CN-119745555-B - Method for constructing osteoarthritis animal model and application thereof

CN119745555BCN 119745555 BCN119745555 BCN 119745555BCN-119745555-B

Abstract

The invention provides a construction method and application of an osteoarthritis animal model. The construction method of the invention selects a standardized experimental Beagle dog and establishes an osteoarthritis model by an improved Hulth method, and optionally adopts a mode of inducing the motion of the experimental dog for further treatment to construct an animal model which is close to the clinical actual situation. The animal model obtained by the construction method disclosed by the invention is attached to the injury arthritis caused by the increasing abrasion of joints in the actual life, has good model stability, and provides a new animal model with important practical value for the research and development of osteoarthritis medicines and medical instruments.

Inventors

OU HUIYU
CHEN JIEJUN
ZHOU CHENGHAO
CHEN JIANZHAO
CHEN DIXIN
JIA DAN
GUO QIUPING

Assignees

广州医药研究总院有限公司

Dates

Publication Date: 20260512
Application Date: 20241223

Claims (12)

1. A method of constructing an animal model of osteoarthritis, the osteoarthritis being knee osteoarthritis, the method comprising the steps of: (1) Taking an experimental dog, shaving Mao Bei skins of a right hind limb knee joint operation area after anesthesia, and sterilizing the skin; (2) Longitudinally cutting a skin incision of 3cm on the inner side of a knee joint, wherein the skin incision is positioned beside a patella on the inner side of a joint capsule at the buckling position of the knee joint, and after the skin incision is cut into a joint cavity layer by layer, the patella is turned to the outer side of the knee joint, the knee joint is buckled, and an anterior cruciate ligament is found and cut off; (3) Resecting the medial meniscus without damaging the articular cartilage surface; (4) Suturing layer by layer, closing joint capsule and skin, injecting penicillin sodium to prevent infection, and allowing the experimental dogs to freely move in a cage without fixing injured limbs; (5) Detecting pathological characteristic indexes at 6-10 weeks after operation to confirm successful modeling, wherein the pathological characteristic indexes comprise articular cartilage pathological section staining or Mankin pathological scoring based on the articular cartilage pathological section staining; And further detecting one or more of behavioral characteristic indicators, joint swelling and pain characteristic indicators, and biological characteristic indicators of the experimental dog, the behavioral characteristic indicators including at least one of lameness, coordination of movement, and joint curvature, the joint swelling and pain characteristic including at least one of joint deformity, knee joint circumference, and pain, the biological characteristic indicators including at least one of inflammatory factors IL-1 beta, IL-6, and TNF-alpha, free radical NO, pain factor SP, and cartilage metabolic marker CTX-II levels.
2. The method of claim 1, wherein the test dogs in step (1) are standardized test Beagle dogs.
3. The method of claim 2, wherein the experimental Beagle dogs are 9-12 months of age.
4. A method of constructing as claimed in any one of claims 1 to 3 wherein pathological characteristics are detected at week 8 post-operation in step (5) to confirm successful modeling.
5. A method of constructing an animal model of osteoarthritis that mimics the clinical condition of an arthritic patient, said osteoarthritis being knee osteoarthritis, said method comprising the steps of: (1) Taking an experimental dog, shaving Mao Bei skins of a right hind limb knee joint operation area after anesthesia, and sterilizing the skin; (2) Longitudinally cutting a skin incision of 3cm on the inner side of a knee joint, wherein the skin incision is positioned beside a patella on the inner side of a joint capsule at the buckling position of the knee joint, and after the skin incision is cut into a joint cavity layer by layer, the patella is turned to the outer side of the knee joint, the knee joint is buckled, and an anterior cruciate ligament is found and cut off; (3) Resecting the medial meniscus without damaging the articular cartilage surface; (4) Suturing layer by layer, closing joint capsule and skin, injecting penicillin sodium to prevent infection, and allowing the experimental dogs to freely move in a cage without fixing injured limbs; (5) Inducing the experimental dogs to exercise for 10-30 minutes every day for 6-10 weeks; (6) Detecting pathological characteristic indexes of the experimental dogs at the 6 th to 10 th weeks after operation to confirm successful modeling, wherein the pathological characteristic indexes comprise joint cartilage pathological section staining or Mankin pathological scoring based on the joint cartilage pathological section staining; And further detecting one or more of behavioral characteristic indicators, joint swelling and pain characteristic indicators, and biological characteristic indicators of the experimental dog, the behavioral characteristic indicators including at least one of lameness, coordination of movement, and joint curvature, the joint swelling and pain characteristic including at least one of joint deformity, knee joint circumference, and pain, the biological characteristic indicators including at least one of inflammatory factors IL-1 beta, IL-6, and TNF-alpha, free radical NO, pain factor SP, and cartilage metabolic marker CTX-II levels.
6. The method of claim 5, wherein the test dogs in step (1) are standardized test Beagle dogs.
7. The method of claim 6, wherein the experimental Beagle dogs are 9-12 months of age.
8. The method of claim 5, wherein step (5) is continued for 8 weeks.
9. The method of claim 5, wherein the step (5) is performed by inducing the experimental dog to move daily to place a toy, playing music, and inducing the experimental dog to move.
10. The method of claim 5, wherein the experimental dogs are induced to exercise for 20 minutes each day in step (5).
11. The method of any one of claims 5 to 10, wherein the pathological characteristic index of the experimental dog is detected at week 8 after the operation in step (6) to confirm successful molding.
12. A method of identifying whether a compound is therapeutically effective against osteoarthritis, said osteoarthritis being knee osteoarthritis, said method comprising the steps of: (1) Constructing an osteoarthritis model dog by the method of any one of claims 1 to 4 or any one of claims 5 to 11, then detecting a pathological characteristic index of the model dog, and further detecting one or more of a behavioral characteristic index, a joint swelling and pain characteristic index and a biological characteristic index of the model dog to obtain first detection data; (2) Administering to the model dog a compound to be identified; (3) Detecting the pathological characteristic index of the model dog again, and further detecting one or more of the behavioral characteristic index, the joint swelling and pain characteristic index and the biological characteristic index of the model dog to obtain second detection data; (4) Comparing the second detection data with the first detection data; The pathological characteristic indexes in the step (1) and the step (3) comprise joint cartilage pathological section staining or Mankin pathological scoring based on the joint cartilage pathological section staining, and after comparing the second detection data with the first detection data in the step (4), the compound is identified as being effective for treating osteoarthritis if the Mankin score of the model dog is found to be reduced; comparing the second test data with the first test data in step (4), and identifying the compound as being effective for osteoarthritis treatment if at least one of a decrease in lameness score, an increase in motor coordination capacity, and an increase in joint curvature is found in the model dog; Comparing the second test data with the first test data in step (4), and identifying the compound as being effective for the treatment of osteoarthritis if the model dog is found to have at least one of reduced joint deformity score, reduced knee joint circumference and reduced pain score; the biological characteristic index in step (1) and step (3) comprises at least one of the levels of inflammatory factors IL-1 beta, IL-6 and TNF-alpha, free radical NO, pain factor SP and cartilage metabolic marker CTX-II, and the compound is identified as being effective for the treatment of osteoarthritis if the model dog is found to have reduced levels of at least one of inflammatory factors IL-1 beta, IL-6 and TNF-alpha, free radical NO, pain factor SP and cartilage metabolic marker CTX-II after comparing the second test data with the first test data in step (4).

