CN106511345A - Construction method of oral submucous fibrosis mouse model - Google Patents
Construction method of oral submucous fibrosis mouse model Download PDFInfo
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Abstract
The invention discloses a construction method of an oral submucous fibrosis (OSF) mouse model. The construction method comprises the following steps: (1) selection and grouping of animals; (2) preparation of an arecoline drinking agent; (3) feeding and killing of the animals; (4) experimental observation; (5) construction of the mouse OSF model. According to the construction method of the OSF mouse model, a Balb/c mouse is fed by the arecoline drinking agent with the concentration of 500 to 2,000 mg/L for 20 weeks, and the obtained mouse OSF model is highly similar to human OSF; the animal model is high in morbidity (100 percent) and has varieties of pathogenic sites; mucous membranes at all parts in an oral cavity can be attacked; an induction method is simple and natural; the mouse drinks water freely, so that influence caused by physical stimulation and stimulation of other chemical medicines is prevented; the effect is ideal, and the induction time is short.
Description
Technical field
It is the present invention relates to a kind of medical domain more particularly to a kind of by administering mode and the process of dosage, structure
Build a kind of method of oral submucosa fibrosises mouse model.
Background technology
Oral submucosa fibrosises (oral submucous fibrosis, OSF) be it is a kind of it is chronic, invisible, with evil
Become the oral mucosal disease of tendency, any part of oral mucosa can occur.Follow up study shows, oral submucosa fibrosises with
The commercialized development of Semen Arecae, sickness rate increases year by year, but the Therapeutic Method that clinic is there is no for OSF at present.Therefore OSF
The research of model is constantly subjected to pay attention to.It is few with regard to the report of the Research of Animal Model for Study of OSF both at home and abroad now, greatly constrain
What OSF was studied further gos deep into.Nineteen sixty, Sirsat etc. carry out surface using 2% capsaicin in rat palate mucosa and apply induction
OSF sample pathological changes are gone out.But its pathological manifestations owes typical case, and without obvious clinical manifestation.Separately there are Chinese scholars all by cheek
Mucosa carries out injecting and surface is coated with the mode of aqueous areca nut extract and induces large and small Mus OSF, but method is loaded down with trivial details and time mistake
It is long, substantially all more than 600 days, and it is showed no obvious later stage vitreous degeneration.Therefore, the structure of existing OSF animal models
The pathological changes relation for being difficult to simulate that people OSF occurs development is built, and infrastest requirement now cannot be met.From current document
See, also fail to obtain gratifying OSF models.If exploring OSF occurs development and prognosis of disease, existing model does not have
There are certain feasibility, reliability.This model is modeled using high concentration arecoline drinking technique first, it is to avoid injection and applied
The Confounding Factor that cloth is likely to result in affects, and reduces into the mould time, improves into and touch success rate, and the OSF for inducing and people OSF has
There is high similarity.
The content of the invention
It is an object of the invention to provide a kind of construction method of oral submucosa fibrosises mouse model, set up mice with
Mankind OSF has the OSF models of high similarity, and the method is simple, and sickness rate is high, and induction time is short, based on the model,
Treatment and preventing and treating for mankind OSF provides strong support.
To achieve these goals, the technical scheme is that：A kind of oral submucosa fibrosises mouse model is provided
Construction method, comprise the following steps：
(1) animal selects and is grouped：It is experiment mice to take 8 week old male Balb/c mices 40, and stochastic averagina is divided into
Experimental group and matched group；
(2) arecoline drinking agent is prepared：By the arecoline preserved under room temperature, 500～2000mg/ of concentration is made into tap water
The arecoline drinking agent of L, arecoline drinking agent is placed in experimental mice drinking bottle and is freely drunk；Control group mice is with originally
Water is freely drunk；
(3) animal feeding and execution：Experimental group is fed 8～20 weeks using arecoline drinking agent, and the matched group same period gives certainly
Water is fed, and random per 4 weeks to put to death 4 mices of each group, remaining mice is all put to death within 20 weeks；
(4) laboratory observation：Carry out overview to experimental group and matched group respectively, after putting to death mice, take oral mucosa group
Knit, take the observation of hematoxylin eosin stain method, MASSON trichrome stains and the observation of Van Gieson stainings, SABC spectrum
I/III collagen types and SABC CD34 dyeing observations；
(5) mice OSF models are built：By step (1) to (4) step, show that arecoline chemical derivatization is successfully established
Mice OSF models, and set up file formatting model.
Preferably, the concentration of the arecoline drinking agent is 2000mg/L.
The experiment mice is：8 week old male Balb/c mices, SPF levels, body weight (18.52 ± 1.15) g, in room temperature 18
～25 DEG C, humidity 30%～50%, fed standard chow, raise under the conditions of 24h Dark-light cycles.
The overview is：Situations such as observing mice mental status, mobility, amount of drinking water daily in experimentation, often
Zhou Jilu mices amount of drinking water and body weight change.After mice was put to death per 4 weeks, the change of observation panel intracavity, mucosa color and luster in recording mouth,
Whether quality etc. changes, if having speckle, ulcer or swollen thing to occur, and observes scope, the limitation of mouth opening situation of fibrous bands.
