A method for constructing a rat model of periodontitis and its application

Abstract

The present invention relates to a method for constructing a rat model of periodontitis and its application. The method comprises the following steps: first, rats are infected with Toxoplasma gondii, and then the rats infected with Toxoplasma gondii are co-infected with Porphyromonas gingivalis and Treponema denticola to establish a rat model of periodontitis. The present invention realizes rapid and stable acquisition of a periodontitis animal model through the synergistic effect between Porphyromonas gingivalis, Treponema denticola and Toxoplasma gondii, and the constructed periodontitis model is highly consistent with the symptoms and signs of human periodontitis, and can reflect the systemicity and severity of periodontitis. The method has the advantages of fast modeling, high success rate and good repeatability, and is convenient for studying the infection, development mechanism and prevention and treatment process of periodontitis.

Description

Technical Field

[0001] The present invention relates to the field of periodontology, and in particular to a method for constructing an animal model for studying the pathogenesis of periodontitis and its associated infectious diseases, specifically a method for constructing a periodontitis rat model and its application in the study of periodontitis-related diseases. Background Art

[0002] Chronic periodontitis (CP) is a chronic infectious disease and the leading cause of tooth loss in adults. It has a high incidence rate worldwide. According to the data from the Fourth National Oral Health Epidemiological Survey, the detection rate of dental calculus in adults aged 35-44 in my country is 96.7%, and the detection rate of gingival bleeding is 87.4%, which is 10.1% higher than 77.3% ten years ago. The prevalence and severity of CP increase with age.

[0003] Porphyromonas gingivalis ( Porphyromonas gingivalis, P. gingivalis ) is the main pathogen of CP and has the ability to activate host inflammation and regulate immune response. Treponema denticola, T. denticola ) as an important bridge bacteria, not only can it be combined with Streptococcus ( Streptococcus ) and Actinomycetes ( Actinomycetes ) and other early colonizing bacteria are colonized on the tooth surface and can also be combined with subsequent P. gingivalis , Tannerella forsythiae Tannerella forsythia, T. forsythia )、Fusobacterium nucleatum( Fusobacterium nucleatum, F. nucleatum ) to copolymerize, promoting the formation of plaque biofilm, and it also has many virulence factors that directly participate in the destruction of periodontal tissues. P. gingivalis It may lead to the occurrence and development of CP. Gingival bleeding is a typical clinical symptom of CP, which provides convenient conditions for oral infection pathogens. Toxoplasma gondii, T. gondii ) is an important food-borne parasite. T. gondii After infecting the body, it can flow with the blood to reach all parts of the body, damaging the brain, heart, etc., causing a decrease in human immunity.

[0004] There are studies in the prior art involving the construction of periodontitis models, such as the paper "Experimental Study on the Immunomodulatory Effect of Th1 / Th2 Cell Response on Periodontitis in Mice" (CNKI: Xie Min. Experimental Study on the Immunomodulatory Effect of Th1 / Th2 Cell Response on Periodontitis in Mice [D]. Jinan University, 2008.), which discloses the establishment of a mouse periodontitis animal model by silk thread ligation plus local bacterial coating, and further infection with ultraviolet-attenuated T. gondii to explore the relationship between the body's immune response and periodontitis. However, this method uses bacteria from periodontitis patients for infection, and the proportion and concentration of bacteria in the dental calculus of different periodontitis patients are different. Therefore, the infection using bacteria from patients cannot be standardized to meet the high success rate and high stability of modeling. In addition, although many periodontitis pathogens are known in the prior art, the properties of different bacteria are quite different, and they will also affect each other when acting on the same subject, so the effect of combined infection is poorly predictable.

