CN108739668A - A kind of method for building up of spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model and application - Google Patents
A kind of method for building up of spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model and application 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/60—Salicylic acid; Derivatives thereof
- A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
- A61K31/616—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid by carboxylic acids, e.g. acetylsalicylic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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Abstract
The invention discloses a kind of method for building up of spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model and applications, including：Method that lower and overstrain is combined is rushed down to model group animal model using eating and drinking without temperance, bitter cold, obtains the animal model of syndrome of deficient cold of spleen and stomach type；With absolute ethyl alcohol and aspirin to syndrome of deficient cold of spleen and stomach type animal model gavage to get.The present invention is building deficiency-cold in spleen and stomach model of a syndrome using " eating and drinking without temperance+bitter cold rush down under+overstrain " compound tcm causa morbi molding method, gastric ulcer model is induced with " absolute ethyl alcohol+aspirin " gavage, gained animal model has the syndrome clinical manifestation of deficiency-cold in spleen and stomach, relatively meet with Mechanism of Gastric Ulcer or the clinical symptoms of recurrence, solves the problems, such as that existing method can not build reliable, stable, favorable reproducibility spleen-stomach deficiency-cold gastric ulcer Disease Syndrome integrated animal model.It can be that clinical research peptic ulcer establishes important foundation, be more conducive to the research of traditional Chinese medicine anti-ulcer effect mechanism and new drug development.
Description
Technical field
The present invention relates to animal model technical fields, and in particular to a kind of spleen-stomach deficiency-cold gastric ulcer disease combination animal is real
Test method for building up and the application of model.
Background technology
Gastric ulcer (gastric ulcer, GU) is a kind of common disease, the frequently-occurring disease of digestive system, typical clinic table
Now be chronic, periodicity, rhythmicity middle Upper abdominal pain can when serious often with symptoms such as glutted belch, pantothenic acid, Nausea and vomitings
See spitting blood and melena symptom.The prevalence of gastric ulcer is up to 5%-10%, just suffers from this there are one people every about ten people
Disease.GU morbidities have apparent seasonal, are common in colder season at the beginning of the Qiu Mozhi spring；This disease can betide any age,
Male's incidence is higher than women.
Not only incidence is high by GU, and the often intractable complication such as recurrent exerbation and adjoint bleeding, perforation, pyloric stenosis,
Wherein 1% intractable GU can also be converted into gastric cancer.Clinical data shows that the latter year recurrence rate of Chinese GU patient's healing is
47.31%, 2 years recurrence rates are 83.22%.As modern society's life rhythm is accelerated, work and ambient pressure, psychiatric
Factor stimulation, bad Diet lifestyle etc. all make morbidity (recurrence) rate of gastric ulcer that the trend risen year by year, serious shadow be presented
Ring physical and mental health, working condition and the quality of life of people.Therefore, the treatment of GU has become the hot spot and difficulty of current medical research
Point problem.
GU can belong to the scopes such as traditional Chinese medicine " epigastric pain ", " feeling of fullness ", " acid regurgitation ", " noisy ", " vomiting ".From cause of disease angle
Analysis, this disease mostly by eating and drinking without temperance, feelings will internal injury, exopathogen violate stomach, ferritic spleen deficiency and cause, clinical Common Syndromes type includes that taste are empty
The cards such as cold, deficiency of stomach-Yin, liver-stomach disharmony, syndrome of liver-stomach heat, stomach network hemostasis.There is clinic of the research to 3422 gastric ulcer Coryza Treated by Syndrome Differentiation
Research Literature is for statistical analysis, it is found that deficiency-cold in spleen and stomach and syndrome of incoordination between liver and stomach are the most common card types of this disease.Pathogenesis is theoretical
Think, deficiency-cold in spleen and stomach is the pathologic basis of GU onsets and recurrence, and deficiency-cold in spleen and stomach is applied among the pathologic process of GU always.Cause
This, establishes spleen-stomach deficiency-cold gastric ulcer Disease Syndrome integrated animal model to the prevention of GU and the combination of Chinese tradiational and Western medicine diagnosis and treatment of GU with important
Meaning.
