阅读说明：本技术 一种模拟蚊虫叮咬症状的动物模型的构建方法 (Method for constructing animal model for simulating mosquito bite symptoms ) 是由 吴葩 李文倩 邓芳清 于 2020-02-07 设计创作，主要内容包括：本发明涉及动物模型技术领域,公开了一种模拟蚊虫叮咬症状的动物模型的构建方法。本发明中直接使用雌性成蚊对豚鼠进行叮咬或免疫唾液蛋白混合物导致豚鼠产生痒觉,并且该模型可以通过抓挠次数量化评估痒觉程度,相对于其他痒觉模型更加直接有效。同时,本发明中联合使用免疫唾液蛋白混合物和雌性成蚊叮咬导致豚鼠的痒觉效应,表现为更显著的皮肤红肿和皮疹症状；本发明动物实验模型有助于高效地筛选和研发防蚊止痒药物。(The invention relates to the technical field of animal models, and discloses a construction method of an animal model for simulating mosquito bite symptoms. According to the invention, the guinea pig is bitten by a female adult mosquito directly or the combination of immune salivary proteins causes the guinea pig to generate itch, and the model can quantitatively evaluate the degree of itch through scratching and is more directly effective compared with other itch models. Meanwhile, the combination of the immune sialoprotein mixture and the bite of female adult mosquitoes in the invention causes the itchy effect of guinea pigs, and the itchy effect is expressed as more obvious symptoms of skin red swelling and rash; the animal experiment model is beneficial to efficiently screening and researching anti-mosquito and itching-relieving medicines.)

1. A method for constructing an animal model simulating mosquito bite symptoms is characterized by comprising the following steps:

anesthetizing a guinea pig, and biting with a female adult mosquito every two days for 3-6 times to obtain the animal model;

or extracting saliva protein mixture from head and chest of female adult mosquito, and immunizing guinea pig twice a week to obtain animal model;

or extracting saliva protein mixture from the head and chest of female adult mosquito to primarily immunize the guinea pig, then performing boosting immunization once every week for 1-2 times, after the last boosting immunization, performing anesthesia on the guinea pig, and biting with the female adult mosquito every two days with the number of bites of 7-12 times to obtain the animal model.

2. The method of claim 1, wherein the saliva protein mixture is a supernatant of the head and chest of female adult mosquitoes after being ground with a buffer or water.

3. The method of claim 2, wherein the buffer is PBS.

4. The construction method according to claim 1, wherein the prime is a prime mixed with a salivary protein mixture with Freund's complete adjuvant; the booster immunization was boosted with incomplete Freund's adjuvant mixed with a salivary protein mixture.

5. The method of claim 1, wherein the area to be bitten is exposed and other areas are covered before the guinea pig bites.

6. The method of claim 5, wherein the guinea pig is exposed to the face and ears before biting, and the other parts are wrapped.

7. The method of claim 1, wherein said female adult mosquito is female Aedes albopictus and/or female Aedes aegypti.

8. The method of claim 1, wherein said female adult mosquitoes account for at least 80.

9. The method of claim 1, wherein said female adult mosquito bites are starved prior to being bitten.

10. Use of the construction method of any one of claims 1 to 9 for screening antipruritic drugs and for basic research on pruritus caused by mosquito bites.

Technical Field

The invention relates to the technical field of animal models, in particular to a construction method of an animal model for simulating mosquito bite symptoms.

Background

Mosquitoes are widely distributed in all parts of the world, and along with global integrated development and climate warming, the distribution and damage range of the mosquitoes are continuously expanded. The aedes albopictus, also called Asian tiger mosquito, has black and silvery white stripes on the body, agile action, great ability to fly, strong adaptability to the environment and severe skin reaction after biting people. The aedes albopictus is originally active in the middle of China and in the southeast coast and is mainly bred in areas with high temperature and heavy rainfall. In the last two decades aedes albopictus began to spread rapidly worldwide as a more harmful invasive species.