Description

Method for constructing osteoarthritis animal model and application thereof Technical Field The invention relates to the technical field of establishment and application of osteoarthritis models, in particular to establishment and application of a related animal model especially fitting knee osteoarthritis clinical conditions. Background Osteoarthritis (OA) is a degenerative joint disease that severely affects the quality of life of a patient, and can lead to joint pain, deformity, and motor dysfunction, thereby increasing the incidence of cardiovascular events and total mortality, especially in knee osteoarthritis. The current animal model of OA mainly comprises spontaneous, inducibility and transgenic animal models, wherein the inducibility model is more stable, the molding time is short, and the model is the most commonly used model. The inducibility model can be divided into two types of operation and non-operation, namely, the molding success rate of the operation model is high, the repeatability is high, the method is suitable for research of post-traumatic OA, is the best choice for short-term research, and has the widest application. The animal used for inducing the OA model is mainly small animals (rats and rabbits), but the joints of the small animals are small, so that the intra-articular operation is not facilitated, the difference between the joint structure and human beings is large, the experimental result is not suitable for being applied to the human body, the joint structure of the large animals is more similar to that of the human body, the operation is convenient, the whole process of occurrence and development of human osteoarthropathy can be better simulated, and the action effect of the medicine can be more accurately predicted when the medicine is used. It is necessary to build a large animal OA surgical model and study its mechanism of action. At present, sheep are mostly adopted in the international reported large animal related documents, and are not experimental animals, and have no standard genetic background and basic data, so that an arthritis model prepared by taking sheep as an experimental object cannot be popularized and applied. The canine animals such as Beagle dogs are fixed in variety, excellent in hereditary property, stable in repeatability, strong in environmental adaptability and disease resistance, mild in character, and widely applied to the fields of medicine research and the like, and especially the world-accepted experimental animals in the fields of new drugs and medical instrument research and development. However, a complete and reliable set of operating technical procedures and specifications for constructing a canine osteoarthritis model are not formed internationally. Therefore, there is an urgent need in the art to obtain a large animal model of osteoarthritis to be applied to the development of human osteoarthritis drugs and medical devices as soon as possible, meeting clinical demands. Disclosure of Invention Aiming at the technical problems, the invention aims to provide a method for constructing an animal model of osteoarthritis, which adopts dogs to construct the animal model so as to solve the limitations of the existing animal model of osteoarthritis. Further, it is another object of the present invention to provide a method for constructing a canine model of osteoarthritis which is more compatible with clinical practice, combining the activity characteristics of clinical patients, and further administering exercise operations after constructing the animal model with dogs, so that the constructed animals can simulate the clinical practice of osteoarthritis in human patients. Furthermore, it is an object of the present invention to provide the use of the animal models obtained by the above two construction methods, in particular in testing whether a compound is therapeutically effective against osteoarthritis. The technical scheme of the invention is as follows. In a first aspect, the present invention provides a method of constructing an animal model of osteoarthritis, the method comprising the steps of: (1) Taking an experimental dog, shaving Mao Bei skins of a right hind limb knee joint operation area after anesthesia, and sterilizing the skin; (2) Longitudinally cutting the inner side of the knee joint, cutting off the anterior cruciate ligament after entering the joint cavity layer by layer; (3) Resecting the medial meniscus without damaging the articular cartilage surface; (4) The joint capsule and skin were closed by suturing layer by layer, penicillin sodium was injected intramuscularly to prevent infection immediately after surgery, and the injured limb was not immobilized after that, and the experimental dogs were free to move in the cage. Preferably, the osteoarthritis is knee osteoarthritis. Preferably, the experimental dogs in step (1) are standardized experimental Beagle dogs, preferably 9-12 months of age. Preferably, in the step (2