Construction method of severe periodontitis mouse model

Abstract

The invention relates to the technical field of animal model construction, in particular to a construction method of a severe periodontitis mouse model. According to the method, ligation and LPS injection are combined for use, the severe periodontitis mouse model is constructed, in the construction process, construction parameters are optimized, and the model construction efficiency is improved, so that a reliable mouse model for severe periodontitis is provided, and the obtained model is more suitable for being used as a mouse model for researching occurrence, development and treatment of severe periodontitis diseases. And the construction method is simple, accurate and rapid.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and in particular to a method for constructing a severe periodontitis mouse model. Background Art

[0002] Periodontitis is one of the most common diseases that threaten the oral health of adults in my country. It is closely related to a variety of systemic diseases and is the main cause of tooth loss in adults in my country. Periodontitis is a chronic local inflammatory disease of periodontal tissues caused by dental plaque biofilm, which can interact with the host oral immune response to aggravate the occurrence and development of inflammation. Severe periodontitis not only leads to tooth loss, but also has a negative impact on other systemic diseases. How to establish a simple, rapid and effective ideal animal model is an important prerequisite and a huge challenge for studying the occurrence, development and treatment of periodontitis.

[0003] The animal models of periodontitis that have been studied so far can be divided into the following categories, including: non-human primates such as rhesus monkeys, miniature pigs, and beagles, rodents mainly including rats and mice, and some other animals such as horses and rabbits. Among them, non-human primates and miniature pigs have oral structures and disease processes similar to those of humans, but their overly expensive experimental costs and high requirements for feeding standards make them difficult to use for large-scale research. Although beagles are relatively less expensive, the care and care conditions required are equally strict, and canine periodontal lesions are self-limiting; more importantly, the above experimental animals all have certain ethical issues. Rodents are low in cost, easy to experimentally induce periodontitis, and have a molar structure that is closer to that of humans. Therefore, considering the comprehensive consideration of experimental costs, animal ethics, and the feasibility of simulating human diseases, rodents are considered to be the most commonly used animal models of periodontitis in current research, mainly including rats and mice. However, rodents have a natural ability to resist periodontitis and have oral flora that is different from that of humans. Currently, there is no consensus on the construction methods and evaluation effects of rodent periodontitis models.

[0004] Common methods for modeling periodontitis in rodents (rats, mice, etc.) include silk thread ligation, local bacterial application, bacterial gavage, and injection of bacterial antigens, among which the most common is silk thread ligation. The silk thread ligation method has a short modeling time, low cost requirements, and little mucosal damage, and promotes acute periodontal inflammation and rapid alveolar bone resorption, but it is difficult to simulate chronic periodontitis. Improper modeling can easily cause mechanical damage. During the modeling process, it is necessary to check whether the silk thread has fallen off several times, and the alveolar bone of mice with a short modeling time is easy to recover after the silk thread is removed. The bacterial injection method can promote progressive alveolar bone resorption, which is consistent with chronic periodontitis, and can also be used to study specific periodontal host-bacteria interactions related to infection; compared with the bacterial injection method, bacterial gavage can be performed without anesthetizing mice, which is easier to operate, but may cause increased pressure and esophageal trauma in mice; however, the method of injecting bacteria or bacterial gavage takes a long time and requires many injections, resulting in relatively insufficient alveolar bone loss. Its modeling effectiveness depends on the strain used, and because it involves bacterial experiments, it must be performed in a microbiology laboratory. The most commonly used bacterial antigen for injection is lipopolysaccharide (Pg LPS) from Porphyromonas gingivalis. This method does not require bacterial culture or inoculation. When lipopolysaccharide is applied for at least 2 weeks, it can induce low-grade systemic inflammation; however, this method has poor modeling effect and still requires repeated injections, and it cannot simulate the interaction between bacteria and the host.