[0005] Rats are widely used as rodent model animals in the study of disease mechanisms, drug metabolism and pharmacokinetics. They have great advantages over mice, such as large size and easy operation, large blood volume and strong tolerance, and large sample size. Given that the effective prevention and treatment of periodontitis has become a hot topic of concern for scholars at home and abroad, the efficient and rapid construction of a rat model of periodontitis is of great significance for the study and prevention of periodontitis. This application unexpectedly discovered through the study of the pathogenesis of periodontitis P. gingivalis, T. denticola and T. gondii Co-infected rats can quickly and stably obtain a periodontitis rat model, which is easy to operate and has a high success rate. Summary of the invention

[0006] The present invention aims to provide a method for modeling periodontitis in rats, aiming to quickly and successfully obtain an animal model of periodontitis for the convenience of research and application.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] 1. T. gondii Establishment of infection model: Each rat was given 100 Toxoplasma gondii cysts by oral gavage to construct T. gondii Infection model.

[0009] 2. Establishment of periodontitis model: 3-0 silk suture was tied through the maxillary first molar and applied intraorally P. gingivalis , T. denticola To simulate the occurrence of periodontitis, the specific steps include rat anesthesia, tooth positioning, silk thread ligation and inoculation of periodontal pathogens.

[0010] 3. Periodontitis+ T. gondii Establishment of the rat composite model of infection: Construction T. gondii After the infection model was established, the infected T. gondii The operation was performed on rats to finally establish a periodontitis rat model.

[0011] 4. Experimental design: The experimental animals were randomly divided into several groups.

[0012] (1) Blank control group: no silk ligature and no periodontal pathogens and T. gondii Infect.

[0013] (2) Periodontitis group: silk thread ligation of maxillary first molars + infection with periodontal pathogens.

[0014] (3) Toxoplasma group: The maxillary first molars were ligated with silk thread after Toxoplasma infection.

[0015] (4) Periodontitis + Toxoplasma gondii group: T. gondii After infection, silk ligation + periodontal pathogen infection was performed on the maxillary first molar.

[0016] Compared with the prior art, the beneficial effects of the present invention are specifically embodied in:

[0017] 1) The present invention is achieved by using P. gingivalis and T. denticola and T. gondii The rats were co-infected with the virus and the periodontitis rat model was successfully obtained in only 7 days. The established periodontitis rat model had edema of the gingival papillae, the gingival margin and the tooth surface could not fit together, the texture was soft, and there were bleeding and periodontal pockets. As the degree of the disease deepened, it would lead to loose teeth and even root exposure, which were consistent with the symptoms, signs and pathological processes of human periodontitis, and could reflect the systemicity and severity of periodontitis. In the prior art, the modeling time of a periodontitis model of similar degree usually takes about one month. It can be seen that the construction method of the present invention greatly reduces the modeling time.

[0018] 2) The present invention not only successfully obtained a periodontitis rat model in a short period of time by screening different combinations of common periodontitis pathogens, but also this method has the advantages of high success rate, good controllability, and good stability, and can be used to study the development mechanism and prevention and treatment process of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The condition of rats on the 5th day after intraperitoneal inoculation of Toxoplasma tachyzoites (a on the upper right: Toxoplasma conservation rats; b on the lower left: control group rats).

[0020] Figure 2 Fix the rat on a rat board.

[0021] Figure 3 Periodontal silk ligation.

[0022] Figure 4 Intraoral inoculation of periodontal pathogens.

[0023] Figure 5 Rats were orally gavaged with Toxoplasma gondii cysts.

[0024] Figure 6 Periodontal conditions of rats before ligation.

[0025] Figure 7 Periodontal conditions of rats in the blank group.

[0026] Figure 8 Periodontal status of the periodontitis group after ligation.

[0027] Fig. 9 Periodontal status of the periodontitis + Toxoplasma group after ligation.

[0028] Fig.10 Toxoplasma cysts were observed in the brain tissue homogenate of SD rats in the Toxoplasma infection group.

[0029] Fig.11 Pathological changes of periodontal tissues in rats of the blank group (A), periodontitis + Toxoplasma group (B), periodontitis group (C), and Toxoplasma group (D).

[0030] Fig.12 Micro-CT scanning results of periodontal tissues of rats in the blank group (A), periodontitis + Toxoplasma group (B), periodontitis group (C), and Toxoplasma group (D). DETAILED DESCRIPTION

[0031] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0032] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources. Unless otherwise specified, the substances described in the present invention are calculated by mass percentage and mass fraction.

