A siRNA targeting PITX2 gene and its medicine and application

Abstract

The technical field of the present invention is medical and dental preparations, and provides a siRNA targeting the PITX2 gene and a medicine and application thereof, wherein the medicine is used to prevent and treat periodontitis, and comprises an siRNA targeting the PITX2 gene, wherein the siRNA specifically binds to and targets the mRNA of the PITX2 gene, thereby blocking the expression of the PITX2 gene, thereby inhibiting the production of inflammatory epithelial cells SAA1+Epi in periodontal tissue. The present invention can significantly reduce the degree of alveolar bone absorption and delay the progression of periodontitis.

Description

Technical Field

[0001] The present invention relates to the technical field of oral medical materials, and more specifically, to a siRNA targeting the PITX2 gene and a drug and application thereof. Background Art

[0002] The results of the fourth national oral epidemiological survey (2015-2017) showed that the prevalence of periodontal disease in my country is high, and the periodontal health rate of adults in each age group (35-44 years old, 55-64 years old and 65-74 years old) is less than 10%. Among them, the periodontal health rate of the 55-64 age group is only 5.0%. Periodontitis is the main cause of tooth loss in adults. At present, the pathogenesis of periodontitis is considered to be the result of the combined effects of microecological imbalance and periodontal tissue immune homeostasis imbalance. Periodontal basic treatment is an important means to prevent and treat microecological imbalance, and the technical progress of periodontal tissue inflammation is insufficient. Periodontitis usually has no better containment method for current clinical treatment. Mechanical removal, i.e. conventional periodontal basic treatment and auxiliary antibacterial drugs are the most commonly used methods at this stage.

[0003] At present, the main treatment method to curb the progression of periodontitis is periodontal basic treatment, that is, through periodontal cleaning and scaling, the periodontal microecological environment is reshaped and the alveolar bone volume is maintained. However, this treatment method requires a lot of manpower and financial resources. If the patient cannot go to the hospital for treatment regularly, the periodontal state cannot be maintained. Summary of the invention

[0004] One of the purposes of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a drug for reducing or inhibiting the occurrence and development of periodontitis by targeting the PITX2 gene and effectively inhibiting its expression, thereby regulating the production of inflammatory epithelial cells SAA1+Epi, thereby inhibiting the progression of periodontitis.

[0005] To achieve the above objectives, one aspect of the present invention provides a drug for preventing and treating periodontitis, comprising siRNA targeting the PITX2 gene, wherein the siRNA specifically binds to and targets the mRNA of the PITX2 gene, thereby blocking the expression of the PITX2 gene, wherein the siRNA specifically binds to and targets the mRNA of the PITX2 gene, and the siRNA consists of the following sequence: positive chain: CAGGCUAUUCCUACAACAA(dT)(dT); and antisense chain: UUGUUGUAGGAAUAGCCUG(dT)(dT).

[0006] In an embodiment, the drug can block the PITX2 gene, a key downstream transcription factor in the TLR2-ERK1 / 2-Smad2 / 3-PITX2 signaling pathway, to regulate the expression of SAA1+Epi in inflammatory epithelial cells in periodontitis, thereby preventing or alleviating periodontitis.

[0007] In an embodiment, the drug can be designed as an injectable small molecule drug, which can be injected subcutaneously into the gingival epithelium of the subject in need, and can act on the area where periodontitis needs to be treated by local injection.

[0008] On the other hand, the present invention provides a siRNA, which specifically binds to and targets the mRNA of the PITX2 gene and is used to inhibit the expression of the PITX2 gene. The siRNA consists of the following sequences: a positive strand: CAGGCUAUUCCUACAACAA(dT)(dT); and an antisense strand: UUGUUGUAGGAAUAGCCUG(dT)(dT).

[0009] In the embodiments, the siRNA can block the PITX2 gene, a key downstream transcription factor in the TLR2-ERK1 / 2-Smad2 / 3-PITX2 signaling pathway, and regulate the expression of SAA1+Epi in inflammatory epithelial cells in periodontitis, thereby preventing or alleviating periodontitis.

[0010] In an embodiment, the siRNA can be injected locally into the area where periodontitis needs to be treated.

