the cytoplasm in the form of vesicle-like structures and responds to lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, hypoxia, hydrogen peroxide, thrombin, thrombin and angiotensin II. Studies have shown that the expression of MIF levels in the serum of patients with common vitiligo is significantly increased, and the expression level of MIF in patients with progressive vitiligo is significantly higher than that in the stable period. In addition, macrophage migration inhibitory factor (MIF) has multiple effects on immune cells and has functions outside the immune system. MIF can promote inflammatory responses by inducing other cytokines, including cytokines of the TNF, IL-6 and IL-1 families.

[0006] Stress is a non-specific defensive adaptive response of the body to various stressors in the internal and external environment. The body can have a series of comprehensive responses including mental, nervous, endocrine and immune systems. Excessive or prolonged stress load can also lead to physiological dysfunction of the body and cause diseases. Human diseases are related to the activation of stress mechanisms. Emotional stress is a comprehensive reaction in the body caused by life events, social failures, tension, pressure, etc. In modern society, with the increase in population, competition and life pressure, emotional stress has an increasing impact on the occurrence and development of human diseases. Vitiligo is an autoimmune disease, and its pathogenesis includes genetic susceptibility theory, autoimmune theory, oxidative stress theory, melanocyte self-destruction, keratinocyte dysfunction theory and psychoneurological theory. WangG showed in a study on vitiligo and depression that among 1,965 vitiligo patients, about 1 / 3 of them had a tendency to depression. Compared with healthy people, the depression and symptoms of vitiligo patients were 4.96 times that of healthy people. Especially in people with darker skin, depression caused by vitiligo is more obvious. In a survey of vitiligo patients by Batty et al., 57% of patients believed that outsiders would stare at them, 20% were victims of rude remarks, and 8% encountered job discrimination. In fact, vitiligo causes more psychological pressure than any other skin disease. In a domestic study, Professor Li Ming et al. conducted a depression scale survey on vitiligo patients and found that the incidence of depression in vitiligo patients was higher than that of ordinary people, and there were stress effect hormones, that is, the serum cortisol (CORT) level of vitiligo patients was reduced, and the levels of the upstream stress-related hormones NPY and ACTH were increased, indicating that the patient's neuroendocrine pathway was abnormal and may play an important role in the pathogenesis of the disease.

[0007] Ancient Chinese medicine also called vitiligo "white spots" and "white spots". Tu Fuhan and others believe that the causes of vitiligo mainly include basic causes and predisposing factors. Basic causes are the root causes of vitiligo, mainly spleen and stomach weakness, kidney deficiency and blood weakness, and qi and blood disharmony. Predisposing factors are the causes of the disease or the factors that aggravate the disease, mainly including wind evil attacking from the outside, and the two together lead to skin malnutrition and the appearance of white spots. Chao Yunfang of the Sui Dynasty recorded in "Treatise on the Causes and Symptoms of Various Diseases" that "this is also caused by wind evil fighting in the skin and blood and qi disharmony"; Wang Qingren of the Qing Dynasty believed that the disease was caused by "blood stasis in the skin" and pioneered Tongqiao Huoxue Decoction to treat vitiligo. Professor Fu Kuixuan, a contemporary Chinese medicine expert, believes that the key pathogenesis of vitiligo is the local stagnation of qi and blood that leads to meridian obstruction, rather than the qi and blood disharmony caused by wind evil fighting on the surface. Liu Fang and others believe that vitiligo is the result of the combined effects of internal and external factors. The external factors are caused by wind evil, and the internal factors are caused by abnormal emotions, which injure the internal organs, liver qi stagnation, poor qi movement, spleen and stomach weakness, liver and kidney deficiency, etc., which lead to qi and blood imbalance, meridian blockage, and skin pores losing nourishment, resulting in white spots. The liver stores blood, is happy to be uplifted, and is responsible for dredging. If the emotions are not smooth, the liver qi will be stagnant, and the qi movement will not be smooth. In addition, the wind evil invades the skin, resulting in local skin qi and blood imbalance and white spots. Moreover, the emotional activities of the human body are closely related to the qi and blood of the internal organs. Abnormal emotional activities can affect the changes in the qi and blood of the corresponding internal organs, damage the internal organs, and cause the seven emotions to cause disease. The seven emotions often cause the qi and blood of the internal organs to be imbalanced and cause the disease; qi is the leader of blood, and blood is the mother of qi. The qi and blood of the internal organs are imbalanced, which affects the human blood, leading to qi and blood disharmony. About 2 / 3 of vitiligo patients have mental trauma or restlessness during the onset and development of skin lesions, which are "diseases caused by depression" or "depression caused by illness". Anger and depression can damage the liver, and liver qi can stagnate; fear and panic can damage the kidneys, and over time, it can deplete the essence and blood; these pathological changes can lead to disharmony between qi and blood in the skin, often manifested as qi stagnation and blood stasis or qi deficiency and blood stasis, and blood stasis in the muscles can cause the skin to lose its nourishment and become ill, as stated in "Medical Errors Correction" that "white spot wind, blood stasis in the skin". Clinically, vitiligo patients often show various abnormal emotional manifestations before the onset of the disease. Experimental studies by Qian Dong and others have also shown that when the same drug is used for treatment at the same time, supplemented by Chinese medicine emotional care plays a key role in the treatment of vitiligo. This shows that emotional stress plays an important role in the onset of vitiligo.

