CN111742883B - Construction method of lung and spleen qi deficiency allergic rhinitis combined animal model - Google Patents
Construction method of lung and spleen qi deficiency allergic rhinitis combined animal model Download PDFInfo
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- CN111742883B CN111742883B CN202010007872.6A CN202010007872A CN111742883B CN 111742883 B CN111742883 B CN 111742883B CN 202010007872 A CN202010007872 A CN 202010007872A CN 111742883 B CN111742883 B CN 111742883B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/35—Animals modified by environmental factors, e.g. temperature, O2
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
Abstract
The invention provides a construction method of an animal model combining lung-spleen qi deficiency allergic rhinitis, which comprises the following steps: the lung-spleen qi deficiency compound syndrome type animal model is constructed by adopting a mode of combining smoking and folium sennae intragastric perfusion, and then the allergic rhinitis animal model is constructed by adopting a mode of egg albumin and aluminum hydroxide suspension intraperitoneal injection and egg albumin nasal drip. The construction method of the invention is based on the traditional Chinese medicine theory that internal and external causes simultaneously cause diseases, comprehensively arranges multi-factor combined modeling, combines physical and chemical factors and disease symptoms, and establishes a compound syndrome disease animal model. Through repeated experimental evaluation, various molding factors are quantified, the molding time process is specified, and the animal has the advantages of standardization, high reproducibility and the like. In addition, the self-made smoking equipment is simple and easy to copy, is low in price and can be prepared by adopting a transparent plastic storage box and self-made stainless steel fittings on the market.
Description
Technical Field
The invention relates to the technical field of animal models, in particular to a construction method of an animal model combining lung-spleen qi deficiency allergic rhinitis.
Background
Allergic Rhinitis (AR) is a global disease affecting health, with an incidence rate of up to 40% and a trend of increasing year by year. The traditional Chinese medicine considers that AR is caused by affection of wind-cold and foreign qi and pathogenic factors on nasal orifices on the basis of deficiency of the lung, spleen and kidney. Modern literature reports that lung and spleen qi deficiency syndrome is a common clinical syndrome of AR. Though the ancient and modern doctors consider AR to be a disease of lung orifice, it is most closely related to spleen deficiency, and as recorded in Su Wen, it is said that "spleen is an isolated organ … … which can not lead to nine orifices.
The construction of an appropriate animal model combining the traditional Chinese medicine symptoms has important significance for perfecting the traditional Chinese medicine theory and developing the traditional Chinese medicine clinic. In recent years, a plurality of students in China construct some syndrome-related animal models including lung qi deficiency, spleen qi deficiency and other AR animal models by adopting different ways and different ideas, and at present, there is no report on lung and spleen qi deficiency compound syndrome type AR models, and the thinking of syndrome modeling and disease modeling is not uniform. The smoking method is the most used modeling method in the lung qi deficiency syndrome experiment at present, but smoking equipment, smoking time and smoking amount are different, the existing smoking equipment in the market at present is high in manufacturing cost, and common laboratories are not configured; the smoke concentration, temperature and ventilation treatment in the self-made smoking box are key problems for preparing the AR model. The application of cold infusion of senna leaves for gastric perfusion is a simple and easy method for preparing spleen-qi deficiency, but the concentration of senna leaves used in the prior art is different.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a construction method of an animal model combining the lung-spleen qi deficiency allergic rhinitis.
In order to achieve the purpose, the invention adopts the technical scheme that:
a construction method of an animal model combining lung and spleen qi deficiency allergic rhinitis comprises the following steps:
step 1: modeling the model group animals by adopting a method combining smoking and folium sennae intragastric perfusion to obtain an animal model with lung-spleen qi deficiency complex syndrome type;
step 2: carrying out intraperitoneal injection on the lung and spleen qi deficiency compound syndrome type animal model obtained in the step 1 by using ovalbumin and aluminum hydroxide suspension; then, carrying out nasal instillation on the animal model by adopting ovalbumin to obtain a lung and spleen qi deficiency type allergic rhinitis disease combined animal model;
and step 3: the obtained lung and spleen qi deficiency allergic rhinitis is verified by combining an animal model.
Preferably, the smoking in step 1 comprises the following specific steps: placing the animal in a smoking box, smoking for 20min with 8 cigarettes each time, ventilating and resting for 10min, continuously smoking for 3 times, smoking time of the animal every day is 1h, and smoking molding period is 60 days.
