CN110419492B - Method for establishing oral mucosa fibrous animal model by adopting areca catechu extract and mechanical friction - Google Patents
Method for establishing oral mucosa fibrous animal model by adopting areca catechu extract and mechanical friction Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified 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; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
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- A—HUMAN NECESSITIES
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- 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; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a method for establishing an oral mucosa fibrous animal model by adopting an areca extract and mechanical friction. Preparing an areca extract by adopting an ethanol reflux extraction method, selecting a healthy SD rat as a model animal, and preparing a control group: no treatment is carried out; betel nut extract smearing group: dipping the betel nut extract into a sterilized cotton swab every day, and uniformly smearing the betel nut extract on the upper jaw and the lower jaw of a rat; mechanical stimulation group: dipping sterilized distilled water by a self-made brush, rubbing and stimulating the upper jaw and the lower jaw of a rat, smearing the betel nut extracting solution and mechanically stimulating the group: smearing the extractive solution and mechanically stimulating; the molding time is 10 weeks in total, after the molding is finished, the free diet feeding is recovered for 2 weeks, and the total time is 12 weeks in total. After the model building is finished, oral mucosa tissues are taken to carry out indexes such as HE staining, Masson staining, immunohistochemistry and the like. Compared with the traditional method, the method is stable and reliable, accords with the pathological process of disease development, and can more comprehensively simulate the influence of chewing areca on the fibrosis of oral mucosa.
Description
Technical Field
The invention relates to a method for establishing an oral mucosa fibrous animal model by adopting an areca extract and mechanical friction, belonging to the technical field of animal experiment model construction.
Background
Submucosal fibrosis of the mouth (OSF) is a chronic, occult disease that is an early marker of oral carcinogenesis. Abnormal deposition of collagen cells under oral mucosa is a main pathological feature of OSF, and the collagen deposition can cause hyperplasia and sclerosis of oral mucosa of a patient, fibrous cords appear, and then the mouth is restricted, the movement is difficult, and the life is seriously influenced. The onset of OSF is closely related to gene, immunity and living habits, and chewing areca is a high risk factor for the onset of OSF. Chewing areca is a dietary habit in southeast Asia and areas such as Hainan and Hunan of China, which are areas with high incidence of oral cancer and OSF. At present, the chewing of betel nuts is generally considered to be directly related to the attack of OSF, and the disease rate of OSF in betel nut chewing people is as high as 0.1% -3.4%, which is far higher than that of other people. The development and establishment of the OSF animal model have important significance for the elucidation of the pathogenesis of OSF and the development of treatment strategies.
Various scholars at home and abroad develop the research of the OSF animal model, and the medicine is induced by using arecoline, chemical preparations and the like, and the medicine is administrated at the cheek of an experimental animal by adopting the modes of medicine feeding, smearing, injection and the like. However, the collagen cell fibroplasia is a complex pathological reaction and is limited by various factors, the areca as a processed food has the characteristics of rough fiber and complex components, the chemical stimulation of one or more components on the local oral mucosa hardly causes continuous influence on the collagen cell fibroplasia, the existing model cannot accurately and stably copy and measure the damage of areca chewing on the collagen cell of the mucosa, and a model making mode is urgently needed to be further improved. The stimulation of rat mucous membrane by comprehensive mechanical and chemical modes can more comprehensively simulate the influence of chewing areca on the proliferation of collagen cells under oral mucosa.
Disclosure of Invention
The purpose is as follows: in order to overcome the defects in the prior art, the invention provides a method for establishing an oral mucosa fibrous animal model by adopting an areca extract and mechanical friction.
The technical scheme is as follows:
the invention aims to provide a method for establishing an oral mucosa fibrous animal model, which can simulate the oral mucosa fibrous deformation caused by betel nuts. The replication method has good stability and repeatability, and accords with the disease course.