[0005] Therefore, in order to better establish a rodent model suitable for studying the treatment of severe periodontitis, this study selected silk thread ligation and Pg LPS injection as two construction methods, and optimized the construction of a severe periodontitis model with typical characteristics such as alveolar bone resorption, bacterial aggregation and inflammatory infiltration, and evaluated and analyzed its effects, aiming to explore a safer, more efficient and simple severe periodontitis mouse model, in order to provide a new optional model for periodontitis research. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing a severe periodontitis mouse model.

[0007] The method for constructing a severe periodontitis mouse model provided by the present invention comprises:

[0008] After silk thread ligature was applied to the molars, (Pg LPS) solution was injected into the gingiva on the palatal side of the molars. After 14 days, a severe periodontitis mouse model was established.

[0009] In the present invention, the molar is the second molar. In a specific embodiment, the second molar is the maxillary left second molar.

[0010] In the experiment, molars in different positions were tried, and it was found that the second molar was easier to operate and easier to achieve a good modeling effect. In addition, different line types were tried, and it was found that the ligature of 5-0 silk thread was more stable and easier to model. In the present invention, the ligature silk thread is 5-0 silk thread.

[0011] Specifically, the ligation step includes: passing a 5-0 non-absorbable silk thread through the interdental space between the maxillary first and second molars, passing the other end of the silk thread through the interdental space between the maxillary second and third molars, and tying a knot on the buccal side of the second molar.

[0012] Preferably, the thread is checked for shedding every 3 days after ligation, and if any shedding is found, the thread is re-ligated using the same method.

[0013] The injectable drug is lipopolysaccharide (Porphyromonas gingivalislipopolysaccharide, Pg LPS) derived from Porphyromonas gingivalis. Different solvents can be used to dissolve Pg LPS to obtain good modeling effects. In order to improve the simplicity of modeling, endotoxin-free water is used as the solvent for injection. In the present invention, the solvent of the Pg LPS solution is water. In the present invention, the concentration of Pg LPS is 8~12μg / μL. As a feasibility case, the concentration of the Pg LPS solution is 8μg / μL, 8.5μg / μL, 9.0μg / μL, 9.5μg / μL, 10μg / μL, 10.5μg / μL, 11μg / μL, 11.5μg / μL or 12μg / μL. In a specific embodiment, the concentration of the Pg LPS solution is 10 μg / μL.

[0014] The present invention optimizes and screens the dosage and frequency of Pg LPS injection. Preferably, the injection dosage is 2 μL / tooth, injected once every 3 days, and injected 4 times in total.

[0015] The injection is carried out using a 10 μL microinjection needle; preferably, the injection needle has a Hamilton ga33 (outer diameter / inner diameter: 0.21×0.11 mm; needle length: 51 mm).

[0016] Preferably, the injection is performed slowly under a stereomicroscope to ensure that the Pg LPS solution does not overflow.

[0017] In a specific embodiment, the first injection is performed immediately after the ligation.

[0018] In the present invention, the ligation step also includes an anesthesia step, and the anesthetic is 1% tribromoethanol.

[0019] In the present invention, in a specific embodiment, the mouse is a 10-week-old C57BL / 6 mouse, and more preferably, a 10-week-old C57BL / 6 male mouse.

[0020] Furthermore, the present invention also provides a mouse model constructed by the method described above.

[0021] The results showed that the dynamic changes of tooth mobility (2.20 ± 0.45) and probing depth (mesial (1.08 ± 0.11 mm), middle (1.04 ± 0.26 mm), distal (1.04 ± 0.26 mm)) of the maxillary left second molar of mice ligated and continuously injected with Pg LPS were more severe than those of mice ligated alone (1.40 ± 0.55). The difference was statistically significant (p < 0.05). Compared with the bone volume fraction (28.97 ± 7.90%), bone density (0.55 ± 0.08 g / cc), distance from cemento-enamel junction to the alveolar bone crest (CEJ-ABC) (0.74 ± 0.12 mm2) and attachment loss area (0.30 ± 0.02 mm2) of the maxillary left second molar of mice with simple ligation, the bone volume fraction (16.44 ± 3.35%) and bone density (0.42 ± 0.04 g / cc) of the maxillary left second molar of mice with ligation and continuous injection of Pg LPS were significantly reduced, and CEJ-ABC (0.88 ± 0.03 mm2) and attachment loss area (0.34 ± 0.01 mm2) were significantly aggravated, and the differences were statistically significant (p < 0.05).