[0008] Most of the existing vitiligo animal models are single-factor pathological animal models, which are often modeled using chemical decolorization methods such as hydroquinone, hydrogen peroxide, and monobenzone cream decolorization. However, the modeling time is long and the efficacy is poor.

[0009] In summary, how to provide a method for constructing a vitiligo model is an urgent problem that technicians in this field need to solve. Summary of the invention

[0010] In view of this, the present invention provides a method for constructing a vitiligo model and its application. The present invention simulates "emotional frustration" through a mouse animal model with restraint stress load, and uses modern molecular biological methods to study the changes in various indicators in vitiligo induced by emotional stress, providing a new theoretical basis for clinical research on the pathogenesis of vitiligo aggravated by emotional stress.

[0011] In order to achieve the above object, the present invention adopts the following technical solution:

[0012] A method for constructing a vitiligo model, wherein mice are smeared with 40% monobenzone cream every day and restrained for 18 hours every other day;

[0013] The restraint stress time is from 16:00 to 10:00 the next day;

[0014] The modeling time was 4 weeks.

[0015] Furthermore, the specific operation of restraint stress is to put the mouse into a 50 ml centrifuge tube with air holes.

[0016] Furthermore, the specific operation of restraint stress is to use an electric soldering iron to iron a hole in the front and back of a 50ml centrifuge tube. The front is the breathing hole for the mouse, and the back is the position for the tail to stick out. A row of 3 holes are ironed on the side, and the 3 rows are the ventilation holes for the mouse.

[0017] Furthermore, 0.2 ml of 40% monobenzone cream was applied daily to a 2×2 cm shaved back of the mouse.

[0018] Furthermore, the mouse is a female 3-week-old SPFC57BL / 6 mouse.

[0019] Further, the preparation method of the Monobenzone cream is as follows:

[0020] Each 75g of Monobenzone cream is prepared by the following method:

[0021] The aqueous phase includes 6 ml of glycerol, 0.156 ml of triethanolamine, 0.0222 g of ethylparaben, 0.0222 g of ethylenediaminetetraacetic acid and 37.59 ml of water;

[0022] The oil phase includes 30 g of monobenzone, 0.9 g of stearic acid, 0.450 ml of laurocapram, 0.9 g of octadecyl alcohol and 3 g of liquid paraffin;

[0023] Heat the water and oil phases to 80°C, slowly add the water phase into the oil phase, and stir at 50°C until coagulation.

[0024] The vitiligo model was constructed using the above method.

[0025] The above-mentioned vitiligo model is used in screening drugs for preventing and / or treating vitiligo.

