CN115251009A - Construction method of rat alopecia areata model - Google Patents

Abstract

The invention provides a method for constructing a rat alopecia areata model, which is characterized in that an MLA (multi-layered line array) is used for carrying out targeted antagonism on alpha 7nAChR so that the model can not play a neuroprotective effect, hair follicles are subjected to neurotrophic loss, and the rat alopecia areata model is obtained in a form of alopecia; the model of the invention adopts intraperitoneal injection, the operation is simple, and the stability is high; the animal welfare and ethics are met, the animal stress response is small, and the tolerance is good; has little influence on other organs and functions of the model animal and can not generate obvious toxic or side effect.

Description

A kind of construction method of rat alopecia areata model

technical field

The invention mainly relates to the medical field, in particular to a method for constructing a rat model of alopecia areata.

Background technique

Alopecia areata is a common non-scarring, hair-loss disorder and a chronic inflammatory skin disorder. So far, the academic circles are still unclear about the pathogenesis of human alopecia areata. It is generally believed that the pathogenesis of alopecia areata is related to the destruction of the mechanism that protects the hair follicle from immune attack and maintains the immune immunity of the hair follicle, and allows cytotoxic immune cells to recognize and generate an immune response to the hair follicle-associated self-antigen. It is an autoimmune disease that is genetically related and stimulated by environmental factors. For the treatment of alopecia areata, there is no effective method at present. Recent studies have shown that the lifetime cumulative incidence of alopecia areata is 2.1%, which is equivalent to 145 million people in the world who have suffered from, are suffering from or will suffer from alopecia areata at some stage in their lives. Alopecia areata can occur at any age, but it is more common in young people. Alopecia areata in children develops rapidly and is severe, and there is no obvious gender difference.

The growth of hair depends on the nutrition of hair follicles, and the nerve endings of the skin can secrete nutrition factors and other regulatory factors to regulate and support the growth of hair follicles. When the function of the nerve is abnormal, the nutritional function of the hair follicle changes, which affects the growth of the hair, and finally leads to the inability to support the hair, resulting in hair loss. There are many receptors distributed on the peripheral nerves, through which neurons exchange information and substances with the outside world continuously, thus playing a regulatory role.

Alopecia areata research is usually carried out using rodent hair loss models, mainly mice, such as C3H/HeJ mice and C57BL/6 mice.

At present, there are four ways to establish a hair loss model: intraperitoneal injection of cyclophosphamide, external application of imiquimod, tail vein injection of interferon, and hair removal:

Cyclophosphamide is a chemotherapeutic drug as well as a cytotoxic drug. While modeling, it will have obvious effects on other organs of the whole body, so it is not suitable for biomedical research.

Imiquimod is a drug for external use, which is easily affected by the operator's technique and animal activities when used. The drug effect is unstable and the consistency is poor, which is not conducive to biomedical research.

Interferon is an immunomodulator, which has a significant impact on the immune system. It has a greater impact on most medical projects that require mechanism research, and is not suitable for biomedical research.

Hair-plucking is a physical modeling method, which is easily affected by the operator's personal technique. The workload is large, and the animal pain is obvious during the hair-plucking process, which does not conform to animal ethics and animal welfare. The stress response is obvious. The indicators have a great impact, and the modeling effect is not standard, which causes unnecessary obstacles to research, and is not suitable for application in biomedical research.

Contents of the invention

For the above-mentioned defect of prior art, the invention provides a kind of construction method of rat alopecia areata model, comprises the steps:

S1: Rat preparation: the rats used in the experiment are taken care of until their physical functions are normal;

S2: Solution preparation: add the required amount of MLA powder to DMSO to dissolve, then add the DMSO solution to medical corn oil to dissolve, control the total volume of DMSO to be less than 10% of the total volume of corn oil, and use a vortex to mix thoroughly after preparation uniform;

S3: Solution injection: take out the rats in S1, appease their emotions, and inject the solution prepared in S2 to the parts that need hair removal, for 30 days, once a day;

S4: Rat pacification: In order to facilitate each injection, the rats need to be pacified before and after a single injection. The way of pacifying the rats by smoothing the hair is to calm the rats and reduce the mood of resistance, so as not to damage or death.

Preferably, in the S1: in the preparation of the rats, SD rats are used for the rats, and an SPF rat room is used for maintenance, the temperature is constant at 20-22°C, the feed is maintenance feed, ultrapure water is used for drinking water, and the light is continuously illuminated Hours are 9A.M. to 9P.M.

