CN113237902A - Method for establishing tree shrew periodontitis model - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a method for establishing a tree shrew periodontitis model, which comprises the following steps: (1) selecting adult healthy male tree shrews for modeling, wherein the weight of the male tree shrews is 130-200 g; dividing 40 tree shrews into a normal control group and an experimental group by adopting a random control method, observing for 1-8 weeks, and each group has 3-8 tree shrews per time point; performing no treatment on the control group, and performing silk thread ligation on the first molars of the right mandible of all the rest tree shrews by using 2-0 silk threads; (2) observing and recording clinical manifestations of the tree shrews at the ligation positions 1-8 weeks after ligation, killing the tree shrews, and collecting mandibles of the tree shrews; (3) Micro-CT scanning is used for detecting bone tissue absorption degree, HE staining is used for detecting inflammatory cell infiltration degree of periodontal tissue, TRAP staining is used for detecting osteoclast infiltration degree quantity, and IHC staining is used for detecting inflammation related proteins TNF-alpha, IL-1 alpha, MPO and osteoclast related protein Cathepsin K expression conditions.
Description
Technical Field
The invention relates to the technical field of oral biology, in particular to a method for establishing a tree shrew periodontitis model.
Background
Periodontitis is one of the most common inflammatory diseases of human beings, which is characterized in that periodontal pathogens in dental plaques infect susceptible hosts to cause immune response of the hosts, so that periodontal supporting tissues (gingiva, periodontal ligament, alveolar bone and cementum) are damaged, periodontal tissue inflammation, periodontal pocket formation, attachment loss and alveolar bone absorption are shown as main reasons of adult tooth loss, and the risk of patients suffering from systemic diseases such as cardiovascular diseases, neurodegenerative diseases, autoimmune diseases, poor pregnancy outcome and cancers is increased, so that the health and the quality of life of the human beings are seriously affected. According to epidemiological investigation data of oral health in the fourth nation in China, the periodontal health rate of the age group of 35-44 years in China is 9.1%, and the periodontal health rate of the age group of 55-64 years in China is only 5.0%, so that serious social and economic burden is caused.
The occurrence and development of periodontitis involve various tissues and pathological changes. In vitro model studies using cells have the problem of single study factor, and the pathogenesis and treatment response of diseases cannot be deeply discussed only by clinical observation and in vitro model studies. Therefore, a stable, reliable and simple periodontitis animal model is established, the pathological process of human periodontitis is reproduced as much as possible, experimental basis can be provided for discussing pathogenesis, pathology, prevention and treatment and the like of periodontitis, and the method has great significance.
At present, the model animals commonly used for constructing periodontitis are mainly: non-human primates, dogs, rats, mice, hamsters, sheep, and the like. Since monkeys, particularly macaques, are a kind of ape primate animal, the number of teeth and periodontal tissue structure are very similar to those of humans, and spontaneous periodontitis and experimental periodontitis occur, in which the causative factors, causative mechanisms, disease manifestations, immunological properties, and the like of experimental periodontitis have many similarities to those of humans, researchers have succeeded in establishing experimental periodontitis models in the oral cavity of monkeys for the study of periodontitis diseases. But the use of the feed is limited by the high price, special feeding requirements, ethics, law and other considerations. Dogs have a tendency to idiopathic periodontitis, but their periodontal anatomy is accessible to humans, e.g., most dogs have no gingival sulcus and gingival crevicular fluid. Rats, which are the most commonly used model animal for experimental periodontitis, have molar periodontal tissue structures similar to those of human beings, but are partially different, such as: rat gingival sulcus epithelium with keratosis and the like; and its inflammatory response is different from that of humans; meanwhile, the rat has stronger healing capacity, and the observation and judgment of experimental results are influenced. Rodents such as mice and hamsters have similar problems as rats. Sheep, rabbits, pigs, etc. are used relatively rarely.
The tree shrew is an animal belonging to a tree shrew in the family of tree shrew, and the Latin name of the tree shrew is as follows: tupaia blanangeri, English name: the whole genome sequencing analysis and a plurality of research results at home and abroad of the Tree Shew show that the genetic relationship of the Tree Shew is closest to that of a primate (about 93.4%), the Tree Shew is similar to that of a human being in the aspects of tissue anatomy, physiology, biochemistry, nervous system (brain function), metabolic system, immune system and the like, and compared with the primate, the Tree Shew has the characteristics of small body size, short reproduction period, easiness in experimental operation, low feeding cost and the like, is a research hotspot of scholars at home and abroad in recent years, is widely applied to the establishment of animal models of human diseases such as hepatitis, depression and the like and mechanism research, but the report that Tree Shrew is used as a model animal for the research of oral periodontitis diseases does not appear.