[0026] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects: the present invention simulates "emotional frustration" through a mouse model of restraint stress load, which is closer to the clinical situation and provides a new basis for clinical research. Moreover, this model can be realized in ordinary mice, which provides more reference basis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The accompanying drawing is a schematic diagram of restraint stress in mice in Example 1 of the present invention;

[0029] Figure 2 The accompanying drawings show the decolorization scores of different restraint stress times in Example 1 of the present invention, wherein A is 12 hours of stress every other day (20:00-8:00), for a total of 6 weeks, and B is 18 hours of stress every other day (16:00-10:00), for a total of 4 weeks; Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+CsA: monobenzone + restraint + CsA group;

[0030] Figure 3 The accompanying drawings are representative images of the back and their scoring results in Example 2 of the present invention, wherein A is a representative image of the back, B is a decolorization score, Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone+restraint group, Mon+RS+CsA: monobenzone+restraint+CsA group;

[0031] Figure 4 The attached figure shows the HE staining results in Example 2 of the present invention, Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+CsA: monobenzone + restraint + CsA group;

[0032] Figure 5 The accompanying drawings are the melanin staining results in Example 2 of the present invention, Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+CsA: monobenzone + restraint + CsA group;

[0033] Figure 6The accompanying drawings are the results of MIF immunofluorescence in Example 2 of the present invention, Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+CsA: monobenzone + restraint + CsA group;

[0034] Figure 7 The accompanying drawings show the test results of the MIF ELISA kit of the present invention, Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone+restraint group, Mon+RS+CsA: monobenzone+restraint+CsA group. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0037] Example 1

[0038] Restraint stress time optimization

[0039] Experimental Animals:

[0040] Female 3-week-old SPFC57BL / 6 mice.

[0041] Experimental groups:

[0042] They were divided into model group, control group and drug-treated group, with 8 mice in each group.

[0043] Experimental protocol:

[0044] The model group was treated with 0.2 ml of 40% monobenzone cream applied to 2×2 cm of shaved back every day; the control group was treated with blank cream applied to 2×2 cm of shaved back every day. Restraint stress was given every other day. The treatment group was subcutaneously injected with cyclosporine A (CsA, 12.5 mg / kg) every other day for 4 weeks. After the experiment, the mice were anesthetized with ether and killed for depigmentation evaluation.

[0045] The preparation method of Monobenzone cream is as follows:

[0046] Each 75g of Monobenzone cream is prepared by the following method:

[0047] The aqueous phase includes 6 ml of glycerol, 0.156 ml of triethanolamine, 0.0222 g of ethylparaben, 0.0222 g of ethylenediaminetetraacetic acid and 37.59 ml of water;

[0048] The oil phase includes 30 g of monobenzone, 0.9 g of stearic acid, 0.450 ml of laurocapram, 0.9 g of octadecyl alcohol and 3 g of liquid paraffin;

[0049] Heat the water and oil phases to 80°C, slowly add the water phase into the oil phase, and stir at 50°C until coagulation.

[0050] Restraint stress: Use an electric soldering iron to make a hole in the front and back of a 50ml centrifuge tube. The front is the breathing hole for the mouse, and the back is where the tail comes out. Make a row of 3 holes on the side. The 3 rows are the ventilation holes for the mouse. Figure 1 shown.

[0051] Restraint stress time:

[0052] 1. Stress for 12 hours every other day (20:00-8:00) for a total of 6 weeks.

[0053] 2. Stress for 18 hours every other day (16:00-10:00), for a total of 4 weeks.

[0054] 3. Daytime stress for 18 hours (6:00-24:00) for a total of 4 weeks.

[0055] Decolorization evaluation method:

[0056] Each exposure site was examined and the degree of depigmentation was estimated as a percentage of the anatomical site. Scoring was performed according to the following scheme: no signs of depigmentation (0%), 0 points; >5%, 1 point; >5-25%, 2 points; >25-50%, 3 points; >50-75%, 4 points; 75-100%, 5 points. The depigmentation score was defined as the sum of the scores.

[0057] The results are as follows Figure 2 shown.

[0058] First, choosing night stress disrupts the circadian rhythm of mice. Under daytime stress (6:00-24:00), mice are more active when they come out of the restraint tube (centrifuge tube) the next day, while mice under night stress are quieter when they come out of the restraint tube, so simulating emotional stress is more appropriate.

[0059] Depend on Figure 2 The results showed that stress for 18 hours every other day had a better effect on the establishment of the emotional stress vitiligo model, and the depigmentation score was more significant. ( ** P<0.01 compared with Con, ## P < 0.01 and #P<0.05 compared with the Mon group, ^^P<0.01 and ^P<0.05 compared with the Mon+RS group)

[0060] Example 2

[0061] Experimental Animals:

[0062] Female 3-week-old SPFC57BL / 6 mice.