[0022] The present invention combines ligation with Pg LPS injection to construct a severe periodontitis mouse model. During the construction process, the construction parameters are optimized to improve the efficiency of model construction, thereby providing a reliable mouse model for severe periodontitis. The obtained model is more suitable as a mouse model for studying the occurrence, development and treatment of severe periodontitis. In addition, the construction method is simple, accurate and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The clinical manifestations of the left oral cavity of the mouse maxilla are shown, wherein Before shows the image observed before the sutures are removed, and After shows the image observed after the sutures are removed;

[0024] Figure 2The clinical indicators of the left oral cavity of the maxillary maxilla of mice are shown: dynamic changes in tooth mobility and probing depth, where A shows the dynamic changes in tooth mobility of the left second molar of the maxilla, and B shows the probing depth;

[0025] Figure 3 Figure 1 shows Micro-CT images of the left maxillary bone of a mouse, where A shows Micro-CT 2D and 3D images of the left maxillary bone of a mouse, B shows the bone volume fraction, C shows the bone density, and D shows the distance between the mesial CEJ-ABC of the second molar;

[0026] Figure 4 Figure 2 shows images of the left maxillary bone of a mouse under a microscope, wherein A shows a representative methylene blue staining of the maxillary bone of a mouse showing the gap between the CEJ and ABC, and B shows the area between the CEJ and ABC;

[0027] Figure 5 HE staining of gingival tissue at the left second molar of the maxillary maxilla of mice in different groups is shown. The red arrows indicate inflammatory cell infiltration, and the yellow arrows indicate tissue edema and necrosis.

[0028] Figure 6 The contents of IL-1β and TNF-α in mouse serum are shown in Figure 1, wherein A shows the level of IL-1β and B shows the level of TNF-α;

[0029] Figure 7 The figures show the results of routine blood examination of mice, wherein A shows the white blood cell level, B shows the lymphocyte level, C shows the monocyte level, D shows the granulocyte level, E shows the red blood cell level, F shows the hematocrit level, G shows the lymphocyte percentage level, H shows the monocyte percentage, I shows the neutrophil percentage, and J shows the hemoglobin level. DETAILED DESCRIPTION

[0030] The present invention provides a method for constructing a severe periodontitis mouse model. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0031] The test materials used in the present invention are all common commercial products and can be purchased on the market. All animal experiments involved in this study have been approved by the Animal Ethics Committee of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (File No. SH9H-2024-A159-SB). All experimental processes were carried out in accordance with the regulations of the "Regulations on the Administration of Experimental Animals".

[0032] The present invention will be further described below in conjunction with embodiments:

[0033] Example

[0034] 1 Materials and methods

[0035] 1.1 Experimental animals

[0036] Fifteen healthy SPF-grade C57BL / 6 male mice of about 10 weeks old, weighing 23-27 g, were selected. The animals were provided by Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0037] 1.2 Experimental reagents and instruments

[0038] Stereo microscope (Olympus Corporation, Japan). Micro forceps (Dumont, Switzerland), microinjection needle (Hamilton, Switzerland). Microplate reader (Life Technologies / Thermo Fisher Scientific, USA). Tribromoethanol (Life Technologies / Thermo Fisher Scientific, USA), Pg LPS (LifeTechnologies / Thermo Fisher Scientific, USA), isoflurane (Shenzhen Reward Life Science Co., Ltd., China), 4% paraformaldehyde (Shanghai Bio-Technology Co., Ltd., China). X-ray microtomography system (Bruker, USA). IL-1β, TNF-α ELISA kits (Xinbosheng Biotechnology Co., Ltd., China).