Preparing areca extract by ethanol reflux extraction, selecting normal and healthy SD rats as model animals, and comparing: drinking water and diet normally without any treatment; betel nut extract smearing group: dipping the betel nut extract into a sterilized cotton swab every day, and uniformly smearing the betel nut extract on the upper jaw and the lower jaw of a rat; mechanical stimulation group: dipping sterilized distilled water by a self-made brush, rubbing and stimulating the upper jaw and the lower jaw of a rat, smearing the betel nut extracting solution and mechanically stimulating the group: applying the extractive solution in the same way as above, and mechanically stimulating in the same way as above; the molding time is 10 weeks in total, after the molding is finished, the rats are not treated, the rats are fed freely and recovered for 2 weeks, and the total time is 12 weeks in total. After the model building is finished, taking oral mucosa tissues to carry out indexes such as HE staining, Masson staining, immunohistochemistry and the like.
The establishment of the model specifically comprises the following steps:
1. SD rats with normal healthy weight of 200-300g are selected as model animals.
2. Preparing areca extract by adopting an ethanol reflux extraction method, crushing and sieving a commercially available areca raw material, adding a proper amount of ethanol solution for ultrasonic treatment, then performing ethanol reflux extraction, filtering, and further concentrating and volatilizing the ethanol extract to obtain the areca extract. Determining the content of effective components arecoline, arecaidine and demethylarecaidine in Arecae semen extract by high performance liquid chromatography.
3. Application method of the betel nut extract: dipping the areca extract (containing 0.1g of extract) with a sterilized cotton swab, uniformly smearing the extract on the upper jaw and the lower jaw of a rat, putting the rat back into a cage after smearing, and keeping the rat from water and fasting for 1 hour.
4. Mechanical stimulation method: the sterilized hemostatic forceps are used for pulling and opening the oral cavity of the rat, a self-made brush is used for dipping sterilized distilled water to rub and stimulate the upper jaw and the lower jaw of the rat for not less than 1min, and then the rat is returned to a cage for normal feeding and diet.
5. The betel nut extract smearing and mechanical stimulation combined method comprises the following steps: dipping the betel nut extract by a sterilized cotton swab every day, uniformly smearing the betel nut extract on the upper jaw and the lower jaw of a rat, putting the rat back into a cage after smearing, stopping water for 1 hour, simultaneously pulling and opening the oral cavity of the rat by sterilized hemostatic forceps every other day, and dipping sterilized distilled water by a self-made brush to rub and stimulate the upper jaw and the lower jaw of the rat for not less than 1 min.
6. And (3) molding recovery period: after the molding was completed, the rats were kept on free diet for 2 weeks without any treatment. During the model building period, the animal condition is observed every day, the weight is weighed and the oral mucosa specimen is collected after 6, 8, 10 and 12 weeks of model building.
7. And (3) pathological sections of oral mucosa, wherein detection indexes comprise HE staining, Masson staining and immunohistochemical alpha SMA, and pathological scoring is carried out.
8. Pathological scoring of oral mucosa fibrosis: 0 minute: the collagen fiber of the intrinsic layer under the epithelium is sparse and thin, has no inflammatory cell infiltration, and the submucosa is not obvious. The lamina propria is dense connective tissue, and the submucous loose connective tissue contains rich blood vessels, glands and lymphatic vessels; 1 minute: thickening the collagen fiber of the inherent layer under the epithelium, infiltrating a little inflammatory cells in the submucosa, proliferating fibrous tissues, and obtaining clear basal layer but orderly arranged cells; and 2, dividing: thickening of collagen fibers of an epithelium inherent layer, infiltration of inflammatory cells of a submucosa, proliferation of fibrous tissues, clear basal layer but regular cell arrangement, expansion and disorderly arrangement of basal layer cells, thickening of vessel walls, congestion with edema; and 3, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, the infiltration of inflammatory cells of the lower mucosal layer, the proliferation of fibrous tissues, the expansion and the increase of basal layer cells, the disorderly arrangement, the thickening of vessel walls and congestion with edema; and 4, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, a large amount of inflammatory cells of the submucosa infiltrate, the fiber tissue proliferates, the expansion of basal layer cells is increased and the arrangement is disordered, the vessel wall is thickened to disappear, and congestion is accompanied with edema.