Disclosure of Invention
The invention aims to solve the existing problems and provides a method for establishing a tree shrew periodontitis model.
In order to achieve the purpose, the invention adopts the following technical scheme: the invention discloses a method for establishing a tree shrew periodontitis model by silk thread ligation, which comprises the following steps: (1) selecting adult healthy male tree shrews for modeling, wherein the weight of the male tree shrews is 130-200 g; dividing 40 tree shrews into a normal control group (hereinafter referred to as a control group) and an experimental group by adopting a random control method, and observing for 1-8 weeks, wherein each group has 3-8 tree shrews per time point; performing no treatment on the control group, and performing silk thread ligation on the first molars of the right mandible of all the rest tree shrews by using 2-0 silk threads;
(2) observing and recording clinical manifestations of the tree shrews at the ligation positions 1-8 weeks after ligation, killing the tree shrews, and collecting the right mandible of the tree shrews;
(3) Micro-CT scanning is used for detecting bone tissue absorption degree, HE staining is used for detecting inflammatory cell infiltration degree of periodontal tissue, TRAP staining is used for detecting osteoclast infiltration quantity, and IHC staining is used for detecting expression conditions of inflammation related proteins TNF-alpha, IL-1 alpha, MPO and osteoclast related protein Cathepsin K.
Further, in step (1), the groups were randomly divided into 9 groups of 3 to 8, and 40 groups were used. Normal control group (hereinafter referred to as control group), 1 week group, 2 week group, 3 week group, 4 week group, 5 week group, 6 week group, 7 week group, and 8 week group, respectively; all the tree shrews are Diancia (Latin's name Tupaiabelangeri chinensis), experimental animals are provided by a tree shrew domestication demonstration application platform of department of animal academia of Kunming medical university, and the tree shrews pass the examination by an animal experiment ethical examination committee of Kunming medical university and agree to carry out experiments (number: KMMU 2020029).
Further, in the step (1), the tree shrew is anesthetized by intraperitoneal injection with 3% sodium pentobarbital according to the ratio of 0.3mL/100g (sodium pentobarbital volume/body weight); after anesthesia, the tongue is gently pulled to the left side, and the right mandibular cuspid, the first molar and the second molar are exposed. Placing the oral cavity probe between two molars, rapidly pressing down between the first molars and the second molars of the lower jaw on the right side of the tree shrew by using a non-absorbable surgical suture line No. 2-0, then placing the oral cavity probe between the first molars and the adjacent canine, rapidly pressing down the silk thread between the first molars and the adjacent canine around the neck of the tooth, and continuously knotting twice on the buccal side.
Further, in the step (2), at a time point of 1-8 weeks after the completion of the silk ligation, the tree shrew is anesthetized by intraperitoneal injection with 3% sodium pentobarbital, and then the tree shrew is killed by cervical dislocation; taking out the lower jaw bone on the right side of the tree shrew, removing incisors and partial canine of the tree shrew, and placing the tree shrew in a 10% neutral formaldehyde solution for fixation.
Further, in step (2), observation was made before sampling every week after the silk ligation, and it was found that 1 week after the ligation, the gingival margin of the first molar and the surrounding periodontal tissues was reddened and the mucosal surface was whitish. The redness and whitening phenomenon deepens after 2 weeks after ligation, and the inflammation becomes worse. 3 and 4 weeks after ligation, the gum is swollen, dark red in color, the gingival sulcus is deepened, and bleeding occurs after probing. After 5 and 6 weeks of ligation, the gum appeared dark red and could reach the superficial periodontal pocket. After 7 and 8 weeks of ligation, the gum is dark red, the depth of the periodontal pocket is increased, the teeth are slightly loosened, and tartar is visible on the root surface of part of the tree shrews;
in the step (3), the fixed tissue is firstly subjected to Micro-CT scanning, wherein the scanning parameters are voltage of 90KV and scanning current of 60 mA; after scanning, washing the tissues overnight with running water, and soaking the tissues in freshly prepared EDTA decalcification solution; the liquid is changed every 2-3 days until the needle tip is stabbed and the needle is taken out without puncture feeling.
