CN110476890A - A kind of preparation method, application and the evaluation method of Collagen-induced Arthritis animal model - Google Patents
A kind of preparation method, application and the evaluation method of Collagen-induced Arthritis animal model Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/10—Animals modified by protein administration, for non-therapeutic purpose
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
Abstract
The present invention provides a kind of preparation method of Collagen-induced Arthritis animal model, chooses 6-8 weeks, male, DBA/1 mouse;It is 20-24 DEG C, relative humidity 50%-60% that mouse selected by step (1), which is placed in temperature, raises, drinking-water of freely ingesting in the alternate cleaning grade animal house of 12 hours light and shades, is used to prepare model after adapting to environment three days;DBA/1 mouse is immunized with chicken II Collagen Type VI and induces CIA model, is immunized to the intracutaneous injection of mouse tail root;It is immunized three times, the present invention is of great significance to the research of arthritic pathologic process mechanism and drug development etc.;Simultaneously, the method of evaluation Collagen-induced Arthritis animal model of the invention, whether succeeded by index verifications models such as anti-CII-IgG antibody levels in osteoarthritis clinical index score, ankle-joint pathological examination, serum, thoroughly evaluating is carried out to Collagen-induced Arthritis animal model.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of preparation side of Collagen-induced Arthritis animal model
Method, application and evaluation method.
Background technique
Rheumatoid arthritis (Rheumatoid arthritis, RA) is by immunocompetent cell and cytokine profiles
The chronic generalized autoimmune disease mediated.This disease is 0 .34%-0 .36%, Nv Xingfa in the illness rate of China human mortality
It is to cause population of China disability and one of the main reason for disable more than sick rate is more three times higher than male.The clinical table of RA
It is now multiple symmetrical arthropathy, arthralgia, swelling, stiff and dyskinesia.Basic pathology variation be synovitis and
Progressive destruction of joint.The disorder of immune system and it is unbalance be the main reason for causing rheumatoid arthritis occurrence and development.T,B
Lymphocyte and its cell factor of secretion play an important role in the morbidity of RA and progression of disease.For the clinic of RA
Treatment mainly controls disease using non-steroid anti-inflammatory drug, improvement state of an illness antirheumatic drug, glucocorticoid and biological agent at present
Feelings, but not can effectively prevent the development of the disease.We still need the pathogenesis for further exploring such disease and are
Patient provides new therapeutic agent;Therefore, model of rheumatoid arthritis is constructed, in the treatment of research rheumatoid arthritis
Play a significant role, research and drug development to arthritic pathologic process mechanism etc. are of great significance.
Summary of the invention
The present invention is to provide a kind of preparation method of Collagen-induced Arthritis animal model, to solve institute in background technique
The problem of proposition.
In order to solve the above technical problems, the embodiment of the present invention provides a kind of system of Collagen-induced Arthritis animal model
Preparation Method, which is characterized in that specifically includes the following steps:
(1) 6-8 weeks, male, DBA/1 mouse are chosen;
(2) mouse selected by step (1) is placed in temperature is 20-24 DEG C, relative humidity 50%-60%, 12 hours light and shade alternatings
Cleaning grade animal house in raise, drinking-water of freely ingesting, adapt to environment three days after be used to prepare model;
(3) chicken is weighed100 μ g of Collagen Type VI, is dissolved in the 0.1 mol/L glacial acetic acid of 50 μ l, and 4 DEG C overnight;Collagen is prepared
Solution A;
(4) it prepares collagen emulsion B: collagen solution is mixed in equal volume with CFA, it is small to be fully ground 1 using glass blender on ice
When, collagen emulsion B solution is made;
(5) it prepares collagen emulsion C: collagen solution A is mixed in equal volume with IFA, it is small to be fully ground 1 using glass blender on ice
When, collagen emulsion C is made;
(6) mouse is fixed using fixator, exposure tail;
(7) it is immunized to the intracutaneous injection of mouse tail root:
(7-1) is immune for the first time: the intradermal 100 μ l of multi-point injection collagen emulsion B solution in mouse tail root;Specifically include following step