9. Oral mucosa collagen deposition pathology score: 0 minute: the collagen fibers of the lower epithelial layer are sparse and thin, inflammatory cells are not infiltrated, and the submucosa is not obvious. The inherent layer is compact connective tissue, and the loose connective tissue under the mucosa contains rich blood vessels, glands and lymphatic vessels; 1 minute: thickening the collagen fiber of the inherent layer under the epithelium, infiltrating a little inflammatory cells in the submucosa, proliferating fibrous tissues, and obtaining clear basal layer but orderly arranged cells; and 2, dividing: thickening of collagen fibers of a lower epithelial layer, infiltration of inflammatory cells of a submucosa, fibrous tissue hyperplasia, clear and orderly arrangement of cells of a basal layer, expansion and disorderly arrangement of cells of the basal layer, thickening of a vessel wall, congestion and edema. And 3, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, the infiltration of inflammatory cells of the lower mucosal layer, the proliferation of fibrous tissues, the expansion and the increase of basal layer cells, the disorderly arrangement, the thickening of vessel walls and congestion with edema; and 4, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, a large amount of inflammatory cells of the submucosa infiltrate, the fiber tissue proliferates, the expansion of basal layer cells is increased and the arrangement is disordered, the vessel wall is thickened to disappear, and congestion is accompanied with edema.
Drawings
Figure 1 is a typical HE stain image of rat oral mucosa.
Figure 2 is the pathological score for fibrosis of rat oral mucosa epithelial cells. P < 0.10, p < 0.05, p < 0.01, compared to the control group.
FIG. 3 shows the results of Masson staining of rat oral mucosa.
Figure 4 is a pathological score of collagen deposition in the oral mucosa of rats. P < 0.10, p < 0.05, p < 0.01, compared to the control group.
FIG. 5 shows the results of the oral mucosa alpha-SMA of rat.
Figure 6 is a rat oral mucosa alpha-SMA pathology score. P < 0.10, p < 0.05, p < 0.01, compared to the control group.
Detailed Description
1. The experimental method comprises the following steps:
SD rats with normal healthy weight of 200-300g were selected as model animals.
Preparing areca extract by ethanol reflux extraction, pulverizing commercially available areca raw material, sieving, adding appropriate amount of ethanol solution, performing ultrasonic treatment, performing ethanol reflux extraction, filtering, and concentrating and volatilizing the ethanol extract to obtain the areca extract. Determining the content of effective components arecoline, arecaidine and demethylarecaidine in Arecae semen extract by high performance liquid chromatography.
Application method of betel nut extract: dipping the areca extract (containing 0.1g of extract) with a sterilized cotton swab, uniformly smearing the extract on the upper jaw and the lower jaw of a rat, putting the rat back into a cage after smearing, and keeping the rat from water and fasting for 1 hour.
Mechanical stimulation method: the sterilized hemostatic forceps are used for pulling and opening the oral cavity of the rat, a self-made brush is used for dipping sterilized distilled water to rub and stimulate the upper jaw and the lower jaw of the rat for not less than 1min, and then the rat is returned to a cage for normal feeding and diet.
Betel nut extract application and mechanical stimulation combined method: dipping the betel nut extract by a sterilized cotton swab every day, uniformly smearing the betel nut extract on the upper jaw and the lower jaw of a rat, putting the rat back into a cage after smearing, stopping water for 1 hour, simultaneously pulling and opening the oral cavity of the rat by sterilized hemostatic forceps every other day, and dipping sterilized distilled water by a self-made brush to rub and stimulate the upper jaw and the lower jaw of the rat for not less than 1 min.
And (3) molding recovery period: after the molding was completed, the rats were kept on free diet for 2 weeks without any treatment.
During the model building period, the animal condition is observed every day, the weight is weighed and the oral mucosa specimen is collected after 6, 8, 10 and 12 weeks of model building.