Hematoxylin eosin stain method (hematoxylin-eosin staining, abbreviation HE are dyeed)
It is viewed as：Take oral cavity tissue mucosa (band part deep muscle tissue) after putting to death mice immediately, esophaguses, stomach, liver,
Spleen, lung, kidney, small intestinal, large intestine etc. are organized, and are fixed with 10% neutral formalin solution, conventional organization process, paraffin embedding, and 4 μm are cut
Piece, haematoxylin eosin stains carry out pathological study.Oral mucosa affection tissue is carried out by stages by OSF pathology standard,
That is earliest period, early stage, mid-term, late period.
The MASSON trichrome stains and Van Gieson dyeing are viewed as：Put to death mice and obtain oral cavity tissue mucosa, use
10% neutral formalin solution is fixed, conventional organization process, paraffin embedding, and 4 μm of sections, respectively by MASSON test kits (Wuhan
Doctor's moral biological engineering company limited) and Van Gieson test kits (Wuhan Boster Biological Technology Co., Ltd.) dyeing observations
Oral mucosa early stage fiber situation of change.
The SABC wide spectrum I/III collagen types observation collagen situation and SABC CD34 dyeing observation epitheliums
Lower blood vessel number is：Using immunohistochemical ABC method, according to routine operation：Murine oral mucous membrane tissue wax stone is cut with 4 μm of thickness
Piece, Jing aquations suppress endogenous peroxydase, after eliminating unspecific staining, one AntiCD3 McAb of difference Deca, 4 (Mouse Polyclonal
Antibody, purchased from Wuhan Boster Biological Technology Co., Ltd., by 1:100 dilution), I-type collagen (Mouse Polyclonal Antibody,
Purchased from Wuhan Boster Biological Technology Co., Ltd., by 1:250 dilution), (Mouse Polyclonal Antibody is purchased from type III collagen protein
Wuhan Boster Biological Technology Co., Ltd., by 1:180 dilution), while, positive control control group mice oral mucosa group
Knit, negative control PBS replaces one to resist, and 4 DEG C overnight；Using SP test kits, (sheep anti mouse anti-rabbit is universal, Beijing for later stage colour developing
Bioisystech Co., Ltd of Zhong Shan Golden Bridge) colour developing, it is dehydrated transparent mounting.Result judgement：CD34 positive reactions thing be light brown extremely
Dark brown granule, is distributed in vascular endothelial cell.Result judgement：I, type III collagen IV-positive reactant are light brown to depth
Brown particle, is distributed in mucosa lamina propria.
The construction method of oral submucosa fibrosises mouse model of the present invention, is drunk using the arecoline of 500～2000mg/L
Water preparation feeds Balb/c mices 20 weeks, obtains mice OSF models and there are high similarity, i.e. pathological changes to occur, develop with mankind OSF
Integrity, it can be observed that the clinical symptoms in earliest period-early stage-mid-term-late period OSF each stages and Pathological.And
Using various detection methods, it is mutually authenticated, eliminates the interference of other related oral fibrosis, improves the accuracy of diagnosis
And repeatability.Animal model sickness rate height (100%), site of pathological change variation, oral cavity Nei Ge positions mucosa can be fallen ill,
The method of induction is easy to be natural, by mice free water, it is therefore prevented that the impact that physical stimulation and other chemicalses stimulate, effect
Fruit is preferable, and induction time is short, and the treatment and preventing and treating for mankind OSF provides strong instrument.
Description of the drawings
Fig. 1：Changes in histopathology (HE × 200) figure of mice different time sections tongue (root of the tongue) mucosa.Each time period
Experimental group (D, E, F) is contrasted with matched group (A, B, C)：It can be seen that epithelial atrophy, mucosa lamina propria, Submucosa collagen deposition and micro-
Vascular lesion, and with the prolongation of administration time, gradually increasing, the obvious vitreous degeneration of last visible lamina propria.Illustrate to build mould
Type success.
Fig. 2：Collagen tissue Pathologic changes (Masson × 200, the V.G × 200) of different time sections mice lingual mucous membrane.It is right
According to group：Masson dyes (A) and V.G dyeing (E), and collagen fiber are elongated, move towards straight, arranges loose (black arrow)；Experiment
Group:8 weeks Masson dyeing (A) and V.G dye (E) and show a small amount of blue dye and red dye region, extend with administration time, 12 weeks
Masson dyeed (C) and the visible comparatively dense collagen fiber bands of V.G dyeing (G), to 16 weeks Masson dyeing (D) and V.G dyes
Color (H), the collagen for being close to epithelium are arranged in the collagen fiber band of obvious densification, and collagen fiber are obvious compared with matched group
Increase, collagen fiber dyed color is deep and obvious, and blue region is mutually echoed with pale red region.
Fig. 3：Different time sections I-type collagen and III collagen protein show (× 200) in tongue.I-type collagen
(A-D), III collagen protein (E-H).Normal lingual mucous membrane (A, E)；8 weeks (B, F)；16 weeks (C, G)；20 weeks (D, H).Type i collagen with
Advancing of disease is consistent, in the way of being in diffusing, be dispersed in is distributed in lamina propria and Submucosa (B-D).8 weeks type III glue
Former (F) is in the slight immuno positive of lamina propria.During by 16 weeks, type III collagen is substantially expressed (G), and is lost in 20 weeks (H) expression completely
Lose.
