A mouse model of vitiligo induced based on chronic stress factors

Abstract

The application discloses a mouse vitiligo induction model based on chronic stress factors, wherein different kinds of stress factors capable of triggering stress response of animals are applied to stimulate the mouse periodically within a certain time period, so as to prevent the mouse from producing habitual response to the stress source and to promote the mouse to become white and the tail epidermis to be flaky. The technical method is different from the existing vitiligo model method, is based on the mechanism of occurrence and development of vitiligo observed in the clinic, and promotes the mouse to truly develop the vitiligo phenomenon from the whole instead of simulating the disease evolution locally. Compared with the existing vitiligo promoting mechanism, the effect is more stable and the authenticity is higher. The technical method is based on the objective fact that the clinical vitiligo patients often combine anxiety and depression to design the stress factors, and considers that the stress factors and the occurrence and development of vitiligo may be inter-caused and viciously circulate, namely, the adverse life events as the stress factors cause the damage and dysfunction of the melanin synthesis system at the micro level and form the vitiligo change at the macro level.

Description

TECHNICAL FIELD

[0001] The present application relates to a model of vitiligo, in particular to a mouse model of vitiligo induced by chronic stress factors. BACKGROUND

[0002] The existing model mice induced by vitiligo are often induced by chemical depigmentation agents, exogenous T cells or transgenic mice.

[0003] Specifically, the existing construction methods have the following disadvantages:

[0004] Chemical depigmentation agents often use 40% monobenzone, hydrogen peroxide, hydroquinone, etc., although the modeling cost is low and the operation is simple, but the pigment loss of modeling is not stable, once it is separated from the depigmentation site of the external preparation, it is replaced by new melanin. And this induction method is limited to local skin pigment loss, cannot fully simulate the pathological changes of autoimmune diseases of vitiligo, and cannot present the pathological changes of vitiligo as an autoimmune disease.

[0005] Exogenous T cell induction needs to be inoculated with B16F10 melanoma cells in advance, then injected with anti-CD4 antibody to deplete Treg cells, activate autoreactive CD8+ T cells to kill epidermal melanocytes, and finally resect the melanoma. Although this method better simulates the characteristics of autoimmune diseases, it has high technical operation requirements, complex modeling method and unstable pigment loss.

[0006] Transgenic mice use genetic engineering technology to construct mice with specific gene mutations or express specific proteins. Although it can simulate the genetic and immune mechanisms of vitiligo, it has the disadvantages of high modeling difficulty, high failure rate and high cost.

[0007] Secondly, whether it is T cell depletion caused by melanoma cells or transgenic mice, it is directly interfering with the whole pathway of melanin synthesis. The above models often appear whole body hair whitening and tail epidermal patchy depigmentation, which is more similar to albinism changes, and does not conform to the local depigmentation performance of clinical vitiligo patients, and vitiligo is not simply a genetic disease. SUMMARY

[0008] The purpose of the present application is to solve the problems in the prior art, and to provide a mouse model of vitiligo induced by chronic stress factors, which is simple and stable.

[0009] To solve the above technical problems, the technical method adopted by the present application is: the present application discloses a mouse model of vitiligo induced by chronic stress factors. In a certain period of time, different types of stress factors that can cause stress reactions of animals are applied to stimulate mice periodically, prevent the mice from developing habitual reactions to the stress source, and promote the mice to turn white and the tail epidermis to be patchy depigmented.

[0010] Further, comprising the following steps:

[0011] S1. Set a plurality of stress factors capable of triggering stress response in animals;

[0012] S2. Randomly select 3-4 stress factors to stimulate mice every day;

[0013] S3. Select 5 days a week, repeat step S2, ensure that the stress source appears in random order and at least interval of one day, prevent the mice from habituation;

[0014] S4. Continue for N weeks to complete the modeling, N is greater than 8 weeks.

