CN109418230A - A kind of sacro-iliitis mouse model - Google Patents
A kind of sacro-iliitis mouse model Download PDFInfo
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- CN109418230A CN109418230A CN201710742546.8A CN201710742546A CN109418230A CN 109418230 A CN109418230 A CN 109418230A CN 201710742546 A CN201710742546 A CN 201710742546A CN 109418230 A CN109418230 A CN 109418230A
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- iliitis
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- articulatio sacroiliaca
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- 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
- 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; AVICULTURE; APICULTURE; PISCICULTURE; 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
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
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- Biodiversity & Conservation Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of sacro-iliitis mouse model of the present invention, belongs to gene engineering technology field.The Immunohistochemical Characterization at the articulatio sacroiliaca position of the sacro-iliitis mouse model is that structure is destroyed at exudate, synovial cell proliferation and articular cartilage in articular cavity.The unconventionality expression of the articulatio sacroiliaca position Wnt/ β-catenin signal path ingredient of the sacro-iliitis mouse model, and by the regulation of mir-29a.The invention has the following advantages that the Research of Animal Model for Study in mouse source can be to avoid the materials of the non-renewable articulatio sacroiliaca of human body；The research of animal model can be to study the sacro-iliitis pathology of appearance, to further investigate to disease incidence mechanism in analogue body；Animal model repeatability is strong, is conducive to stablize and carries out the function assessments research such as signal path regulation.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of sacro-iliitis mouse model.
Background technique
Joint of vertebral column inflammation (Spondyloarthritis, SpA) includes ankylosing spondylitis (Ankylosing
Spondylitis, AS) it is chronic inflammation disease of the major class based on articulatio sacroiliaca and backbone involvement, person between twenty and fifty are apt to occur in,
Have potential crippling, characteristic pathological change is sacro-iliitis.
Articulatio sacroiliaca is it is now recognized that belong to non-renewable tissue, from the pathologic sampling of human body and research by ethics
Limitation.Building sacro-iliitis animal model facilitate carry out disease morbidity research, for disease condition monitoring with
And the following new drug development is provided fundamental basis and foundation.
Current study show that Wnt/ β-catenin signal path plays a crucial role in AS skeletonization, Dkk-1 is that its is important
Negative regulatory factor, the unconventionality expression in AS block Dkk-1 to can promote the fusion of patient's articulatio sacroiliaca, are formed to the spur of AS patient
It shields.MiR-29a is the positive regulatory factor of Osteoblast Differentiation, and it is direct to be that bioinformatics and In vitro cell experiment confirm
Target the important microRNA of Dkk-1.Wnt/ β-catenin signal path is the Wnt signal pathway being found earliest, when being in
When the state not being activated, Axin, APC and GSK3 β form the β-in a huge protein complexes phosphorylation cytoplasm
Catenin, the β-catenin being phosphorylated finally are degraded by proteasome in turn by ubiquitination.When extracellular ligand Wnt with
After seven-transmembrane receptor Frizzled (FZD) and single-transmembrane receptor LRP5/6 on cell membrane are combined, Wnt/ β-catenin signal is logical
Road is activated.Signal is further passed to the related system albumen in cytoplasm, including Dishevelled by the receptor of activation
(Dsh), Axin, APC and GSK3 β etc. causes Axin-APC-GSK3 β complex to be corrupted such that β-catenin from this drop
It releases in solution complex and is accumulated in cytoplasm.A large amount of β-catenin are subsequently into nucleus and transcription factor LEF/TCF
Deng combination, Osteoblast Differentiation is realized in the transcription of activation downstream target gene Runx2, alkaline phosphatase (ALP), osteocalcin (OC) etc.
Regulation.
Summary of the invention
The characteristic pathology that the art of this patent confirms that sacro-iliitis can occur in TNF transgenic mice changes.Exempt from simultaneously
Epidemic disease groupization confirms that the unconventionality expression of Wnt/ β-catenin signal path ingredient locally occurs in lesion for the first time, and by the tune of mir-29a
Control.
It is an object of the invention to disclose a kind of sacro-iliitis mouse model.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of sacro-iliitis mouse model, wherein exempt from the articulatio sacroiliaca position of the sacro-iliitis mouse model
Epidemic disease group feature is that structure is destroyed at exudate, synovial cell proliferation and articular cartilage in articular cavity.