Preferably, in the S2: in the preparation of the solution, the total volume of the appropriate amount of MLA solution sucked by the syringe is calculated according to the weight of 0.1ml/100g of the rat, and the dose of the MLA solution can be 4mg/kg.

Preferably, in the preparation of the S2: solution, the final prepared solution should be stored in a refrigerator at 4°C, and rewarmed at room temperature for 1 hour before use.

Preferably, in the S3: solution injection, the injection site is generally the abdomen, and after the syringe quickly pierces the abdominal wall, it needs to be withdrawn to ensure that there is no blood before injecting the MLA solution.

Preferably, S3: In solution injection, the syringe may be a 1ml syringe, or/and, the syringe may be a syringe with a screw interface.

Preferably, in said S4: in appeasement of the rat, the appeasement time is 1-2 min.

Beneficial effects of the present invention:

1. The model adopts intraperitoneal injection, which is easy to operate and has high stability.

2. It conforms to animal welfare and ethics, and the animals have little stress response and good tolerance.

3. It has little impact on other organs and functions of model animals, and will not produce obvious toxic side effects.

Description of drawings

Fig. 1 is a normal SD rat cell structure diagram under an electron microscope of the present invention;

Fig. 2 is the SD rat cell structure diagram after injecting 1mg/kgMLA solution under the electron microscope of the present invention;

Fig. 3 is the SD rat cell structure diagram after injecting 4mg/kgMLA solution under the electron microscope of the present invention;

Fig. 4 is the comparative figure of α7nAChR cell and GAPDH cell expression level of SD rat depilatory area cell in the present invention under different MLA solution concentration;

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, and to make the above-mentioned features, objectives and advantages of the present invention clearer and easier to understand, the present invention will be further described below in conjunction with embodiments. The examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

The present invention comprises: following steps:

S1: Rat preparation: the rats used in the experiment are taken care of until their physical functions are normal;

S2: Solution preparation: add the required amount of MLA powder to DMSO to dissolve, then add the DMSO solution to medical corn oil to dissolve, control the total volume of DMSO to be less than 10% of the total volume of corn oil, and use a vortex to mix thoroughly after preparation uniform;

S3: Solution injection: take out the rats in S1, appease their emotions, and inject the solution prepared in S2 to the parts that need hair removal, for 30 days, once a day;

S4: Rat pacification: In order to facilitate each injection, the rats need to be pacified before and after a single injection. The way of pacifying the rats by smoothing the hair is to calm the rats and reduce the mood of resistance, so as not to damage or death.

Set up the above structure:

In step S1, the physical skills of the rats are restored to normal. The duration is generally one week, which can be extended according to the actual situation of the rats.

In step S2, the MLA powder is methylcaconitine powder. Methyllycaconitine (MLA) is a specific antagonist of α7nAChR, and MLA is used to target and antagonize α7nAChR so that it cannot exert its neuroprotective effect, resulting in hair follicle denervation nutrition, resulting in hair loss.

DMSO is dimethyl sulfoxide, and is used as a solvent for MLA powder.

Medical corn oil is used as a medical grade solvent to dilute and quantify the mixed solution of DMSO and MLA.

In S3: In the solution injection, select the site to be depilated by holding the plunger of the syringe, and quickly puncture the abdominal wall with the syringe, then draw back to confirm that there is no blood, and then inject the MLA solution. When puncturing, avoid inserting the needle too deeply to damage the abdominal viscera, resulting in the death of the rat. In case of excessive resistance, the injection should be stopped immediately.

In S4: Rat Soothe, rats can be soothed by stroking the hair, which is efficient and simple.

As a preferred option:

S1: In the preparation of rats, SD rats are used as rats, and SPF-grade rat rooms are used for maintenance. The temperature is constant at 20-22°C, the feed is maintenance feed, the drinking water is ultra-pure water, and the lighting time is 9A.M. .to 9 P.M.

Alopecia areata research is usually carried out using rodent hair loss models, mainly mice, such as C3H/HeJ mice and C57BL/6 mice, with gray hair and a high incidence rate. And the effects, especially with interferons or cytotoxins, are extremely unstable.

SD rats are a strain of rats bred from Wistar rats, with albino hair, long and narrow head, tail length close to body length, many litters, and rapid growth and development. The cost of selecting SD rats is low, and the effect is remarkable in the subsequent experiment process. The above-mentioned conditioning environment is the routine living environment of SD rats, which can be adjusted in due course according to the actual situation.

