Application of porphyromonas gingivalis extract in preparation of embryo cleft palate model related products

Abstract

The invention discloses application of a porphyromonas gingivalis extract in preparation of products related to an embryo cleft palate model, relates to the technical field of molecular biology, and particularly discloses application of the porphyromonas gingivalis extract in construction of the embryo cleft palate model. The research finds that in the embryonic development period, after a porphyromonas gingivalis extract is applied to a parent body by simulating the condition that a periodontitis patient is infected with the porphyromonas gingivalis, a certain probability of cleft palate of an embryo can be caused, and then the application of the porphyromonas gingivalis to construction of related products of an embryo cleft palate model is provided. The invention provides a novel method for constructing a cleft palate embryo, the application field of the porphyromonas gingivalis is expanded, the research on periodontal diseases and systemic toxic and side effects caused by oral infection of the porphyromonas gingivalis is deepened, and a theoretical basis is provided for the occurrence and prevention research of offspring cleft palate of maternal periodontitis patients.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to application of a Porphyromonas gingivalis extract in preparing products related to an embryonic cleft palate model. Background Art

[0002] Oral microorganisms are one of the three major bacterial reservoirs in the human body. In addition to being related to oral diseases such as caries and periodontal disease, their relationship with systemic diseases such as cardiovascular and cerebrovascular diseases, diabetes, adverse pregnancy outcomes, respiratory diseases, etc. is also receiving increasing attention. It is currently believed that oral microorganisms can spread to different parts of the body through the oral mucosa and periodontal pockets through multiple pathways such as direct dissemination, hematogenous, and immune, thereby causing systemic or local infections. The fourth national oral health survey in 2017 showed that the prevalence of periodontal disease in adults in my country is as high as over 80%, and it is more common in pregnant women. Therefore, periodontal disease caused by periodontal pathogens represented by Porphyromonas gingivalis has become one of the most common public health problems today. With the advancement of research methods and technologies, Porphyromonas gingivalis has been found to be associated with a variety of diseases in recent years, such as Alzheimer's disease, diabetes, tumors, atherosclerosis, etc. It contains a variety of pathogenic factors, which are mainly divided into two categories, including its own components, such as lipopolysaccharide, pili, cilia, outer membrane, capsule, heat shock protein, etc., and its secretory products such as gingipain, extramembranous vesicles, etc. Cleft palate is the most common craniofacial birth defect in humans. Its etiology is complex, and both genetic and environmental factors are involved. Since the complex uterine microenvironment is an environmental factor that directly affects embryonic development, disrupting the uterine microenvironment may cause embryonic malformations. Therefore, maternal infection with bacteria or viruses has always been considered an important pathogenic factor for the occurrence of cleft palate. Traditionally, it is believed that the fetus develops in the sterile environment of the uterus. However, recent evidence shows that maternal oral microorganisms can be vertically transmitted to the uterus and affect the uterine microenvironment. Among them, those related to intrauterine infection are mostly periodontal pathogens such as Porphyromonas gingivalis. A case-control study also revealed a possible strong association between maternal periodontal disease and cleft palate in offspring, but the exact relationship is still unclear. Therefore, clarifying the impact of Porphyromonas gingivalis infection on palate development and cleft palate and its main pathways can not only provide a basis for clinical genetic counseling and disease prevention, but also has important significance for eugenics and improving the quality of my country's newborn population.

[0003] Due to the difficulty in obtaining maternal or embryonic samples of cleft palate, and based on embryological and etiological studies, mice are an easily available experimental animal with a high litter size and a high degree of gene sequence homology with humans (99%), and are generally recognized as a suitable animal model for most studies. Therefore, mice are often used as an important model for in vivo experimental studies of cleft palate. However, there are no reports on the effects of Porphyromonas gingivalis on palate development and cleft palate occurrence, including in mice. Summary of the invention

[0004] The purpose of the present invention is to provide a method for using a Porphyromonas gingivalis extract to construct a cleft palate animal model, and further propose a new application of microorganisms represented by Porphyromonas gingivalis.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for constructing an embryonic cleft palate model using a Porphyromonas gingivalis extract, which is constructed by administering the Porphyromonas gingivalis extract to pregnant mice every other day from day 2.5 to day 14.5 of pregnancy, wherein the pregnant mice are pregnant mice with normal fetuses before administration.