[0015] Further, the stress factors include environmental stimuli, physiological stimuli and sensory stimuli;

[0016] The environmental stimuli include:

[0017] Exchange cage method, exchange the cages of two mice but not the bedding, and put them back in the original cage after 24 hours;

[0018] Inclined cage method, place the cage on a fixed triangular iron stand inclined at 45 degrees for 6 hours;

[0019] Shaking cage method, tilt the cage to the left and right by 45 degrees with the center as the axis, one back and forth for every 1 second, for 20 minutes;

[0020] Wet cage method, pour 200 milliliters of pure water evenly into each cage to make the bedding completely wet, and replace the new dry bedding after 24 hours;

[0021] Pulsed flashing light method, select a spontaneous pulsed flashing light source, flash 5 times per second, maintain a dark environment, and last for 1 hour;

[0022] Water bath method, pour 300 milliliters of pure water evenly into each empty cage, without bedding, and put the mice back in the original cage after 1 hour;

[0023] The physiological stimuli include: restraint method, take a clean 50ml centrifuge tube, use an electric iron to make several small holes as the mouse's breathing holes, place the mouse's head on the conical side of the centrifuge tube, and place it vertically for 4 hours;

[0024] Cold water swimming method, slowly put the mouse into 2 liters of 4℃ cold water, place another empty cage on the surface to prevent escape, take it out after 5 minutes, and completely dry the water;

[0025] Tail suspension method, suspend the mouse's tail to 20cm above the ground, and last for 5 minutes;

[0026] The sensory stimulation method includes: a predator sound stimulation method, selecting a sound file of a mouse natural enemy to stimulate at 80 decibels, with a 90-second cycle, for 1 hour;

[0027] A predator smell stimulation method, selecting a smell analog of a mouse natural enemy to stimulate the innate fear / defense behavior of a laboratory mouse, i.e., the instinctive freezing response; 20ul of TMT is dropped into a cotton swab head in each cage using a 1ml syringe, and the cotton swab is removed after 20 minutes of sealing.

[0028] Further, in the step S2, 1-2 kinds of stress factors are randomly selected from environmental stimulation, physiological stimulation and sensory stimulation to stimulate the mouse every day, and the total number of stimulations does not exceed 4.

[0029] Further, the calls of various owls are selected as the calls of the mouse natural enemy.

[0030] Further, the fox smell analog 2,4,5-trimethyl-3-thiazoline TMT is selected as the smell analog of the mouse natural enemy.

[0031] Further, the modeling is continued for 12 weeks.

[0032] Further, the mouse is a male mouse.

[0033] Further, the mouse is a five-week-old mouse. Beneficial effects

[0034] 1. The present technical method is different from the existing vitiligo model method, based on the mechanism of the development of vitiligo observed in the clinic, starting from the whole rather than simulating the evolution of the disease locally, and prompting the mouse to truly develop vitiligo phenomenon. Compared with the existing vitiligo promotion mechanism, the effect is more stable and the authenticity is higher.

[0035] 2. The present technical method is based on the objective fact that clinical vitiligo patients often combine anxiety and depression to design stress factors, considering that stress factors and vitiligo occurrence and development may be mutually causal and vicious, i.e. adverse life events as stress factors cause damage and dysfunction of the melanin synthesis system at the micro level, and form vitiligo changes at the macro level. Such changes in skin color that are different from ordinary people become new stress factors that increase the psychological burden of patients, leading to continuous or escalating anxiety and depression, and ultimately promoting the occurrence of vitiligo. Therefore, this method innovatively uses mental and psychological stress factors to induce such vitiligo model, abandoning the existing conventional drug local induction, which can make the mouse model more realistic simulation of clinical vitiligo patients with adverse life events. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1The mouse tail skin photo under the wood lamp of the control group and the model group in the embodiment of the present application;

[0037] Figure 2 The mouse back skin photo under the natural light and the wood lamp of the white hair part of the model group mouse in the embodiment of the present application;

[0038] Figure 3 The blood analysis table of the reference group and the control group mouse in the embodiment of the present application;

[0039] Figure 4 The Masson-Fontana melanin staining pathological section of the reference group and the control group mouse in the embodiment of the present application. DETAILED DESCRIPTION

[0040] The present application is described in further detail below in connection with the description of the drawings and the specific embodiments.

