CN105769371A - Animal model of inflammation complicated with fracture complex disease as well as construction method and application of animal model - Google Patents
Animal model of inflammation complicated with fracture complex disease as well as construction method and application of animal model Download PDFInfo
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- CN105769371A CN105769371A CN201410837178.1A CN201410837178A CN105769371A CN 105769371 A CN105769371 A CN 105769371A CN 201410837178 A CN201410837178 A CN 201410837178A CN 105769371 A CN105769371 A CN 105769371A
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Abstract
The invention provides an animal model of inflammation complicated with fracture complex disease as well as a construction method and application of the animal model. According to the technical scheme, the construction method provided by the invention comprises the following steps: constructing an animal model with rheumatoid arthritis; and then, constructing a fracture model on the basis of the animal model with the rheumatoid arthritis, so that the animal model of the inflammation complicated with fracture complex disease is successfully constructed; and the animal model is beneficial for simulating disease state of a clinical RA patient after fracture.
Description
Technical field
The present invention relates to medical care evaluation, medical treatment detection field, be specifically related to a kind of inflammation and occur together fracture combined condition animal model and construction method thereof and application.
Background technology
Rheumatoid arthritis (RA) is a kind of chronic, inflammatory, systematic autoimmune disease, with joint and periarticular tissue nonsuppurative inflammation for principal character.Its basic pathological changes is synovitis, acute stage synovial membrane swelling, oozes out, neutrophil infiltration；Chronic phase synovial hyperplasia is plump, forms pannus；The latter be cause destruction of joint, joint deformity, dysfunction, make disease enter the irreversible stage pathologic basis.
Traumatic fracture is a kind of most commonly seen orthopaedic disease, account for the great majority of clinical findings fracture, the most of fracture patient of clinical discovery there is also the pain in fracture and injury place and other joints after clinical union of bone, and pain, swelling, the series of symptoms performance such as rubescent become apparent from when running into Changes in weather.On inspection, these symptoms are mostly caused by RA.With regard to current bibliographical information, whether fracture is still not can determine that caused by RA, and the relevant animal models that RA occurs with fracture simultaneously also still has been reported that.
At present, independent rheumatoid arthritis animal model and individually animal fracture model all have been reported that, these models are simply under given conditions, choose some or certain several stress factor and set up, can not reflect that all properties of RA, different arthritis animal model are likely to only one side with regard to disease and further investigate completely, but, in clinic, several symptoms occur simultaneously often, be also many contacts simultaneously, interact on pathology, physiology.
For verifying the rheumatoid arthritis mechanism with aspects such as the cause of disease of fracture generation, pathology, immunology, clinics, find the scheme of more effective treatment rheumatoid arthritis and damaging fracture, it is necessary to build the animal model that rheumatoid arthritis occurs with fracture.
Summary of the invention
For solving the problems referred to above, the invention provides a kind of inflammation and occur together fracture combined condition animal model and construction method thereof and application.Technical scheme provided by the invention first builds rheumatoid arthritis animal model, then on the basis of rheumatoid arthritis animal model, build fracture model, successfully construct inflammation to occur together fracture combined condition animal model, be conducive to the morbidity state after with this combined condition model for the clinical patient's RA fracture of object of study simulation.
First aspect, the invention provides a kind of inflammation occur together fracture combined condition animal model construction method, comprise the steps:
1) rheumatoid arthritis animal model is built；
2) build fracture model with constructed rheumatoid arthritis animal for object, obtain described inflammation occur together fracture combined condition animal model.
As described in the present invention, described " inflammation the occur together fracture combined condition animal model " animal model referring to that rheumatoid arthritis occurs with fracture, it is preferred to the animal model that rheumatoid arthritis occurs with open fracture.
In an embodiment of the present invention, in described step (1), when building rheumatoid arthritis animal model, the animal of employing is rat, mice, rabbit, dog or monkey, but is not limited to this.
When the present invention builds rheumatoid arthritis animal model, adopt the structure rheumatoid arthritis Animal models that those skilled in the art commonly use, and according to different mode animal, or different animal strains, adopt the modeling method matched.
In a preferred embodiment, in described step (1), build rheumatoid arthritis animal model and be preferably but not limited to the rheumatoid arthritis animal model constructing method summarized in " Contemporary Chinese medicine " (volume the 4th phase February the 19th in 2012) such as Cai Wenhong.Such as, a) Collagen-Induced Arthritis (CIA) method can be adopted for rat kind rheumatic arthritis modeling method, it is preferred to use DA, BB, DR or LEW rat；B) arthritis that cartilage oligomer body stromatin (COMP) is induced, it is preferred to use DA, LEW rat；C) arthritis of adjuvant induction, it is preferred to use DA, LEW rat；D) arthritis of Avridine induction, it is preferred to use DA, LEW rat；E) arthritis of pristane induction, it is preferred to use DA, LEW rat.