Preferably, the step 1 of performing stomach irrigation by senna leaves comprises the following specific steps: the administration is carried out by intragastric administration from 31 days of smoking according to the physical quality of 1ml/100g, 1 time per day, and the intragastric molding cycle is 30 days.
Preferably, the preparation method of the senna leaf in the step 1 is as follows: soaking folium sennae in boiling water for 1 hr, filtering to obtain medicinal liquid, and storing in refrigerator at 4 deg.C for use, wherein the ratio of folium sennae to water is 1: 10.
Preferably, the step 2 of intraperitoneal injection of the ovalbumin and aluminum hydroxide suspension comprises the following specific steps: preparing suspension of ovalbumin and aluminum hydroxide, performing intraperitoneal injection from the 40 th day of smoking, 1 time every other day and 7 times in total, wherein the intraperitoneal injection molding period is as follows: and 14 days.
Preferably, the preparation method of the egg white protein and aluminum hydroxide suspension comprises the following steps: 0.3mg of ovalbumin and 30mg of aluminum hydroxide were added to 1ml of physiological saline to prepare a suspension.
Preferably, the smoking of step 2 starts with the application of 10% physiological saline solution of ovalbumin to the bilateral nasal cavities at 54 days, each time, 20 μ l of the solution is applied to each nasal cavity, and the application is carried out 1 time per day for 7 consecutive days.
Preferably, the basic sensitization of the intraperitoneal injection of the egg white protein and aluminum hydroxide suspension is further included before the nasal drip of the egg white protein physiological saline.
Preferably, the step 3 verification method includes: food intake, body mass, nasal symptom score, weight bearing swimming time, ELISA test for serum IgE levels, and HE staining of nasal mucosal pathological sections.
Preferably, the animal employed in the animal model is an SD rat.
The invention also provides a smoking box for constructing the lung-spleen qi deficiency allergic rhinitis combined animal model, a storage box (1) and smoking accessories (3); the smoking accessory comprises a cylinder (32) with a chimney (31) and a base (33); the chimney (31) is positioned at the top of the cylinder (32) and communicated with the top; the bottom of the column body is open and not closed, and the side surface of the column body close to the bottom is provided with a vent hole (34); the base (33) is open at the top and closed at the bottom, and the column (32) is nested on the base (33).
Preferably, the smoking accessory (3) is made of stainless steel; the column (32) and the base (33) are cylindrical; the vent hole (34) on the side surface of the column body is square; the storage box (1) is made of plastic materials, and the size of the storage box is 60cm (length) multiplied by 40cm (width) multiplied by 35cm (height); the diameter of the cylindrical stainless steel column body (32) is 7cm, and the height of the cylindrical stainless steel column body is 18 cm; the diameter of the chimney (31) is 2cm, and the height of the chimney is 2 cm; the size of the vent hole (34) is 4cm multiplied by 3.5 cm.
The invention has the beneficial effects that: the disease and spleen qi deficiency combined animal model is a lung and spleen qi deficiency compound syndrome type allergic rhinitis animal model, the adopted animal species rat is an SD rat, a syndrome model and a disease model are created synchronously, a self-made smoking box is placed in a fume hood of a laboratory, the model is manufactured based on the simultaneous pathogenicity of internal and external causes, the combination of physical and chemical factors and multi-factor, various model manufacturing factors and time are quantized, verification indexes including food intake, body quality, weight bearing swimming time, nasal symptom score, serum IgE level and a nasal mucosa pathological section are adopted, repeated experiment verification shows that the animal model manufacturing death rate is low, mass operation can be carried out, the symptom expression of the lung and spleen qi deficiency compound syndrome type AR can be well simulated, and the method has the advantages of standardization, reproducibility and the like. The smoking equipment is simple and easy to copy, has low price, and can be realized by adopting a transparent plastic storage box and self-made stainless steel fittings on the market.
Drawings
FIG. 1 is a flow chart of the use of the smoking device of the present invention.
FIG. 2 is a flow chart of the lung-spleen qi deficiency allergic rhinitis combined with animal model modeling.
FIG. 3 is a graph showing the variation trend of the three ABC groups of masses.
FIG. 4 is a graph showing the variation trend of the intake of three groups ABC.
FIG. 5 is a comparison graph of ABC three groups of weight swimming experiment time.
FIG. 6 is a graph comparing the levels of serum IgE in three ABC groups.