[0041] Experimental animals: clean level C57BL / 6 mice, male, 5 weeks old, 18-20g per mouse, purchased from Nanjing Medical University Animal Experimental Center. The animals are randomly divided into model group and control group, of which 20 are in the model group and 10 are in the normal control group, and both are subjected to depilation treatment. The experimental animals are fed in separate cages, 5 per cage.

[0042] Basic feed bedding: the mouse feed bedding is provided by Nanjing Medical University Animal Experimental Center.

[0043] Mouse treatment method: 11 kinds of stress factors are formulated for the model group mice, specifically:

[0044] Exchange cage method: exchange the cages of two mice but not the bedding, and then put them back into the original cages after 24 hours;

[0045] Inclined cage method: place the cage on a fixed triangular iron stand inclined at 45 degrees for 6 hours;

[0046] Shaking cage method: tilt the cage to the left and right by 45 degrees with the center as the axis, one way for every 1 second, for 20 minutes;

[0047] Moisture cage method: pour 200 milliliters of pure water evenly into each cage to completely wet the bedding, and replace the new dry bedding after 24 hours;

[0048] Binding method: take a clean 50ml centrifuge tube and use an electric iron to make several small holes on the conical side as the breathing hole of the mouse, place the mouse's head on the conical side of the centrifuge tube, and place it vertically for 4 hours;

[0049] Pulsed flickering light method: select a spontaneous pulsed flickering light source, flicker 5 times per second, maintain a dark environment, and last for 1 hour;

[0050] Water bath method, evenly pour 300 milliliters of pure water into each empty cage, no padding, after 1 hour, put the mice back into the original cage;

[0051] Restraint method, take a clean 50ml centrifuge tube, use an electric iron to make several small holes on the conical side as the mouse's breathing holes, place the mouse's head on the conical side of the centrifuge tube, and place it vertically for 4 hours;

[0052] Cold water swimming method, slowly put the mouse into 2 liters of 4°C cold water, place another empty cage on the surface to prevent escape, continue for 5 minutes, then take out and dry completely;

[0053] Tail suspension method, suspend the mouse tail at a height of 20 cm from the ground, and continue for 5 minutes;

[0054] Predator sound stimulation method, select various owl sound files to the player, 80 decibels, cycle every 90 seconds, for 1 hour;

[0055] Predator odor stimulation method, after pre-experiment, finally select fox odor analog 2,4,5-trimethyl-3-thiazoline TMT, which is used to arouse the innate fear / defense behavior of laboratory mice, i.e. the instinctive freezing reaction; use a 1ml syringe to take 20ul TMT to the cotton swab head in each cage, seal for 20 minutes, then take out the cotton swab.

[0056] Randomly select 3-4 kinds of stressors every day (if you choose a 24-hour stressor, only perform this stress operation on the same day). Stress operation is performed for 5 days a week, ensuring that the order of stressors appears randomly and at least one day apart to prevent habituation of the mice. The modeling ends after 12 weeks. The experimental results are as follows:

[0057] Time (weeks) Control group 1 Control group 2 Experimental group 1 Experimental group 2 Experimental group 3 Experimental group 4 Number of white mice 1 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 4 0 0 1 0 1 0 2 5 0 0 1 0 1 0 2 6 0 0 1 0 1 1 3 7 0 0 2 1 1 1 5 8 0 0 2 1 1 1 5 9 0 0 2 1 1 2 6 10 0 0 3 1 1 2 7 11 0 0 3 2 2 2 9 12 0 0 3 2 3 3 11

[0058] Through the above experiments, it is found that the probability of white hair in the experimental group mice is 55%. In this experiment, 5-week-old male mice were selected to exclude the influence of female hormones and adapt to the environment for 1 week. After 6 weeks, the male mice matured and the modeling began.