And (3) pathological section of the oral mucosa, wherein detection indexes comprise HE staining and Masson staining, and pathological scoring is carried out.
Oral mucosal fibrosis scoring: 0 minute: the collagen fiber of the intrinsic layer under the epithelium is sparse and thin, has no inflammatory cell infiltration, and the submucosa is not obvious. The lamina propria is dense connective tissue, and the submucous loose connective tissue contains rich blood vessels, glands and lymphatic vessels; 1 minute: thickening the collagen fiber of the inherent layer under the epithelium, infiltrating a little inflammatory cells in the submucosa, proliferating fibrous tissues, and obtaining clear basal layer but orderly arranged cells; and 2, dividing: thickening of collagen fibers of an epithelium inherent layer, infiltration of inflammatory cells of a submucosa, proliferation of fibrous tissues, clear basal layer but regular cell arrangement, expansion and disorderly arrangement of basal layer cells, thickening of vessel walls, congestion with edema; and 3, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, the infiltration of inflammatory cells of the lower mucosal layer, the proliferation of fibrous tissues, the expansion and the increase of basal layer cells, the disorderly arrangement, the thickening of vessel walls and congestion with edema; and 4, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, a large amount of inflammatory cells of the submucosa infiltrate, the fiber tissue proliferates, the expansion of basal layer cells is increased and the arrangement is disordered, the vessel wall is thickened to disappear, and congestion is accompanied with edema.
Oral mucosa collagen deposition pathology scoring: 0 minute: the collagen fiber of the intrinsic layer under the epithelium is sparse and thin, has no inflammatory cell infiltration, and the submucosa is not obvious. The lamina propria is dense connective tissue, and the submucous loose connective tissue contains rich blood vessels, glands and lymphatic vessels; 1 minute: thickening the collagen fiber of the inherent layer under the epithelium, infiltrating a little inflammatory cells in the submucosa, proliferating fibrous tissues, and obtaining clear basal layer but orderly arranged cells; and 2, dividing: thickening of collagen fibers of a lower epithelial layer, infiltration of inflammatory cells of a submucosa, fibrous tissue hyperplasia, clear and orderly arrangement of cells of a basal layer, expansion and disorderly arrangement of cells of the basal layer, thickening of a vessel wall, congestion and edema. And 3, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, the infiltration of inflammatory cells of the lower mucosal layer, the proliferation of fibrous tissues, the expansion and the increase of basal layer cells, the disorderly arrangement, the thickening of vessel walls and congestion with edema; and 4, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, a large amount of inflammatory cells of the submucosa infiltrate, the fiber tissue proliferates, the expansion of basal layer cells is increased and the arrangement is disordered, the vessel wall is thickened to disappear, and congestion is accompanied with edema.
The oral mucosa pathological specimen is subjected to immunohistochemistry, and the detection index comprises alpha SMA.
2. Experimental results and analysis:
2.1 the content of each effective component in the Arecae semen extract is determined by high performance liquid chromatography
Table per gram of areca extract
2.2 general conditions and oral mucosal observations
1) Control group: the rat has good general condition, smooth and glossy hair and normal activity food intake, the jaw mucosa of the rat is light red, smooth, particle-free and elastic, the mucosa has no plaque, ulcer and erosion, the folds run smoothly, the boundary is clear, the arrangement is neat and symmetrical, and the rat is covered by the epithelium with high cornification.
2) Experimental groups:
and (4) group T: the whole is still, the conditions of dull and dull hair and poor spirit appear in the later period of few molding, but no obvious hair erection phenomenon exists, the food intake has no obvious change, the symptom of dry mouth is obvious, the water intake is increased rapidly, and the weight gain is not obvious. The mucosa of the jaw part is complete and has no obvious scratch, the mucosa has obvious uniform thin-layer white sample change and mucosa thickening after 8 weeks, the white sample change range of the mucosa is enlarged after 10 weeks, the fold is rough, the mucosa of individual rats has local extravasated blood and mucosa particles, but the whole mucosa is still complete, the fold running is not obviously disturbed, the molding is stopped, and the improvement is not obvious after 12 weeks.