Fig. 4：Different time sections vascular counts.Experimental group blood vessel number is first slowly increased, and has system to two groups of contrasts in the 12nd week
Meter learns meaning, and then experimental group blood vessel number declines, to two groups of contrasts in 20 weeks statistically significant (pairing " t " inspection, *<
0.05)。
Specific embodiment
Embodiment 1
(1) animal selects and is grouped：It is experiment mice to take 8 week old male Balb/c mices 40, and stochastic averagina is divided into
Experimental group and matched group；
The condition of experiment mice is：Described experiment mice is：8 week old male Balb/c mices, SPF levels, body weight
(18.52 ± 1.15) g, raises under the conditions of 18～25 DEG C of room temperature, humidity 30%～50%, fed standard chow, 24h Dark-light cycles
Support；
(2) arecoline drinking agent is prepared：By the arecoline preserved under room temperature (arecoline), concentration is made into tap water
2000mg/L medicines are placed in experimental mice drinking bottle and freely drink.Control group mice is freely drunk with tap water；
(3) animal feeding and execution：Experimental group is fed 20 weeks using arecoline drinking agent, and the matched group same period gives tap water
Feed, random per 4 weeks to put to death 4 mices of each group, remaining mice is all put to death within 20 weeks；
(4) laboratory observation：Carry out overview to experimental group and matched group respectively, after putting to death mice, take oral mucosa group
Knit, take hematoxylin eosin stain method (hematoxylin-eosin staining, abbreviation HE are dyeed) observation, MASSON tri-
Color is dyeed and the observation of Van Gieson stainings, and SABC composes I/III collagen types with SABC CD34 dyeing observations；
Overview is：
Situations such as observing mice mental status, mobility, amount of drinking water daily in experimentation, records weekly mice amount of drinking water
And body weight change.After mice was put to death per 4 weeks, observation panel intracavity changes, mucosa color and luster in recording mouth, and whether quality etc. changes,
Whether there are speckle, ulcer or swollen thing to occur, observe scope, the limitation of mouth opening situation of fibrous bands.
HE dyeing is viewed as：
Take oral cavity tissue mucosa (band part deep muscle tissue) after putting to death mice immediately, esophaguses, stomach, liver, spleen, lung, kidney,
Small intestinal, large intestine etc. are organized, and are fixed with 10% neutral formalin solution, conventional organization process, paraffin embedding, 4 μm of sections, haematoxylin
Eosin stains, carry out pathological study.Oral mucosa affection tissue carry out by stages by OSF pathology standard, i.e., earliest period,
In early days, mid-term, late period.
MASSON trichrome stains and Van Gieson dyeing are viewed as：
Put to death mice and obtain oral cavity tissue mucosa, fixed with 10% neutral formalin solution, conventional organization process, paraffin bag
Bury, 4 μm of sections, respectively by MASSON test kits (Wuhan Boster Biological Technology Co., Ltd.) and Van Gieson test kits
(Wuhan Boster Biological Technology Co., Ltd.) dyeing observation oral mucosa early stage fiber situation of change.
SABC wide spectrum I/III collagen types observe collagen situation with SABC CD34 dyeing observation epithelium hematochezia
Pipe quantity is：
Using immunohistochemical ABC method, according to routine operation：Murine oral mucous membrane tissue wax stone is cut into slices with 4 μm of thickness, Jing
Aquation, suppresses endogenous peroxydase, after eliminating unspecific staining, respectively one AntiCD3 McAb 4 of Deca (Mouse Polyclonal Antibody,
Purchased from Wuhan Boster Biological Technology Co., Ltd., by 1:100 dilution), (Mouse Polyclonal Antibody, purchased from force for I-type collagen
Han Boshide biological engineering company limited, by 1：250 dilutions), type III collagen protein (Mouse Polyclonal Antibody, it is rich purchased from Wuhan
Shi De biological engineering company limited, by 1:180 dilutions), while, positive control control group mice oral cavity mucous membrane tissue is cloudy
Property control replaces one anti-with PBS, 4 DEG C are overnight；Using SP test kits, (sheep anti mouse anti-rabbit is universal, China fir in Beijing for later stage colour developing
Bioisystech Co., Ltd of Golden Bridge) colour developing, it is dehydrated transparent mounting.Result judgement：CD34 positive reactions thing is light brown to dark brown
Coloured particles, are distributed in vascular endothelial cell.Result judgement：I, type III collagen IV-positive reactant are light brown to dark-brown
Granule, is distributed in mucosa lamina propria.