[0059] To verify that the white mouse is a symptom of vitiligo, the following verification is performed:

[0060] Use a Canon 30D camera to take general photos, and use a handheld dermatoscope (3Gen DermLite DL4) to take photos of the mouse's back and tail skin under natural light and under a wood lamp.

[0061] As shown in Figure 1 and 2 , compared with the blank control group mice, after 12 weeks of experiment, the back of the modeling group mice appeared clusters of white hair, and the tail appeared patchy pigment loss.

[0062] Masson-Fontana melanin staining of the mouse tail skin showed that the number of epidermal melanin granules in the model group was significantly reduced.

[0063] In summary, this method can indeed cause pigment loss in mice from both macroscopic and microscopic perspectives.

[0064] As shown in Figure 3 , blood analysis of white spot related cytokines was performed on the reference and control mice. The Serum IL-6 (serum interleukin-6), Serum IFNγ (serum interferon gamma), and Serum CXCL10 (serum chemotactic cytokine 10) levels in the control mice were much higher than those in the control group, further confirming that the experimental mice indeed had symptoms of white spot.

[0065] As shown in Figure 4 , Figure 4 The Masson-Fontana melanin staining pathology of the left control group and the right experimental group can be seen. The number of melanin granules from the basal layer to the keratin layer in the experimental group was significantly reduced (Fontana-Masson staining ×400 magnification), further confirming that the experimental mice indeed had symptoms of white spot.

[0066] Theoretical analysis of white spot:

[0067] Neuroendocrine dysfunction is now considered one of the potential mechanisms of neurospsychological factors in the pathogenesis of white spot. In clinical diagnosis and treatment, it is found that the occurrence and development of skin lesions in white spot patients are often related to recent stressful life events. Similarly, more and more studies have shown that white spot patients have early stress events and specific personality characteristics.

[0068] Jianzhe et al. investigated 10,000 cases of white spot patients and found that 2,133 patients (21.33%) believed that there were inducing factors for the onset, including: 628 cases of trauma (29.44%); 471 cases of mental stress (22.08%); 379 cases of mental depression (17.77%); 357 cases of fatigue (16.74%); 221 cases of sun exposure (10.36%); 73 cases of chemical exposure (3.42%); and 101 patients mentioned three or more inducing factors.

[0069] Hu Xuehui et al. conducted a study including 137 cases of white spot patients and 1,388 normal adult normative subjects, and measured their basic information, mental health, and personality characteristics.

[0070] The personality characteristics of the vitiligo patients can be summarized as follows: the mental health status of the vitiligo patients is generally poor; the mental health status is closely related to the disease progression and the area of the white spots; compared with the patients in the stable period, the patients in the active period have more obvious changes in negative emotions such as depression, anxiety, obsessive-compulsive symptoms, hostility, terror, paranoia and psychosis; in interpersonal communication, they are more likely to have large emotional fluctuations, have a strong desire for stimulation and adventure, and are difficult to adapt to the external environment.

[0071] Stress refers to a state in which a human being consumes physiological and psychological energy in a challenging or fearful environment, and is a process in which the body produces corresponding reactions through various adaptive mechanisms when the dynamic balance of the physiological state is disturbed by external or internal stressors.

[0072] After stress is perceived by the human body and transmitted to the central nervous system, it activates the brain to release biological signals, thereby affecting the health of multiple organs such as the cardiovascular system, the nervous system, and the digestive system. Among them, the skin, as the largest organ of the human body, has the ability to synthesize and transport melanin, thereby protecting the human body from ultraviolet damage. Similarly, the skin can also release a variety of neurotransmitters, and the surface of each layer of cells in the dermis also has a large number of corresponding neurotransmitter receptors distributed.