[0063] Experimental groups:

[0064] They were divided into model group, control group and drug-treated group, with 8 mice in each group.

[0065] Experimental protocol:

[0066] The model group was treated with 0.2 ml of 40% monobenzone cream applied to 2×2 cm shaved back every day; the control group was treated with blank cream applied to 2×2 cm shaved back every day for 4 weeks. Restraint stress was given for 18 hours (16:00-10:00) every other day for a total of 4 weeks. The drug group was subcutaneously injected with cyclosporine A (CsA, 12.5 mg / kg) every other day for a total of 4 weeks. After the experiment, the mice were anesthetized with ether and killed, and the skin tissue and blood of the mice were collected.

[0067] Restraint stress: Use an electric soldering iron to make a hole in the front and back of a 50ml centrifuge tube. The front is the breathing hole for the mouse, and the back is where the tail comes out. Also, make a row of 3 holes on the side. The 3 rows of holes are the ventilation holes for the mouse.

[0068] The detection indicators and methods are as follows:

[0069] 1. Skin lesions: evaluation of depigmentation. Each exposed site was examined and the degree of depigmentation was estimated as a percentage of the anatomical site. Scoring was based on the following scheme: no signs of depigmentation (0%), 0 points; >5%, 1 point; >5-25%, 2 points; >25-50%, 3 points; >50-75%, 4 points; 75-100%, 5 points. The depigmentation score was defined as the sum of the scores.

[0070] 2. HE staining: Take out the skin tissue sample and fix it in 4% paraformaldehyde. Then embed and slice it. Put the cut paraffin slices into xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% alcohol for 5 minutes, 50% alcohol for 5 minutes, and 25% alcohol for 5 minutes. Wash it with distilled water for 3 to 5 times to dewax it. Then stain it with hematoxylin and eosin and seal it with neutral gum. Check and measure the basal layer, cortical thickness, cell morphology, etc. under a fluorescent inverted microscope.

[0071] 3. Melanin staining: Put the cut paraffin sections into xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% alcohol for 5 minutes, 50% alcohol for 5 minutes, and 25% alcohol for 5 minutes, and then rinse with distilled water for 3 to 5 times for dewaxing. Then immerse the sections in the melanin working solution and cover them, place them at 4℃ in the dark for 12 to 18 hours, and then rinse with distilled water for 3 times. (The melanin working solution is prepared by taking 10 ml of melanin dye solution A, adding concentrated ammonia water drop by drop until precipitation occurs, and then continue to add ammonia water, shaking while dropping, so that the precipitation produced will be dissolved again and the solution will become clear. Then add a few drops of melanin dye solution A to make the solution slightly turbid, and finally add 20 ml of distilled water, and filter it before use.) Then re-stain VG (9 ml of melanin dye solution B is added to 1 ml of melanin dye solution C to mix into VG dye solution), dye for 1 minute, quickly wash with water, and quickly dehydrate with three cylinders of anhydrous ethanol for 3s, 5s, and 30s. Then transparently seal the slides, make two cylinders of clean xylene transparent for 5 minutes each, and wet-seal with neutral gum. Microscope examination, and image acquisition and analysis.

[0072] 4. Immunofluorescence staining: Put the cut paraffin sections into xylene I for 20 min-xylene II for 20 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min-50% alcohol for 5 min-25% alcohol for 5 min, and wash with distilled water for 3 to 5 times for dewaxing. After antigen repair and membrane permeabilization, add 3% hydrogen peroxide to treat catalase in the tissue, and finally block with goat serum. Then, add primary antibody MIF and other antibodies and incubate at 4°C overnight (antibody concentration 1:200). After the target section is washed with TBST three times, AlexaFluorTM488goatanti-rabbitlgG (H+L) and AlexaFluorTM555goatanti-mouselgG (H+L) fluorescent secondary antibodies (1:1000) are added to the target tissue and incubated at 37°C in the dark for 60 min. Subsequently, each target tissue is washed three times with PBS, each time for 5 min. The target tissue was stained with DAPI (1:1000) at 37°C in the dark for 10 minutes. Similarly, the target tissue was washed three times with PBS, 5 minutes each time. Finally, each target slice was sealed with anti-fluorescence quenching sealing agent, and the slice was kept away from light and dried. The tissue staining was recorded using a laser confocal imaging system. During the operation, consecutive slices in the same slice were taken as background controls, and the controls were incubated with goat serum without primary antibody when incubating the primary antibody.