Observation result (such as Fig. 1,2,3,4)：
Test rise within the 2nd week after there is weight loss, but the 4th week in existing experimental mice find body weight be slowly increased, treat reality
After testing the 8th week, experimental mice body weight tends towards stability, and average weight is (17.52 ± 1.15) g.Control group mice average weight
For (27.52 ± 1.23) g.It is low and deep that hair color occurs in the experimental mice later stage, and mobility is reduced, and is reluctant feed etc..Control group mice
Hair color is vivid, appetite, mobility are good.Experimental mice oral mucosa start to engender the 8th week from experiment turn white, it is stiff
Firmly, the different degrees of above-mentioned change of appearance of all mice mucosas in the 20th week is extremely tested, but has no that obvious fibrous bands are formed.
Matched group oral mucosa color, quality change be not obvious.As time goes on, experimental mice mouth opening is gradually reduced, right
Gradually increase according to a group mice mouth opening, from the beginning of experiment the 16th week, the mouth opening significant difference of two groups of mices.Experimental mice
From 8 weeks, tongue tissue starts to occur in that OSF samples change, and after 12 weeks, cheek palatine tissue also occurs in that OSF samples change.Experimental group is little
Mus early stage Oral mucosa keratinocyte starts abnormal positive keratinization, cuticular layer atrophy, the visible full nucleus of fibrocyte, lymph is presented
Cellular infiltration, lamina propria are thickened, and blood vessel number increases, and substantially, caliber changes for the congestion of blood vessel.Extend with administration time, epithelial peg
It is prominent gradually become shallower as, mice, lamina propria thickness thickens all the more substantially, upper veins beneath the skin there is first congested expansion after atresia gradually
Change process, drug-induced to the 20th week, epithelial thickness is persistently reduced, it is seen that atrophic outward appearance.Lamina propria collagen is in unordered, wavy
Profile sequence fiber it is fine and close, cell quantity is reduced, and basal layer cell destruction is obvious.Collagen fiber density increase regions, it is seen that
Class vitreous degeneration.Lamina propria blood vessel lacks, and is wrapped by fibrous tissue, the visible fibers encapsulation of muscle under mucosa, and majority of case can
See muscle layer atrophy.Masson dyeing shows that collagen is distributed in bluish violet, the bluish violet elastic fiber of a small amount of stained positive of matched group
Mucosa lamina propria is distributed in, collagen fiber are elongated, moves towards straight, arrange loose, thicker elastic fiber sends many fine length
Fiber extend in nipple connective tissue.The fine eventually end of these elastic fibers epithelium basal cell weaves beneath into
Elastic webs.Experimental group cheek mucosa lamina propria thickness broadens, and collagen fiber deposit thick and fine and close herein, thickness,
The collagen for being close to epithelium is arranged in the collagen fiber band of obvious densification, increases (8 weeks, 12 weeks, 16 weeks) over time, glue
Fibril showed increased compared with matched group, collagen fiber dyeing after color also become it is deeper.Additionally, being contaminated by Van Gieson
Color shows that cherry intensive collagen is corresponding with Masson dyeing, and has similar collagen change, mutually confirms.Tie by more than
Fruit understands, when 8 weeks, OSF early lesions occurs in tongue tissue, and when 12 weeks, cheek, palatine tissue occur OSF early lesions in succession, extremely
16 weeks, there are OSF mid-term pathological changes in tongue, palate, buccal tissue, and induction is caused 20 weeks, and OSF advanced lesions occur in tongue, palatine tissue.Pass through
Veins beneath the skin quantity on CD34 countings, is carried out to blood vessel under experiment, control group mice oral mucosa under microscope high power field
Find after counting：Increase over time, 2 groups of blood vessel numbers gradually increase, and experimental group increase becomes apparent from, by 12 weeks
Afterwards, 2 groups of blood vessel number obvious differences, show as experimental group vascular counts and are significantly more than matched group；But as administration time prolongs
Long, experimental group blood vessel number starts decline occur on the contrary, has statistical significance, experimental group table to 2 groups of blood vessel differences when 20 weeks
It is now to significantly reduce.It is consistent with clinical people OSF Vascular changes.By I, type III collagen content semi-quantitative analyses, I types are found
Collagen extends with induction time, and content substantially increases, and matched group obvious difference, and type III collagen is without significant change, this and people
OSF I, type III pathological changes are consistent, show as collagen deposition increase, the collagen pathological changes situation degraded based on reducing.
(5) mice OSF models are built：By step (1) to (4), show that arecoline chemical derivatization is successfully established mice
OSF models, and set up file formatting model.
Embodiment 2
(1) animal selects and is grouped：It is experiment mice to take 8 week old male Balb/c mices 40, and stochastic averagina is divided into
Experimental group and matched group；
The condition of experiment mice is：Described experiment mice is：8 week old male Balb/c mices, SPF levels, body weight
(18.52 ± 1.15) g, raises under the conditions of 18～25 DEG C of room temperature, humidity 30%～50%, fed standard chow, 24h Dark-light cycles
Support；
(2) arecoline drinking agent is prepared：By the arecoline preserved under room temperature (arecoline), concentration is made into tap water
500mg/L medicines are placed in experimental mice drinking bottle and freely drink.Control group mice is freely drunk with tap water；
(3) animal feeding and execution：Experimental group is fed 20 weeks using arecoline drinking agent, and the matched group same period gives tap water
Feed, random per 4 weeks to put to death 4 mices of each group, remaining mice is all put to death within 20 weeks；
(4) laboratory observation：Carry out overview to experimental group and matched group respectively, after putting to death mice, take oral mucosa group
Knit, take hematoxylin eosin stain method (hematoxylin-eosin staining, abbreviation HE are dyeed) observation, MASSON tri-
Color is dyeed and the observation of Van Gieson stainings, and SABC composes I/III collagen types with SABC CD34 dyeing observations；
Overview is：
Situations such as observing mice mental status, mobility, amount of drinking water daily in experimentation, records weekly mice amount of drinking water
And body weight change.After mice was put to death per 4 weeks, observation panel intracavity changes, mucosa color and luster in recording mouth, and whether quality etc. changes,
Whether there are speckle, ulcer or swollen thing to occur, observe scope, the limitation of mouth opening situation of fibrous bands.