In another preferred embodiment of the present invention, in described step (1), the method building rheumatoid arthritis animal model is Collagen-Induced Arthritis (CIA) method, adopts the modeling of wistar rat.
In an embodiment of the present invention, in described step (2), during with constructed rheumatoid arthritis animal for object structure fracture model, adopt the structure animal fracture model method that those skilled in the art commonly use, and according to different mode animal, or different animal strains, adopt the modeling method matched.
In a preferred embodiment, in described step (2), it is preferably but not limited to the fracture model that constructed rheumatoid arthritis animal builds for object: long bone shaft fracture model, closed fracture model, compound fracture model, gene fracture model.
In another preferred embodiment of the present invention, in described step (2), the fracture model built with constructed rheumatoid arthritis animal for object is for compound fracture model, and method is: after anesthetized animal, taking animal right lower extremity, Lateral approach exposes femoral shaft；Femur is walked crosswise sawed-off in femur stage casing, with Kirschner wire fixation, successively close incisions sewing up.
Further preferably, described method is particularly as follows: after anesthetized animal, choose animal right lower extremity and animal wool is shaved off, first cut a minute opening on lower limb knee ligament limit but its ligament can not be cut off, then Kirschner wire is inserted otch and passes from greater trochanter of femur along marrow cavity of femur from animal femoral knee end, drive in the wrong direction extract out Kirschner wire but be not fully drawn out, lateral side of femur otch expose femoral shaft；Femur is walked crosswise sawed-off in femur stage casing, then reseting fracture, Kirschner wire direct motion is inserted fracture far-end.Confirmation is fixing can rearward, and Aculeata is close to greater trochanter root bending, and redundance is wiped out, successively close incisions careful stitching.
The present invention builds rheumatoid arthritis with open fracture animal model, simulating human rheumatoid arthritis is with the clinical symptoms of multiple fracture, rheumatoid arthritis and the two many contact offer data on physiology, pathology of fracture are provided for research, also provide certain reference frame for risk of bone fracture after the occurring or reduce rheumatoid arthritis of rheumatoid arthritis after preventing fracture clinically simultaneously.
Second aspect, the invention provides a kind of inflammation and occurs together fracture combined condition animal model, and the described inflammation fracture combined condition animal model that occurs together builds by the following method: 1) build rheumatoid arthritis animal model；2) build fracture model with constructed rheumatoid arthritis animal for object, obtain described inflammation occur together fracture combined condition animal model.
In a preferred embodiment, described inflammation occur together fracture combined condition animal model adopt as described in relation to the first aspect construction method constructed by.
The third aspect, the invention provides inflammation as described in relation to the first aspect occur together fracture combined condition animal model construction method preparing the application that rheumatoid arthritis occurs together in animal fracture model.
The invention provides inflammation and occur together fracture combined condition animal model and construction method and application have the advantages that
(1) according to the respective pathological characteristic of rheumatoid arthritis and open fracture for same animal, model of rheumatoid arthritis and animal fracture model are successively built.The combined animal model obtained, has mankind's rheumatoid arthritis simultaneously and occurs together the clinical symptoms of fracture, compare existing, independent model of rheumatoid arthritis or animal fracture model only simulate, reacted RA in a certain respect or several factor；The two is then integrated by combined animal model provided by the invention, more meets mankind's rheumatoid arthritis and occurs together the feature of the clinical symptoms of fracture, pathology and iconography, is a kind of rheumatoid arthritis combined condition animal model of adding fracture.
Accompanying drawing explanation
Figure1 is that the inflammation that the embodiment of the present invention provides occurs together the signal of construction method of fracture combined condition animal modelFigure；
FigureThe clinical symptoms of the 2 immune sequela rats provided for the embodiment of the present invention；
FigureThe order of severity Clinical points of the 3 immunological pathogenesis rats provided for the embodiment of the present invention；
Figure4 is relevant inflammatory factors content detection result in the rat blood of embodiment of the present invention offer；
FigureThe 5 postoperative results carrying out X-light detection provided for the embodiment of the present invention；
FigureThe 6 postoperative results carrying out CT detection provided for the embodiment of the present invention；
Figure7 dye observed result with the new bone growth situation HE (Hematoxylin-eosin) of fracture model and matched group for arthritis.