[0011] Another aspect of the present invention provides the use of the above-mentioned drug or the above-mentioned siRNA in blocking the PITX2 gene to prevent and treat periodontitis.

[0012] On the other hand, the present invention provides a gene regulation method targeting the PITX2 gene, which comprises delivering the drug as described above or the siRNA as described above, targeting the PITX2 gene, a key transcription factor in the downstream of the periodontitis-related signaling pathway TLR2-ERK1 / 2-Smad2 / 3-PITX2, and inhibiting the expression of the PITX2 gene.

[0013] In an embodiment, the method targets the PITX2 gene and exerts its effect by regulating the expression of SAA1+Epi in inflammatory epithelial cells in periodontitis.

[0014] In another aspect, the present invention provides a kit comprising a probe or an antibody for detecting the expression level of PITX2 gene in patients with periodontitis.

[0015] Compared with the prior art, the beneficial effects of the present invention include: it can be designed as an injectable small molecule drug to curb the progression of periodontitis. Compared with basic periodontal treatment, small molecule drugs consume less manpower, have a short single treatment time, a simple and convenient treatment method, a simple configuration, and significant effects. In addition, the local injection method can accurately act on the part of the periodontitis that needs to be treated, and has little effect on the whole body. And due to the effect of siRNA modification, it can remain locally for a long time and play a continuous role. Compared with the non-injected group, the injection of siPITX2 can significantly reduce the absorption of alveolar bone and delay the progression of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings illustrate exemplary embodiments of the inventive concept and, together with the description, serve to explain the principles of the inventive concept. The accompanying drawings are included to provide a further understanding of the inventive concept and are incorporated in and constitute a part of this specification.

[0017] Figure 1 Schematic diagram of UMAP (Unified Manifold Approximation and Projection) for epithelial cell subtype identification by single-cell transcriptome sequencing.

[0018] Figure 2 This is a bubble chart of GO (Gene Ontology) functional enrichment analysis of 6 types of epithelial cell subtypes.

[0019] Figure 3 This is the SCENIC (single-cell regulatory network inference and clustering) analysis diagram of single-cell sequencing of 6 types of epithelial cell subtypes.

[0020] Figure 4 This is the SAHMI (Single-cell Analysis of Host-Microbiome Interactions) analysis of single-cell sequencing of Porphyromonas gingivalis in healthy and periodontitis patients. The vertical axis represents the UMI (unique molecular marker) value of bacteria detected in the cells, and the horizontal axis represents the grouping of periodontitis and healthy patients.

[0021] Figure 5 This is a schematic diagram of the expression of pattern recognition receptors in 6 types of epithelial cell subtypes. The horizontal axis is the 6 types of epithelial cell subtypes, and the vertical axis is the name of the pattern recognition receptor.

[0022] Figure 6 It is a mouse model of periodontitis. Figure 6 A is a Micro-CT (micro-computed tomography) three-dimensional reconstruction pattern of a healthy mouse jaw. Figure 6B is a Micro-CT three-dimensional reconstruction of the jaw of a mouse with periodontitis.

[0023] Figure 7 It is a bar graph of alveolar bone resorption level.

[0024] Figure 8 Characterization of the inflammation-related epithelial cell subtype SAA1+Epi in periodontitis and healthy mice. DAPI marked the cell nucleus, KRT5 marked the mouse epithelial cells, and MMP13+ marked the inflammation-related epithelial cell subpopulation SAA1+Epi.

[0025] Fig. 9 It is a mouse model of periodontitis. Fig. 9 A is a Micro-CT three-dimensional reconstruction model of the healthy mouse jaw; Fig. 9 B is a Micro-CT 3D reconstruction of the jaw of a healthy mouse with local injection of PITX2+siRNA; Fig. 9 C is a Micro-CT three-dimensional reconstruction of the jaw of a mouse with periodontitis; Fig. 9 D is a Micro-CT three-dimensional reconstruction of the jaw of a periodontitis mouse after local injection of PITX2+siRNA.

[0026] Fig.10 It is a bar graph of alveolar bone resorption level.

[0027] Fig.11 It is a bar graph of alveolar bone density and bone volume fraction.

[0028] Fig.12 Characterization diagram of the inflammation-related epithelial cell subtype SAA1+Epi in each group of mice.