Suddenly;
(7-1-1) first scrapes the hair at root of the tail position completely, and root of the tail position part routine disinfection, left hand thumb and index finger pin mouse
Root of the tail portion skin is allowed to tighten, and between two fingers, connects 4.5 injection needles with micro syringe and punctures, syringe needle enters mouse tail
Root skin shallow-layer, then piercing is provoked upwards, medical fluid is injected intradermal;
There is a small skin mound of white in mouse tail root skin after (7-1-2) injection, selects 4-5 point injection, collagen emulsion B solution
Accumulated dose is 100 μ l;
(7-2) is immune for the second time: the 21st day after immune for the first time, the 100 μ l conduct of mouse peritoneal injectable collagen emulsion C solution
Secondary immune solution;Specifically include following steps;
(7-2-1) grabs mouse, its abdomen is made slightly to let droop towards head portion;
(7-2-2), which firmly grasps back of mice skin, keeps skin of abdomen tight, in two thigh root lines and ventrimeson crosspoint side about 1
The position cm is pierced into subcutaneous;
(7-2-3) promotes syringe needle 3-5 mm in subcutaneous parallel ventrimeson, then with 45° angle to intraperitoneal piercing;
After (7-2-4) needle point passes through abdominal muscle, resistance disappears, and without reflux when pumpback, is pushed into drug;Collagen emulsion C solution is total
Dosage is 100 μ l;
(7-3) third time is immune: the 28th day after immune for the first time, injecting 20 μ l lipopolysaccharides, the lipopolysaccharides in mouse peritoneal
Concentration is 1 mg/ml, and Collagen-induced Arthritis animal model is made.
Further, the preparation method of the glacial acetic acid, comprising the following steps: it is bis- that the 5 pure glacial acetic acid of μ g are dissolved in 870 ul
It steams in water, 0.1 mol/L glacial acetic acid is made in mixing.
Further, the preparation method of the lipopolysaccharides, comprising the following steps: it is bis- that 10 mg lipopolysaccharides powder are dissolved in 1 ml
Water is steamed, mixing is made 1 mg/ml lipopolysaccharides solution, and when use is diluted using PBS.
A kind of application of Collagen-induced Arthritis animal model, which is characterized in that by the Collagen-induced Arthritis
Animal model is used for chickenCollagen Type VI participates in research and the drug development of arthritic pathologic process mechanism.
A method of evaluation Collagen-induced Arthritis animal model, which comprises the following steps:
S1, clinical index scoring: since the 21st day, every three days to the small of the Collagen-induced Arthritis animal model of preparation
Mouse four sufficient pawls score, and have carried out 19 scorings altogether, and standards of grading are as follows: 0 point: without redness;1 point: slight is red and swollen;
2: significant rubefaction and arthroncus;3 points: anchylosis, limb activity are limited;Four sufficient pawls are scored, and mouse is most
Higher assessment is divided into 12 points, and lowest score is 0 point;Experiment is double-dummy design;It is higher than 8 timesharing when scoring, shows that model may be created as
Function;
S2, pathology sections observation: model group is established to first time immune latter 37th day mouse ankle joint frozen section, and will
Healthy mice ankle-joint frost of the same age as a control group, carries out HE dyeing to model group and control group, will complete the slice of dyeing
It is placed under microscope and carries out observation film making, comparison model group and control group situation;If synovial membrane in the mouse ankle-joint of model group mouse
There is obvious hyperplasia in tissue, articular cavity has a large amount of inflammatory cell infiltrations, articular surface cartilage to have destruction, shows that model may be created as
Function;
Anti- C in S3, ELISA method detection serumIgG antibody is horizontal: extracting immune latter 37th day mice serum for the first time and builds
Vertical model group, and as a control group by healthy mice serum of the same age, anti-C in ELISA method detection serumIgG antibody is horizontal,
If anti-C in model group serumIgG antibody level is higher than anti-C in control group serum3 times of IgG antibody level, show mould
Type may be successfully established;
S4, at least meet requirement in the above S1 step or S2 step, that is, can be shown that model foundation success.
Further, the step S4 is model foundation success when meeting the requirement of step S1, S2 and S3.