Detailed description of the invention
The following stated is the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Without outside illustrating in the embodiment of the present invention, agents useful for same and consumptive material are commercial goods.
Embodiment 1
Figure1 is that the inflammation that the embodiment of the present invention provides occurs together the flow process of construction method of fracture combined condition animal modelFigure, in conjunction withFigure1, present embodiments provide a kind of inflammation occur together fracture combined condition animal model construction method, comprise the steps:
(1) II Collagen Type VI Emulsion is joined
Cattle Type Ⅱ collagen (ImmunizationGradeTypeIICollagen, Chondrex, Inc.2002) and not formula Freund's incomplete adjuvant (IncompleteFreund ' sAdjuvant article No. 7002) is purchased from Chondrex, Inc. company, is emulsified into milkiness drop after being mixed by the two equal-volume in emulsator.
(2) animal immune
8 weeks healthy wistar rats, male and female, body weight 180-220g, adaptability is fed 1 week, and rat rat fixator is fixed, and at distance rat root of the tail position 2, centimeters carries out subcutaneous injection, and dosage is 200ul, and this is initial immunity.The method same after the 10 days offside subcutaneous injection 100ul mixed liquor at initial injection position carries out booster injection, and every day observes, and joint occurs that redness person is modeling success rat (CIA).Matched group simultaneous equal injecting normal saline.
Result is shown inFigure2～4；Result shows, rat has the joint of animal of about 10% redness occur for latter 7 days in second time immunity, and the animal disease time of 90% concentrates on after secondary immunity 10-14 days.?FigureIn 3, Clinical points is all animal averages in group,FigureIn 2, AFigureFor matched group intact animal, BFigureFor immune group affected animal, it is seen that B treated animal hind leg occurs in that obvious redness and the red and swollen Symptoms last after morbidity.
Figure4 is the content detection result of inflammatory factor in rat blood, byFigure4 it can be seen that morbidity after 7 days, 20 days, it is all significantly raised that IL-6, IL-1beta and the TNF-a factor in immunological pathogenesis treated animal body compares matched group.
Above-mentioned test result indicate that, the present invention adopts the Emulsion containing II Collagen Type VI that wistar rat root of the tail portion is carried out intradermal injection immunity twice, and rat model, with normal rat compared with, substantially foot pawl redness occurs, and Clinical points relatively matched group is increased significantly trend.
The embodiment of the present invention 1 additionally provides the detections such as modeling success rat (CIA) relevant inflammatory factors (TNF-α, IL-1 β, IL-6).Result is shown inFigure4:A, B, C.
(3) arthritis index assessment
Arthritis index is used to the development degree of assessment arthritis, and particular content is as follows: 0: natural joint；1:1-2 interphalangeal joint is red and swollen；2: involve 3-4 interphalangeal joint or 1 big joint；3: red and swollen joint is more than 4；4: arthritis involves whole sufficient pawl.Every rats with bilateral hind leg can obtain the 0-8 assessed value divided with this score-system.
(4) arthritis makes with fracture model (composite model)
After the success of CIA model construction, after the morbidity of immune group animal, the last fortnight is inflammatory phase, within 15th day, carry out open fracture operation: take the CIA rat model of step (2) gained, after using pentobarbital sodium (0.5ml/100g) intraperitoneal anesthesia of 1%, choose animal right lower extremity and animal wool is shaved off, first cut a minute opening on lower limb knee ligament limit but its ligament can not be cut off, then Kirschner wire is inserted otch and passes from greater trochanter of femur along marrow cavity of femur from animal femoral knee end, drive in the wrong direction extract out Kirschner wire but be not fully drawn out, lateral side of femur otch exposes femoral shaft；Femur is walked crosswise sawed-off in femur stage casing, then reseting fracture, Kirschner wire direct motion is inserted fracture far-end.Confirmation is fixing can rearward, and Aculeata is close to greater trochanter root bending, and redundance is wiped out, successively close incisions careful stitching.Animal being put back to Animal House raise, every day prevents wound inflammation to operative site sterilization.
Matched group 2: adopt the rat of the matched group 1 of step (2), according to above-mentioned open fracture surgical procedure.
For absolutely proving beneficial effects of the present invention, the embodiment of the present invention 1 provides the postoperative result carrying out X-light detection and the result of the X-light detection of comparative example.AsFigureShown in 5.?FigureIn 5, A is operation X-ray check result on the same day；BFigureFor after performing the operation 8 weeks composite model rat, bFigureFor matched group 2 rat after performing the operation 8 weeks；CFigureFor after performing the operation 18 weeks composite model rat, cFigureFor matched group 2 rat after performing the operation 18 weeks.