A kind of sacro-iliitis mouse model described in above-mentioned technical proposal, wherein the sacro-iliitis mouse model
Articulatio sacroiliaca position Wnt/ β-catenin signal path ingredient unconventionality expression, and by the regulation of mir-29a.
The invention has the following advantages:
1, the Research of Animal Model for Study in mouse source can be to avoid the materials of the non-renewable articulatio sacroiliaca of human body.
2, the research of animal model can be to study the sacro-iliitis pathology of appearance, thus to disease incidence machine in analogue body
System further investigation.
3, animal model repeatability is strong, is conducive to stablize and carries out the function assessments research such as signal path regulation.
Detailed description of the invention:
1, Fig. 1 is articulatio sacroiliaca materials.
2, Fig. 2 is control group mice pathological manifestations.
3, Fig. 3 is the pathological manifestations that transgenosis group is sliced side articulatio sacroiliaca.
4, Fig. 4 is the pathological manifestations that transgenosis group is sliced other side articulatio sacroiliaca.
5, the enlarged drawing (i.e. the enlarged drawings of transgenosis group slice other side articulatio sacroiliaca pathological manifestations) that Fig. 5 is Fig. 4.
6, Fig. 6 is the mRNA expression of miR-29a after three kinds of miRNA tail vein injections.
7, Fig. 7 is Wnt signal correlation factor Dkk-1, LRP6 and GSK-3 β, β-after three kinds of miRNA tail vein injections
The mRNA of catenin is expressed.
8, Fig. 8 is Wnt signal correlation factor Collagen X and ALP, OC and Runx2 after three kinds of miRNA tail vein injections
MRNA expression.
9, Fig. 9 is Collagen X Immunohistochemical Expression after tail vein injection inhibitor.
10, Figure 10 is Collagen X Immunohistochemical Expression after tail vein injection mimics.
11, Figure 11 is β-cateinin Immunohistochemical Expression after tail vein injection inhibitor.
12, Figure 12 is β-cateinin Immunohistochemical Expression after tail vein injection mimics.
Specific embodiment:
In order to make the technical solution of the present invention easy to understand, below in conjunction with specific test example to a kind of sacro-iliitis of the present invention
Mouse model is further described.
Embodiment 1:The preparation of sacro-iliitis mouse model:
One, mouse source and modeling process:
Source of people tumor necrosis factor α (hTNF α) transgenic mouse lines TgTC mouse originates from using the people TNF/ prepared
β-globin (TNFglobin) recombination carrier (the 2.8kb piece of promoter and code area overall length comprising a hTNF α gene
Section, is fused to the length of 3 ' non-translational regions (UTR) and polyadenylation site including people β-globin of a substitution hTNF α gene
(EMBO is J.1991Dec for the segment of 0.77kb；10 (13): 4025-31.), it is provided by the monitoring of Guangdong experimental animal.
Then, people TNF/ β-globin (TNFglobin) recombination carrier enters FVB/J inbreeding by microinjection
The protokaryon of the fertilized eggs of germline.Finally, the fertilized eggs after injection are implanted the fallopian tubal of 8 week old female false pregnancy ICR mouse.Pass through
Transgenosis propositus individual and the backcrossing of the germline of FVB/J inbreeding are established to the strain of transgenosis.Pass through conventional coda gene
Type and pcr gene type screening transgenic animal, obtain TgTC mouse.
The PCR primer of hTNF α transgenic sequence specificity is:
Upstream primer: 5 '-GA Α CTCCCTCGATGTTAACCA-3 '
Downstream primer: 5 '-TTCAATCCCCAAATCCTAGCC-3 '.
PCR reaction system is as follows:
94 DEG C 4 minutes；95 DEG C 30 seconds 35 circulation, 57 DEG C 40 seconds；72 DEG C 40 seconds；72 DEG C 10 minutes.
Two, model is identified:
Articulatio sacroiliaca obtains (as shown in Figure 1): putting to death TgTC mouse after 4 weeks, removes mouse skin, opens abdominal cavity, removal
Internal organ and peritonaeum.Backbone is cut between lumbar vertebrae and sacral, and cuts off thigh and tail.The stern of pelvis back side is removed as far as possible
Then big flesh muscle goes to abdomen and surveys the muscle removed on the inside of pelvis；It is careful not to damage articulatio sacroiliaca.