[0006] The cleft palate of the fetal mouse is caused by the use of Porphyromonas gingivalis extract, which leads to abnormal macrophage efferocytosis and abnormal palatine process osteogenesis during the palate development stage, thereby causing abnormal palate fusion.

[0007] The present invention administers a Porphyromonas gingivalis extract to pregnant mice during a specific period of pregnancy, which can effectively induce embryonic cleft palate and successfully construct a cleft palate animal model. The method is simple and has a high success rate, and provides a new method for the microbial infection of the mother represented by Porphyromonas gingivalis during pregnancy and the subsequent influence on the maxillofacial development of the offspring and related research.

[0008] In the construction method of the present invention, the administration method is tail vein injection combined with oral gavage, and the daily injection dose is 4 mg / kg.

[0009] The method of tail vein injection combined with oral gavage is easy to operate, has strong explanatory experimental results, and has a low embryonic mortality rate.

[0010] The model constructed by the present invention can observe the phenotypic changes of the palate of fetal mice after birth, and detect the macrophage burial and palatine cell osteogenesis at the fusion stage, so as to better illustrate the effect of the Porphyromonas gingivalis extract on the occurrence of cleft palate.

[0011] In a second aspect, the present invention provides the use of a Porphyromonas gingivalis extract in constructing a drug for treating embryonic cleft palate.

[0012] In the application of the present invention, the active ingredient of the drug exists in a free form or in a pharmaceutically acceptable salt form.

[0013] The active ingredient or its pharmaceutically acceptable salt may also be used in the form of a hydrate or other solvents as required for the application.

[0014] In the application of the present invention, the medicine contains at least one pharmaceutically acceptable carrier.

[0015] In the application of the present invention, the drug is administered orally or by intravenous injection.

[0016] The drug of the present invention can be prepared by conventional methods, such as by conventional mixing, granulation, sugar coating, dissolution or freeze drying. It can contain a therapeutically effective amount of pharmacologically active ingredients or can also contain one or more pharmaceutically acceptable carriers. The preferred route of administration of the drug of the present invention is intravenous injection and oral gavage.

[0017] The dosage form of the drug of the present invention can be a sugar-coated tablet, tablet or capsule. The unit content of the active ingredient contained in a single dose in each dosage form does not necessarily need to constitute an effective amount, and the required effective amount can be achieved by administering multiple dosage units, i.e., intravenous injection of 2 mg / kg + oral gavage of 2 mg / kg.

[0018] In preparing drugs for oral dosage forms, any conventional pharmaceutical media may be used, such as water, glycols, oils or alcohols; or in the case of oral solid preparations such as powders, capsules and tablets, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used.

[0019] In the application of the present invention, the drug is administered during embryogenesis to palate development (fusion of palate processes).

[0020] The beneficial effects of the present invention are at least: The present invention provides a new method for constructing an embryonic cleft palate model using a Porphyromonas gingivalis extract, which expands the application field of microorganisms represented by Porphyromonas gingivalis and the connection between periodontal disease and systemic diseases, deepens the understanding of how maternal microorganisms affect embryonic development, and provides a theoretical basis for the study of the mechanism of cleft palate occurrence and prevention and treatment. The embryonic cleft palate animal model of the present invention has a short modeling cycle, simple operation, low cost, and a cleft palate rate of 12.5%. Instruction Manual

[0021] Figure 1 Part of the stereomicroscope observation results of the blank control group (control) and the experimental group (P. g).

[0022] Figure 2 Part of the HE staining results of the blank control group (control) and the experimental group (P. g).

[0023] Figure 3 Part of the Masson staining results of the blank control group (control) and the experimental group (P. g) (the blue-stained part is collagen fiber). The decreased bone formation ability in the experimental group is one of the causes of cleft palate.

[0024] Figure 4 The results of Masson staining quantitative analysis of the blank control group (control) and the experimental group (P. g).