Mosquito bites cause local skin symptoms including itching, rubella, redness and swelling, and burning. In severe cases, severe inflammatory reactions or Athery reactions such as edema and hemorrhagic necrosis occur on the local skin of the bite position within 2-6 hours after the bite of mosquitoes. The erythema and pimple with pruritus caused by mosquito bites seriously affect the life quality of people and interfere the normal work and rest of people. Therefore, establishing a reasonable animal experimental model has important significance for researching and developing the erythema pimple with pruritus caused by mosquito bite.

The current animal model for researching and developing antipruritic and detumescent medicaments for mosquito bites comprises the following components: 1. animal model of xylene-induced ear swelling in mice. Xylene is a carcinogenic chemical, which can cause skin dryness, chapping and dermatitis when contacted, and is used as an inducer in mouse ear swelling models. The mechanism of dermatitis induced by xylene is mediated inflammatory mediator release, causing local vasodilation, capillary permeability increase, inflammatory cell infiltration, and thus skin exudative inflammatory edema. The erythema and pimple with pruritus caused by mosquito bites is a hypersensitivity reaction involving IgE, IgG and T lymphocytes, so that the xylene is not suitable for evaluating the effectiveness of the mosquito itching-relieving medicine; 2. exogenous histamine-induced skin itch model. Namely, the skin of a healthy mouse is injected with histamine to induce the itch (from the patent: the study on the mechanism of peripheral receptors of mice which are induced by estrogen-facilitated histamine). Although mosquito bites can induce histamine production in human bodies and generate itching reaction, histamine receptor antagonists cannot effectively inhibit itching caused by mosquito bites, and the fact that itching caused by the bites is a complex response synergistically induced by various components including the histamine is suggested. Thus, histamine can induce the phenotype closest to mosquito bites, but is not the most ideal model for developing and evaluating mosquito antipruritic drugs; 3. the traditional Chinese medicine combination floral water is directly applied to human subjects (from the patent: a traditional Chinese medicine combination for relieving itching and detumescence after mosquito bites and a preparation method thereof). Direct recruitment of human subjects can most directly and truly assess the efficacy of drugs, however, ethical review and management of human experiments is becoming more and more stringent. The research and development mode of the medicine is commonly used for improving the prescription of the Chinese herbal medicine in the ancestral formula and is not suitable for screening effective single mosquito itching-relieving and swelling-diminishing new medicines on a large scale; 4. the low concentration formic acid induces the pain of the mouse with the pimple model (from the patent: a blister eliminating and itching relieving liquid for mosquito bites). The back of the white mouse is unhaired, a small amount of low-concentration formic acid is injected into the epidermis by an injector, and the unhaired back condition and the activity condition of the white mouse are observed. Formic acid is the main effective component of the venom of the formicaceae insects, and mainly causes pain sensation to vertebrates so as to achieve the defense effect. Pain caused by low-concentration formic acid and itching caused by mosquito bites have completely different signal conduction pathways and molecular mechanisms, so a mouse pain sensation model induced by the low-concentration formic acid is not a suitable research and development model of mosquito itching-relieving medicines.

In conclusion, in order to relieve erythema, pimple and pruritus caused by mosquito bites in summer, an effective animal experimental model needs to be established so as to efficiently screen and research anti-mosquito and itching-relieving medicines.

Disclosure of Invention

In view of the above, the invention aims to provide a construction method of an animal model for simulating mosquito bite symptoms, so that the construction method can construct an animal model with highly similar red rash and wheal accompanied by itching symptoms caused by mosquito bite on human skin, and is helpful for efficiently screening and researching anti-mosquito and itching-relieving medicines by objectively quantifying scratching times and number of times of animals.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method for constructing an animal model simulating mosquito bite symptoms comprises the following steps:

anesthetizing a guinea pig, and biting with a female adult mosquito every two days for 3-6 times to obtain the animal model;

or extracting saliva protein mixture from head and chest of female adult mosquito, and immunizing guinea pig twice a week to obtain animal model;

or extracting saliva protein mixture from the head and chest of female adult mosquito to primarily immunize the guinea pig, then performing boosting immunization once every week for 1-2 times, after the last boosting immunization, performing anesthesia on the guinea pig, and biting with the female adult mosquito every two days with the number of bites of 7-12 times to obtain the animal model.

Preferably, the bite is 1 hour at a time.