[0039] 1.3 Experimental methods

[0040] 1.3.1 Mouse grouping and establishment of periodontitis model

[0041] Fifteen mice were randomly divided into three groups:

[0042] Healthy control group (labeled as group N);

[0043] Simple silk ligature periodontitis modeling group (marked as group L);

[0044] Silk ligation and Pg LPS injection combined with periodontitis modeling group (labeled as LP group), 5 rats in each group, were housed in separate cages.

[0045] Among them, the N group did not receive any treatment. The L group was fixed on the surgical board under 1% tribromoethanol anesthesia, and the maxillary left second molar was ligated with 5-0 silk thread. The silk thread was checked for shedding every 3 days. If it was shedding, it was re-ligated with the same method. The modeling time was 14 days. The LP group was fixed on the surgical board under 1% tribromoethanol anesthesia, and the maxillary left second molar was ligated with 5-0 silk thread. Then, 2 μL of 10 μg / μL Pg LPS solution was injected into the palatal gingiva of the maxillary left second molar of the mouse with a microinjection needle. Then, it was injected once every 3 days for a total of 4 times. The modeling time was 14 days.

[0046] 1.3.2 Clinical observation indicators

[0047] After 14 days of modeling, the ligature was removed, and the following indicators were observed under a stereomicroscope and the results were recorded.

[0048] (1) Tooth mobility: Grade I (loose in the buccal-palatal direction); Grade II (loose in both the buccal-palatal and mesiodistal directions); Grade III (loose in the buccal-palatal, mesiodistal and vertical directions).

[0049] (2) Bleeding on probing: Use a No. 20 gutta-percha tip to gently insert it into the periodontal pocket and then remove it. Observe for 10 seconds to see if there is any bleeding.

[0050] (3) Probing depth: Use a marked No. 20 gutta-percha tip to gently insert it into the mesial, middle, and distal locations on the palatal side of the left maxillary second molar of the mouse. After removal, record the depth of the periodontal pocket detected by the gutta-percha tip.

[0051] (4) Attachment loss area: After the mice were sacrificed and sampled, the maxillary bones were stained with methylene blue, and the palatal side of the maxillary bones of the mice were photographed under a stereomicroscope. The attachment loss area of ​​the second molar was calculated using Image J software.

[0052] (5) Routine blood test: After killing, blood was collected from mice for routine blood test.

[0053] 1.3.3 Enzyme linked immunosorbent assay (ELISA)

[0054] Fourteen days after modeling, blood was collected from mice, and the serum was collected after standing and centrifugation. The serum was diluted at a ratio of 1:10 and then subjected to ELISA tests for interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The absorbance at 450 nm was measured using an enzyme reader.

[0055] 1.3.4 Micro computed tomography (Micro-CT)

[0056] The left maxillary bone with the gingiva stripped off was fixed in 4% paraformaldehyde for 24 h, and then the maxillary bone was scanned using Micro-CT technology to quantitatively analyze the bone volume fraction, bone density and CEJ-ABC.

[0057] 1.3.5 Hematoxylin-eosin (HE) staining

[0058] The left maxillary bone and gingiva were fixed with 4% paraformaldehyde and dehydrated with alcohol. After paraffin embedding, serial sections were taken along the mesiodistal direction of the second molar and the horizontal direction of the gingiva. After sealing, the degree of periodontal bone tissue absorption and inflammation level were observed under a microscope and photographed for preservation.

[0059] 1.4 Statistical analysis

[0060] The data were recorded and analyzed using Prism software, and the quantitative data were expressed as mean ± standard deviation ( ±s) was used to evaluate the statistical significance, and p < 0.05 indicated that the difference was statistically significant.

[0061] 2 Results

[0062] 2.1 Clinical manifestations of periodontal disease in mice

[0063] 14 days after modeling, before killing the mice, the ligature was removed under a stereomicroscope and the periodontal clinical manifestations of the mice were observed: the second molar gums of the mice in group N were pink, with thin edges, close to the tooth neck, and tough texture; the second molar gums of the mice in group L were light red and soft; the second molar gums of the mice in group LP were dark red, soft, and easy to bleed during the removal process ( Figure 1 ).