[0029] Fig.13 It is a bar graph of SAA1, SAA2, CSF3, MMP13 and PITX2 mRNA expression. Fig.13 A is a bar graph showing the expression of SAA1 mRNA. Fig.13 B is a bar graph showing the expression of SAA2 mRNA. Fig.13 C is a bar graph of MMP13 mRNA expression. Fig.13 D is a bar graph showing CSF3 mRNA expression. Fig.13 E is a bar graph of PITX2 mRNA expression.

[0030] Fig.14 It is a bar graph of TLR2 mRNA expression.

[0031] Fig.15 This is a diagram of the ERK1 / 2 phosphorylation process and Smad2 / 3 phosphorylation protein detection.

[0032] Fig.16 The bar graph shows the relative expression of SAA1, SAA2, CSF3, MMP13 and PITX2 mRNA. Fig.16 A is a bar graph showing the relative expression of SAA1 mRNA. Fig.16 B is a bar graph showing the relative expression of SAA2 mRNA. Fig.16 C is a bar graph showing the relative expression of MMP13 mRNA. Fig.16 D is a bar graph showing the relative expression of CSF3 mRNA. Fig.16 E is a bar graph showing the relative expression of PITX2 mRNA. DETAILED DESCRIPTION

[0033] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0034] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] In this document, the terms "contain" or "include" are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0036] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0037] Given that the loss of gingival homeostasis is a key factor in periodontitis, studying the mediators that cause the destruction of gingival barrier homeostasis will not only help reveal the mechanism of periodontitis progression, but also help develop innovative treatments for this common disease. In particular, with the popularization of the three-level prevention concept, how to use blocking drugs to inhibit the progression of inflammation in the early stages of periodontitis and maintain the stability of periodontal support tissues is a new idea for the prevention and treatment of periodontitis.

[0038] Resident cells in the gingiva play a vital role in regulating immune responses, repairing and regenerating periodontal tissues, and maintaining barrier homeostasis. For example, epithelial cells perform antigen presentation functions to maintain periodontal homeostasis. Fibroblasts support alveolar bone regeneration by differentiating into bone-forming cells or producing cytokines that promote bone regeneration. In addition, immune cells play a key role in removing pathogenic bacteria and releasing cytokines that promote tissue repair. However, the key signaling pathways and effective genes in the gingiva during the progression of periodontitis and its main effector cells are still unclear.

[0039] Based on the development of modern sequencing technology, the present invention firstly uses a combination of single-cell transcriptome sequencing and spatial transcriptome sequencing to sequence and analyze gingival tissue samples collected from clinical periodontitis and healthy people. Through deconvolution and dimensionality reduction clustering, the cells of gingival tissue are divided into 6 major categories, including epithelial cells (KRT5+), endothelial cells (PLVAP+), B / plasma cells (CD79A+), T / NK cells (CD3D+), fibroblasts (COL3A1+) and myeloid cells (LYZ+). The epithelial cells are further identified and analyzed as subgroups. Figure 1 As shown in , epithelial cells are divided into six subclasses in total, COL17A1+Epi, KRT6C+Epi, IGLC2+Epi, VIM+Epi, SAA1+Epi, and SPRR1B+Epi.

[0040] GO functional analysis showed that the main functions of SAA1+Epi were phagocytosis, activation of immune cells and other inflammatory response-related functions. Therefore, it was predicted that SAA+Epi might be a subtype of epithelial cells with significant inflammatory characteristics (see Figure 2 ).

[0041] Through SCENIC analysis of single-cell sequencing, the transcription factors that induce the formation of the SAA+epi epithelial subpopulation were predicted. According to the prediction results, the top three transcription genes are FOXC2, PITX2, and CEPBEP. Among them, the transcription gene PITX2 regulates more regulons, so it is predicted that it may have a stronger induction effect on the formation of the SAA1+epi subpopulation (see Figure 3 ).