Preferably, the saliva protein mixture is the supernatant of female adult mosquito heads and chests after grinding with buffer or water, preferably PBS; in a specific embodiment of the present invention, the method for extracting the saliva protein respiratory mixture comprises the following steps:

30 female adult mosquitoes, not fed blood, were used and their heads and chests (containing salivary glands) were ground in 500ul PBS. Centrifuging at 4 deg.C for 10min at 1000g, and collecting supernatant, i.e. the mixture of mosquito head saliva proteins. The protein concentration was measured using a kit from 5ul of the supernatant of the salivary protein mixture.

Preferably, the protein concentration of the saliva protein mixture during immunization is 0.4mg/m L. in the case of priming, the saliva protein mixture is mixed with Freund's complete adjuvant in a ratio of 1:1, and in the case of boosting, the saliva protein mixture is mixed with Freund's incomplete adjuvant in a ratio of 1: 1.

Preferably, the area to be bitten is exposed before the guinea pig bites, and the other areas are wrapped. In a specific embodiment of the invention, the guinea pig is exposed to the face and ears before biting and the other parts are wrapped.

Preferably, the female adult mosquito is female aedes albopictus and/or female aedes aegypti; the number of the female adult mosquitoes is at least 80 when the guinea pig is bitten, and the female adult mosquitoes are subjected to starvation treatment before being bitten (raisins fed to the female adult mosquitoes are taken out earlier for starvation treatment one night before the experiment).

Through experimental verification of the animal model, the itch degree can be quantitatively evaluated according to the scratching frequency of the experimental animal; the guinea pig itch assessment model established by the invention is obviously superior to a mouse model, the mouse model still has no scratching phenomenon after being bitten for 7 times under the same construction method, the scratching times and scratching return times of the model continuously increase along with the increase of the biting times and reach the highest after being bitten for 6 times, and the animal model can simulate the symptoms of human skin erythema, wheal and the like caused by mosquito bites to a certain extent.

In addition, injection of guinea pigs with a mosquito saliva-associated antigen mixed adjuvant prior to mosquito bites can enhance the prurigo effect caused by mosquito bites, manifested by more pronounced symptoms of skin redness and rash.

According to the technical scheme, the experimental model for the guinea pig bitten by the mosquitoes and the mosquitoes, which is established by the invention, is highly similar to the symptom of the red rash and wheal accompanied with pruritus caused by the human skin bitten by the mosquitoes and the mosquitoes, the guinea pig is caused to generate the itch by directly using the female adult mosquitoes to bite the guinea pig or immunizing the saliva protein mixture, and the model can quantitatively evaluate the itch degree through scratching, and is more direct and effective compared with other itch models. Meanwhile, the combination of the immune sialoprotein mixture and the bite of female adult mosquitoes in the invention causes the itchy effect of guinea pigs, and the itchy effect is expressed as more obvious symptoms of skin red swelling and rash; the animal experiment model is beneficial to efficiently screening and researching anti-mosquito and itching-relieving medicines.

Drawings

FIG. 1 shows the results of the number of scratches after biting of 3 groups of Aedes albopictus;

FIG. 2 shows the results of the number of scratching returns after biting by 3 groups of Aedes albopictus;

FIG. 3 shows the results of the number of scratching and ticking of mice bitten by Aedes albopictus;

FIG. 4 shows the results of scratching times and number of ticks after the Aedes aegypti bites the guinea pig;

FIG. 5 shows the results of the number of scratching and the number of returns of guinea pigs injected with the Aedes aegypti protein saliva mixture;

FIG. 6 shows the results of scratching times and recountries of guinea pigs injected with the Aedes albopictus sialoprotein mixture + bites;

fig. 7 shows the facial erythema, pimple and wheal appearance in the guinea pig bite group and the guinea pig injection + bite group; wherein A is guinea pig bite group, B and C are guinea pig injection + bite group.

Detailed Description

The invention discloses a method for constructing an animal model for simulating mosquito bite symptoms, and a person skilled in the art can appropriately improve process parameters by referring to the content of the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method of construction of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations and modifications, as appropriate, may be made in the method of construction described herein to practice or use the techniques of the present invention without departing from the spirit and scope of the invention.

The construction method of the animal model for simulating the symptoms of mosquito bites provided by the invention is further explained below.