FIG. 7 is a graph showing the results of HE staining in group A.
FIG. 8 is a graph showing the results of HE staining in group B.
FIG. 9 is a graph showing the results of HE staining in group C.
FIG. 10 is a schematic view of the self-made smoke box of the present invention.
Fig. 11 is a schematic view of the overall structure of the present invention.
FIG. 12 is a schematic view of the smoking device of the present invention.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Main molding equipment and reagents: the smoking box comprises a plastic storage box (with the size of 60cm long, 40cm wide and 35cm high) and a cylindrical stainless steel small part (with the diameter of 7cm and the height of 18cm, see attached figure 10) with a small chimney. Fume hood (Eustace corporation), ovalbumin (OVA grade V, Sigma corporation), aluminum hydroxide dry powder (Sigma corporation), Daqianmen brand cigarette, senna leaf (Kangmei corporation).
Example 1
The embodiment provides a smoking box for constructing an animal model combining lung-spleen qi deficiency allergic rhinitis, which comprises a storage box (1) and smoking accessories (3); the smoking accessory comprises a cylinder (32) with a chimney (31) and a base (33); the chimney (31) is positioned at the top of the cylinder (32) and communicated with the top; the bottom of the column body is open and not closed, and the side surface of the column body close to the bottom is provided with a vent hole (34); the base (33) is open at the top and closed at the bottom, and the column (32) is nested on the base (33) (fig. 12).
In this embodiment, the smoking accessories are made of stainless steel; the cylinder and the base are cylindrical; the vent hole positioned on the side surface of the column body is square; the storage box is made of plastic materials, the size of the storage box is 60cm (length) multiplied by 40cm (width) multiplied by 35cm (height), the diameter of the cylindrical stainless steel column body is 7cm, and the height of the cylindrical stainless steel column body is 18 cm; the diameter of the chimney is 2cm, and the height of the chimney is 2 cm; the size of the square hole is 4cm multiplied by 3.5 cm.
Smoking process (fig. 1): firstly, a cover (1) of a plastic storage box is opened, a cage (2) (46.4cm (length) × 30cm (width) × 18cm (height)) provided with rats is placed in the whole cage (6 rats) at one corner of the plastic storage box (2 rats can be placed at most), and a smoking accessory (3) is placed at the diagonal position. The top of a cylindrical stainless steel base (33) is upwards arranged in a plastic box; (figure 11) opening a transparent window of a laboratory fume hood, and placing the plastic storage box (1) with the rat in the table top of the laboratory fume hood; thirdly, bundling 8 cigarettes into a cylinder with the same horizontal bottom by using a rubber band, wherein the bundling part of the rubber band is positioned at a cigarette filter tip, and then igniting the 8 cigarettes and vertically placing the 8 cigarettes in the center of a cylindrical stainless steel base; embedding a cylindrical stainless steel cylinder (32) with a chimney (31) into a cylindrical stainless steel base (33); the ventilation of the post (32) near the ventilation holes (34) on the bottom side is ensured to ensure that the cigarette burns sufficiently in the stainless steel fitting. Covering the cover of the plastic storage box (not fastened), and closing the transparent window of the fume hood; when smoking, the ventilation equipment is closed, and smoking is carried out for 20 minutes; starting ventilation equipment after smoking is finished for 20 minutes, pumping out smoke, pulling up a transparent window of a fume hood, opening a cover of the plastic storage box, taking out a cylindrical stainless steel cylinder with a chimney, removing cigarette butts, and ventilating for 10 minutes; sixthly, carrying out second smoking on the same batch of rats. The smoking process and time are the same as above, the ventilation equipment is started after smoking for 20 minutes, and the third smoking of the same batch of rats is carried out after ventilation for 10 minutes. The lung-spleen qi deficiency model group AR rats smoked for 60 minutes per day.
Example 2 construction of a Lung-spleen Qi deficiency allergic rhinitis combined animal model
Step 1: a method for constructing a lung-spleen qi deficiency compound syndrome type rat by combining fumigation and folium sennae intragastric perfusion comprises the following specific steps:
1) after 3 days of quarantine of SD rats, the SD rats are placed in a squirrel cage and then placed in a self-made smoking box, the smoking box is placed in a laboratory fume cupboard (a storage box cover is covered but is not fastened and sealed), 8 cigarettes are smoked for 20min each time, then ventilation and rest are carried out for 10min, 3 times of continuous smoking are carried out, the smoking time of the rats every day is 1h, 24 cigarettes are used in common, and the smoking and molding cycle is 60 days.