And (S) group: rats in the early molding period (less than 4 weeks) are dysphoria, upright and dry hair, and in the later molding period (8 weeks later) are listened, hair is dull, food consumption is reduced, and weight is not obviously increased. The jaw mucosa shows that the depth is uniform, partial rats have mucosal surface granular sensation in 6 weeks, blood stasis under the mucosa, whitening and irregular hyperplasia at the scratched part, partial rats have rough mucosa, uneven hyperplasia of folds in 8 weeks, walking disorder, obvious mucosa thickening, obvious hyperplasia at the scratched part, higher than the mucosa plane, most rats have the above performance in 10 weeks, molding is stopped, and no obvious improvement is realized in 12 weeks.
Group T + S: rats in the early molding stage (4-6 weeks) are seriously dysphoria, upright and dry hair, the water intake is greatly increased, and rats in 8 weeks are listened, dark hair, the food intake is reduced, and the body weight is obviously reduced. The jaw mucosa shows that the depth is uniform, the hyperplasia of the mucosa surface is obviously accompanied with granular sensation, large-area whitening local blood stasis, uneven hyperplasia at the scratch and fold, running disorder, ulcer of most rats in 10 weeks, great reduction of food intake, listlessness, obvious slower weight rise than that of a control group, individual rats can not eat food by opening mouth, the model building is stopped, and no obvious improvement is realized at 12 weeks.
2.3 HE staining results:
compared with a control group, the mucosa is obviously damaged as a whole, the keratinized layer is not uniform, the part of the mucosa is obviously thickened, the nucleolus is clear, the epithelial cells are actively proliferated, the epithelial nail process is disordered and obviously thickened, the collagen fiber cells of the intrinsic layer under the epithelium are proliferated, the inflammatory cells of the submucosa are infiltrated, the fiber tissues are proliferated, the expansion of basal layer cells is increased, the arrangement is disordered and the congestion is caused. Severity: betel nut extract smearing + mechanical stimulation group (T + S group) > betel nut extract smearing group (T group) ≈ mechanical stimulation group (S) > control group (CON group).
Oral mucosal epithelial cell lesion scoring:
by adopting the method, pathological scoring is carried out on the fibrosis of the epithelial cells of the oral mucosa of the rat respectively at 6 weeks, 8 weeks and 10 weeks of molding and 2 weeks (12 weeks) after the molding is finished, and the injury of the oral mucosa of the rat can be caused by chemical (smearing of an extract), mechanical stimulation (a brush) and combination of the chemical stimulation and the mechanical stimulation. But with the progress of molding, the injury caused by the three stimulation modes has certain difference, namely the stimulation caused by chemical smearing is the most rapid, the initial injury degree is strong, but the injury is unstable, and the subsequent oral mucosa injury self-repair can occur; the mechanical stimulation is most stable, a certain damage can be maintained after the model is manufactured for six weeks, the chemical stimulation is simultaneously given on the basis of the mechanical stimulation, the oral mucosa damage is gradually obvious and more stable, certain deterioration still exists after the damage is stopped, and the pathological physiological process of the development of the oral diseases is more met.
Masson staining results:
compared with a control group, the experimental group obviously shows that the mucosa of the mucous membrane is obviously damaged integrally, the keratinized layer is not uniform, the thickening is obvious, the nail protrusion is not obvious, meanwhile, the fibrous tissue is proliferated, the blood vessel is thickened, the inflammatory cells of the submucosa are infiltrated, the fibrous tissue is proliferated, the basal layer cells are expanded and arranged disorderly, the blood vessel wall is thickened, and the closure is serious. Severity: betel nut extract smearing + mechanical stimulation group (T + S group) > betel nut extract smearing group (T group) ≈ mechanical stimulation group (S) > control group (CON group).