S2: In the preparation of the solution, the appropriate amount of MLA solution absorbed by the syringe, the total volume is calculated according to the weight of the rat at 0.1ml/100g, and the dose of the MLA solution can be 4mg/kg.

Methyllycaconitine (MLA) is a specific antagonist of α7nAChR. In the present application, MLA is used to target and antagonize α7nAChR so that it cannot exert its neuroprotective effect, resulting in neurotrophy of hair follicles and hair loss.

As shown in Fig. 1, the structure of the nerve fiber barrier in the skin tissue of the rats in the normal group was uniform, neatly arranged, and without breakage.

As shown in Figure 2, the nerve fiber barrier structure in the skin tissue of the 1 mg/kg injection group had scattered fractures and deformation, and the overall structure existed.

As shown in Figure 3, the nerve fiber barrier structure in the skin tissue of the 4mg/kg group had multiple fractures and deformations, and the overall structure was disordered.

As shown in FIG. 4 , compared with the control group (con group), the expression levels of α7nAChR protein and GAPDH protein gradually decreased with the increase of MLA solution.

Therefore, the best solution is to use 4 mg/kg of MLA solution.

S2: During the preparation of the solution, the final prepared solution should be stored in a refrigerator at 4°C, and rewarmed at room temperature for 1 hour before use to prevent the composition of the solution from changing due to external factors during subsequent injections.

S3: In the solution injection, the injection site is usually the abdomen. After the syringe quickly pierces the abdominal wall, it needs to be withdrawn to ensure that there is no blood before injecting the MLA solution to prevent the puncture of internal organs or blood vessels.

S3: In the solution injection, the syringe can be 1ml syringe, or/and, the syringe can be a syringe with a screw interface. Since the corn oil solution is slippery, the syringe and the needle will fall off. It is safer to use a syringe with a screw interface. The use of 1ml syringes can minimize the pain response of rats.

S4: During rat appeasement, the appeasement time is 1-2min, which is simple and efficient.

The above-mentioned embodiments only illustrate the principle and effect of the present patent application, but are not intended to limit the present patent application. Any person familiar with this technology can modify or change the above-mentioned embodiments without departing from the spirit and scope of this patent application. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in this patent application should still be covered by the claims of this patent application.

Claims (7)

1. a construction method of rat alopecia areata model, is characterized in that, comprises the steps: S1: Rat preparation: the rats used in the experiment are taken care of until their physical functions are normal; S2: Solution preparation: add the required amount of MLA powder to DMSO to dissolve, then add the DMSO solution to medical corn oil to dissolve, control the total volume of DMSO to be less than 10% of the total volume of corn oil, and use a vortex to mix thoroughly after preparation uniform; S3: Solution injection: take out the rats in S1, appease their emotions, and inject the solution prepared in S2 to the parts that need hair removal, for 30 days, once a day; S4: Rat pacification: In order to facilitate each injection, the rats need to be pacified before and after a single injection. The rats are calmed by smoothing the hair, and the rebellious emotions are reduced, so as to avoid the formation of injury or death. 2. the construction method of a kind of rat alopecia areata model according to claim 1, is characterized in that: described S1: in rat preparation, rat adopts SD rat, take good care of and take good care of adopts the mouse room of SPF level, and temperature is 20 The temperature is -22°C, the feed is maintenance feed, the drinking water is ultra-pure water, and the lighting time is 9A.M. to 9P.M. 3. the construction method of a kind of rat alopecia areata model according to claim 1, is characterized in that: described S2: in solution is equipped with, the appropriate MLA solution that syringe draws, total volume calculates according to rat body weight 0.1ml/100g , the dose of MLA solution can be 4mg/kg. 4. The construction method of a kind of alopecia areata rat model according to claim 1, characterized in that: said S2: in the preparation of the solution, the final configured solution should be stored in a refrigerator at 4°C, and it should be reconstituted at room temperature for 1 hour before use. temperature. 5. The construction method of a kind of rat alopecia areata model according to claim 1, characterized in that: said S3: in solution injection, the injection site is generally the abdomen, after the syringe quickly punctures the abdominal wall, it needs to be withdrawn first Make sure there is no blood before injecting the MLA solution. 6. the construction method of a kind of rat alopecia areata model according to claim 1, is characterized in that: described S3: in solution injection, syringe can adopt 1ml syringe, or/and, The syringe can be a syringe with a screw interface. 7. The construction method of a kind of alopecia areata rat model according to claim 1, characterized in that: said S4: in appeasing the rat, the appeasing time is 1-2min.

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