Pelvic tissue is taken, 4% formalin fixes 12 hours, and 2 μm of 14%EDTA decalcification at least 1 week after bone tissue softening
The case where continuous paraffin section, understanding articulatio sacroiliaca synovial membrane inflammation and damage of articular surface.
Model pathological examination: paraffin wax flaking HE decoration method
(1), experiment equipment and reagent:
Experiment equipment and reagent difference are as shown in Table 1 and Table 2:
1 experiment equipment of table
2 major experimental reagent of table
(2), tissue paraffin embedded section experimental procedure:
1, draw materials: flesh tissue be fixed on 4% paraformaldehyde for 24 hours more than.Tissue is taken out in draught cupboard from fixer
It is with scalpel that purpose site tissue equating is whole, the tissue cut will be repaired and corresponding label is put in dewatering box；
2, it is dehydrated: putting dewatering box into hanging basket in successively graded ethanol is dehydrated in dewaterer.75% alcohol 4h-
85% alcohol 2h-90% alcohol 2h-95% alcohol 1h- dehydrated alcohol I 30min- dehydrated alcohol II 30min- alcohol benzene 5-
10min- dimethylbenzene I 5-10min- dimethylbenzene II 5-10min- wax I 1h- wax II 1h- wax III 1h；
3, it embeds: being organized in embedding machine for wax will have been soaked and embedded.The wax of thawing is first put into embedding frame, it is solidifying to wax
Gu tissue is taken out out of dewatering box before and is put into embedding frame according to the requirement in embedding face and sticks corresponding label.Freeze in -20 °
Platform is cooling, takes out wax stone from embedding frame after wax solidification and modifies wax stone；
4, it is sliced: the wax stone trimmed being placed on paraffin slicing machine and is sliced, piece is 4 μm thick.Slice floats on booth piece machine 40
On DEG C warm water will tissue flattening, tissue is picked up with glass slide, and put into and bake piece in 60 DEG C of baking ovens.It is taken after water dries wax roastingization
It is stored at room temperature out spare.
(3), HE Coloration experiment step:
1, paraffin section de-waxing is to water: slice being successively put into I 20min- dimethylbenzene of dimethylbenzene, the II anhydrous second of 20min-
I 10min- dehydrated alcohol of alcohol, II 10min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol
5min- distillation washing.
2, bush uniformly dyeing nucleus: being sliced into Harris bush uniformly dyeing 3-8min, originally wash, 1% hydrochloride alcohol point
Change the several seconds, tap water rinses, and 0.6% ammonium hydroxide returns indigo plant, and flowing water rinses.
3, Yihong contaminates cytoplasm: being sliced in eosin stain and dyes 1-3min.
4, it is dehydrated mounting: slice is sequentially placed into 95% alcohol I 5min-95% alcohol II 5min- dehydrated alcohol I
It is dehydrated transparent in II 5min- dimethylbenzene of 5min- dehydrated alcohol, I 5min- dimethylbenzene, II 5min, slice is taken out from dimethylbenzene
It slightly dries, neutral gum mounting.
5, sediments microscope inspection, Image Acquisition analysis.
Coloration result: nucleus blue, cytoplasm are red.
Four, pathological characters:
As a result as shown in Fig. 2~Fig. 5, wherein Fig. 2 is control group mice pathological manifestations, and Fig. 3 is that transgenosis group is sliced side
The pathological manifestations of articulatio sacroiliaca, Fig. 4 are the pathological manifestations that transgenosis group is sliced other side articulatio sacroiliaca, and Fig. 5 is the enlarged drawing of Fig. 4
(i.e. the enlarged drawings of transgenosis group slice other side articulatio sacroiliaca pathological manifestations).
The present invention has been successfully established the mouse model of sacro-iliitis, and immunohistochemistry shows experimental group sample visible joint chamber
Structure is destroyed at interior exudate, synovial cell proliferation and articular cartilage；And control group sample articular cavity has no that obvious inflammation is anti-
It answers, synovial cell has no obvious hyperplasia.