The advantageous effects of the above technical solutions of the present invention are as follows: Collagen-induced Arthritis animal model of the invention
Preparation method is immunized DBA/1 mouse with chicken II Collagen Type VI and induces CIA model, to the research of arthritic pathologic process mechanism and
Drug development etc. is of great significance;Meanwhile the method for evaluation Collagen-induced Arthritis animal model of the invention, pass through pass
Save scorching clinical index scoring, in ankle-joint pathological examination, serum the index verifications model such as anti-CII-IgG antibody level whether at
Function carries out thoroughly evaluating to Collagen-induced Arthritis animal model.
Detailed description of the invention
Fig. 1 is the schematic diagram of the fixed mouse of mouse fixing device of the invention and mouse injection;
Fig. 2 is the result figure of clinical index scoring in the present invention;
Fig. 3 is that figure is compared in control group and model group mouse ankle joint HE dyeing in the present invention;
Wherein, Fig. 3 A is control group mice ankle-joint HE colored graph;Fig. 3 B is model group mouse ankle joint HE colored graph;Fig. 3 C is
Local enlarged drawing in Fig. 3 A, Fig. 3 D are partial enlarged view in Fig. 3 B;
Fig. 4 is anti-C in ELISA method detection control group in the present invention and model group mice serumIgG antibody relative level ratio
Compared with figure.
Specific embodiment
To keep the technical problem to be solved in the present invention, technical solution and advantage clearer, below in conjunction with specific implementation
Example is described in detail.
A kind of preparation method of Collagen-induced Arthritis animal model, specifically includes the following steps:
(1) 6-8 weeks, male, DBA/1 mouse are chosen.
(2) mouse selected by step (1) is placed in temperature is 20-24 DEG C, relative humidity 50%-60%, 12 hours light and shades
It is raised in alternate cleaning grade animal house, drinking-water of freely ingesting is used to prepare model after adapting to environment three days.
(3) chicken is weighed100 μ g of Collagen Type VI, is dissolved in the 0.1 mol/L glacial acetic acid of 50 μ l, and 4 DEG C overnight;It is prepared
Collagen solution A.Wherein, the preparation method of the glacial acetic acid, comprising the following steps: the 5 pure glacial acetic acid of μ g are dissolved in the bis- steamings of 870 ul
In water, 0.1 mol/L glacial acetic acid is made in mixing.
(4) it prepares collagen emulsion B: collagen solution being mixed in equal volume with CFA, is fully ground on ice using glass blender
1 hour, collagen emulsion B solution is made.
(5) it prepares collagen emulsion C: collagen solution A being mixed in equal volume with IFA, is sufficiently ground using glass blender on ice
Mill 1 hour, is made collagen emulsion C.
(6) mouse is fixed using fixator, exposure tail.
(7) it is immunized to the intracutaneous injection of mouse tail root.
(7-1) is immune for the first time: the intradermal 100 μ l of multi-point injection collagen emulsion B solution in mouse tail root;It specifically includes following
Step;
(7-1-1) first scrapes the hair at root of the tail position completely, and root of the tail position part routine disinfection, left hand thumb and index finger pin mouse
Root of the tail portion skin is allowed to tighten, and between two fingers, connects 4.5 injection needles with micro syringe and punctures, syringe needle enters mouse tail
Root skin shallow-layer, then piercing is provoked upwards, medical fluid is injected intradermal.
There is a small skin mound of white in mouse tail root skin after (7-1-2) injection, selects 4-5 point injection, collagen emulsion B
Solution accumulated dose is 100 μ l.
(7-2) is immune for the second time: the 21st day after immune for the first time, 100 μ l of mouse peritoneal injectable collagen emulsion C solution
As secondary immune solution;Specifically includes the following steps:
(7-2-1) grabs mouse, its abdomen is made slightly to let droop towards head portion.
(7-2-2), which firmly grasps back of mice skin, keeps skin of abdomen tight, in two thigh root lines and ventrimeson crosspoint one
About 1 position cm of side is pierced into subcutaneous.
(7-2-3) promotes syringe needle 3-5 mm in subcutaneous parallel ventrimeson, then with 45° angle to intraperitoneal piercing.
After (7-2-4) needle point passes through abdominal muscle, resistance disappears, and without reflux when pumpback, is pushed into drug;Collagen emulsion C is molten
Liquid accumulated dose is 100 μ l.