The most pure simulation gastric ulcer of experimental animal model of modern medicine study gastric ulcer, can not embody spleen
The stomach cold of insufficiency type ulcer fall ill and recur in pathology value and significance, i.e., how to embody this Chinese medical discrimination characteristic of deficiency-cold in spleen and stomach at
For the key of problem.Currently, there is scholar to propose through the reserpine to SD rats by intraperitoneal injection 0.2mg/kg/d dosage, continuous 13
It, rat show weight mitigates, becomes thin, hair is loose, curl up it is sleeping flock together, the deficiency-cold in spleen and stomach symptom such as diarrhea, temperature decline.To the greatest extent
Manage that this method intervention factor is simple, easily controllable, repeatable, but its modeling principle is bad anti-using the gastrointestinal tract of reserpine
It answers, does not meet Traditional Chinese Medicine teiology modeling method, it is different from the pathogenesis of deficiency-cold in spleen and stomach, syndrome of deficient cold of spleen and stomach cannot be reacted
Essence does not also match with the clinical symptoms of spleen-stomach deficiency-cold patients w ith peptic ulcer disease.
Therefore, TCM Syndrome Features can be embodied on same animal body but also react modern medicine disease by how establishing one kind
Sick pathological characters and the experimental model that can symbolize inner link between the two become the key of research.
Invention content
The main purpose of the present invention is to provide a kind of building for spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model
Cube method and application can not build reliable, stable, favorable reproducibility taste void at least to solve modeling method in the prior art
The technical issues of cold type gastric ulcer disease combination animal experimental model.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of spleen-stomach deficiency-cold gastric ulcer disease
In conjunction with the method for building up of animal experimental model, include the following steps：
(1), the method that lower and overstrain is combined is rushed down using eating and drinking without temperance, bitter cold and modeling is carried out to model group animal,
Obtain the animal model of syndrome of deficient cold of spleen and stomach type；
(2), gavage is carried out to the animal model of syndrome of deficient cold of spleen and stomach type obtained by step (1) with absolute ethyl alcohol and aspirin,
Obtain spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model.
Further, in step (1), method that lower and overstrain is combined is rushed down to model using eating and drinking without temperance, bitter cold
Group animal carries out modeling, including：
(1.1), first day, the free diet drinking-water of model group animal filled model group animal with folium sennae water decoction
Then stomach makes model group animal swim powerless to four limbs stroke, is picked up immediately when head is submerged in water；
(1.2), second day, the fasting of model group animal, free water carried out gavage with pig fat to model group animal, then
Gavage is carried out to model group animal with folium sennae water decoction, so that model group animal is swum powerless to four limbs stroke, head does not have
It is picked up immediately when entering in water；
(1.3), it repeats step (1.1) and step (1.2) carries out continuous modeling, establish the animal mould of syndrome of deficient cold of spleen and stomach type
Type.
Further, in step (1.1), with folium sennae water decoction to model group animal carry out gavage, in particular to：To mould
Type group animal gives 0.8-1.2ml folium sennaes water decoction/100g weights and carries out folium sennae water decoction gavage.
Further, in step (1.2), with pig fat to model group animal carry out gavage, in particular to：It is dynamic to model group
Object gives 1.8-2.2ml pig fats/100g weights and carries out pig fat gavage；In step (1.2), with folium sennae water decoction to mould
Type group animal carry out gavage, in particular to：To model group animal give 0.8-1.2ml folium sennaes water decoction/100g weights into
Row folium sennae water decoction gavage.
Further, folium sennae water decoction is prepared via a method which to obtain：Folium sennae is added in distilled water and decocts 1-
Folium sennae Aqueous extracts are collected in 2h, filtering, then same amount of folium sennae is taken to be added in the folium sennae Aqueous extracts collected, and are continuously added
Distilled water, decocts 1-2h, and filtering collects filtrate to get folium sennae water decoction, a concentration of 0.8- of the folium sennae water decoction
1.2g/ml。
Further, total number of days of modeling is -12 days 10 days.
Further, in step (2), syndrome of deficient cold of spleen and stomach type obtained by step (1) is moved with absolute ethyl alcohol and aspirin
Object model carries out gavage, including：0.8-1.2ml absolute ethyl alcohols/200g weights are given to the animal model of syndrome of deficient cold of spleen and stomach type
Absolute ethyl alcohol gavage is carried out, then gives 180-220mg aspirin aqueous solution/kg weights again and carries out aspirin aqueous solution
Gavage.
Further, the number of days for carrying out gavage to the animal model of syndrome of deficient cold of spleen and stomach type with absolute ethyl alcohol and aspirin is
- 6 days 4 days.
Further, the model group animal used in the present invention specifically refers to：SPF grades of male SD rats, weight are
160-200g；First adaptable fed 3 days, during which freely ingest and drink water before modeling, and raising temperature control is at 18-23 DEG C, phase
To humid control in 45%-55%, the alternation of light and darkness period is 12 hours.
According to another aspect of the present invention, a kind of animal experimental model obtained by above-mentioned method for building up is provided to exist
Application in the clinical research of peptic ulcer or in the drug development of peptic ulcer.