Embodiment 2:Influence of the miR-29a to Wnt signal path in sacro-iliitis mouse peripheral blood:
Influence after miR-29a injection to Wnt signal path:
By 2 × 107Unit carries miR-29a mimcis, and the slow virus of miR-29a inhibitor, NC control carries
Body passes through tail vein injection respectively and enters in TgTC197 Mice Body, takes peripheral blood after 7 days, qPCR detect peripheral blood miR-29a,
Dkk-1, LRP6 and GSK-3 β, β-catenin, Collagen X and ALP, OC and Runx2 mRNA and/or protein expression.
Experimentation and method:
One, qPCR is detected:
(1), reagent and instrument:
Reagent and instrument are as shown in Table 3 and Table 4:
3 experiment reagent of table
4 laboratory apparatus of table
(2), qPCR detecting step:
1, RNA is extracted:
(1), appropriate cell is taken, 1ml Trizol is added, concussion mixes and is placed at room temperature for 5min；
(2), 0.2ml chloroform is added, acutely shakes 15s, stands 3min；
(3), 4 DEG C of 12000rpm are centrifuged 10min, upper strata aqueous phase are transferred in new pipe；
(4), isometric isopropanol is added, mixes, stands 20min；4 DEG C of 12000rpm are centrifuged 10min, remove supernatant；
(5), it is precipitated with 1ml 75%DEPC ethanol wash；
(6), 4 DEG C of 8000rpm are centrifuged 5min, discard liquid；
(7), after room temperature is dried, the 30 processed ddH of μ l DEPC are added2O water dissolves RNA, freezes after identification in -80 DEG C
It deposits spare.
(8), liquid 2ul electrophoresis in 2% Ago-Gel, identification are sampled；
2, reverse transcription:
Using 1 μ g total serum IgE as template, reverse transcription reaction system is prepared according to Bestar qPCR RT Kit specification, it is overall
System is 10 μ l, synthesizes the first chain of cDNA:
(1), reaction solution is prepared first, in accordance with following system:
(2), then 65 DEG C of 5min, on ice chilling；
(3), reaction solution is prepared according still further to following systems:
(4),37℃,60min；98 DEG C, 10min, the first chain of cDNA is synthesized, and collect spare.
3, PCR detection primer:
QPCR reaction system:
The reaction system of Real time PCR amplification is 20 μ l (DBISybrGreen qPCRmasterMix),
Reaction system is prepared according to following table:
PCR reaction condition: 94 DEG C of 2min, 94 DEG C of 20s, 58 DEG C of 20s, 72 DEG C of 20s, 40 circulations.
Melt curve analysis analysis: 62 DEG C -95 DEG C of temperature.
Each sample is repeated 3 times.
Fluorescent quantitative PCR experiment is carried out with Agilent Stratagene fluorescence quantitative PCR instrument Mx3000P.
4, data processing:
Experiment is according to 2-△△CtMethod handles data.Wherein, the average value ± mark of △ Ct=(target gene Ct- internal reference Ct)
Quasi- deviation；Average value ± the standard of △ △ Ct=(target gene △ Ct in target gene △ Ct- reference sample in sample to be tested)
Deviation (selects the maximum sample of Ct to be calculated for reference) if without reference sample；Relative sample original template amount=(2- △
△ Ct) average value ± standard deviation.
5, result:
29a mimics is compared with 29a inhibitor group and our NC control group, miR29a in mouse peripheral blood
Expression it is significant respectively increase and reduce, show miR-29a content having significantly to raise and reduce in vivo and (scheme in mouse
6).It comparing with NC control group, the expression of miR-29a group Dkk-1 and GSK β reduce, LRP6, β-catenin, Collagen X,
The expression of ALP, OC, Runx2 increase.The expression of the above-mentioned factor of miR-29a inhibitor group is then opposite with mimics group (to be schemed
7, Fig. 8).Confirm that miR-29a can promote the activation of Wnt signal and the correlation effect factor.
Experimental example 3:The shadow that miR-29a expresses sacro-iliitis mouse articulatio sacroiliaca β-catenin, Collagen X
It rings:
One, experimentation and method:
Materials: by 2 × 107Unit carries miR-29a mimcis, miR-29a inhibitor, NC control it is slow
Viral vectors passes through tail vein injection respectively and enters in TgTC197 Mice Body, puts to death mouse after 4 weeks, takes pelvic tissue, 4% Fu Er
Malin fixes, 2 μm of continuous paraffin sections after bone tissue softening of 14%EDTA decalcification, immunohistochemistry detection β-catenin,
The expression (Fig. 9-Figure 12) of Collagen X.