Further, in the step (3), washing the sample completely decalcified by running water overnight, putting the sample into an automatic come card dehydration instrument, dehydrating, soaking wax in a transparent way, embedding the wax block, and storing the wax block in a ventilating shade at room temperature; after exposing the complete tooth root of the first molar in the coronal plane direction, remaining slices according to the thickness of 3 mu m/piece, and sticking 2-3 slices on each slide; baking in a 65 ℃ oven for 12h, taking out, recovering to room temperature, storing for later use, and slicing for histopathological staining; the pathological staining is one or a combination of HE staining, TRAP staining or IHC staining.
Further, in the step (3), the HE staining comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3 staining with hematoxylin for 10min, washing with water for 5s, differentiating with hydrochloric acid for 5s, washing with water for 5s, and bluing with PBS for 15 s; step 470% ethanol for 2min, and eosin dip dyeing for 30 s; step5 dehydrating with low-concentration to high-concentration gradient alcohol; and Step6, air drying and sealing.
Further, in step (3), the TRAP staining comprises the steps of: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3, preparing TRAP staining solution according to the kit instructions, and then putting the slices into the staining solution to incubate for 60 min; washing with warm water at step437 deg.C, counterstaining with hematoxylin, differentiating, and bluing; and Step5, air drying and sealing.
Further, in step (3), the IHC staining comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3 selects proper antigen repairing method according to the antibody instruction to expose antigen, and washing with PBS solution for 3 times and 3-5 min/time; step 43% H2O2After sealing for 10min, washing with PBS solution for 3 times, 3-5 min/time; step5, adding primary antibody dropwise, and standing overnight at 4 ℃; the antibody was used at the following concentrations: MPO (Ready-to-use, RAB-0379, Mexican), TNF- α (1:100, GTX110520, GeneTex), IL-1 α (1:1000, ab124962, abcam), Cathepsin K (1:200, sc-48353, Santa); step6 PBS washing 3 times, 3-5 min/time; adding secondary antibody dropwise, and incubating for 15 min. Washing with PBS for 3 times, 3-5 min/time; step7 DAB color development, washing, hematoxylin counterstaining, differentiation and bluing; step8 air drying, and sealing.
Has the advantages that: the observation and recording clinical manifestations of the tree shrew at the ligation part and the experimental detection results prove that the method for establishing the periodontitis model of the tree shrew through silk thread ligation is feasible and effective, and compared with the existing commonly used periodontitis models of rats, mice and the like, the method has the manifestations of probing bleeding, dental calculus and the like which are more similar to those of human periodontitis in the occurrence and development processes.
The Micro-CT scan results show that the distance between the mandibular first molar enamel cementum boundary and the crest of the alveolar ridge of the tree shrew gradually increases with increasing ligation time, increases to a maximum value by 4 weeks, and then decreases with increasing ligation time. However, the distance between the mandibular first molar enamel cementum and the crest of the alveolar ridge of the tree shrew was increased compared to the control group.
The results of TRAP staining in combination with IHC staining for Cathepsin K protein showed that the number of osteoclasts was the greatest in the first 2 weeks, followed by a sharp decrease, and bone resorption lacunae appeared at 6 weeks, suggesting that the tree shrew was ligated by silk ligation to the first molar of the mandible of 1-8W, and alveolar bone was destroyed by resorption to a varying degree.
The HE staining is combined with the IHC staining results of MPO, TNF-alpha and IL-1 alpha proteins of inflammation to show that compared with a control group, periodontal tissues have inflammatory cell infiltration with different degrees, a large amount of inflammatory cell infiltration is achieved in 1-2 weeks, acute inflammatory cells are mainly used, inflammatory cells infiltrate deeply in 3-4 weeks, and shallower periodontal pockets appear in 6 weeks, so that after the first molar tooth of the lower jaw of a shrew tree is ligated by a silk ligation method, acute inflammation is mainly used in the early stage, and the inflammatory cells continuously infiltrate and aggregate in the direction of tooth roots and around osteoclasts along with the increase of the ligation time.