(7-3) third time is immune: the 28th day after immune for the first time, injecting 20 μ l lipopolysaccharides, the rouge in mouse peritoneal
Polysaccharide concentration is 1 mg/ml, and Collagen-induced Arthritis animal model is made.Wherein, the preparation method of the lipopolysaccharides, including
Following steps: 10 mg lipopolysaccharides powder are dissolved in 1 ml distilled water, and mixing is made 1 mg/ml lipopolysaccharides solution, and when use uses
PBS dilution.
In the present invention, the source of required reagent is as shown in table 1 below.
The source table of required reagent in 1 present invention of table
The Collagen-induced Arthritis animal model is used for chicken by a kind of application of Collagen-induced Arthritis animal model
Collagen Type VI participates in research and the drug development of arthritic pathologic process mechanism.
A method of evaluation Collagen-induced Arthritis animal model, comprising the following steps:
S1, clinical index scoring: since the 21st day, every three days to the small of the Collagen-induced Arthritis animal model of preparation
Mouse four sufficient pawls score, and have carried out 19 scorings altogether, and standards of grading are as follows: 0 point: without redness;1 point: slight is red and swollen;
2: significant rubefaction and arthroncus;3 points: anchylosis, limb activity are limited;Four sufficient pawls are scored, and mouse is most
Higher assessment is divided into 12 points, and lowest score is 0 point;Experiment is double-dummy design;It is higher than 8 timesharing when scoring, shows that model may be created as
Function.
S2, pathology sections observation: establishing model group to first time immune latter 37th day mouse ankle joint frozen section,
And as a control group by healthy mice ankle-joint of the same age frost, HE dyeing is carried out to model group and control group, dyeing will be completed
Slice, which is placed under microscope, carries out observation film making, comparison model group and control group situation;If in the mouse ankle-joint of model group mouse
There is obvious hyperplasia in synovial tissue, articular cavity has a large amount of inflammatory cell infiltrations, articular surface cartilage to have destruction, shows that model may be built
It stands successfully.
Wherein, pathology sections observation specifically includes the following steps:
S2-1, perfusion: with compound anesthetic after immune to first time the 37th day mouse carry out intraperitoneal anesthesia, through left ventricle to liter
0.9% physiological saline of aorta quick filling, 100 ml, perfusion contains the phosphate buffer of 4% formaldehyde later, first quick and back slow, to mouse
After twitch stops, control drop speed according to 1 drop/s, continues 20 min;After mouse ankle joint is removed skin, taking-up is placed in 4%
24 hours in paraformaldehyde solution;37 DEG C of 10% EDTA decalcification 15 days is moved it into again, it is rear to move into the phosphorus for containing 20% and 30% sucrose
Serial dehydration is carried out in phthalate buffer.
S2-2, slice: mouse ankle joint is placed in and freezes cryofixation on platform, in -20 DEG C of downlink sagittal plains of freezing microtome
Slice;Ankle-joint piece is 15 μm thick, and slice is directly affixed on the processed glass slide of gelatin, is dried.
S2-3, HE dyeing: slice washing 1-2s, haematoxylin 30s wash 5-10s, and 1% acidic alcohol breaks up liquid 1-3s, stream
Water rinses 5-10min, Yihong 10s, washes 1-2s, 80% ethyl alcohol 1-2s, 95% ethyl alcohol 1-2s, dehydrated alcohol 1-2s, dimethylbenzene 2-
The slice for completing dyeing is placed under microscope and carries out observation film making by 3s, neutral gum mounting.
HE coloration result is as shown in Figure 3, wherein Fig. 3 A is control group mice ankle-joint HE colored graph, hollow arrow in Fig. 3 A
Signified head is control group mice ankle-joint single layer synovial tissue, articular cavity (double asterisk is signified) interior no inflammation cellular infiltration, joint
Thin-skinned bone (black arrows are signified in figure) is smooth.Fig. 3 B is model group mouse ankle joint HE colored graph, and model group (d37) is small
The obvious hyperplasia of synovial tissue (hollow arrow is signified in figure) in mouse ankle-joint, and articular cavity has a large amount of inflammatory cell infiltrations, joint
Thin-skinned bone has a degree of destruction.