[0033] Porphyromonas gingivalis: number: ATCC 33277, provided by the laboratory of School of Stomatology, Guangzhou Medical University.

[0034] Treponema denticola: number: DSM 14222, provided by the laboratory of the School of Stomatology, Guangzhou Medical University.

[0035] Toxoplasma gondii: No.: Toxoplasma gondii ( T. gondii)Me49, provided by the Parasitology Laboratory, College of Veterinary Medicine, South China Agricultural University.

[0036] SD rats: purchased from the Animal Experiment Center of Southern Medical University, production experimental animal license number: SCXK (Guangdong) 2021-0041.

[0037] The experiment was conducted in the animal laboratory of the College of Veterinary Medicine of South China Agricultural University, and the experimental animal use license number is SYXK (Guangdong) 2024-0136.

[0038] The present invention provides a P. gingivalis and T. denticola Collaboration T. gondii The method for establishing the infection rat model will be described in detail below with regard to the specific implementation steps of the method.

[0039] 1. Preparation of Experimental Materials

[0040] 1.1 Experimental animals: 4-week-old SPF male SD rats weighing 200±20 g were selected. The animals were housed in a specific pathogen-free (SPF) central clean animal room with an ambient temperature of 22±2°C and a humidity of 50%±10%.

[0041] 1.2 Bacteria: Porphyromonas gingivalis ATCC 33277 standard strain and Treponema denticola DSM 14222 standard strain.

[0042] 1.3 Toxoplasma gondii: Toxoplasma gondii T. gondii Me49 strains.

[0043] 1.4 Main materials and reagents: bovine heart brain infusion (BHI) culture medium, hemin chloride, paraformaldehyde, vitamin K1.

[0044] 1.5 Main experimental instruments: including anaerobic incubator, automatic dehydrator, paraffin embedding machine, paraffin tissue slicer, etc.

[0045] 1.6 Preparation of main reagents: Prepare BHI solid culture medium and liquid culture medium according to the reagent instructions.

[0046] 2. Experimental Methods

[0047] 2.1 Periodontitis group: rats were ligated and smeared on the maxillary first molars. P. gingivalis and T. denticola to induce periodontitis.

[0048] Toxoplasma group: Rats were infected by oral administration of Toxoplasma cysts.

[0049] Periodontitis + Toxoplasma group: Combining the above two methods, inducing periodontitis and T. gondii Infect.

[0050] Blank group: physiological saline was used for each infection step and the mice were raised in the same environment and under the same conditions.

[0051] Comparison group 1: Use F. nucleatum replace T. denticola The other conditions were the same as those of the periodontitis + Toxoplasma gondii group.

[0052] Comparison group 2: Use T. forsythia replace T. denticola The other conditions were the same as those of the periodontitis + Toxoplasma gondii group.

[0053] Comparative group 3-4: Ligated and smeared on the maxillary first molar of rats P. gingivalis and T. denticola to induce periodontitis, and the difference between the periodontitis + Toxoplasma group was that the P. gingivalis and T. denticola Usage ratio.

[0054] The Fusobacterium nucleatum ( F. nucleatum ) ATCC 2558, Tannerella forsythiae ( T. forsythia ) ATCC700198 was provided by the Oral Microbiology Laboratory of Guangzhou Medical University.

[0055] 2.2 Sample collection and testing methods: including periodontal symptom examination, imaging examination, pathological examination, etc.

[0056] Example 1 Periodontitis + Toxoplasma gondii group

[0057] Establishment of periodontitis rat model:

[0058] (1) Select clean-grade SD rats and feed them for 7 days before infection;

[0059] (2) Chronic infection T. gondii The Kunming rats were killed, the whole brains were removed, ground and homogenized, and diluted with normal saline to 100 brain cysts / mL to obtain the infection fluid. After the rats were adaptively fed for 7 days, each rat was gavaged with 1 mL of the infection fluid.