[0073] 5. MIF kit: Set up standard wells and sample wells to be tested, and add 50μL of standard of different concentrations to each standard well. Set up blank control wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and sample wells to be tested. First add 40μL of sample diluent to the sample wells to be tested on the enzyme-labeled plate, and then add 10μL of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix. Add 100μL of enzyme-labeled reagent to each well, except the blank control well. Seal the plate with a sealing film and incubate at 37℃ for 60 minutes. Then dilute the 20-fold concentrated washing solution with 20-fold distilled water for use. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and discard, repeat this 5 times, and pat dry. First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color at 37℃ in the dark for 15 minutes. Add 50μL of stop solution to each well to stop the reaction (the blue color turns yellow immediately). Use the blank control well as zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0074] The results are as follows Figures 3 to 7 shown.

[0075] Figure 3 The following is a representative picture of the back and its scoring results: The Mon group showed hair depigmentation on the back compared to the Con group. After restraint, the hair depigmentation on the back of the Mon+RS group worsened. Then, CsA (12.5 mg / kg) was subcutaneously injected on this basis, and the hair depigmentation on the back of the Mon+RS+CsA group improved. The back score was performed in the fourth week after administration, and statistics were collected. ( ** P<0.01 compared with Con, ## P<0.01 compared with the Mon group,^^P<0.01 compared with the Mon+RS group)

[0076] Figure 4 HE staining results: Compared with the Con group, the Mon group had inflammatory cell infiltration. After restraint, the inflammatory cell infiltration in the Mon+RS group was aggravated, while the inflammatory cell infiltration in the Mon+RS+CsA group was improved.

[0077] Figure 5 Melanin staining results: Compared with the Con group, the Mon group had epidermal melanin loss. After restraint, the epidermal melanin loss in the Mon+RS group was aggravated, while the epidermal melanin loss in the Mon+RS+CsA group was improved.

[0078] Figure 6The results of MIF immunofluorescence were as follows: in the epidermis of the skin, the positive expression of MIF in the Mon group was higher than that in the Con group. After restraint, the positive expression of MIF in the Mon+RS group was aggravated, while the positive expression of MIF in the Mon+RS+CsA group was reduced.

[0079] Figure 7 The results of the MIF ELISA kit test: In the serum, the MIF expression in the Mon group increased compared with the Con group. After restraint, the MIF expression in the Mon+RS group increased, and the MIF expression in the Mon+RS+CsA group decreased. ( * P<0.05 compared with Con, # P<0.05 compared with the Mon group,^^P<0.01 compared with the Mon+RS group)

[0080] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0081] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a vitiligo model. It is characterized in that Mice were treated with 40% monobenzone cream every day and restrained for 18 h every other day. The restraint stress time is from 16:00 to 10:00 the next day; The modeling time was 4 weeks; The specific operation of restraint stress is to put the mouse into a 50 ml centrifuge tube with air holes.

2. A method for constructing a vitiligo model as claimed in claim 1, It is characterized in that Apply 0.2 ml of 40% monobenzone cream daily to a 2 × 2 cm area of ​​the shaved back of the mouse.

3. The method for constructing a vitiligo model according to claim 1, It is characterized in that The mice were female 3-week-old SPF C57BL / 6 mice.

4. The method for constructing a vitiligo model according to claim 1, It is characterized in that The preparation method of the Monobenzone cream is as follows: Each 75g of Monobenzone cream is prepared by the following method: The aqueous phase includes 6 ml of glycerol, 0.156 ml of triethanolamine, 0.0222 g of ethylparaben, 0.0222 g of ethylenediaminetetraacetic acid and 37.59 ml of water; The oil phase includes 30 g of monobenzone, 0.9 g of stearic acid, 0.450 ml of laurocapram, 0.9 g of octadecyl alcohol and 3 g of liquid paraffin; Heat the water and oil phases to 80°C, slowly add the water phase into the oil phase, and stir at 50°C until coagulation.

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