HE dyeing is viewed as：
Take oral cavity tissue mucosa (band part deep muscle tissue) after putting to death mice immediately, esophaguses, stomach, liver, spleen, lung, kidney,
Small intestinal, large intestine etc. are organized, and are fixed with 10% neutral formalin solution, conventional organization process, paraffin embedding, 4 μm of sections, haematoxylin
Eosin stains, carry out pathological study.Oral mucosa affection tissue carry out by stages by OSF pathology standard, i.e., earliest period,
In early days, mid-term, late period.
MASSON trichrome stains and Van Gieson dyeing are viewed as：
Put to death mice and obtain oral cavity tissue mucosa, fixed with 10% neutral formalin solution, conventional organization process, paraffin bag
Bury, 4 μm of sections, respectively by MASSON test kits (Wuhan Boster Biological Technology Co., Ltd.) and Van Gieson test kits
(Wuhan Boster Biological Technology Co., Ltd.) dyeing observation oral mucosa early stage fiber situation of change.
SABC wide spectrum I/III collagen types observe collagen situation with SABC CD34 dyeing observation epithelium hematochezia
Pipe quantity is：
Using immunohistochemical ABC method, according to routine operation：Murine oral mucous membrane tissue wax stone is cut into slices with 4 μm of thickness, Jing
Aquation, suppresses endogenous peroxydase, after eliminating unspecific staining, respectively one AntiCD3 McAb 4 of Deca (Mouse Polyclonal Antibody,
Purchased from Wuhan Boster Biological Technology Co., Ltd., by 1:100 dilution), (Mouse Polyclonal Antibody, purchased from force for I-type collagen
Han Boshide biological engineering company limited, by 1：250 dilutions), type III collagen protein (Mouse Polyclonal Antibody, it is rich purchased from Wuhan
Shi De biological engineering company limited, by 1:180 dilutions), while, positive control control group mice oral cavity mucous membrane tissue is cloudy
Property control replaces one anti-with PBS, 4 DEG C are overnight；Using SP test kits, (sheep anti mouse anti-rabbit is universal, China fir in Beijing for later stage colour developing
Bioisystech Co., Ltd of Golden Bridge) colour developing, it is dehydrated transparent mounting.Result judgement：CD34 positive reactions thing is light brown to dark brown
Coloured particles, are distributed in vascular endothelial cell.Result judgement：I, type III collagen IV-positive reactant are light brown to dark-brown
Granule, is distributed in mucosa lamina propria.
Observation result (such as Fig. 1,2,3,4)：
Test rise within the 6th week after there is weight loss, but the 8th week in existing experimental mice find body weight be slowly increased, treat reality
After testing the 10th week, experimental mice body weight tends towards stability, and average weight is (19.31 ± 0.87) g.Control group mice average weight
For (26.89 ± 1.57) g.It is low and deep that hair color occurs in the experimental mice later stage, and mobility is reduced, and is reluctant feed etc..Control group mice
Hair color is vivid, appetite, mobility are good.Experimental mice oral mucosa start to engender the 14th week from experiment turn white, it is stiff
Firmly, the different degrees of above-mentioned change of appearance of all mice mucosas in the 20th week is extremely tested, but has no that obvious fibrous bands are formed.