The present invention theoretical foundation and have the beneficial effect that：
The present invention is to provide the production methods of spleen-stomach deficiency-cold gastric ulcer Disease Syndrome integrated animal model.According to traditional Chinese medicine
The theory of " the chronic diarrhea impairment of the spleen ", " bitter cold impairment of yang ", the purgatives medicines such as folium sennae bitter cold in nature, QI of the spleen and stomach can be injured by crossing clothes, with the passing of time then
Impairing the spleen and stomach yang-energy, the functional disturbance that makes that The spleen has the function to transport and transform nutrients form deficiency-cold in spleen and stomach syndrome.《The Yellow Emperor's Canon of Internal Medicine》It says：" human diet's overstrain is hindered
Spleen ", " overstrain " are used as the cause of disease, excessive fatigue, excessive thinking and anxiety and excess of sexual intercourse,indulgence in sexual activities to be easy the impairment of the spleen and damage stomach, cause spleen deficiency.Animal is swum
Swimming is exhausted to power, not only its power of labor but also labor its god.《Plain Questions numbness opinion》There is speech：" eating twice as usual, taste are wound ".Famine it is full immoderate and
Overfeeding fatness, sweet, thick and oily (such as pig fat) can impairing the spleen and stomach yang-energy, lead to deficiency-cold in spleen and stomach.
The making of syndrome of deficient cold of spleen and stomach of the present invention is using Basic Theories of Chinese Medicine as foundation, it then follows the differentiation of pathogen and pathology of tcm is advised
Rule, can reflect the substance and intension of tcm syndrome.Using aspirin plus alcohol induced gastric ulcer model, the incidence of ulcer is high,
Mechanism is clear, and absolute ethyl alcohol corrosivity is strong, directly destroys gastric mucosal barrier, causes mucosal lesion, constructed ulcer shape,
Pathologic Characteristics, healing and relapsing course are similar with people's mucosal lesion.
Model operability, the favorable repeatability that the present invention makes, model validation can maintain the long period, under satisfaction
The Indexs measure required time of one step.This method modeled according to traditional Chinese medicine etiology and pathogenesis and the modern medicine cause of disease and existing
Disease performance caused by real environment changes is very much like, meets the apparent reliability of animal model evaluation；It is to animal symptom and body
Changing caused by sign has direct causality, meets the inherent reliability of animal model evaluation；It is many clinical drug trials, new
This model can be used in medicine exploitation, and confirms reliable, the effective and stability of model, meets the Forecasting reliability of model evaluation.
The animal model that the present invention makes can more embody the general process and feature that gastric ulcer occurs and lapses to, animal
Model sings and symptoms and clinical patients are very much like, can provide reference for the combination of Chinese tradiational and Western medicine diagnosis and treatment of canker, are traditional Chinese medicine
Scientific basis is provided in the popularization and application of clinical treatment ulcer disease, the animal model that the present invention makes also contributes to traditional Chinese medicine
The research of anti-ulcer effect mechanism and new drug development.
Description of the drawings
The accompanying drawings which form a part of this application are used to provide further understanding of the present invention, and of the invention shows
Meaning property embodiment and its explanation are not constituted improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is that normal group is right with the model group rats local appearance (tongue picture, muffle, auricle, pawl toe) of the embodiment of the present invention
Than figure.
Fig. 2 is the model group rats weight comparison diagram of normal group and the embodiment of the present invention.
Fig. 3 is the model group rats food-intake comparison diagram of normal group and the embodiment of the present invention.
Fig. 4 is the model group rats amount of drinking water comparison diagram of normal group and the embodiment of the present invention.
Fig. 5 is the model group rats anus temperature comparison diagram of normal group and the embodiment of the present invention.
Fig. 6 is the model group rats stomach lining morphological contrast map of normal group and the embodiment of the present invention.
Fig. 7 is that the model group rats stomach lining HE staining versus of normal group and the embodiment of the present invention schemes.
Fig. 8 is that the model group immunohistochemistry of normal group and the embodiment of the present invention detects TLR-2 protein expression situations.
Fig. 9 is that the model group immunohistochemistry of normal group and the embodiment of the present invention detects MyD88 protein expression situations.
Specific implementation mode
To facilitate the understanding of the present invention, the present invention is made below in conjunction with Figure of description and preferred embodiment more complete
Face meticulously describes, but the protection scope of the present invention is not limited to the following specific embodiments.
Unless otherwise defined, all technical terms used hereinafter and the normally understood meaning of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection domain.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through doctor
Medicine company or other enterprises are commercially available or can be prepared by existing method.