[0060] (3) The rats treated in step (3) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0061] (4) After anesthetization, use 3-0 silk thread to ligate the cervical part of the maxillary first molars on both sides, and then use 1*10 9 CFU / mL P. gingivalis Bacterial liquid and 2*10 9 CFU / mL T. denticolaThe bacterial solution was inoculated on the ligature and in the gingival sulcus of rats at a dose of 0.1 mL / tooth; the inoculation frequency was once a day for 7 consecutive days;

[0062] (5) Whether the modeling is successful is determined by measuring the periodontal symptoms of the rats treated in step (4) and measuring the alveolar bone absorption. The alveolar bone absorption rate is ≥20% and the periodontal pocket probing depth is ≥1 mm. If both meet the requirements, the modeling is successful.

[0063] (6) Perform oral, imaging and pathological examinations on the rats treated in step (4).

[0064] Example 2 Periodontitis group:

[0065] Establishment of periodontitis rat model:

[0066] (1) Select clean-grade SD rats and feed them for 7 days before infection;

[0067] (2) Each rat was gavaged with 1 mL of normal saline;

[0068] (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0069] (4) After anesthetization, use 3-0 silk thread to ligate the cervical part of the maxillary first molars on both sides, and then use 1*10 9 CFU / mL P. gingivalis Bacterial liquid and 2*10 9 CFU / mL T. denticola The bacterial solution was inoculated on the ligature and in the gingival sulcus of rats at a dose of 0.1 mL / tooth; the inoculation frequency was once a day for 7 consecutive days;

[0070] (5) Whether the modeling is successful is determined by measuring the periodontal symptoms of the rats treated in step (4) and measuring the alveolar bone absorption. The alveolar bone absorption rate is ≥20% and the periodontal pocket probing depth is ≥1 mm. If both meet the requirements, the modeling is successful.

[0071] (6) Perform intraoral, imaging, and pathological examinations on the rats treated in step (4).

[0072] Example 3: Toxoplasma gondii group:

[0073] (1) Select clean-grade SD rats and feed them for 7 days before infection;

[0074] (2) Chronic infection T. gondiiThe Kunming rats were killed, the whole brains were removed, ground and homogenized, and diluted with normal saline to 100 brain cysts / mL to obtain the infection fluid. After the rats were adaptively fed for 7 days, each rat was gavaged with 1 mL of the infection fluid.

[0075] (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0076] (4) After anesthetization, the rats were ligated at the neck of the maxillary first molars using 3-0 silk thread, and then normal saline was inoculated on the ligature and into the gingival sulcus of the rats at a dose of 0.1 mL / tooth. The inoculation frequency was once a day for 7 consecutive days.

[0077] (5) Whether the modeling is successful is determined by measuring the periodontal symptoms of the rats treated in step (4) and measuring the alveolar bone absorption. The alveolar bone absorption rate is ≥20% and the periodontal pocket probing depth is ≥1 mm. If both meet the requirements, the modeling is successful.

[0078] (6) Perform oral, imaging and pathological examinations on the rats treated in step (4).

[0079] Example 4: Blank group:

[0080] (1) Select clean-grade SD rats and feed them for 7 days before infection;

[0081] (2) Each rat was gavaged with 1 mL of normal saline;

[0082] (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital;

[0083] (4) After anesthetization, the rats were ligated at the neck of the maxillary first molars using 3-0 silk thread, and then normal saline was inoculated on the ligature and into the gingival sulcus of the rats at a dose of 0.1 mL / tooth. The inoculation frequency was once a day for 7 consecutive days.

[0084] (5) Whether the modeling is successful is determined by measuring the periodontal symptoms of the rats treated in step (4) and measuring the alveolar bone absorption. The alveolar bone absorption rate is ≥20% and the periodontal pocket probing depth is ≥1 mm. If both meet the requirements, the modeling is successful.

[0085] (6) Perform oral, imaging and pathological examinations on the rats treated in step (4).

[0086] The preparation methods of comparison group 1 and comparison group 2 are the same as those of Example 1, and the only difference from Example 1 is that the aforementioned comparison group 1 and comparison group 2 are respectively limited in bacterial species.