Matched group oral mucosa color, quality change be not obvious.As time goes on, experimental mice mouth opening is gradually reduced, right
Gradually increase according to a group mice mouth opening, to experiment the 20th week, the mouth opening significant difference of two groups of mices.Experimental mice the 12nd week
Rise, tongue tissue starts to occur in that OSF samples change, after 16 weeks, cheek palatine tissue also occurs in that OSF samples change.Experimental mice
Early stage Oral mucosa keratinocyte starts abnormal positive keratinization is presented, and cuticular layer atrophy, the visible full nucleus of fibrocyte, lymph are thin
Born of the same parents infiltrate, and lamina propria is thickened, and blood vessel number increases, and substantially, caliber changes for the congestion of blood vessel.Extend with administration time, epithelial peg is dashed forward
Gradually become shallower as, mice, lamina propria thickness thickens all the more substantially, and the gradual change of atresia after first congested expansion occurs in upper veins beneath the skin
Process, drug-induced to the 20th week, epithelial thickness is persistently reduced, it is seen that atrophic outward appearance.Lamina propria collagen is in unordered, wavy
Profile sequence fiber is fine and close, and cell quantity is reduced, and basal layer cell destruction is obvious.Collagen fiber density increase regions, but have no
Obvious class vitreous degeneration.Lamina propria blood vessel lacks, and is wrapped by fibrous tissue, the visible fibers encapsulation of muscle under mucosa, most of feelings
The visible muscle layer atrophy of condition.Masson dyeing shows that collagen is distributed in bluish violet, the bluish violet elasticity of a small amount of stained positive of matched group
Fiber is distributed in mucosa lamina propria, and collagen fiber are elongated, moves towards straight, arranges loose, and thicker elastic fiber sends many micro-
Elongated fiber is extended in nipple connective tissue.Hand over below the basal cell of epithelium at the fine end eventually of these elastic fibers
It is made into elastic webs.Experimental group cheek mucosa lamina propria thickness broadens, and collagen fiber deposit thick and fine and close herein, and thickness is not
Deng, the collagen for being close to epithelium is arranged in the collagen fiber band of obvious densification, increase over time (12 weeks, 16 weeks, 20
Week), collagen fiber showed increased compared with matched group, collagen fiber dyeing after color also become it is deeper.Additionally, passing through Van
Gieson dyeing shows that cherry intensive collagen is corresponding with Masson dyeing, and has similar collagen change, mutually confirms.
From result above, when 12 weeks, there are OSF early lesions in tongue tissue, and when 16 weeks, cheek, palatine tissue occur OSF morning in succession
Phase pathological changes, to 20 weeks, there are OSF mid-term pathological changes in tongue, palatine tissue.By veins beneath the skin quantity on CD34 countings, in microscope
Find after counting to blood vessel under experiment, control group mice oral mucosa under high power field：Increase over time, 2 groups of blood vessels
Quantity gradually increases, and experimental group increase becomes apparent from, and to after 16 weeks, 2 groups of blood vessel number obvious differences show as experiment
Group vascular counts are significantly more than matched group；But as administration time extends, experimental group blood vessel number starts decline occur on the contrary,
There is statistical significance to 2 groups of blood vessel differences when 20 weeks, experimental group shows as significantly reducing.With clinical people OSF Vascular change phases
Unanimously.By I, type III collagen content semi-quantitative analyses, it is found that type i collagen extends with induction time, content substantially increases, with
Matched group obvious difference, and type III collagen, without significant change, this is consistent with people OSF I, type III pathological changes, shows as collagen heap
Product increases, the collagen pathological changes situation based on degraded reduction.
(5) mice OSF models are built：By step (1) to (4), show that arecoline chemical derivatization is successfully established mice
OSF models, and set up file formatting model.
Embodiment 3
(1) animal selects and is grouped：It is experiment mice to take 8 week old male Balb/c mices 40, and stochastic averagina is divided into
Experimental group and matched group；
The condition of experiment mice is：Described experiment mice is：8 week old male Balb/c mices, SPF levels, body weight
(18.52 ± 1.15) g, raises under the conditions of 18～25 DEG C of room temperature, humidity 30%～50%, fed standard chow, 24h Dark-light cycles
Support；
(2) arecoline drinking agent is prepared：By the arecoline preserved under room temperature (arecoline), concentration is made into tap water
1000mg/L medicines are placed in experimental mice drinking bottle and freely drink.Control group mice is freely drunk with tap water；
(3) animal feeding and execution：Experimental group is fed 20 weeks using arecoline drinking agent, and the matched group same period gives tap water
Feed, random per 4 weeks to put to death 4 mices of each group, remaining mice is all put to death within 20 weeks；
(4) laboratory observation：Carry out overview to experimental group and matched group respectively, after putting to death mice, take oral mucosa group
Knit, take hematoxylin eosin stain method (hematoxylin-eosin staining, abbreviation HE are dyeed) observation, MASSON tri-
Color is dyeed and the observation of Van Gieson stainings, and SABC composes I/III collagen types with SABC CD34 dyeing observations；
Overview is：
Situations such as observing mice mental status, mobility, amount of drinking water daily in experimentation, records weekly mice amount of drinking water
And body weight change.After mice was put to death per 4 weeks, observation panel intracavity changes, mucosa color and luster in recording mouth, and whether quality etc. changes,
Whether there are speckle, ulcer or swollen thing to occur, observe scope, the limitation of mouth opening situation of fibrous bands.
HE dyeing is viewed as：
Take oral cavity tissue mucosa (band part deep muscle tissue) after putting to death mice immediately, esophaguses, stomach, liver, spleen, lung, kidney,
Small intestinal, large intestine etc. are organized, and are fixed with 10% neutral formalin solution, conventional organization process, paraffin embedding, 4 μm of sections, haematoxylin
Eosin stains, carry out pathological study.Oral mucosa affection tissue carry out by stages by OSF pathology standard, i.e., earliest period,
In early days, mid-term, late period.
MASSON trichrome stains and Van Gieson dyeing are viewed as：
Put to death mice and obtain oral cavity tissue mucosa, fixed with 10% neutral formalin solution, conventional organization process, paraffin bag
Bury, 4 μm of sections, respectively by MASSON test kits (Wuhan Boster Biological Technology Co., Ltd.) and Van Gieson test kits
(Wuhan Boster Biological Technology Co., Ltd.) dyeing observation oral mucosa early stage fiber situation of change.