2) Performing intragastric administration from 31 days of smoking, soaking folium sennae in boiling water for 1 hr (1 g (0.1g/ml) folium sennae in 10ml water), filtering to obtain medicinal liquid, and storing in refrigerator at 4 deg.C. The rats are administered 1 time daily by intragastric administration according to the physical quality of 1ml/100 g. The period of the stomach filling and molding is 30 days.
Step 2: an allergic rhinitis animal model is constructed by adopting a method of egg white protein and aluminum hydroxide suspension intraperitoneal injection and egg white protein nasal drip, and the specific steps are as follows:
1) intraperitoneal injection was started on day 40 after smoking, ovalbumin (OVA grade V, Sigma), 0.3mg and 30mg of aluminum hydroxide dry powder (Sigma) were simultaneously added to 1ml of physiological saline to prepare a suspension (this is the amount of one rat), and then the rat prepared in step 1 was subjected to intraperitoneal injection for basic sensitization, 1 time every other day, for 7 times in total. The intraperitoneal injection molding cycle is 14 days.
2) Nasal drops are made from 54 days of smoking, and two nasal cavities are dropped with 10% ovalbumin physiological saline, 20 mul is dropped into each nasal cavity, 1 time per day, and 7 days are continued.
And step 3: verification is carried out on the constructed lung-spleen qi deficiency allergic rhinitis combined animal model
Comparing the rats in the lung and spleen qi deficiency allergic rhinitis group (hereinafter referred to as group A), the rats in the allergic rhinitis group (hereinafter referred to as group B) and the blank control group (hereinafter referred to as group C), verifying that the rats in the lung and spleen qi deficiency allergic rhinitis group (group A) obtained by the construction of the invention have pathological symptoms, wherein the comparison result is as follows:
1. general conditions and nasal symptoms
Group A has sneezing, frequent nasal scratching and nasal discharge, and is accompanied by symptoms of qi deficiency of lung and spleen, such as urgent breath, reduced activity, lusterless hair, easy falling, listlessness, reduced appetite, preference for curling and gathering, loose stool, etc.; sneezing, marked nasal obstruction, watery nasal discharge, occasional loose stool in group B; occasionally, the patient in group C has a nasal scratchback, with no signs of yang symptoms. Nasal symptom scores for each group are shown in table 1. Nasal symptom scores in rats in groups A and B were statistically significant (P < 0.05) compared to group C, but in groups A and B, there was no statistical significance (P > 0.05).
Table 1 nasal symptoms score (
n＝8)
*P＜0.05
2. Comparison of molded body masses
A. B, C the comparison between groups was statistically significant (P < 0.05), and further two-by-two comparisons showed statistical significance between group A and group B, C (P < 0.05), and no statistical significance between group B and group C (P >0.05) (Table 2). As can be seen from the trend graph of the change in body mass, the body mass of rats in group A increased more slowly than that of rats in group B, C (FIG. 3).
TABLE 2 comparison of body Mass
g(n＝8)
*P＜0.05
3. Food intake
After the model is made, the differences among the three groups have statistical significance (P is less than 0.05), and further the two groups are compared, the group A has statistical significance (P is less than 0.05) compared with the group B, C, and the group B has no statistical significance (P is more than 0.05) compared with the group C (Table 3). As can be seen from the trend graph of food intake, the food intake of rats in group A was less than that of B, C (FIG. 4).
TABLE 3 food intake comparison
g(n＝8)
*P＜0.05
4. Weight bearing swimming experiment
Group a swim times were shorter than group B, C (fig. 5). Through calculation, the comparison between three groups has statistical significance (P is less than 0.05), and further two-by-two comparison shows that the comparison between the group A and the group B, C has statistical significance (P is less than 0.05), and the comparison between the group B and the group C has no statistical significance (P is more than 0.05). The rats in group A were suggested to show the appearance of qi deficiency.