The experimental animal used in following embodiment is：Male SD rat, is purchased from lake by SPF grades, weight (180 ± 20) g
The Lakes Nan Si scape reaches experimental animal Co., Ltd, credit number SCXK (Hunan) 2016-0002.First adaptable fed before formal experiment
It 3 days, during which freely ingests and drinks water, 18-23 DEG C of raising temperature, relative humidity 45%-55%, alternation of light and darkness period are 12 small
When.
Animal experimental model establishes the main agents used in the process and source includes：(the great safe food of Feng Yuan is special for pig fat
Battalion shop)；Absolute ethyl alcohol (Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd)；Aspirin (the limited public affairs of Bayer medicines and health protection
Department).
Embodiment：
A kind of method for building up of spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model of the present invention, including following step
Suddenly：
Step 1, the animal model for preparing syndrome of deficient cold of spleen and stomach type
Using " eating and drinking without temperance+bitter cold rush down under+overstrain " method modeling.110g folium sennaes are added in 550ml distilled water
Conventional to decoct 1-2h, filtered through gauze collects 220ml folium sennae Aqueous extracts, then takes 110g folium sennaes to be added and walk the 220ml filtered out
In folium sennae Aqueous extracts, continuously adds 330ml distilled water and decoct 1-2h, filter out 220ml folium sennae water decoctions, dispense, be placed in 4
DEG C refrigerator saves backup.The concentration of the folium sennae water decoction is about 1.0g/ml.
1st day, the free diet drinking-water of rat.Model group rats daily afternoon 13:00 give 1ml folium sennaes water decoction/
100g weights carry out folium sennae water decoction gavage, and model group rats are put into 1.0m (length) × 0.8m (width) × 0.8m after 2h
(height) contains the sink went swimming of full tap water at normal temperature, until the stroke of rat four limbs is powerless, head is submerged in water to be picked up immediately.
2nd day, Rat Fast, free water.The morning 10:30, which give 2ml pig fats/100g weights, carries out pig fat filling
Stomach, afternoon 13:00, which gives 1ml folium sennaes water decoction/100g weights, carries out folium sennae water decoction gavage, after 2h that model group is big
Mouse is put into 1.0m (length) × 0.8m (width) × 0.8m (height), the sink went swimming of full tap water at normal temperature is contained, until rat four limbs
Stroke is powerless, and head is submerged in water to be picked up immediately.
3rd day modeling method was with the 1st day, and the 4th day modeling method was with the 2nd day, i.e., rat meets even-numbered days to give 2ml/100g pigs
Grease stomach is raised, fasting, free water.Odd-numbered day using Chinese medical etiology " bitter cold rush down under+overstrain " method modeling, during even-numbered days use
Doctor teiology " eating and drinking without temperance+bitter cold rush down under+overstrain " method modeling, continuous modeling 10 days build syndrome of deficient cold of spleen and stomach mould with this
Type.
Step 2 prepares gastric ulcer animal model
Carry out the preparation of gastric ulcer model immediately after preparing deficiency-cold in spleen and stomach model of a syndrome.It is anhydrous that model group rats give 1ml
Ethyl alcohol/200g weights carries out absolute ethyl alcohol gavage, give again after 1h 200mg aspirin aqueous solution/kg weights carry out Ah
Take charge of a woods aqueous solution gavage, successive administration 4 days.As a result, prepared by spleen-stomach deficiency-cold gastric ulcer Disease Syndrome integrated animal model to complete.
Gained spleen-stomach deficiency-cold gastric ulcer Disease Syndrome integrated animal model has down in spirits, slow movement inability, reaction slow
Blunt, burnout flocks together, narrows eye, eyes without refreshing, body flesh is very thin, weight mitigates, dorsal body setae evacuation is without pool (or even erect and turn to be yellow), stool
It is half congealed rush down, the performance of the deficiency-cold in spleen and stomach syndrome such as anus filth.
Experiment effect detects：
Main agents used in effect detection and source include：Chloraldurate (Beijing Suo Laibao Science and Technology Ltd)；
Sodium chloride injection (Hunan Cologne Pharmaceutical Co., Ltd)；Absolute ethyl alcohol (Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd)；
PBS phosphate buffers (pH 7.2-7.4, Beijing Suo Laibao Science and Technology Ltd)；Malonaldehyde (MDA) assay kit (Nanjing
Build up Bioengineering Research Institute)；Nitric oxide (NO) assay kit (Bioengineering Research Institute is built up in Nanjing)；Rat PGE
ELISAKIT (the Shanghai bio tech ltd Ji Ning)；Rabbit anti-TLR2/HRP conjugated antibody
(Beijing Bo Aosen Bioisystech Co., Ltd)；(Beijing is rich by Rabbit anti-MyD88/HRP conjugated antibody
Ao Sen Bioisystech Co., Ltd)；(Beijing Bo Aosen biotechnologys have Goat anti-rabbit IgG/HRP antibody
Limit company)；DAB colour reagents box (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；Eosin stain (Beijing Zhong Shan Golden Bridge biology
Technology Co., Ltd.)；Hematoxylin (Shandong West Asia chemical industry Co., Ltd).