Drawings
FIG. 1 is a schematic diagram of a method for establishing a tree shrew periodontitis model by animal picture and silk thread ligation, which is used in the present invention;
FIG. 2 is a clinical representation diagram of a method for establishing a tree shrew periodontitis model by silk thread ligation according to the present invention;
FIG. 3 is a micro-CT result diagram of the method for establishing a tree shrew periodontitis model by silk thread ligation provided by the invention;
FIG. 4 is a histological staining result diagram of a method for establishing a tree shrew periodontitis model by silk thread ligation according to the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
As shown in figures 1-3, the method for establishing a tree shrew periodontitis model by silk thread ligation and the verification method thereof comprise the following steps: (1) selecting adult healthy male tree shrews for modeling, wherein the weight of the male tree shrews is 140 g; dividing 40 tree shrews into a normal control group (hereinafter referred to as a control group) and an experimental group by adopting a random control method, and observing for 1-8 weeks, wherein each group has 3-8 tree shrews per time point; performing no treatment on the control group, and performing silk thread ligation on the first molars of the right mandible of all the rest tree shrews by using 2-0 silk threads; the groups were randomly divided into 9 groups of 3-8, 40 in total. Normal control group (hereinafter referred to as control group), 1 week group, 2 week group, 3 week group, 4 week group, 5 week group, 6 week group, 7 week group, and 8 week group, respectively; all the tree shrews are Diancia (Latin's name Tupaiabelangeri chinensis), experimental animals are provided by a tree shrew domestication demonstration application platform of department of animal academia of Kunming medical university, and the tree shrews pass the examination by an animal experiment ethical examination committee of Kunming medical university and agree to carry out experiments (number: KMMU 2020029).
3% sodium pentobarbital is used for anesthetizing the tree shrew by intraperitoneal injection according to the proportion of 0.3mL/100g (the volume of the sodium pentobarbital/the body weight); after anesthesia, the tongue is gently pulled to the left side, and the right mandibular cuspid, the first molar and the second molar are exposed. Placing the oral cavity probe between two molars, rapidly pressing down between the first molars and the second molars of the lower jaw on the right side of the tree shrew by using a non-absorbable surgical suture line No. 2-0, then placing the oral cavity probe between the first molars and the adjacent canine, rapidly pressing down the silk thread between the first molars and the adjacent canine around the neck of the tooth, and continuously knotting twice on the buccal side.
(2) At a time point of 1-8 weeks after the silk ligation is completed, 3% sodium pentobarbital is used for anesthetizing the tree shrew through intraperitoneal injection, and then the tree shrew is killed by a cervical vertebra dislocation method; taking out the lower jaw bone on the right side of the tree shrew, removing incisors and partial canine of the tree shrew, and placing the tree shrew in a 10% neutral formaldehyde solution for fixation. Observations were made weekly after ligation of the silk before sampling and 1 week after ligation, the gingival margin of the first molar and surrounding periodontal tissues was found to be red and the mucosal surface was white. The redness and whitening phenomenon deepens after 2 weeks after ligation, and the inflammation becomes worse. 3 and 4 weeks after ligation, the gum is swollen, dark red in color, the gingival sulcus is deepened, and bleeding occurs after probing. After 5 and 6 weeks of ligation, the gum appeared dark red and could reach the superficial periodontal pocket. After 7 and 8 weeks of ligation, the gum is dark red, the depth of the periodontal pocket is increased, the teeth are slightly loosened, and tartar is visible on the root surface of part of the tree shrews;
(3) Micro-CT scanning is used for detecting bone tissue absorption degree, HE staining is used for detecting inflammatory cell infiltration degree of periodontal tissue, TRAP staining is used for detecting osteoclast infiltration degree quantity, and IHC staining is used for detecting inflammation related proteins TNF-alpha, IL-1 alpha, MPO and osteoclast related protein Cathepsin K expression conditions.
Carrying out Micro-CT scanning on the fixed tissue, wherein the scanning parameters are voltage 90KV and scanning current 60 mA; after scanning, washing the tissues overnight with running water, and soaking the tissues in freshly prepared EDTA decalcification solution; the liquid is changed every 2-3 days until the needle tip is stabbed and the needle is taken out without puncture feeling. Washing the sample completely decalcified overnight with running water, placing into an automatic come card dehydrating instrument, dehydrating, soaking wax, embedding into wax block, and storing in a cool place with ventilation at room temperature; after exposing the complete tooth root of the first molar in the coronal plane direction, remaining slices according to the thickness of 3 mu m/piece, and sticking 2-3 slices on each slide; baking in a 65 ℃ oven for 12h, taking out, recovering to room temperature, storing for later use, and slicing for histopathological staining; the pathological staining is HE staining or TRAP staining or IHC staining.