SABC wide spectrum I/III collagen types observe collagen situation with SABC CD34 dyeing observation epithelium hematochezia
Pipe quantity is：
Using immunohistochemical ABC method, according to routine operation：Murine oral mucous membrane tissue wax stone is cut into slices with 4 μm of thickness, Jing
Aquation, suppresses endogenous peroxydase, after eliminating unspecific staining, respectively one AntiCD3 McAb 4 of Deca (Mouse Polyclonal Antibody,
Purchased from Wuhan Boster Biological Technology Co., Ltd., by 1:100 dilution), (Mouse Polyclonal Antibody, purchased from force for I-type collagen
Han Boshide biological engineering company limited, by 1：250 dilutions), type III collagen protein (Mouse Polyclonal Antibody, it is rich purchased from Wuhan
Shi De biological engineering company limited, by 1:180 dilutions), while, positive control control group mice oral cavity mucous membrane tissue is cloudy
Property control replaces one anti-with PBS, 4 DEG C are overnight；Using SP test kits, (sheep anti mouse anti-rabbit is universal, China fir in Beijing for later stage colour developing
Bioisystech Co., Ltd of Golden Bridge) colour developing, it is dehydrated transparent mounting.Result judgement：CD34 positive reactions thing is light brown to dark brown
Coloured particles, are distributed in vascular endothelial cell.Result judgement：I, type III collagen IV-positive reactant are light brown to dark-brown
Granule, is distributed in mucosa lamina propria.
Observation result (such as Fig. 1,2,3,4)：
Test rise within the 3rd week after there is weight loss, but the 5th week in existing experimental mice find body weight be slowly increased, treat reality
After testing the 6th week, experimental mice body weight tends towards stability, and average weight is (18.12 ± 0.32) g.Control group mice average weight
For (27.24 ± 1.43) g.It is low and deep that hair color occurs in the experimental mice later stage, and mobility is reduced, and is reluctant feed etc..Control group mice
Hair color is vivid, appetite, mobility are good.Experimental mice oral mucosa start to engender the 12nd week from experiment turn white, it is stiff
Firmly, the different degrees of above-mentioned change of appearance of all mice mucosas in the 20th week is extremely tested, but has no that obvious fibrous bands are formed.
Matched group oral mucosa color, quality change be not obvious.As time goes on, experimental mice mouth opening is gradually reduced, right
Gradually increase according to a group mice mouth opening, from the beginning of experiment the 18th week, the mouth opening significant difference of two groups of mices.Experimental mice
From 8 weeks, tongue tissue starts to occur in that OSF samples change, and after 12 weeks, cheek palatine tissue also occurs in that OSF samples change.Experimental group is little
Mus early stage Oral mucosa keratinocyte starts abnormal positive keratinization, cuticular layer atrophy, the visible full nucleus of fibrocyte, lymph is presented
Cellular infiltration, lamina propria are thickened, and blood vessel number increases, and substantially, caliber changes for the congestion of blood vessel.Extend with administration time, epithelial peg
It is prominent gradually become shallower as, mice, lamina propria thickness thickens all the more substantially, upper veins beneath the skin there is first congested expansion after atresia gradually
Change process, drug-induced to the 20th week, epithelial thickness is persistently reduced, it is seen that atrophic outward appearance.Lamina propria collagen is in unordered, wavy
Profile sequence fiber it is fine and close, cell quantity is reduced, and basal layer cell destruction is obvious.Collagen fiber density increase regions, palatine
It can be seen that class vitreous degeneration.Lamina propria blood vessel lacks, and is wrapped by fibrous tissue, the visible fibers encapsulation of muscle under mucosa, most of feelings
The visible muscle layer atrophy of condition.Masson dyeing shows that collagen is distributed in bluish violet, the bluish violet elasticity of a small amount of stained positive of matched group
Fiber is distributed in mucosa lamina propria, and collagen fiber are elongated, moves towards straight, arranges loose, and thicker elastic fiber sends many micro-
Elongated fiber is extended in nipple connective tissue.Hand over below the basal cell of epithelium at the fine end eventually of these elastic fibers
It is made into elastic webs.Experimental group cheek mucosa lamina propria thickness broadens, and collagen fiber deposit thick and fine and close herein, and thickness is not
Deng, the collagen for being close to epithelium is arranged in the collagen fiber band of obvious densification, increase over time (8 weeks, 12 weeks, 16
Week), collagen fiber showed increased compared with matched group, collagen fiber dyeing after color also become it is deeper.Additionally, passing through Van
Gieson dyeing shows that cherry intensive collagen is corresponding with Masson dyeing, and has similar collagen change, mutually confirms.