Key instrument equipment and source used in effect detection include：KD-BM II computer biological tissue embedding machines (Zhejiang
The Jinhuas Jiang Sheng section enlightening experimental instruments and equipment limited)；CM1900 freezing-microtomes (German LEICA companies)；YD-A biological tissues
Spread out piece machine (Jinhua, Zhejiang Province city Yi Di Medical Devices Co., Ltd)；Tri- mesh biomicroscope (Mikes of Moticam Pro 205A
Audi (Xiamen) medical diagnosis system Co., Ltd)；ME204 electronic balances (METTLER TOLEDO companies of Switzerland)；
Centrifuge 5810R high speed freezing centrifuges (German Eppendorf companies)；SmartSpecTM plus nucleic acid-proteins are surveyed
Determine instrument, SDS-PAGE electrophoresis systems, ChemiDocTM XRS+Imaging System (BIO-RAD companies of the U.S.)；Cytation
Imaging reader (BioTek companies of the U.S.)；UV-1750 ultraviolet-visibles photometer (Japanese Shimadzu Corporation).
Experiment effect detection method includes with lower part：
(1) the general symptom of rat is acquired with sign
It is the state of mind of the whole observation each group rat of experiment, mass activity situation, appetite state, hair luster, fecal
Shape carries out the careful observation of emphasis to tongue picture, muffle, auricle, pawl toe.Weight, food-intake, the drinking-water of a rat are measured every other day
Amount, anus temperature.
The result shows that the normal rat state of mind is good before modeling, agile, freely, body flesh is healthy and strong, dorsal body setae light for activity
Bright Mi Ze, it clings to the body, stool is normal；Model group rats down in spirits after modeling, slow movement is powerless, slow in reacting, burnout
It flocks together, narrows eye, eyes without god, body flesh is very thin, and weight mitigates, and dorsal body setae evacuation is without pool (or even erectting jaundice), diarrhea.Greatly
Mouse mass activity situation proves spleen-stomach deficiency-cold gastric ulcer model modeling success of the present invention.
Fig. 1 is of moderate size the results show that normal rat red tongue body is lived bright profit before modeling, thin white fur of tongue；Muffle is light red micro- wet
Profit；Auricle is red and white, and train of thought is clear；Pawl toe is dark red, and activity flexibly, is grabbed and climbed effectively.Model Rat Tongue matter is livid purple after modeling, dark
It is dim unglazed；Muffle is purple dark；Auricle is light white or even pale, and auricle train of thought is shunk；Pawl toe is not received, livid purple to send out cool, and activity is powerless.Rat
Topical manifestations prove spleen-stomach deficiency-cold gastric ulcer animal model modeling of the present invention successfully.
Fig. 2 and table 1 are the results show that normal rats weight keeps steady growth trend, model group rats to exist before modeling
Modeling starts weight on the 1st day and downward trend is just presented, and starts weight within the 7th day and is decreased obviously, the P compared with normal group<0.05,
With statistical significance.
Table 1：Rat stomach body mass ratio is compared with (means standard deviation)
Note：*P<0.05, compared with normal group
Fig. 3 and table 2 normally organize the food-intake kept stable with model group rats the results show that 6 days before modeling,
Start within 8th day, model group rats food-intake significantly reduces, the P compared with normal group<0.05, there is statistical significance.
Table 2：Rats eating amount compares (means standard deviation)
Note：*P<0.05, compared with normal group
The results show that 8 days before modeling, each group rat amount of drinking water is consistent substantially for Fig. 4 and table 3, and the 9th day starts model
Reduction trend is presented in group rat amount of drinking water.
Table 3：Rat amount of drinking water compares (means standard deviation)
Fig. 5 and table 4 the result shows that, since modeling the 8th day, model group rats anus temperature was begun to decline, measured value twice
Respectively (34.96 ± 0.23) DEG C and (35.17 ± 0.09) DEG C are significantly lower than normal rats anus temperature value (P<0.05), have
It is statistically significant.Result above further demonstrates that, spleen-stomach deficiency-cold gastric ulcer disease Binding experiment model construction of the present invention at
Work(.