[0064] The mobility, probing bleeding and palatal probing depth of the maxillary left second molars of mice were analyzed. The second molars of group N were all physiologically mobile and no probing bleeding was observed (Table 1).

[0065] Table 1 Clinical indicators of the left oral cavity of the mouse maxilla

[0066]

[0067] Note: BOP: bleeding on probing; M2 Mobility: Change of M2 mobility (dynamic change of tooth mobility of the maxillary left second molar); PD: probing depth.

[0068] The results showed that compared with the dynamic changes in tooth looseness (1.40 ± 0.55) and probing depth (mesial (0.86 ± 0.14 mm), middle (0.33 ± 0.47 mm), distal (0.55 ± 0.33 mm)) of mice with simple ligation, the dynamic changes in tooth looseness (2.20 ± 0.45) and probing depth (mesial (1.08 ± 0.11 mm), middle (1.04 ± 0.26 mm), distal (1.04 ± 0.26 mm)) of the maxillary left second molar of mice with ligation and continuous injection of Pg LPS were more severe, and the difference was statistically significant (p < 0.05). The dynamic changes in the second molar tooth looseness of the three groups of mice were analyzed, and it was found that the average tooth looseness of the LP group was more severe than that of the L group, and the difference was statistically significant (Table 1 and Figure 2 Compared with group N, both group L and group LP showed bleeding on probing (Table 1). Compared with group N, the average probing depth of group L and group LP was significantly increased, and the average probing depth of group LP was more serious, showing a very significant difference (p = 0.0012), indicating that the combined method of silk thread ligation and Pg LPS injection caused more serious damage to the periodontal tissue of mice than the simple silk thread ligation method (Table 1 and Figure 2 Middle B).

[0069] 2.2 Alveolar bone resorption in mice

[0070] After the mice were killed 14 days after modeling, the Micro-CT results of the left alveolar bone in the maxilla were analyzed: the bone volume fraction, bone density and CEJ-ABC of the second molar area were measured based on the two-dimensional images, and the alveolar bone resorption of the mice was analyzed. Compared with the N group, the bone volume fraction and bone density of the L group and the LP group were significantly reduced, and the distance of the CEJ-ABC of the second molar mesial was significantly increased, and the difference was statistically significant ( Figure 3Compared with the bone volume fraction (28.97 ± 7.90 %), bone density (0.55 ± 0.08 g / cc) and the distance from the cemento-enamel junction to the alveolar bone crest (CEJ-ABC) (0.74 ± 0.12 mm) of the L group, the bone volume fraction (16.44 ± 3.35 %) and bone density (0.42 ± 0.04 g / cc) of the LP group were further reduced, and the distance from the mesial CEJ-ABC of the second molar (0.88 ± 0.03 mm) was further increased, and the difference was statistically significant ( Figure 3 ). Analyze the palatal image of the left alveolar bone in the maxilla observed and photographed under a stereo microscope ( Figure 4 The results showed that compared with the N group, the attachment loss area in the L group (0.30 ± 0.02 mm 2 ) and LP groups (0.34 ± 0.01 mm 2 ) was significantly reduced, and the difference was statistically significant ( Figure 4 This suggests that the combination of silk ligation and Pg LPS injection caused more severe alveolar bone loss in mice than silk ligation alone.

[0071] 2.3 Periodontal inflammation in mice

[0072] HE staining was used to observe the gingival inflammation. The results showed that the periodontal tissue structure of group N was intact, there was a small amount of inflammatory cell infiltration in the gingiva of group L (indicated by the red arrow), and there was obvious inflammatory cell infiltration, tissue edema and necrosis in the gingiva of group LP (indicated by the yellow arrow), indicating that the combination of silk thread ligation and Pg LPS injection caused more severe local periodontal inflammatory response in mice than silk thread ligation alone ( Figure 5 ).