[0042] The main bacteria that disrupt periodontal homeostasis are Porphyromonas gingivalis ( Porphyromonas gingivalis , P.gingivalis ), of which its lipopolysaccharides (LPS) are important virulence factors. SAHMI analysis of single-cell sequencing showed that the number of Porphyromonas gingivalis invading in patients with periodontitis was significantly higher than that in healthy people (see Figure 4). Bacteria activate cells mainly by activating pattern recognition receptors on the cell surface. By analyzing the expression of pattern recognition receptors on the surface of various cell subtypes through single-cell sequencing, it was found that TLR2 receptors were most expressed on the surface of SAA1+Epi cells (see Figure 5 ). This receptor is a receptor associated with inflammatory activation and can be P.gingivalis LPS activation. Through analysis and research, it was found that TLR2 receptor can activate downstream ERK1 / 2 phosphorylation, further regulating downstream Smad2 / 3 phosphorylation to activate transcription factor PITX2. Therefore, it is speculated that P.gingivalis LPS activates the TLR2 receptor on the surface of epithelial cells, leading to ERK1 / 2 phosphorylation, further activating Smad2 / 3 phosphorylation, inducing increased PITX2 expression and thus inducing the appearance of the inflammation-related epithelial cell subtype SAA1+Epi, namely the TLR2-ERK1 / 2-Smad2 / 3-PITX2 axis.

[0043] One aspect of the present invention provides a gene related to periodontitis, which is the PITX2 gene. The PITX2 gene acts as a downstream key transcription factor in the TLR2-ERK1 / 2-Smad2 / 3-PITX2 signaling pathway, and regulates the periodontitis response by regulating the expression of SAA1+Epi in inflammatory epithelial cells in periodontitis.

[0044] The inventors of the present invention analyzed through single-cell sequencing technology that the key transcription factor induced in the inflammatory epithelium in gingival epithelial cells is PITX2, and found that Porphyromonas gingivalis LPS can activate the TLR2 receptor on the surface of epithelial cells, activate ERK1 / 2 phosphorylation, and further activate downstream smad2 / 3 phosphorylation, thereby activating the PITX2 transcription factor, further inducing the production of inflammatory epithelial cell subtypes, and promoting the occurrence and development of periodontitis.

[0045] Key nodes in the signaling pathway (such as TLR2, ERK, Smad, PITX2) are defined as potential drug targets. Drugs targeting these targets, or inhibitors / agonists screened based on the targets.

[0046] Another aspect of the present invention provides the biological effect of PITX2 gene in the treatment of periodontitis and its application.

[0047] Another aspect of the present invention provides the use of epithelial cell subpopulation SAA1+Epi marker genes in the treatment and diagnosis of periodontitis, wherein the SAA1+Epi marker genes include one or more of SAA1, SAA2, MMP13 and CSF3.

[0048] Another aspect of the present invention provides a drug for preventing and treating periodontitis, wherein the drug contains siRNA targeting the PITX2 gene, and the siRNA specifically binds to and targets the mRNA of the PITX2 gene, thereby blocking the expression of the PITX2 gene, thereby inhibiting the production of inflammatory epithelial cells SAA1+Epi in periodontal tissues and inhibiting the occurrence and development of periodontitis.

[0049] According to a specific embodiment of the present invention, the sequence of siRNA may include a sense strand and an antisense strand.

[0050] Sense strand: CAGGCUAUUCCUACAACAA(dT)(dT).

[0051] Antisense strand: UUGUUGUAGGAAUAGCCUG(dT)(dT).

[0052] By using the above siRNA, the expression of PITX2 gene can be effectively inhibited, thereby reducing the inflammatory response associated with periodontitis.

[0053] Another aspect of the present invention provides a siRNA, which specifically binds to and targets the mRNA of the PITX2 gene and is used to inhibit the expression of the PITX2 gene. The siRNA consists of the following sequences: a positive strand: CAGGCUAUUCCUACAACAA(dT)(dT); and an antisense strand: UUGUUGUAGGAAUAGCCUG(dT)(dT).

[0054] The present invention also provides the use of the above-mentioned drug or siRNA in blocking the PITX2 gene.

[0055] The present invention also provides the use of the above-mentioned medicine or siRNA in the preparation of medicine for preventing or alleviating periodontitis.

[0056] The drugs or siRNA of the embodiments of the present invention can also play an important role in preventing or alleviating periodontitis by blocking the PITX2 gene in the TLR2-ERK1 / 2-Smad2 / 3-PITX2 signaling pathway and regulating the expression of SAA1+Epi in inflammatory epithelial cells in periodontitis.