Table 4：Rat anus temperature compares (means standard deviation)
Note：*P<0.05, compared with normal group
(2) stomach lining ulcer index measures
Mucosal lesion degree is visually observed, for the pathology damages such as the erosion of gastric epithelial, ulcer, hemorrhagic focus, ginseng
According to Guth standards, mucosal lesion integral is calculated：Normal is 0 point, and lesion length≤1mm counts 1 point, 1mm < lesion lengths≤2mm
2 points of meter, 2mm < lesion lengths≤3mm count 3 points, and 3mm < lesion lengths≤4mm counts 4 points.When lesion length > 4mm, segmentation meter
Point, if lesion width G T.GT.GT 2mm, score are multiplied by 2.Finally referred to using the mean value of stomach lining Lesion score total value as stomach lining ulcer
Number.
Fig. 6 and table 5 have no apparent ulcer the results show that normal rat stomach lining finishing is intact.Model group rats stomach lining
There is acute injury, it is seen that apparent blutpunkte or streak bleeding band, ulcer index is up to 28.67 ± 5.43, the P compared with normal group
<0.01, there is statistical significance；It is taste that result above demonstrates the model constructed by the present invention from mucous membrane macroscopic form level
Asthenic cold type gastric ulcer experimental model.
Table 5：Rat gastric ulcer index (means standard deviation)
| Group | N | Ulcer index |
| Normal group | 12 | 0.42±0.51 |
| Model group | 12 | 28.67±5.43** |
Note：**P<0.01, compared with normal group
(3) gastric mucosa tissue morphological observation
After the last administration for 24 hours, rat is put to death, full stomach is won, stomach bottom point is wiped out, with 4% poly after physiological saline cleaning
Formaldehyde is fixed, and takes the tissue of 1cm × 1cm sizes to body of stomach ulcer spot in antrum, using Gradient elution using ethanol, routine paraffin wax packet
Slice is buried, hematoxylin-eosin (hematoxylin and eosin, HE) dyes, after neutral gum mounting under light microscope
Carry out Histomorphological.
Fig. 7 is the results show that normal rat gastric tissue is well arranged, structural integrity；Epithelial cell marshalling has no damage
Hinder, fall off；Mucous membrane surface foveolae gastricae is high-visible, and intrinsic body of gland queueing discipline is close, has no apparent congested and cell infiltration.
There is apparent damage in model group rats stomach lining, and a large amount of epithelial cell denaturation, fall off at erosion；Foveolae gastricae structure is destroyed, body of gland knot
Structure is disorderly or lacks；(lymphocyte, neutrophil(e) granule are thin for lamina propria and the visible different degrees of oedema of muscularis mucosae and inflammatory cell
Born of the same parents) it infiltrates, submucosa thickens.Result above confirms that rat gastric ulcer model construction is successful, and Chinese medicine from microscopic pattern level
Card type is syndrome of deficient cold of spleen and stomach.
(4) gastric mucosa nitric oxide (NO), malonaldehyde (MDA), prostaglandin E (PGE) content
Using Nitric Oxide in Rats (NO) assay kit, malonaldehyde (MDA) assay kit, prostaglandin E (PGE)
ELISA kit is tested according to kit operational manual step, is detected using BioTek cytation5 microplate reader
PGE and NO contents use Shimadzu UV-1750 ultraviolet-visible photometric determination MDA contents.PGE measures suction at wavelength 450nm
Luminosity makees calibration curve equation, and sample P GE contents are calculated according to the equation of linear regression of standard curve.The detection wave of NO and MDA
Long is respectively 550nm, 532nm, and NO contents=(measuring OD values-blank OD values/standard OD values-blank OD values) × standard items are dense
Degree；MDA contents=(measuring OD values-control OD values/standard OD values-blank OD values) × standard concentration.
PGE, NO, MDA are the important indicators for evaluating gastric ulcer and occurring, healing.Wherein PGE and NO is the mucosa protection factor,
MDA is mucous membrane attack factor.For table 6 the results show that compared with normal group, model group PGE, NO content is remarkably decreased (equal P<
0.01)；Compared with normal group, model group rats stomach lining MDA contents significantly increase (P<0.01).The above results are from cell factor
Level confirms that the experimental model constructed by the present invention is spleen-stomach deficiency-cold gastric ulcer animal model.