[0073] Therefore, if the mental stress is too great, it can cause changes in hormones and neurotransmitters, which in turn can damage the normal pigment metabolism of the skin to varying degrees. For this reason, it can also induce the occurrence of skin pigment diseases such as vitiligo in susceptible populations.

[0074] Based on the above theory, it can also be proved that the present application conforms to the objective law and has repeatability.

[0075] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A mouse vitiligo induction method based on chronic stress factors, characterized by, Within a certain period of time, without using drug local induction, periodically applying different kinds of stress factors that can trigger animal stress reaction to stimulate mice, prevent mice from developing habitual response to stress source, and promote mice hair turning white and tail epidermis flaky depigmentation; The method comprises the following steps: S1. setting multiple stress factors that can trigger animal stress reaction; S2. randomly selecting 1-2 stress factors from environmental stimulation, physiological stimulation and perceptual stimulation every day to stimulate the mice, and the total number of stimulation is not more than 4; S3. selecting 5 days every week, repeating step S2, ensuring that the order of stress source appears randomly and at least one day apart, preventing the mice from developing habituation; S4. continuing for N weeks to end the modeling, and N is greater than 8 weeks; The stress factors include environmental stimulation, physiological stimulation and perceptual stimulation; The environmental stimulation includes: The exchange cage method, exchanging the cages of two mice but not the bedding, and then returning them to the original cages after 24 hours; The inclined cage method, placing the cage on a 45-degree inclined fixed triangular iron stand for 6 hours; The shaking cage method, tilting the cage 45 degrees to the left and right with the center as the axis, one back and forth every 1 second, and continuing for 20 minutes; The wet cage method, evenly pouring 200 milliliters of pure water into each cage to completely wet the bedding, and replacing the new dry bedding after 24 hours; The pulsed flashing light method, selecting a self-pulsed flashing light source, flashing 5 times per second, maintaining a dark environment, and continuing for 1 hour; The water bath method, evenly pouring 300 milliliters of pure water into each empty cage without bedding, and returning the mice to the original cages after 1 hour; The physiological stimulation includes the restraint method, using a clean 50ml centrifuge tube, and using an electric iron to make several small holes as the breathing holes of the mice, placing the head of the mice in the conical side of the centrifuge tube, and vertically placing for 4 hours; The cold water swimming method, slowly putting the mice into 2 liters of 4℃ cold water, placing another empty cage on the surface to prevent escape, and taking out after 5 minutes, and completely wiping off the water; The tail suspension method, suspending the mouse tail to 20cm above the ground, and continuing for 5 minutes; The perceptual stimulation includes the predator sound stimulation method, selecting the call sound file of the mouse enemy, 80 decibels, and cycling once every 90 seconds for 1 hour; The predator smell stimulation method, selecting the smell analog of the mouse enemy to arouse the innate fear / defense behavior of the laboratory mice, i.e., the instinctive freezing reaction; using a 1ml syringe to extract 20ul TMT from each cage and dropping it on a cotton swab, and placing the cotton swab in the cage, and taking out the cotton swab after sealing for 20 minutes.

2. The mouse vitiligo induction method based on chronic stressors according to claim 1, characterized in that, Selecting various owl calls as the call of the mouse enemy.

3. The mouse vitiligo induction method based on chronic stressors according to claim 1, characterized in that, Selecting fox smell analog 2, 4, 5-trimethyl-3-thiazoline TMT as the smell analog of the mouse enemy.

4. The mouse vitiligo induction method based on chronic stressors according to claim 1, characterized in that, Continuing for 12 weeks to end the modeling.

5. The mouse vitiligo induction method based on chronic stressors according to claim 1, characterized in that, The mice are male mice.

6. The mouse vitiligo induction method based on chronic stressors according to claim 5, characterized in that, The mice are five-week-old mice.

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