[0057] The drug can be designed as an injectable small molecule drug and injected subcutaneously into the gingival epithelium of the subject in need, so as to precisely act on the area that needs treatment for periodontitis through local injection.

[0058] Specifically, the siRNA or the drug may be subcutaneously injected into the gingival epithelium of a subject in need thereof to inhibit the occurrence or development of periodontitis.

[0059] Another aspect of the present invention provides a method for blocking the PITX2 gene, the method comprising delivering the above-mentioned drug or the above-mentioned siRNA to the PITX2 gene.

[0060] Exemplarily, a gene regulation method targeting the PITX2 gene is provided, the method comprising delivering the drug as described above or the siRNA as described above, targeting the PITX2 gene, a key transcription factor downstream of the periodontitis-related signaling pathway TLR2-ERK1 / 2-Smad2 / 3-PITX2, and inhibiting the expression of the PITX2 gene.

[0061] The present invention also relates to a kit for detecting the expression level of the PITX2 gene or the epithelial cell subpopulation SAA1+Epi marker gene associated with periodontitis, which can be used for the early diagnosis or prognosis evaluation of periodontitis.

[0062] Exemplarily, the kit may include a probe or an antibody for detecting the expression level of the PITX2 gene in patients with periodontitis.

[0063] According to the present invention, in vitro P.gingivalis LPS stimulates gingival epithelial cells. In vivo, an animal model of periodontitis was selected to explore the role of TLR2-ERK1 / 2-Smad2 / 3-PITX2 axis in activating SAA1+Epi, and the important role of blocking PITX2 in inhibiting the development of periodontitis.

[0064] In order to better understand the above exemplary embodiments of the present invention, they are further described below with reference to specific examples, where m / v% is mass / volume percentage, and v / v is volume / volume percentage.

[0065] In vivo: (1) An animal model of periodontitis in mice was established by periodontal ligation. Micro-CT was used to verify the successful establishment of periodontitis, and immunofluorescence was used to demonstrate that the inflammation-related epithelial subtype SAA1+Epi was increased in mice with periodontitis. (2) PITX2 siRNA was injected into mice with periodontitis ligation to block PITX2 expression in local epithelial tissues. Micro-CT was used to verify the severity of periodontitis and the expression of SAA1+Epi in mice injected with PITX2 siRNA.

[0066] Example 1

[0067] The in vivo experiment included the characterization of the SAA1+Epi subtype of inflammatory epithelial cells in the gingival epithelium of mice with periodontitis, including the following steps:

[0068] S110. Construction of a mouse model of periodontitis

[0069] 8-week-old C57BL / 6 mice were treated with antibiotics for 3 days and fed with specific pathogen-free (SPF) feed. After antibiotic treatment, they continued to be fed with SPF drinking water and feed. Using isoflurane gas anesthesia, when the mice were fully anesthetized (3-5 minutes), they were fixed, the maxillary teeth were exposed, and a nylon thread (6-0) of about 10-15 cm long was cut. The thread was passed from the mesial to the distal of the maxillary second molar, and the two ends of the thread were tied with a surgical knot on the palatal side of the maxillary second molar, and the excess thread was cut off. P.gingivalis After overnight anaerobic culture, the bacterial solution was centrifuged at 4000 g for 10 minutes and then resuspended in saline containing 2% carboxymethyl cellulose to a concentration of 1×10 7 CFU / mL, and applied around the ligature silk thread every day. After 4 weeks, the animals were killed and the silk thread and the mandible tied with the thread were removed.

[0070] S120. Micro-CT scanning and analysis of maxillary alveolar bone resorption in mice: The Micro-CT scan data was imported into mimics 21.0 software for three-dimensional construction and analysis. Based on the constructed three-dimensional image and the corresponding longitudinal section of the distal root apical foramen of the maxillary first molar of mice, the distance between the cemento-enamel junction (CEJ) of the buccal distal root of the first molar and the alveolar bone crest (ABC) was measured (CEJ-ABC). Repeat the measurement twice to obtain the average distance in microns.