[0025] Figure 5 Partial immunofluorescence staining of the blank control group (control) and the experimental group (P. g) shows some observation results of the macrophages performing the efferocytosis function on apoptotic epithelial cells at E15 (MerTK shows macrophages performing efferocytosis function, TUNEL shows apoptotic epithelial cells, and abnormal efferocytosis is the main reason for the occurrence of cleft palate caused by Porphyromonas gingivalis extract).

[0026] Figure 6 The results of quantitative analysis of cell burial function in the blank control group (control) and the experimental group (P. g). Implementation

[0027] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0029] In the specific implementation of the present invention, some experimental materials and reagents used are as follows: C57BL / 6 mice, female mice: healthy, 6-8 weeks, 22-25g; male mice: healthy, 8-10 weeks, 25-28g.

[0030] Reagents used: Porphyromonas gingivalis (ATCC33277), sterile defibrinated sheep blood (KJ-DB-0010S, Kejing Biotechnology), trypticase soy agar (CM0131, Oxoid, UK), anti-MerTK (ab300136, Abcam), one-step TUNEL cell apoptosis detection kit (C1089, Biotech).

[0031] Preparation method of Porphyromonas gingivalis extract: Porphyromonas gingivalis was cultured at 37°C under anaerobic conditions in a culture medium consisting of trypticase soy agar supplemented with 10% defibrinated sheep blood. 9After centrifugation of the bacterial suspension of CFU / mL, the culture medium was discarded and resuspended in physiological saline. The cells were broken on ice using an ultrasonic cell disruptor (JYD-150, Zhixin Instrument Co., Ltd.) (300W, on 10s, off 10s, 90 cycles) to ensure complete bacterial lysis. The ultrasonic extract was sterilized using a filter with a pore size of 0.22 μm (PR03683, Millipore, USA). The total protein concentration was determined by the BCA method and stored at -80°C. Example

[0032] This example verifies the effect of Porphyromonas gingivalis extract in constructing an embryonic cleft palate model. The experimental process is as follows: 1. Select 8-10 week old C57BL / 6 male and female mice, and cage them together at 9 pm on the same day in a ratio of 1:2. After about 10-12 hours, separate the female mice from the male mice and observe the formation of vaginal plugs in the female mice. The female mice with vaginal plugs were recorded as 0.5 days of pregnancy (i.e. E0.5) and their body weights were recorded. 2. Pregnant mice were randomly divided into two groups. The experimental group was given saline containing P. gingivalis extract (P. g group, n = 12), and the control group was given pure saline (control group, n = 12). From E2.5 to E14.5, saline containing 4 mg / kg of extract or an equal amount of pure saline was given to both groups of pregnant mice every other day via tail vein injection and oral instillation (2 mg / kg for each administration method).

[0033] 3. Pregnant mice were killed by cervical dislocation at E16.5, and palate tissue samples were collected from fetal mice. The development of the palate was observed under a stereomicroscope.

[0034] Specifically, the stereomicroscope observation is to visually observe the development of the palate at E16.5 under a stereomicroscope, and the observation steps are as follows: Pregnant mice were killed by cervical dislocation at E16.5, and the embryos were collected and placed in pre-cooled PBS. The heads of all fetuses were removed with microsurgery scissors, and the mandibles and tongues were removed from the corners of the mouth to expose the palate and observe under a stereo microscope. Some observation photos are shown in Figure 1 .

[0035] Figure 1 Part of the stereomicroscope observation results of the blank control group (control) and the experimental group (P. g). The scale in the figure represents 1 mm; the arrow indicates the palate tissue.

[0036] As can be seen from the figure, at E16.5, the palates of the control group mice were completely fused, while some fetal mice in the experimental group (P. g) had obvious cleft palates.

[0037] Finally, based on the above experiments, the statistical results are shown in Table 1.

[0038] Table 1 Grouping Number of pregnant mice Number of cleft palate / total number of embryos Control 6 0 / 38 P.g 6 4 / 32 The specific results of this embodiment are: the fetal rats in the experimental group (P. g) had a cleft palate rate of 12.5%, which was statistically different from that in the control group. Example

[0039] According to the experimental research results of Example 1, an experimental group was constructed again in this example to further verify the effect of constructing an embryonic cleft palate model with the extract of Porphyromonas gingivalis.