The HE staining method comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3 staining with hematoxylin for 10min, washing with water for 5s, differentiating with hydrochloric acid for 5s, washing with water for 5s, and bluing with PBS for 15 s; step 470% ethanol for 2min, and eosin dip dyeing for 30 s; step5 dehydrating with low-concentration to high-concentration gradient alcohol; and Step6, air drying and sealing. The TRAP staining comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3, preparing TRAP staining solution according to the kit instructions, and then putting the slices into the staining solution to incubate for 60 min; washing with warm water at step437 deg.C, counterstaining with hematoxylin, differentiating, and bluing; and Step5, air drying and sealing. The IHC staining comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3 selects proper antigen repairing method according to the antibody instruction to expose antigen, and washing with PBS solution for 3 times and 3-5 min/time; sealing with step 43% H2O2 for 10min, washing with PBS solution for 3 times (3-5 min/time); step5, adding primary antibody dropwise, and standing overnight at 4 ℃; the antibody was used at the following concentrations: MPO (Ready-to-use, RAB-0379, Mexican), TNF- α (1:100, GTX110520, GeneTex), IL-1 α (1:1000, ab124962, abcam), Cathepsin K (1:200, sc-48353, Santa); step6 PBS 3 times, 3-5 in/times; adding secondary antibody dropwise, and incubating for 15 min. Washing with PBS for 3 times, 3-5 min/time; step7 DAB color development, washing, hematoxylin counterstaining, differentiation and bluing; step8 air drying, and sealing.
Example 2
Example 2 differs from example 1 in that:
the invention relates to a method for establishing a tree shrew periodontitis model by silk thread ligation and a verification method thereof, wherein the method comprises the following steps: in the step (1), adult healthy male tree shrews are selected for modeling, and the weight of the male tree shrews is 130 g. Each group had 4 per time point.
Example 3
Example 3 differs from example 1 in that:
the invention relates to a method for establishing a tree shrew periodontitis model by silk thread ligation and a verification method thereof, wherein the method comprises the following steps: in the step (1), adult healthy male tree shrews are selected for modeling, and the weight of the male tree shrews is 200 g. Each group had 3 per time point.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. The method for establishing the tree shrew periodontitis model by silk thread ligation and the verification thereof are characterized by comprising the following steps: (1) selecting adult healthy male tree shrews for modeling, wherein the weight of the male tree shrews is 130-200 g; dividing the tree shrews into a normal control group and an experimental group by adopting a random control method, observing for 1-8 weeks, and allowing 3-8 tree shrews to be in each group at each time point; performing no treatment on the control group, and performing silk thread ligation on the first molars of the right mandible of all the rest tree shrews by using 2-0 silk threads;
(2) observing and recording clinical manifestations of the tree shrews at the ligation positions 1-8 weeks after ligation, killing the tree shrews, and collecting mandibles of the tree shrews;
(3) Micro-CT scanning is used for detecting bone tissue absorption degree, HE staining is used for detecting inflammatory cell infiltration degree of periodontal tissue, TRAP staining is used for detecting osteoclast infiltration degree quantity, and IHC staining is used for detecting inflammation related proteins TNF-alpha, IL-1 alpha, MPO and osteoclast related protein Cathepsin K expression conditions.
2. The method for establishing a tree shrew periodontitis model according to claim 1, wherein: in the step (1), the raw materials are randomly divided into 9 groups, and each group comprises 3-8, and 40 in total; the normal control groups are respectively referred to as a control group, a 1-week group, a 2-week group, a 3-week group, a 4-week group, a 5-week group, a 6-week group, a 7-week group and an 8-week group; all the tree shrews are Diancia (Latin's name Tupaiabelangeri chinensis), experimental animals are provided by a tree shrew domestication demonstration application platform of department of animal academia of Kunming medical university, and the tree shrews pass the examination by an animal experiment ethical examination committee of Kunming medical university and agree to carry out experiments (number: KMMU 2020029).
3. The method for establishing a tree shrew periodontitis model according to claim 1, wherein: in the step (1), 3% sodium pentobarbital is used for carrying out anesthesia on the tree shrews by intraperitoneal injection according to the proportion of 0.3mL/100g (the volume of the sodium pentobarbital/the body weight); after anesthesia, slightly poking the tongue to the left side to expose the right mandibular cuspid, the first molar and the second molar; placing the oral cavity probe between two molars, rapidly pressing down between the first molars and the second molars of the lower jaw on the right side of the tree shrew by using a non-absorbable surgical suture line No. 2-0, then placing the oral cavity probe between the first molars and the adjacent canine, rapidly pressing down the silk thread between the first molars and the adjacent canine around the neck of the tooth, and continuously knotting twice on the buccal side.