5. Serum IgE levels
A. The serum IgE levels of the two groups B are increased and are consistent with the serum IgE increase of allergic disease organisms (including human bodies and animals) reported in the literature (the analysis of total serum IgE and specific IgE of carambola patients, the measurement and clinical significance of the total serum IgE of allergic rhinitis, Zhanghuhuhui, the initial detection of the total serum IgE, INF-gamma and IL-4 as objective evaluation indexes established by an allergic rhinitis animal model, the establishment and evaluation of Lijiafeng and an allergic rhinitis rat animal model). Prompting the successful establishment of the allergic rhinitis rat model. A. B, C the comparison between three groups was statistically significant (P < 0.05), and further two-by-two comparison showed that the difference between group C and group A, B was statistically significant (P < 0.05) and that the difference between group A and group B was not statistically significant (P >0.05) (FIG. 6).
6. HE staining of nasal mucosa pathology
The cilia of the nasal mucosa can be detached, the arrangement is disordered, the glands are increased, and local inflammatory cells are infiltrated in the A group (figure 7) and the B group (figure 8); group C (fig. 9) nasal mucociliary was neat, intact, and no inflammatory cell infiltration.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (4)
1. A construction method of an animal model combining lung and spleen qi deficiency allergic rhinitis is characterized by comprising the following steps:
step 1: modeling the model group animals by adopting a method combining smoking and folium sennae intragastric perfusion to obtain an animal model with lung-spleen qi deficiency complex syndrome type;
the smoking in the step 1 comprises the following specific steps: placing the animal in a smoking box, smoking for 20min with 8 cigarettes each time, then ventilating and resting for 10min, continuously smoking for 3 times, wherein the smoking time of the animal is 1h every day, and the smoking molding period is 60 days; the step 1 of performing gastric lavage on senna leaves comprises the following specific steps: starting from 31 days of smoking, performing intragastric administration according to the physical quality of 1ml/100g, 1 time per day, and performing intragastric administration for 30 days;
step 2: carrying out intraperitoneal injection on the lung and spleen qi deficiency compound syndrome type animal model obtained in the step 1 by using ovalbumin and aluminum hydroxide suspension; then, carrying out nasal instillation on the animal model by adopting ovalbumin to obtain a lung and spleen qi deficiency type allergic rhinitis disease combined animal model;
before the step 2, the basic sensitization of the egg white protein and aluminum hydroxide suspension is adopted for intraperitoneal injection before the egg white protein physiological saline is dripped into the nose;
the step 2 of performing intraperitoneal injection on the ovalbumin and aluminum hydroxide suspension comprises the following specific steps: adding 0.3mg of ovalbumin and 30mg of aluminum hydroxide into 1ml of normal saline to prepare suspension, carrying out intraperitoneal injection from the 40 th day of smoking, 1 time every other day and 7 times in total, wherein the intraperitoneal injection molding period is as follows: 14 days; in the step 2, the nasal cavities at both sides are dripped with 10% ovalbumin physiological saline at 54 days of smoking, 20 mu l of ovalbumin physiological saline is dripped into each nasal cavity at each time, 1 time per day, and 7 days are continued;
and step 3: verifying the obtained lung-spleen qi deficiency allergic rhinitis by combining an animal model;
the step 3 verification method comprises the following steps: food intake, body mass, nasal symptom score, weight bearing swimming time, ELISA test for serum IgE levels, and HE staining of nasal mucosal pathological sections.
2. The method of claim 1, wherein the senna leaf of step 1 is prepared by the following method: soaking folium sennae in boiling water for 1 hr, filtering to obtain medicinal liquid, and storing in refrigerator at 4 deg.C for use, wherein the ratio of folium sennae to water is 1: 10.
3. The method of constructing of claim 1, wherein the smoke box comprises: a storage box (1) and a smoking accessory (3); the smoking accessory comprises a cylinder (32) with a chimney (31) and a base (33); the chimney (31) is positioned at the top of the cylinder (32) and communicated with the top; the bottom of the column body is open and not closed, and the side surface of the column body close to the bottom is provided with a vent hole (34); the base (33) is open at the top and closed at the bottom, and the column (32) is nested on the base (33).
4. A method of construction according to claim 3, wherein the smoking accessory (3) is of stainless steel; the column (32) and the base (33) are cylindrical; the vent hole (34) on the side surface of the column body is square; the storage box (1) has the dimensions of 60cm in length, 40cm in width and 35cm in height; the diameter of the cylindrical stainless steel column body (32) is 7cm, and the height of the cylindrical stainless steel column body is 18 cm; the diameter of the chimney (31) is 2cm, and the height of the chimney is 2 cm; the size of the vent hole (34) is 4cm multiplied by 3.5 cm.
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