[0040] The specific experimental process is the same as that of Example 1, except that: In step 3, pregnant mice were killed by cervical dislocation at E16.5, and palate tissue samples of fetal mice were collected. The E16.5 samples were stained with HE and Masson staining.

[0041] HE staining is to observe the development of the palate at E16.5 under a microscope after paraffin-embedded sections. The specific steps are as follows: (1) Tissue sections were baked in an oven at 65°C for 3 h; (2) Dewaxing with xylene twice, 10 min each time; (3) Gradient alcohol hydration: 100% alcohol twice, 2 min each time; 95% alcohol twice, 2 min each time; 80% alcohol - 2 min, 70% alcohol - 2 min, 50% alcohol - 2 min; (4) Rinse with PBS three times, 5 min each time. Gently wipe the sections clean and place them in a hematoxylin staining tank for 2-3 min; (5) Rinse with PBS three times, 5 min each time. Gently wipe the sections clean and place them in an eosin staining tank for staining for 30 s to 1 min; (6) Rinse with PBS three times, 5 min each time. Dehydrate with graded alcohol: 70% alcohol for 2 min, 80% alcohol for 2 min, 95% alcohol for 2 min, and 100% alcohol twice, 2 min each time; (7) Xylene was used twice, each time for 5 min; (8) After the transparency is completed, seal the slide. Use a pipette to drop neutral gum onto the tissue specimen, remove the excess gum and cover with a coverslip, taking care not to create bubbles.

[0042] After HE staining, the tissue sections were observed under an optical microscope. Some of the observation results are shown in Figure 2 .

[0043] Figure 2The following are the results of partial HE staining observation of the control group (control) and the experimental group (P. g). The scale bar in the figure represents 200μm, "PS" represents the palatine process of the fetal mouse, and "T" represents the tongue of the fetal mouse. As can be seen from the figure, at E16.5, the palate of the control group mice has been completely fused, and some fetal mice in the experimental group (P. g) have obvious cleft palate.

[0044] Masson staining is to observe the osteogenesis of the palate at E16.5 under a microscope after paraffin-embedded sections. The specific steps are as follows: (1) Tissue sections were baked in an oven at 65°C for 3 h; (2) Dewaxing with xylene twice, 10 min each time; (3) Gradient alcohol hydration: 100% alcohol twice, 2 min each time; 95% alcohol twice, 2 min each time; 80% alcohol - 2 min, 70% alcohol - 2 min, 50% alcohol - 2 min; (4) Rinse with PBS three times, 5 min each time. Gently wipe the sections clean and place them in a Masson complex stain for staining for 5-10 min. (5) Rinse briefly with distilled water. Treat with 1% tungsten phosphate for about 5 minutes; (6) No need to wash with water. Gently place the sections in aniline blue solution and counterstain for 5 minutes; (7) 1% glacial acetic acid water treatment for 1 min; (8) Rinse with PBS 3 times, 5 min each time. Dehydrate with gradient alcohol: 70% alcohol for 2 min, 80% alcohol for 2 min, 95% alcohol for 2 min, and 100% alcohol twice, 2 min each time; (9) Xylene was used twice, each time for 5 min; (10) After the transparency is completed, seal the slide. Use a pipette to drop neutral gum onto the tissue specimen, remove the excess gum and cover with a coverslip, taking care not to create bubbles.

[0045] After Masson staining, the tissue sections were observed under an optical microscope. Some of the observation results are shown in Figure 3 , 4.

[0046] Figure 3 The Masson staining results of the control group (control) and the experimental group (P. g) are shown in Figure 1. The scale bar represents 200 μm, and the arrows in the figure point to the blue-stained collagen fibers. Figure 4For quantitative analysis of the palate ossification area, GraphPad Prism 8 software was used for statistical analysis, and there was a statistical difference. As can be seen from the figure, at E16.5, the palate ossification center of the control group mice had been formed, and the palate ossification area was larger, while the experimental group (P. g) had a smaller palate ossification area and poor osteoplastic development. Example

[0047] According to the experimental research results of Examples 1 and 2, an experimental group was constructed again in this example to further verify the effect of the Porphyromonas gingivalis extract on the endocytosis ability of macrophages in the fusion stage of the palate of fetal mice.