From result above, when 8 weeks, there are OSF early lesions in tongue tissue, and when 12 weeks, cheek, palatine tissue occur OSF morning in succession
Phase pathological changes, to 16 weeks, there are OSF mid-term pathological changes in tongue, palatine tissue, and induction is caused 20 weeks, and tongue, buccal are in OSF mid-term pathological changes, palate
There are OSF advanced lesions in portion's tissue.By veins beneath the skin quantity on CD34 countings, under microscope high power field to experiment, it is right
Find after being counted according to blood vessel under group murine oral mucosa：Increase over time, 2 groups of blood vessel numbers gradually increase, and
Experimental group increase becomes apparent from, and to after 12 weeks, 2 groups of blood vessel number obvious differences show as experimental group vascular counts and are significantly more than
Matched group；But as administration time extends, experimental group blood vessel number starts decline occur on the contrary, poor to 2 groups of blood vessels when 20 weeks
It is different with statistical significance, experimental group shows as significantly reducing.It is consistent with clinical people OSF Vascular changes.By I, type III glue
Former content semi-quantitative analyses, it is found that type i collagen extends with induction time, and content substantially increases, and matched group obvious difference, and
, without significant change, this is consistent with people OSF I, type III pathological changes for type III collagen, shows as collagen deposition increase, and degraded is reduced to
Main collagen pathological changes situation.
(5) mice OSF models are built：By step (1) to (4), show that arecoline chemical derivatization is successfully established mice
OSF models, and set up file formatting model.
The construction method of oral submucosa fibrosises mouse model of the present invention be respectively adopted concentration 500mg/L, 1000mg/L,
The arecoline drinking agent of 2000mg/L feeds mice, by the result of three kinds of embodiments and regular injection and coating aqueous areca nut extract
Inducing mouse is contrasted, and correction data is as shown in table 1, as can be seen from Table 1：Using oral submucosa fibrosises mice of the present invention
The construction method of model successfully obtains mice OSF models and mankind OSF there are high similarity, i.e. pathological changes to occur, development it is complete
Whole property, animal model sickness rate height (100%), and it is short into the mould time.
Table is 1 present invention compared with conventional induction data using three embodiments
Above disclosed is only presently preferred embodiments of the present invention, can not limit certainly the right of the present invention with this
Scope, therefore the equivalent variations made according to the claims in the present invention, still fall within the scope covered by the present invention.
Claims (3)
1. a kind of construction method of oral submucosa fibrosises mouse model, it is characterised in that comprise the following steps：
(1) animal selects and is grouped：It is experiment mice to take 8 week old male Balb/c mices 40, and stochastic averagina is divided into experiment
Group and matched group；
(2) arecoline drinking agent is prepared：By the arecoline preserved under room temperature, it is made into 500～2000mg/L's of concentration with tap water
Arecoline drinking agent, arecoline drinking agent is placed in experimental mice drinking bottle and is freely drunk；Control group mice is with tap water
Freely drink；
(3) animal feeding and execution：Experimental group is fed 8～20 weeks using arecoline drinking agent, and the matched group same period gives tap water
Feed, random per 4 weeks to put to death 4 mices of each group, remaining mice is all put to death within 20 weeks；
(4) laboratory observation：Carry out overview to experimental group and matched group respectively, after putting to death mice, take oral cavity mucous membrane tissue, adopt
Take the observation of hematoxylin eosin stain method, MASSON trichrome stains and the observation of Van Gieson stainings, SABC spectrum I/III
Collagen type and SABC CD34 dyeing observations；
(5) mice OSF models are built：By step (1) to (4), show that arecoline chemical derivatization is successfully established mice OSF moulds
Type, and set up file formatting model.
2. the construction method of oral submucosa fibrosises mouse model as claimed in claim 1, it is characterised in that：The Semen Arecae
The concentration of alkali drinking agent is 2000mg/L.
3. the construction method of oral submucosa fibrosises mouse model as claimed in claim 1, it is characterised in that the experiment
Mice is：8 week old male Balb/c mices, SPF levels, 18.52 ± 1.15g of body weight, 18～25 DEG C of room temperature, humidity 30%～
50%th, raise under the conditions of fed standard chow, 24h Dark-light cycles.
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| CN108236198A (en) * | 2018-01-23 | 2018-07-03 | 湖南中医药大学第附属医院（中医临床研究所） | Prepare the brush of rat oral submucosa fibrosis experimental model |
| CN111165428A (en) * | 2019-11-01 | 2020-05-19 | 江汉大学 | Construction method and application of animal model addicted to chewing areca nuts |
| CN112715474A (en) * | 2020-12-25 | 2021-04-30 | 江汉大学 | Method for forming animal model with conditional site preference by induction of arecoline and menthol and application |
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2016
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| CN108236198A (en) * | 2018-01-23 | 2018-07-03 | 湖南中医药大学第附属医院（中医临床研究所） | Prepare the brush of rat oral submucosa fibrosis experimental model |
| CN111165428A (en) * | 2019-11-01 | 2020-05-19 | 江汉大学 | Construction method and application of animal model addicted to chewing areca nuts |
| CN111165428B (en) * | 2019-11-01 | 2021-12-28 | 江汉大学 | Construction method and application of animal model addicted to chewing areca nuts |
| CN112715474A (en) * | 2020-12-25 | 2021-04-30 | 江汉大学 | Method for forming animal model with conditional site preference by induction of arecoline and menthol and application |
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