Pathological scoring of collagen deposition:
according to the scoring standard, the pathological degree (A) of the epithelial cell pathological changes and the fibroplasia (B) of the pathological section of the rat are scored, compared with a control group, the pathological scores of a betel nut extracting solution smearing group (T), a mechanical stimulation group (S) and a betel nut extracting solution smearing group and mechanical stimulation group (T + S) are all increased in 6 weeks, 8 weeks and 10 weeks, and the scores are not obviously reduced in 2-week (12-week) recovery, but along with the modeling, the injuries caused by three stimulation modes are different to a certain extent, namely the stimulation caused by chemical smearing is the most rapid, the initial injury degree is strong, but the injury is unstable, and the subsequent collagen deposition is not obvious; the mechanical stimulation is stable, a certain damage can be maintained within a period of time, the chemical stimulation is simultaneously given on the basis of the mechanical stimulation, the collagen deposition of the oral mucosa is maintained to a certain degree, the collagen deposition is maintained after the damage is stopped, and the pathological physiological process of the development of the oral diseases is met.
Results and scores of immunohistochemistry for alpha-SMA
Over time, the experimental group showed a significant increase in α -SMA expression compared to the control group, with overall scores: betel nut extract smearing + mechanical stimulation group (T + S group) > betel nut extract smearing group (T group) ≈ mechanical stimulation group (S) > control group (CON group).
And (4) implementing a conclusion:
analysis of the above results
The research researches the pathological damage of chemical and mechanical stimulation factors to the oral mucosa of a rat, and finds that the extract of the betel nut and the crude fiber components in the betel nut can cause stimulation to the oral mucosa and interfere the spirit and normal dietary state of the rat. After 10 weeks of modeling, the oral mucosa of the rat has hyperkeratosis, squamous epithelial cell hyperplasia, disordered arrangement of basal layer cells, collagen fiber hyperproliferation, edema, vasodilation, inflammatory cell infiltration and other reactions, the alpha-SMA expression is increased, and the model accords with the pathological development process of oral diseases. Overall severity of various molding process comparisons: betel nut extract application + mechanical stimulation group (T + S group) > betel nut extract application group (T group) ≈ mechanical stimulation group (S) > control group (CON group), which shows that the betel nut extract application + mechanical stimulation molding method and the pathological development process of oral diseases
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (8)
1. A method for establishing an oral mucosa fibrous animal model by adopting an areca extract and mechanical friction is characterized by comprising the following steps of: preparing areca extract by ethanol reflux extraction, selecting normal and healthy SD rats as model animals, and comparing: drinking water and diet normally without any treatment; betel nut extract smearing group: dipping the betel nut extract into a sterilized cotton swab every day, and uniformly smearing the betel nut extract on the upper jaw and the lower jaw of a rat; mechanical stimulation group: dipping sterilized distilled water by a self-made brush, and rubbing to stimulate the upper jaw and the lower jaw of a rat; betel nut extract smearing and mechanical stimulation group: dipping a sterilized cotton swab into the betel nut extract every day, uniformly smearing the betel nut extract on the upper jaw and the lower jaw of a rat, putting the rat back into a cage after smearing, stopping water and fasting for 1 hour, simultaneously using sterilized hemostatic forceps to pull and open the oral cavity of the rat every other day, and dipping sterilized distilled water by a self-made brush to rub and stimulate the upper jaw and the lower jaw of the rat for not less than 1 min; the molding time is 10 weeks, after molding, the rats are not treated, free diet feeding is recovered for 2 weeks, the total time is 12 weeks, after molding, oral mucosa tissues are taken for HE dyeing, Masson dyeing and immunohistochemical indexes, the betel nut extract is prepared by an ethanol reflux extraction method, commercially available betel nut raw materials are crushed and sieved, a proper amount of ethanol solution is added for ultrasonic treatment, then ethanol reflux extraction is carried out, filtration is carried out, the ethanol extract is further concentrated and volatilized to obtain the betel nut extract, and the contents of effective components of the betelnut extract, arecoline and demethylarecoline are determined by a high performance liquid chromatography.