[0073] 2.4 Systemic inflammation levels in mice

[0074] The levels of IL-1β and TNF-α in the serum of the three groups of mice were studied 14 days after modeling (n = 4): compared with the N and L groups, the levels of IL-1β and TNF-α in the serum of the LP group were significantly increased, and the results were statistically significant ( Figure 6 ). 14 days after modeling, the blood of the three groups of mice was tested for routine blood tests: compared with the N group, the percentage of lymphocytes in the L group and the LP group was significantly reduced, and the percentage of neutrophils was significantly increased, and the results were statistically significant ( Figure 7 This suggests that the combination of silk ligation and Pg LPS injection caused more severe systemic inflammation in mice than silk ligation alone.

[0075] 3 Discussions

[0076] At present, the research on the treatment of periodontitis still lacks an ideal experimental animal model, which has become a bottleneck problem restricting the treatment and pathogenesis research of periodontal disease. In recent years, the most commonly used modeling method for experimental animal models simulating human periodontitis is the mouse periodontal silk thread ligation method, and the modeling time is usually 10-14 days. Although this method can cause acute alveolar bone resorption, after removing the ligature silk thread, due to the ability of mice to naturally restore alveolar bone resorption, the severity of the modeling is often not enough for research on the treatment of periodontitis. Therefore, the present invention further optimizes the silk thread ligation method, constructs a modeling method for a mouse periodontitis model of silk thread ligation combined with injection of Pg LPS, and performs a short-term rapid modeling for 14 days to observe the changes in the degree of alveolar bone resorption and local and systemic inflammation levels in mice.

[0077] In previous studies, in order to make up for the shortcomings of simple silk thread ligation modeling, such as the inability to simulate the pathogenic flora of human periodontitis and the difficulty in studying the interaction between the host and bacteria, two or more periodontitis modeling methods are often combined. The more common one is the modeling method of silk thread ligation combined with bacterial gavage or local application. However, these combined methods require a microbiological laboratory to operate because bacteria are selected for modeling. At the same time, the effectiveness of the selected periodontal pathogenic strains is required to be high, and the operation is difficult and dangerous. The present invention selects to inject lipopolysaccharide from Porphyromonas gingivalis, a virulence factor of relatively safer pathogenic bacteria. The results show that the scheme of injecting lipopolysaccharide and ligation combined modeling can more effectively induce the occurrence of periodontitis in mice. In addition, preliminary experiments show that the selection of PgLPS can construct a safer and faster mouse periodontitis model than other bacteria and virulence factors (such as Porphyromonas gingivalis).

[0078] In addition, by comparing and analyzing the previous studies on periodontitis modeling in mice such as simple silk thread ligation, simple injection of lipopolysaccharide, or ligation combined with bacterial gavage, the present invention screened and optimized the dosage, interval, and number of injections during the modeling process. As shown in the previous embodiment, a short-term modeling time of 14 days was used, during which Pg LPS was injected once every 3 days, for a total of 4 injections, and each injection dose was 20 μg. After 14 days of the experiment, the clinical manifestations of the oral cavity of mice observed under the microscope showed that the degree of periodontal tissue damage and inflammation in mice with silk thread ligation combined with injection of Pg LPS was higher than that in mice with simple silk thread ligation modeling, indicating that this method can reduce the damage to mice caused by frequent (such as every other day) anesthesia and injection, and can achieve the effect of severe periodontitis modeling.

[0079] An important feature of periodontitis is attachment loss. The present invention uses the periodontal probing depth of mice and the calculated area of ​​alveolar bone attachment loss as the main investigation indicators, and finds that compared with simple silk thread ligation, the method of silk thread ligation combined with Pg LPS injection can cause mice to form deeper periodontal pockets and more severe attachment loss, resulting in a mouse periodontitis model with clinical manifestations closer to severe human periodontitis.