The embodiment of the present invention 1 additionally provides the postoperative result carrying out CT detection and the result of the CT detection of comparative example.AsFigureShown in 6.?FigureIn 6, AFigureFor control rats, a after performing the operation 8 weeksFigureFor the composite model rat after performing the operation 8 weeks；BFigureFor control rats, b after performing the operation 8 weeksFigureFor the composite model rat after performing the operation 8 weeks.
Above-mentioned the results show, detect through X-ray check and CT scan and find, postoperative simple fracture model (matched group 2) is different from RA+ fracture model group (composite model group) healing, these characteristics are closer to the rheumatoid arthritis of people, and II Collagen Type VI induction rheumatoid arthritis provided by the invention is the ideal model studying its pathology and screening drugs for rheumatoid arthritis with fracture model.
For further illustrating beneficial effects of the present invention, the embodiment of the present invention 1 provide step (4) arthritis with the New born formation bone of fracture model and matched group dye (Hematoxylin-eosin) result asFigure7,AFigureFor control rats, a after performing the operation 8 weeksFigureFor the composite model rat after performing the operation 8 weeks, byFigure7 it can be seen that relative to matched group, the freshman bone tissue of composite model is few, and healing is relatively low, closer to the rheumatoid arthritis symptom of people.
Comparative example: inventor has done the contrast tests such as SD rat, Wistar rat, DBA/1 mice, C57 mice when building model of rheumatoid arthritis, and wherein SD rat and C57 mice modeling success rate are relatively low, general only have about 10%, non-sensitive strain.But DBA/1 mice CIA modeling success rate relatively high mice fracture model manipulation is more difficult, so the present invention has selected Wistar rat.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all any amendment, equivalent replacement and improvement etc. made within the spirit and principles in the present invention, should be included within protection scope of the present invention.
Claims (8)
1. an inflammation occurs together the construction method of fracture combined condition animal model, it is characterised in that comprise the steps:
1) rheumatoid arthritis animal model is built；
2) build fracture model with constructed rheumatoid arthritis animal for object, obtain described inflammation occur together fracture combined condition animal model.
2. inflammation as claimed in claim 1 occurs together the construction method of fracture combined condition animal model, it is characterised in that in described step (1), when building rheumatoid arthritis animal model, the animal of employing is rat, mice, rabbit, dog or monkey.
3. inflammation as claimed in claim 2 occurs together the construction method of fracture combined condition animal model, it is characterised in that described rat is wistar rat.
4. inflammation as claimed in claim 1 occurs together the construction method of fracture combined condition animal model, it is characterized in that, in described step (1), when building rheumatoid arthritis animal model, adopt the modeling method of collagen-induced arthritis, cartilage oligomer body stromatin (COMP) induced arthritis, adjuvant induced arthritis, Avridine induced arthritis or pristane induced arthritis.
5. inflammation as claimed in claim 1 occurs together the construction method of fracture combined condition animal model, it is characterized in that, in described step (2), the fracture model of structure is long bone shaft fracture model, closed fracture model, compound fracture model or gene fracture model.
6. the inflammation described in claim 5 occur together fracture combined condition animal model construction method, it is characterised in that the construction step of described compound fracture model includes: after anesthetized animal, takes animal right lower extremity, Lateral approach expose femoral shaft；Femur is walked crosswise sawed-off in femur stage casing, with Kirschner wire fixation, successively close incisions sewing up.
7. an inflammation occurs together fracture combined condition animal model, it is characterised in that the described inflammation fracture combined condition animal model that occurs together builds by the following method: 1) build rheumatoid arthritis animal model；2) build fracture model with constructed rheumatoid arthritis animal for object, obtain described inflammation occur together fracture combined condition animal model.
8. the occur together construction method of fracture combined condition animal model of inflammation as claimed in claim 1 is preparing the application that rheumatoid arthritis occurs together in animal fracture model.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110604098A (en) * | 2019-09-23 | 2019-12-24 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for constructing animal model of rheumatoid arthritis combined with interstitial lung disease |
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- 2014-12-26 CN CN201410837178.1A patent/CN105769371A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110604098A (en) * | 2019-09-23 | 2019-12-24 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for constructing animal model of rheumatoid arthritis combined with interstitial lung disease |
| CN110604098B (en) * | 2019-09-23 | 2021-09-07 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for constructing animal model of rheumatoid arthritis combined with interstitial lung disease |
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Application publication date: 20160720 |