[0087] The preparation method of comparative group 3 is different from that of example 1 in that in step (4) P. gingivalis The concentration of bacterial solution is 1.5*10 9 CFU / mL, T. denticola The concentration of bacterial solution is 1.5*10 9 CFU / mL, and the rest of the steps and parameters were exactly the same.

[0088] The preparation method of comparative group 4 is different from that of Example 1 in that in step (4) P. gingivalis The concentration of bacterial solution is 2*10 9 CFU / mL, T. denticola The concentration of bacterial solution is 1*10 9 CFU / mL, and the rest of the steps and parameters were exactly the same.

[0089] There were eight experimental groups, with 20 rats in each group.

[0090] 3. Sample collection and intraoral examination, imaging and pathology testing:

[0091] Intraoral examination: Check the changes in the color and texture of the rats' gums, the probing depth, and the looseness of the teeth. Observe the sample rats 1 week after modeling to check the color changes of the free gingiva and gingival papilla, whether they are bright red or dark red, whether they affect the attached gingiva; whether the gingival tissue is swollen and the swelling; the changes in the bright color of the gingival surface; whether the gingival margin is thickened and whether it fits the tooth surface; whether there is pus or erosion in the ligature; the texture of the gums, whether they lack elasticity or are soft. Whether the gums bleed during probing, whether the periodontal pocket can be probed and its depth; whether the experimental teeth are loose and the looseness.

[0092] The verification criteria for whether the model was successfully tested in the intraoral examination were as follows: the free gingiva and gingival papilla were bright red or dark red and affected the attached gingiva; the gingival tissue was swollen, the gingival margin was thickened and not in close contact with the tooth surface; there was pus or erosion at the ligature; the gingiva was soft or lacked elasticity; the gingiva bled during probing; there were certain periodontal pockets; and the experimental teeth were loose.

[0093] Preparation of specimens required for imaging and pathological examination: Maxillary bone samples and periodontal tissue structure specimens were collected from rats that met the criteria for imaging and pathological examination after treatment in step (5). In order to preserve the structure of the periodontal tissue of the first molar to the greatest extent, the cardiac perfusion method was used to fix the tissue. The cardiac perfusion method is as follows: 1.5% sodium pentobarbital solution 1 mL was injected intraperitoneally to give general anesthesia, and the rat's breathing and heartbeat were observed at any time. Rubber bands were wrapped around the incisors of the limbs and head, and the rat was fixed on a mouse board and placed in a supine position, and cardiac perfusion fixation was started. After the rat was anesthetized, the chest wall was cut open along the axillary line on the surface of the body below the ribs, and a 5 cm incision was made laterally. The liver and diaphragm were carefully separated with scissors. After cutting, the chest wall was turned up to expose the heart. The ventricle was gently clamped and fixed with hemostatic forceps, and then the tip of the syringe needle (the tip of the needle should be blunted) was gently inserted into the ascending aorta at the apex of the heart for about 1 mm, and the right atrial appendage was cut open with scissors. 50 mL of normal saline was injected. After clear liquid flowed out from the right auricle and the rat liver turned white, 4% paraformaldehyde fixative equivalent to the rat body weight was continued to be perfused. The maxilla of the rat was dissected and cut off, and the periodontal tissue was collected and preserved in 4% paraformaldehyde.

[0094] Imaging examination: Micro-CT scanning and three-dimensional reconstruction were performed on the maxillary specimens to measure the alveolar bone resorption.

[0095] Pathological examination: HE staining was used to observe the pathological morphological changes of periodontal tissues. The HE staining method was specifically referred to the instruction manual of the HE staining kit. The HE staining kit was purchased from Solebow Technology Co., Ltd., item number: G1120.

[0096] Modeling success rate = (number of mice with successful modeling / total number of mice in the group) × 100%

[0097] Average alveolar bone resorption rate = (sum of alveolar bone resorption rates of mice in this group / total number of mice in this group) × 100%

[0098] Average periodontal pocket probing depth = sum of periodontal pocket probing depths of the group of mice / total number of mice in the group

[0099] All the above results are rounded to two significant figures.