[0080] Alveolar bone absorption is one of the main pathological changes of periodontitis. The present invention analyzes the attachment loss area, CEJ-ABC and bone volume fraction of mice modeled by different methods by microscopic observation and Micro-CT, and uses them as indicators to judge the degree of alveolar bone absorption in mice. The results obtained all indicate that the degree of alveolar bone absorption in mice with silk thread ligation combined with Pg LPS injection is significantly higher than that in mice with silk thread ligation after 14 days. This shows that in a short period of time, the modeling method of silk thread ligation combined with Pg LPS injection can effectively promote the degree of alveolar bone absorption, resulting in more severe attachment loss and obvious root bifurcation lesions, and is a mouse periodontitis model with more severe pathological and imaging manifestations. Previous studies have reported that the periodontitis modeling method of silk thread ligation combined with oral smearing of a single pathogen does not aggravate the degree of alveolar bone absorption in mice. Although the modeling method of silk thread ligation combined with oral smearing of multiple mixed pathogens has been reported to aggravate the degree of alveolar bone absorption in mice, mixed pathogens are more difficult to obtain and culture and are more dangerous. Therefore, using Pg LPS instead of mixed pathogens can more safely and efficiently construct a mouse periodontitis model with more severe alveolar bone absorption.

[0081] The present invention simultaneously detected the periodontal and systemic inflammation levels of periodontitis mice. The results showed that the periodontal inflammation of mice modeled by silk thread ligation combined with Pg LPS injection was more severe, and the expression of inflammatory factors TNF-α and IL-1β in the serum was higher, indicating that this modeling method can create a mouse periodontitis model with more severe local oral and systemic inflammation levels.

[0082] Clinically, periodontitis is mainly diagnosed by probing for the presence of periodontal pocket formation and the degree of attachment loss, and imaging manifestations are often used as a means of auxiliary diagnosis. The present invention detects the clinical manifestations of mice after modeling, including tooth mobility, probing bleeding, and probing depth. The results suggest that compared with simple silk ligation, the method of silk ligation combined with injection of Pg LPS caused more severe clinical manifestations of periodontitis in mice. In previous studies on the treatment of periodontitis, the periodontal recovery of mice was mainly judged by comparison with the control group, and there was a lack of comparison of the periodontal recovery of the experimental group before and after treatment. The test results of the clinical manifestations of mice in the present invention suggest that when it is difficult to perform oral live imaging of mice, the clinical manifestations of the periodontium of mice can be used as a basis for judging the severity of periodontitis in modeling.

[0083] In summary, the silk thread ligation combined with Pg LPS injection method for modeling periodontitis in mice can induce periodontal inflammation in mice in a safer, faster and more effective manner in a short period of time, causing more severe attachment loss and alveolar bone resorption in mice, and constructing a severe mouse periodontitis model that is more suitable for periodontitis treatment and other research.

[0084] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Method for constructing a severe periodontitis mouse model, including: After silk thread ligature was applied to the molars, LPS solution was injected into the gingiva on the palatal side of the molars. A severe periodontitis mouse model was established 14 days later.

2. The construction method according to claim 1, characterized in that: The molar is a second molar.

3. The construction method according to claim 1 or 2, characterized in that: The second molar is the maxillary left second molar.

4. The construction method according to claim 1, characterized in that: The ligature is made of 5-0 silk thread.

5. The construction method according to claim 1, characterized in that: The solvent of the LPS solution is water, and the concentration is 10 μg / μL.

6. The construction method according to claim 1 or 5, characterized in that: The injection dose was 2 μL / tooth, and the injection was performed once every 3 days, for a total of 4 injections.

7. The construction method according to any one of claims 1 to 6, characterized in that: The first injection is completed after the ligation.

8. The construction method according to any one of claims 1 to 7, characterized in that: The ligation step also includes an anesthesia step, and the anesthetic is 1% tribromoethanol.

9. The construction method according to any one of claims 1 to 8, characterized in that: The mice were 10-week-old male mice.

10. The construction method according to claim 9, characterized in that: The mice are C57BL / 6 mice.

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