4. The method for establishing a tree shrew periodontitis model according to claim 1, wherein: in the step (2), the tree shrew is anesthetized by intraperitoneal injection by 3% pentobarbital sodium at a time point of 1-8 weeks after the silk ligation is finished, and then the tree shrew is killed by a cervical dislocation method; taking out the lower jaw bone on the right side of the tree shrew, removing incisors and partial canine of the tree shrew, and placing the tree shrew in a 10% neutral formaldehyde solution for fixation.
5. The method for establishing the tree shrew periodontitis model according to claim 4, wherein: in step (2), observations were made weekly before sampling after ligation of the silk thread and 1 week after ligation, the gingival margin of the first molar and surrounding periodontal tissues was found to be red and the mucosal surface was white. The redness and whitening phenomenon deepens after 2 weeks after ligation, and the inflammation becomes worse. 3 and 4 weeks after ligation, the gum is swollen, dark red in color, the gingival sulcus is deepened, and bleeding is caused after probing; after 5 to 6 weeks of ligation, the gum is dark red and can reach the superficial periodontal pocket; after 7 and 8 weeks of ligation, the gum is dark red, the depth of the periodontal pocket is increased, the teeth are slightly loosened, and tartar is visible on the root surface of part of the tree shrews;
in the step (3), the fixed tissue is firstly subjected to Micro-CT scanning, wherein the scanning parameters are voltage of 90KV and scanning current of 60 mA; after scanning, washing the tissues overnight with running water, and soaking the tissues in freshly prepared EDTA decalcification solution; the liquid is changed every 2-3 days until the needle tip is stabbed and the needle is taken out without puncture feeling.
6. The method for establishing a tree shrew periodontitis model according to claim 1, wherein: in the step (3), washing the sample completely decalcified overnight with running water, putting the sample into an automatic come card dehydration instrument, dehydrating, soaking wax in a transparent way, embedding the wax into a wax block, and storing the wax block in a ventilating and shady place at room temperature; after exposing the complete tooth root of the first molar in the coronal plane direction, remaining slices according to the thickness of 3 mu m/piece, and sticking 2-3 slices on each slide; baking in a 65 ℃ oven for 12h, taking out, recovering to room temperature, storing for later use, and slicing for histopathological staining; the pathological staining is one or a combination of HE staining, TRAP staining or IHC staining.
7. The method for establishing the tree shrew periodontitis model according to claim 6, wherein: in step (3), the HE staining comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3 staining with hematoxylin for 10min, washing with water for 5s, differentiating with hydrochloric acid for 5s, washing with water for 5s, and bluing with PBS for 15 s; step 470% ethanol for 2min, and eosin dip dyeing for 30 s; step5 dehydrating with low-concentration to high-concentration gradient alcohol; and Step6, air drying and sealing.
8. The method for establishing a tree shrew periodontitis model according to claim 6 or 7, wherein: in step (3), the TRAP staining comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3, preparing TRAP staining solution according to the kit instructions, and then putting the slices into the staining solution to incubate for 60 min; washing with warm water at step437 deg.C, counterstaining with hematoxylin, differentiating, and bluing; and Step5, air drying and sealing.
9. The method for establishing a tree shrew periodontitis model according to claim 6 or 7, wherein: in step (3), the IHC staining comprises the following steps: step1 taking out the stored slices, baking in an oven at 65 deg.C for 40min to melt the wax; step2 paraffin removal from the sections using xylene, rehydration with high to low concentration gradient alcohol; step3 selects proper antigen repairing method according to the antibody instruction to expose antigen, and washing with PBS solution for 3 times and 3-5 min/time; step 43% H2O2After sealing for 10min, washing with PBS solution for 3 times, 3-5 min/time; step5, adding primary antibody dropwise, and standing overnight at 4 ℃; the antibody was used at the following concentrations: MPO, TNF-alpha, IL-1 alpha, Cathepsin K; step6 PBS washing 3 times, 3-5 min/time; adding secondary antibody dropwise, and incubating for 15 min. Washing with PBS for 3 times, 3-5 min/time; step7 DAB color development, washing, hematoxylin counterstaining, differentiation and bluing; step8 air drying, and sealing.
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| CN114699202A (en) * | 2022-04-07 | 2022-07-05 | 中山大学附属口腔医院 | Composite periodontitis and tooth movement mouse modeling method |
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| CN113841658A (en) * | 2021-10-13 | 2021-12-28 | 昆明医科大学 | Rapid building method of periodontitis model and treatment medicine |
| CN114699202A (en) * | 2022-04-07 | 2022-07-05 | 中山大学附属口腔医院 | Composite periodontitis and tooth movement mouse modeling method |
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