[0100] 4. Experimental Results

[0101] Table 1 Detection of model rat samples

[0102] Experimental Group Average alveolar bone resorption rate / % Significance (compared with the blank group / compared with the periodontitis + Toxoplasma group) Average periodontal pocket probing depth / mm Significance (compared with the blank group / compared with the periodontitis + Toxoplasma group) Modeling success rate / % Periodontitis group 12.4 * / # 0.72 * / # 5 Toxoplasma group 8.5 * / # 0.52 * / # 0 Periodontitis + Toxoplasma group 31.2 *** / 1.65 *** / 95 Blank Group 3.2 / # 0.22 / # 0 Comparison group 1 21.5 * / # 1.12 * / # 50 Comparison group 2 20.4 * / # 1.04 * / # 40 Comparison group 3 23.5 * / # 1.26 * / # 70 Comparison group 4 24.2 * / # 1.32 * / # 75

[0103] Note: "*" indicates that the experimental group is significantly different from the blank group, "#" indicates that the experimental group is significantly different from the periodontitis + Toxoplasma group; "*" indicates p <0.05, “**” representsp <0.01, “***” represents p <0.001; “#” represents p <0.05, “ns” means no significant difference compared with the blank group or the periodontitis + Toxoplasma group.

[0104] The above experimental results show that the P. gingivalis and T. denticola and T. gondii The establishment method of the periodontitis rat model of co-infected rats can achieve a modeling success rate of more than 95% in 7 days, with a short modeling time and a high success rate. In addition, intraoral examinations, imaging and pathological examinations were performed on the 19 rats in the group that successfully established the model. The results of the intraoral examination showed that the 19 rats in the periodontitis + Toxoplasma group that successfully established the model all showed dark red gingival papillae, swollen gingival tissue, erosion of the gingiva at the ligature, soft gingival texture, and bleeding gums when probing.

[0105] HE staining showed that the blank group ( Fig.11 The junctional epithelium of rats in the periodontitis group (A) had no attachment loss and was located at the cementum-enamel junction, with intact tooth structure. A small part of the epithelium of rats in the periodontitis group proliferated into the connective tissue, and there was a small amount of absorption of the alveolar ridge top and the alveolar bone proper, and there were fewer osteoclasts in the alveolar bone. Fig.11 The periodontal tissue of rats in the C group showed loss of junctional epithelial attachment, alveolar bone destruction, and osteoclasts. Fig.11 D) in the periodontitis + toxoplasma group ( Fig.11 The periodontal tissue of the periodontitis group (B) showed a significant loss of junctional epithelial attachment, significant destruction of alveolar bone, and a large number of osteoclasts. Fig.11 C), Toxoplasma gondii group rats ( Fig.11 D) in the figure was significantly increased compared to the previous one.

[0106] Micro-CT results showed that compared with the blank group ( Fig.12 A in the periodontitis group ( Fig.12 C in), Toxoplasma group ( Fig.12 D), periodontitis + Toxoplasma gondii group ( Fig.12 The height, integrity and fullness of the alveolar bone were significantly reduced in B).

[0107] Specifically, by comparing the results of the rat model of periodontitis + Toxoplasma with those of the periodontitis group and Toxoplasma group, we can see that:

[0108] (1) Using periodontitis pathogens alone or T. gondiiThe infection made it almost impossible to obtain a periodontitis rat model 7 days after infection. Only 1 of the 20 rats in the periodontitis group had obvious symptoms of periodontitis, as evidenced by periodontal redness, slight gingival bleeding with a probe, and obvious swelling of the gingival tissue. No obvious symptoms were observed in the remaining 19 rats in this group or in the Toxoplasma gondii group.

[0109] (2) Toxoplasma infection can aggravate periodontitis, which is manifested by worsening clinical symptoms and increased alveolar bone resorption on imaging. This aggravating effect is significant in rat models ( P <0.001).

[0110] By comparing the results of the rat model of periodontitis + Toxoplasma gondii group with those of control group 1-2, it can be seen that the infection effects of different periodontitis pathogens on rats vary greatly. P. gingivalis and T. denticola The combined infection effect was significantly better than P. gingivalis and F. nucleatum combination of P. gingivalis and T. forsythia The combination of P. gingivalis and T. denticola Infection is more suitable for T. gondii The infected rats synergistically acted to quickly obtain a periodontitis rat model, and had a significant impact on the success rate of modeling.