Table 6：Gastric mucosa NO, MDA, PGE content (means standard deviation)
| Group | N | PGE(pg/mL) | MDA(nmol/mL) | NO(μmol/L) |
| Normal group | 6 | 63.46±6.27 | 5.09±1.06 | 76.69±6.27 |
| Model group | 6 | 38.87±4.15** | 9.46±2.17** | 28.57±3.98** |
Note：**P<0.01, compared with normal group
(5) gastric mucosa TLR-2 protein expressions
It takes antrum to the tissue of body of stomach mucosa injury position 1cm × 1cm sizes, is cleaned with 4 DEG C of physiological saline, 4% poly
Formaldehyde fixes, is dehydrated, paraffin embedding.By gastric mucosa tissue paraffin section de-waxing, aquation, 70 μ l 3%H are added dropwise in every slice2O2
Room temperature is closed, and antigen is repaired, and heterogenetic antigen is closed with 5% fetal calf serum.With TLR-2 primary antibodies (1：100) it was incubated for 4 DEG C
Night, dropwise reaction enhancement solution react at room temperature 20min, with Goat anti-rabbit secondary antibodies (1 after PBS flushings：2000) room temperature is incubated
Educate 2h.DAB solution develops the color, and haematoxylin is redyed, and acidic alcohol color separation, dimethylbenzene is transparent, neutral gum mounting.Every slice is aobvious
4 high power fields are shot under micro mirror, and the OD value of TLR-2 expression is measured using Image Pro Plus image analysis softwares.
TLR-2/MyD88 signal cascade wide expressions are distributed in gastrointestinal mucosa tissue, are the weights of the gastrointestinal mucosa innate immunity
Want molecule, gastrointestinal tract is inflamed or TLR-2/MyD88 expression drastically increases when ulcer.Fig. 8 and table 7 are the results show that TLR-2
Differential expression is mainly reflected in the gastric epithelial of rat.Normal rat stomach lining TLR-2 relative expression quantities (IOD values) are
13.69 ± 3.89, model group rats stomach lining TLR-2 relative expression quantity (IOD values) are 53.35 ± 6.55, are significantly higher than normal
Group (P<0.01).Result above confirms that the present invention successfully constructs spleen-stomach deficiency-cold gastric ulcer animal model from molecular level.
Table 7 stomach lining TLR-2 protein expressions optical density (IOD) value
| Group | N | Optical density (IOD) value |
| Normal group | 6 | 13.69±3.89 |
| Model group | 6 | 53.35±6.55** |
Note：**P<0.01, compared with normal group
(6) gastric mucosa MyD88 protein expressions
4% paraformaldehyde of learning from else's experience fixes gastric mucosa tissue for 24 hours, with paraffin embedding.Gastric mucosa tissue is routinely dewaxed, ladder
Spend dehydration of alcohol processing.Every slice is closed with the hydrogen peroxide room temperature of 70 μ l 3%, PBS cleanings.Slice is immersed in PBS,
Micro-wave oven is heated to boiling, and repairs antigen.Heterogenetic antigen, PBS cleanings are closed using 5% fetal calf serum.MyD88 mono- is added
Anti- (1：100), in 4 DEG C of overnight incubations, PBS cleanings are added increased response liquid, are placed at room temperature for 20min.PBS is cleaned, and Goat is added dropwise
Anti-rabbit secondary antibodies (1：2000) it is incubated at room temperature 2h.PBS is cleaned, with DAB reagent colour developments, microscopically observation colour developing knot
Fruit, tap water terminate reaction.Haematoxylin is respectively adopted to redye, acidic alcohol color separation, dimethylbenzene is transparent, neutral gum mounting.Often
It opens slice and respectively shoots 4 visuals field under 10 × 20 times of mirrors, positive expression is detected using Image Pro Plus image analysis softwares
Optical density (IOD) value in region, i.e. MyD88 albumen relative expression quantity.
Fig. 9 and table 8 the result shows that, when damage occurs for stomach lining or when ulcer (model group), MyD88 protein expressions significantly increase
Add, the P compared with normal group<0.01.Result above further discloses spleen-stomach deficiency-cold gastric ulcer experiment of the present invention from molecular level
The successful structure of model.
Table 8 stomach lining MyD88 protein expressions optical density (IOD) value
| Group | N | Optical density (IOD) value |
| Normal group | 6 | 13.43±3.43 |
| Model group | 6 | 48.70±6.43** |
Note：**P<0.01, compared with normal group
The foregoing is merely the preferred embodiment of the present invention, are not intended to restrict the invention, for those skilled in the art
For member, the invention may be variously modified and varied.Any modification made by all within the spirits and principles of the present invention,
Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method for building up of spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model, includes the following steps：
(1), the method that lower and overstrain is combined is rushed down using eating and drinking without temperance, bitter cold and modeling is carried out to model group animal, obtained
The animal model of syndrome of deficient cold of spleen and stomach type；
(2), gavage is carried out to the animal model of syndrome of deficient cold of spleen and stomach type obtained by step (1) with absolute ethyl alcohol and aspirin, obtained
Spleen-stomach deficiency-cold gastric ulcer disease combination animal experimental model.