Immunohistochemistry step:
1, experiment equipment
2, major experimental reagent:
Two, paraffin section immunohistochemical experiment step:
1, paraffin section de-waxing is to water: slice being successively put into I 15min- dimethylbenzene of dimethylbenzene, II 15min- dimethylbenzene
I 5min- dehydrated alcohol of III 15min- dehydrated alcohol, II 5min-85% alcohol 5min-75% alcohol 5min- distillation washing；
2, antigen retrieval: histotomy is placed in Yu Weibo in the reparation box for filling with EDTA antigen retrieval buffer (PH9.0)
Furnace is interior to carry out antigen retrieval, and moderate heat 5min is to boiling, and truce 5min heat preservation turns low fire 10min again, this should prevent buffer in the process
Excessive evaporation is sure not dry plate.Slide is placed in PBS (PH7.4) on decolorization swinging table after natural cooling and shakes washing 3 times, often
Secondary 5min；
3, block endogenous peroxydase: slice is put into 3% hydrogen peroxide solution, and room temperature, which is protected from light, is incubated for 25min, by slide
It is placed in PBS (PH7.4) on decolorization swinging table and shakes washing 3 times, each 5min；
4, serum is closed: 3%BSA uniform fold tissue is added dropwise in group change circle, room temperature closes 30min；(primary antibody is goat
Source is closed with rabbit anteserum, other sources are closed with BSA)；
5, add primary antibody: gently getting rid of confining liquid, the primary antibody that PBS is prepared by a certain percentage is added dropwise on slice, slice is laid flat
In 4 DEG C of overnight incubations in wet box；(in wet box plus a small amount of water prevents antibody from evaporating)；
6, add secondary antibody: slide, which is placed in PBS (PH7.4) on decolorization swinging table, shakes washing 3 times, each 5min.Slice is slightly
The secondary antibody (HRP label) that kind corresponding to primary antibody is added dropwise after drying in circle covers tissue, is incubated at room temperature 50min；
7, DAB develops the color: slide, which is placed in PBS (PH7.4) on decolorization swinging table, shakes washing 3 times, each 5min；Slice is slightly
The DAB developing solution of Fresh is added dropwise after drying in circle, developing time is controlled under microscope, the positive is brown color, tap water
Rinse slice color development stopping；
8, redye nucleus: haematoxylin redyes 3min or so, originally washes, and haematoxylin breaks up liquid and breaks up several seconds, tap water
It rinses, haematoxylin returns blue liquid and returns indigo plant, and flowing water rinses；
9, it is dehydrated mounting: slice is sequentially placed into 75% alcohol 5min-85% alcohol 5min-I 5min of dehydrated alcohol-nothing
It is dehydrated transparent in II 5min of water-ethanol-dimethylbenzene, I 5min, slice is taken out from dimethylbenzene and slightly dried, neutral gum mounting；
10, sediments microscope inspection, Image Acquisition analysis.
Three, paraffin section ImmunohistochemistryResults Results interpretation:
Bush uniformly dyeing nucleus is blue, and the positive expression that DAB is showed is brown color.
Experimental result: tail vein injection mimics has differences compared with the expression of inhibitor two indices, and inflammatory cell soaks
It is obvious compared with the latter to moisten the former.
The above, only presently preferred embodiments of the present invention, not to the present invention make in any form with substantial limit
System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above skill
Art content, and the equivalent variations for a little variation, modification and evolution made, are equivalent embodiment of the invention；Meanwhile it is all according to
According to the variation, modification and evolution of substantial technological any equivalent variations to the above embodiments of the invention, this is still fallen within
In the range of the technical solution of invention.
Claims (2)
1. a kind of sacro-iliitis mouse model, it is characterised in that: the articulatio sacroiliaca position of the sacro-iliitis mouse model
Immunohistochemical Characterization be exudate in articular cavity, structure is destroyed at synovial cell proliferation and articular cartilage.
2. a kind of sacro-iliitis mouse model according to claim 1, it is characterised in that: the sacro-iliitis mouse
The unconventionality expression of the articulatio sacroiliaca position Wnt/ β-catenin signal path ingredient of model, and by the regulation of mir-29a.
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