[0111] By comparing the results of the rat model of periodontitis + Toxoplasma gondii group with those of the control group 3-4, it can be seen that different ratios of the same bacteria will also affect the modeling effect to a certain extent. P. gingivalis Bacterial liquid and T. denticola The ratio of bacterial liquid is 1:2, which has the best infection effect and can further improve the success rate of modeling.

[0112] Conclusion: This invention successfully established a rat model of periodontitis, and according to the experimental results, T. gondii Infection and P. gingivalis + T. denticola There is a certain synergistic effect between combined infections, and the model is simple to build, short in construction time, highly repeatable, and can be mass-produced. This model can effectively simulate human P. gingivalis + T. denticola Companion T. gondii The pathological process of infection is T. gondii The impact of infection on the course of periodontitis and its mechanisms provides a powerful tool.

[0113] The above is only an embodiment of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for establishing a periodontitis rat model, characterized in that: The following steps are involved: (1) Select clean-grade Sprague-Dawley (SD) rats and feed them for 7 days before infection; (2) Kunming rats chronically infected with Toxoplasma gondii were killed, the whole brain was removed, ground and homogenized, and diluted with physiological saline to a count of 100 brain cysts / mL to obtain the infection fluid. After the rats were adaptively fed for 7 days, 1 mL of the infection fluid was administered orally to each rat; (3) The rats treated in step (2) were anesthetized by intraperitoneal injection of 1 mL of 1.5% sodium pentobarbital; (4) After anesthetization, 3-0 silk thread was used to ligate the cervical part of the maxillary first molars on both sides, and then 1×10 9 CFU / mL Porphyromonas gingivalis liquid and 2×10 9 CFU / mL of Treponema denticola liquid was inoculated on the ligature and in the gingival sulcus of rats at a dose of 0.1 mL / tooth; the inoculation frequency was once a day; Vaccination continued for 7 days; (5) After 7 days, measure and observe the alveolar bone and periodontal pocket of the rats treated in step (4) to determine whether the modeling is successful; The alveolar bone and periodontal pocket conditions in step (5) are as follows: the alveolar bone absorption rate is ≥20%, and the periodontal pocket probing depth is ≥1 mm. Both of these conditions meet the requirement, indicating that the periodontitis rat model is successfully established.

2. The method for establishing a periodontitis rat model according to claim 1, characterized in that: The body weight of the rats was in the range of 200±20 g.

3. The method for establishing a periodontitis rat model according to claim 1, characterized in that: The method further comprises: after the model is successfully established, the rats treated in step (4) are subjected to oral, imaging and pathological examinations.

4. The method for establishing a periodontitis rat model according to claim 3, characterized in that: The steps of the oral, imaging and pathological examinations are as follows: A1: 1 mL of 1.5% sodium pentobarbital solution is injected intraperitoneally to induce general anesthesia; A2: Wrap rubber bands around the limbs and the incisors of the head, fix them on the mouse board and place them in the supine position; A3: After the rat is anesthetized, the chest wall is cut open along the anterior axillary line on the surface of the body below the ribs, and a 5-6 cm incision is made laterally to separate the liver and diaphragm. After cutting, the heart is exposed; A4: Fix the ventricle and then rotate the tip of the syringe needle at the apex of the heart to 1 mm into the ascending aorta; A5: Cut the right atrial appendage with scissors, inject 50 mL of normal saline, and continue to perfuse 4% paraformaldehyde fixative according to the rat's body weight after the liquid flows out of the right atrial appendage and the rat liver turns white. After the filling is completed, dissect and cut off the rat's maxilla, collect the periodontal tissue, and store it in 4% paraformaldehyde; A6: Micro-CT scanning and three-dimensional reconstruction of maxillary specimens; A7: HE staining was used to observe the pathological morphological changes of periodontal tissues.

5. Use of the rat model obtained by the method according to any one of claims 1 to 4 in the study of the mechanism of periodontitis.

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