Fig. 3 C is the enlarged drawing of Blocked portion in Fig. 3 A, and Fig. 3 D is the enlarged drawing of Blocked portion in Fig. 3 B;It can be seen in Fig. 3 C
Out, control group shows fibroblast-like Normal synovial cell (black arrows are signified in figure).It can be seen that in Fig. 3 D, mould
Type group mouse shows that there are a large amount of inflammatory cells (black wedge shape arrow is signified in figure).
Anti- C in S3, ELISA method detection serumIgG antibody is horizontal: extracting immune latter 37th day mouse blood for the first time
Model group is established clearly, and as a control group by healthy mice serum of the same age, anti-C in ELISA method detection serumIgG antibody water
It is flat, if anti-C in model group serumIgG antibody level is higher than anti-C in control group serum3 times of IgG antibody level, show
Model may be successfully established.
Wherein, anti-C in ELISA method detection serumIgG antibody level specifically includes following procedure: claiming chickenCollagen Type VI
100 μ g are dissolved in 50 μ l glacial acetic acid (0.1 mol/L), and concentration is 2 mg/ml, and 4 DEG C of dissolutions are overnight.With 0.01 mol/L
PBS with 1:20 dilute chickenThe diluted chicken of 100 μ l is added in every hole of 96 orifice plates in Collagen Type VICollagen Type VI, 4 DEG C of mistakes
Night.Go the chicken in 96 orifice platesCollagen Type VI, 100 μ l PBST(PBS+Tween) washing 3 times.It is added with 0. 01 mol/L's
Serum 100 μ l, 37 DEG C of 30 min of incubation PBS detected with 1:500 dilution.PBST is washed 3 times, and horseradish peroxidase is added
The secondary antibody (1:5000) of enzyme label, 37 DEG C of 1 h of incubation.96 orifice plates are washed, 100 μ l substrates (TMB) are added, stand 10 min.It is added
100 μ l terminate liquids.Microplate reader surveys OD value (450 nm).The relative value of control group and model group is taken every time, is ginseng with control group
It examines.
As shown in figure 4, wherein anti-C in model groupThe relative value of IgG antibody level is higher than anti-C in control group-
5 times of the relative value of IgG antibody level.
S4, at least meet requirement in the above S1 step or S2 step, that is, can be shown that model foundation success.When step S4 is full
When the requirement of sufficient step S1, S2 and S3, model foundation success successful is higher.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, it can also make several improvements and retouch, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (6)
1. a kind of preparation method of Collagen-induced Arthritis animal model, which is characterized in that specifically includes the following steps:
Choose 6-8 weeks, male, DBA/1 mouse;
It is 20-24 DEG C that mouse selected by step (1), which is placed in temperature, and relative humidity 50%-60%, light and shade is alternately clear within 12 hours
It is raised in clean grade animal house, drinking-water of freely ingesting, is used to prepare model after adapting to environment three days;
Weigh chicken100 μ g of Collagen Type VI, is dissolved in the 0.1 mol/L glacial acetic acid of 50 μ l, and 4 DEG C overnight;Collagen solution A is prepared;
It prepares collagen emulsion B: collagen solution being mixed in equal volume with CFA, is fully ground 1 hour using glass blender on ice,
Collagen emulsion B solution is made;
It prepares collagen emulsion C: collagen solution A being mixed in equal volume with IFA, is fully ground 1 hour using glass blender on ice,
Collagen emulsion C is made;
Mouse is fixed using fixator, exposure tail;
It is immunized to the intracutaneous injection of mouse tail root:
(7-1) is immune for the first time: the intradermal 100 μ l of multi-point injection collagen emulsion B solution in mouse tail root;Specifically include following step
Suddenly;
(7-1-1) first scrapes the hair at root of the tail position completely, and root of the tail position part routine disinfection, left hand thumb and index finger pin mouse
Root of the tail portion skin is allowed to tighten, and between two fingers, connects 4.5 injection needles with micro syringe and punctures, syringe needle enters mouse tail
Root skin shallow-layer, then piercing is provoked upwards, medical fluid is injected intradermal;
There is a small skin mound of white in mouse tail root skin after (7-1-2) injection, selects 4-5 point injection, collagen emulsion B solution
Accumulated dose is 100 μ l;