2. The method of establishing according to claim 1, wherein: SD rats with normal healthy weight of 200-300g are selected as model animals.
3. The method of establishing according to claim 1, wherein: application method of the betel nut extract: dipping the areca extract with a sterilized cotton swab, uniformly smearing the areca extract on the upper jaw and the lower jaw of a rat, putting the rat back into a cage after smearing, and keeping the rat from water and fasting for 1 hour.
4. The method of establishing according to claim 1, wherein: mechanical stimulation method: the sterilized hemostatic forceps are used for pulling and opening the oral cavity of the rat, a self-made brush is used for dipping sterilized distilled water to rub and stimulate the upper jaw and the lower jaw of the rat for not less than 1min, and then the rat is returned to a cage for normal feeding and diet.
5. The method of establishing according to claim 1, wherein: and (3) molding recovery period: after the model building is finished, the rats are not treated, the rats are fed in free diet and recovered for 2 weeks, the animal condition is observed every day during the model building period, the weight is weighed and the oral mucosa specimen is collected after 6, 8, 10 and 12 weeks of model building.
6. The method of establishing according to claim 1, wherein: and (3) pathological sections of oral mucosa, wherein detection indexes comprise HE staining, Masson staining and immunohistochemical alpha SMA, and pathological scoring is carried out.
7. The method of establishing according to claim 1, wherein: pathological scoring of oral mucosa fibrosis: 0 minute: the collagen fiber of the subepithelial layer is sparse and thin, has no inflammatory cell infiltration, the submucosa layer is not obvious, the natural layer is compact connective tissue, and the submucous loose connective tissue contains rich blood vessels, glands and lymphatic vessels; 1 minute: thickening the collagen fiber of the inherent layer under the epithelium, infiltrating a little inflammatory cells in the submucosa, proliferating fibrous tissues, and obtaining clear basal layer but orderly arranged cells; and 2, dividing: thickening of collagen fibers of an epithelium inherent layer, infiltration of inflammatory cells of a submucosa, proliferation of fibrous tissues, clear basal layer but regular cell arrangement, expansion and disorderly arrangement of basal layer cells, thickening of vessel walls, congestion with edema; and 3, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, the infiltration of inflammatory cells of the lower mucosal layer, the proliferation of fibrous tissues, the expansion and the increase of basal layer cells, the disorderly arrangement, the thickening of vessel walls and congestion with edema; and 4, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, a large amount of inflammatory cells of the submucosa infiltrate, the fiber tissue proliferates, the expansion of basal layer cells is increased and the arrangement is disordered, the vessel wall is thickened to disappear, and congestion is accompanied with edema.
8. The method of establishing according to claim 1, wherein: oral mucosa collagen deposition pathology score: 0 minute: the collagen fiber of the subepithelial layer is sparse and thin, has no inflammatory cell infiltration, the submucosa layer is not obvious, the natural layer is compact connective tissue, and the submucous loose connective tissue contains rich blood vessels, glands and lymphatic vessels; 1 minute: thickening the collagen fiber of the inherent layer under the epithelium, infiltrating a little inflammatory cells in the submucosa, proliferating fibrous tissues, and obtaining clear basal layer but orderly arranged cells; and 2, dividing: thickening of collagen fibers of a lower epithelial lamina, infiltration of inflammatory cells of a submucosa, fibrous tissue hyperplasia, clear basal layer but regular cell arrangement, expansion and disorderly arrangement of basal layer cells, thickening of vessel walls, congestion with edema, 3 points: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, the infiltration of inflammatory cells of the lower mucosal layer, the proliferation of fibrous tissues, the expansion and the increase of basal layer cells, the disorderly arrangement, the thickening of vessel walls and congestion with edema; and 4, dividing: the proliferation of collagen fiber cells of the lower epithelial layer is obvious, a large amount of inflammatory cells of the submucosa infiltrate, the fiber tissue proliferates, the expansion of basal layer cells is increased and the arrangement is disordered, the vessel wall is thickened to disappear, and congestion is accompanied with edema.
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