[0048] The specific experimental process is the same as that of Examples 1 and 2, except that: In step 3, pregnant mice were killed by cervical dislocation at E15 (fetal palate fusion stage), and fetal palate tissue samples were collected for TUNEL+ immunofluorescence detection of macrophage efferocytosis.

[0049] The specific steps of TUNEL+ immunofluorescence staining are as follows: (1) Dewaxing: Dewaxing in xylene for 10 minutes. Switch to fresh xylene and dewaxing for another 10 minutes. Dewaxing in anhydrous ethanol for 5 minutes, then switch to fresh anhydrous ethanol for 3 minutes. Dewaxing in 95% ethanol for 3 minutes. Dewaxing in 85% ethanol for 3 minutes. Dewaxing in 75% ethanol for 3 minutes. Dewaxing in 50% ethanol for 3 minutes. Rinse in PBS 3 times, 5 minutes each time. (2) Antigen retrieval: Add distilled water to a bath, preheat to 99°C together with the retrieval solution, add the sections, boil for 30 min, let stand to room temperature, and rinse with PBS three times, 5 min each time; (3) Follow the instructions of the one-step TUNEL cell apoptosis detection kit: according to the number of slices and tissue size, take an appropriate amount of reagent 1 (TdT) and reagent 2 (dUTP) in the Tunel kit and mix them in a ratio of 2:29, add them to the circle to cover the tissue, lay the slices flat in a humidified box, incubate at 37°C for 11 hours, and add a small amount of water to the humidified box to maintain humidity. Rinse with PBS three times, 5 minutes each time; (4) Blocking: Block with goat serum working solution at 37° or room temperature for 30-60 min; incubate at room temperature in the dark for 30 min; (5) Add the primary antibody (anti-MerTK antibody, 1:100) and incubate in a humidified chamber at 4°C overnight; (6) Remove from the well at 4°C and return to room temperature (30 min), then rinse with PBS three times, 5 min each time; (7) Add secondary antibody, goat anti-rabbit ALexa Fluor 488 (1:300) fluorescent secondary antibody and incubate at room temperature in the dark; (8) Use DAPI-containing sealing solution to stain the cell nucleus and seal the slides. Make sure no small bubbles remain in the tissue area, let it dry naturally, and take pictures.

[0050] Observe under a fluorescence microscope. Some of the observation results are shown in Figure 5 , 6.

[0051] from Figure 5 It can be seen that the TUNEL experiment showed that there were obvious apoptosis signals in the epithelial suture of the middle palate ridge during the palatine process fusion period (E15) in both the control group (control) and the experimental group (P. g). MerTK is very important in the function of macrophages in performing cell burial. However, in the control group (control), apoptotic cells were mainly located in the MerTK-positive macrophage area, while in the experimental group (P. g), apoptotic cells free from MerTK-positive macrophages increased.

[0052] Figure 6 Yes Figure 5 The free apoptotic cells and apoptotic epithelial cells associated with MerTK macrophages were quantified and statistically analyzed using GraphPad Prism 8 software, and there was a statistical difference. This indicates that the efferocytosis ability of fetal palate macrophages in the experimental group (Pg) was weakened.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of Porphyromonas gingivalis extract in the construction of embryonic cleft palate related products.

2. Application of Porphyromonas gingivalis extract in the preparation of embryonic cleft palate phenotype animal model.

3. The use according to claim 1 or 2, characterized in that: The active ingredient of the drug exists in free form or a pharmaceutically acceptable salt thereof.

4. The use according to any one of claims 2 to 3, characterized in that: The drug is administered orally, intravenously, or in combination.

5. The use according to any one of claims 2 to 4, characterized in that: The drug is administered during embryogenesis to mid-palate development.

6. A method for constructing a cleft palate embryo model, characterized in that: The method was constructed by administering a Porphyromonas gingivalis extract to pregnant mice at intervals of one day from day 2.5 to day 14.5 of pregnancy, wherein the pregnant mice were pregnant with normal embryos before the administration of the drug.

7. The construction method according to claim 6, characterized in that: The administration method is tail vein injection combined with oral gavage, with a daily injection dose of 4 mg / kg (2 mg / kg by tail vein injection and 2 mg / kg by oral gavage).

Images