(7-2) is immune for the second time: the 21st day after immune for the first time, the 100 μ l conduct of mouse peritoneal injectable collagen emulsion C solution
Secondary immune solution;Specifically include following steps;
(7-2-1) grabs mouse, its abdomen is made slightly to let droop towards head portion;
(7-2-2), which firmly grasps back of mice skin, keeps skin of abdomen tight, in two thigh root lines and ventrimeson crosspoint side about 1
The position cm is pierced into subcutaneous;
(7-2-3) promotes syringe needle 3-5 mm in subcutaneous parallel ventrimeson, then with 45° angle to intraperitoneal piercing;
After (7-2-4) needle point passes through abdominal muscle, resistance disappears, and without reflux when pumpback, is pushed into drug;Collagen emulsion C solution is total
Dosage is 100 μ l;
(7-3) third time is immune: the 28th day after immune for the first time, injecting 20 μ l lipopolysaccharides, the lipopolysaccharides in mouse peritoneal
Concentration is 1 mg/ml, and Collagen-induced Arthritis animal model is made.
2. a kind of preparation method of Collagen-induced Arthritis animal model according to claim 1, which is characterized in that institute
State the preparation method of glacial acetic acid, comprising the following steps: the 5 pure glacial acetic acid of μ g are dissolved in 870 ul distilled waters, mixing is made 0.1
Mol/L glacial acetic acid.
3. a kind of preparation method of Collagen-induced Arthritis animal model according to claim 1, which is characterized in that institute
State the preparation method of lipopolysaccharides, comprising the following steps: 10 mg lipopolysaccharides powder are dissolved in 1 ml distilled water, and 1 mg/ml is made in mixing
Lipopolysaccharides solution is diluted using PBS when use.
4. a kind of application of Collagen-induced Arthritis animal model according to claim 1, which is characterized in that will be described
Collagen-induced Arthritis animal model is used for chickenCollagen Type VI participates in the research of arthritic pathologic process mechanism and drug is opened
Hair.
5. a kind of method for the Collagen-induced Arthritis animal model that evaluation is prepared according to the method for claim 1,
It is characterized in that, comprising the following steps:
S1, clinical index scoring: since the 21st day, every three days to the small of the Collagen-induced Arthritis animal model of preparation
Mouse four sufficient pawls score, and have carried out 19 scorings altogether, and standards of grading are as follows: 0 point: without redness;1 point: slight is red and swollen;
2: significant rubefaction and arthroncus;3 points: anchylosis, limb activity are limited;Four sufficient pawls are scored, and mouse is most
Higher assessment is divided into 12 points, and lowest score is 0 point;Experiment is double-dummy design;It is higher than 8 timesharing when scoring, shows that model may be created as
Function;
S2, pathology sections observation: model group is established to first time immune latter 37th day mouse ankle joint frozen section, and will
Healthy mice ankle-joint frost of the same age as a control group, carries out HE dyeing to model group and control group, will complete the slice of dyeing
It is placed under microscope and carries out observation film making, comparison model group and control group situation;If synovial membrane in the mouse ankle-joint of model group mouse
There is obvious hyperplasia in tissue, articular cavity has a large amount of inflammatory cell infiltrations, articular surface cartilage to have destruction, shows that model may be created as
Function;
Anti- C in S3, ELISA method detection serumIgG antibody is horizontal: extracting immune latter 37th day mice serum for the first time and builds
Vertical model group, and as a control group by healthy mice serum of the same age, anti-C in ELISA method detection serumIgG antibody is horizontal,
If anti-C in model group serumIgG antibody level is higher than anti-C in control group serum3 times of IgG antibody level, show model
It may be successfully established;
S4, at least meet requirement in the above S1 or S2 step, that is, can be shown that model foundation success.
6. a kind of evaluation method of Collagen-induced Arthritis animal model according to claim 5, which is characterized in that institute
Stating step S4 is model foundation success when meeting the requirement of step S1, S2 and S3.
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