2. method for building up according to claim 1, which is characterized in that described using eating and drinking without temperance, bitter cold in step (1)
It rushes down the method that lower and overstrain is combined and modeling is carried out to model group animal, including：
(1.1), first day, the free diet drinking-water of model group animal carried out gavage, so with folium sennae water decoction to model group animal
After so that model group animal is swum powerless to four limbs strokes, picked up immediately when head is submerged in water；
(1.2), second day, the fasting of model group animal, free water carried out gavage with pig fat to model group animal, then with kind
It rushes down leaf water decoction and gavage is carried out to model group animal, so that model group animal is swum powerless to four limbs stroke, water is submerged on head
It is picked up immediately when middle；
(1.3), it repeats step (1.1) and step (1.2) carries out continuous modeling, establish the animal model of syndrome of deficient cold of spleen and stomach type.
3. method for building up according to claim 2, which is characterized in that described to use folium sennae water decoction pair in step (1.1)
Model group animal carry out gavage, in particular to：0.8-1.2ml folium sennaes water decoction/100g weights are given to model group animal
Carry out folium sennae water decoction gavage.
4. method for building up according to claim 2, which is characterized in that
In step (1.2), it is described with pig fat to model group animal carry out gavage, in particular to：Model group animal is given
1.8-2.2ml pig fats/100g weights carry out pig fat gavage；
In step (1.2), it is described with folium sennae water decoction to model group animal carry out gavage, in particular to：To model group animal
Give 0.8-1.2ml folium sennaes water decoction/100g weights and carries out folium sennae water decoction gavage.
5. method for building up according to claim 2, which is characterized in that the folium sennae water decoction is prepared via a method which
It obtains：
Folium sennae is added in distilled water and decocts 1-2h, is filtered, folium sennae Aqueous extracts are collected, then same amount of folium sennae is taken to be added
In the folium sennae Aqueous extracts of collection, distilled water is continuously added, decocts 1-2h, filtering collects filtrate to get the folium sennae decocting
Agent, a concentration of 0.8-1.2g/ml of the folium sennae water decoction.
6. method for building up according to claim 2, which is characterized in that total number of days of the modeling is 10-12 days.
7. method for building up according to claim 1, which is characterized in that described to use absolute ethyl alcohol and A Si in step (2)
Woods carries out gavage to the animal model of syndrome of deficient cold of spleen and stomach type obtained by step (1), including：
0.8-1.2ml absolute ethyl alcohols/200g weights are given to the animal model of syndrome of deficient cold of spleen and stomach type and carry out absolute ethyl alcohol gavage,
Then give 180-220mg aspirin aqueous solution/kg weights again and carry out aspirin aqueous solution gavage.
8. method for building up according to claim 7, which is characterized in that with absolute ethyl alcohol and aspirin to syndrome of deficient cold of spleen and stomach
The number of days that the animal model of type carries out gavage is 4-6 days.
9. method for building up according to claim 1, which is characterized in that the model group animal refers to：SPF grades of male SD are big
Mouse, weight 160-200g；First adaptable fed 3 days, during which freely ingest and drink water before modeling, raising temperature control
At 18-23 DEG C, in 45%-55%, the alternation of light and darkness period is 12 hours for relative humidity control.
10. a kind of animal experimental model that method for building up as claimed in any one of claims 1-9 wherein obtains is in peptic ulcer
Clinical research in or the drug development of peptic ulcer in application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810909308.6A CN108739668B (en) | 2018-08-10 | 2018-08-10 | Method for establishing animal experiment model by combining spleen and stomach deficiency-cold type gastric ulcer disease and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201810909308.6A CN108739668B (en) | 2018-08-10 | 2018-08-10 | Method for establishing animal experiment model by combining spleen and stomach deficiency-cold type gastric ulcer disease and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108739668A true CN108739668A (en) | 2018-11-06 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113016714A (en) * | 2021-03-04 | 2021-06-25 | 湖南中医药大学 | Preparation method of animal model with diarrhea and dyspepsia gastrointestinal syndrome and method for evaluating animal model with diarrhea and dyspepsia gastrointestinal syndrome |
| CN116983334A (en) * | 2023-07-11 | 2023-11-03 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Animal model combining spleen deficiency and dampness encumbering with allergic rhinitis symptoms and construction method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113016714A (en) * | 2021-03-04 | 2021-06-25 | 湖南中医药大学 | Preparation method of animal model with diarrhea and dyspepsia gastrointestinal syndrome and method for evaluating animal model with diarrhea and dyspepsia gastrointestinal syndrome |
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| CN116983334A (en) * | 2023-07-11 | 2023-11-03 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Animal model combining spleen deficiency and dampness encumbering with allergic rhinitis symptoms and construction method and application thereof |
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