[0071] The difference between the CEJ-ABC values ​​of the experimental group and the CEJ-ABC values ​​of the group ΔCEJ-ABC represents the alveolar bone resorption in experimental periodontitis in mice:

[0072] ΔCEJ-ABC=CEJ-ABC(Left)-CEJ-ABC(Right)

[0073] like Figure 6 and Figure 7 As shown in the results, the periodontitis model was successfully established in the mice in the silk tethering group. The Micro-CT results showed that the alveolar bone of mice with periodontitis was significantly absorbed. The statistical analysis of the degree of alveolar bone absorption showed that there was a significant difference in the degree of alveolar bone absorption in mice with periodontitis.

[0074] S130, Immunofluorescence

[0075] The samples were fixed with paraformaldehyde at 4 degrees overnight. The mouse jaws were decalcified with decalcification solution for 3-4 weeks and then embedded in paraffin. The paraffin sections were washed in sequence in xylene for 10 minutes, xylene for 10 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, and distilled water. Antigen repair was performed with pH 6.0 citric acid repair solution, and 3% hydrogen peroxide solution was incubated at room temperature in the dark for about 25 minutes to block endogenous peroxidase. The samples were washed in phosphate buffered saline (PBS) for 3 times, each time for 5 minutes. 3m / v% bovine serum albumin (BSA) was blocked at room temperature for 30 minutes, the primary antibody was incubated at 4 degrees overnight, and the secondary antibody of the corresponding species to the primary antibody was added to cover the tissue and incubated at room temperature for 50 minutes. DAPI (4',6-diamidino-2-phenylindole) staining solution was incubated at room temperature in the dark for 10 minutes. PBS was washed 3 times, each time for 5 minutes. The images were collected after sealing with anti-fluorescence quenching mounting medium.

[0076] The results showed that KRT5 marked mouse gingival epithelial cells, and MMP13 marked the inflammatory epithelial cell subpopulation SAA1+Epi. In mice with periodontitis, the number of inflammatory epithelial cell subpopulation SAA1+Epi increased significantly (see Figure 8 ).

[0077] Example 2

[0078] The in vivo experiment involved blocking the expression of PITX2 in the inflammatory epithelial cell subtype SAA1+Epi in the mouse gingival epithelium, specifically including the following steps:

[0079] S210, model construction: divided into control group, periodontitis group, control group + siPITX2 group, periodontitis + siPITX2 group. During the model construction process, a microsyringe was used to subcutaneously inject PITX2 siRNA into the mouse gingival epithelium. The injection was once every three days, and each injection was 5μM. The control group was injected with a corresponding volume of PBS. Micro-CT scanning was used to analyze the resorption of maxillary alveolar bone in mice, and immunofluorescence was used to characterize the expression of SAA1+Epi.

[0080] The results showed that the degree of alveolar bone resorption in periodontitis mice injected with PITX2 siRNA was significantly alleviated compared with that in periodontitis mice, and the bone density was significantly increased. Immunofluorescence results showed that the expression of the inflammatory epithelial cell subtype SAA1+Epi in periodontitis mice injected with PITX2 siRNA was significantly reduced compared with that in periodontitis mice (see Figures 9 to 12 ). It is confirmed that blocking epithelial PITX2 expression plays an important role in inhibiting the occurrence and development of periodontitis.

[0081] Example 3

[0082] In vitro: (1) by using P.gingivalisLPS stimulated gingival epithelial cells, and the expression of SAA1+Epi marker genes was verified by RT-qPCR. (2) The effect of TLR2-ERK1 / 2-Smad2 / 3-PITX2 axis on inducing SAA1+epi production was verified by using TLR2 inhibitors, ERK1 / 2 phosphorylation inhibitors, Smad2 / 3 phosphorylation inhibitors, and PITX2 siRNA. (3) A PITX2 overexpression cell line was constructed to verify that PITX2 is a key transcription factor in the production of SAA1+epi.

[0083] in vitro: P.gingivalis LPS stimulates gingival epithelial cells to produce inflammation-related epithelial cell subpopulation SAA1+Epi, specifically including the following steps:

[0084] S310. Culture human gingival epithelial cells in a high glucose medium (DMEM) + 10v / v% fetal bovine serum at 37 degrees Celsius in a cell incubator containing 5v / v% carbon dioxide. Six-well plates were plated and stimulated for 24 hours with an LPS concentration of 10μg / ml. RNA extraction: remove the culture medium, wash twice with PBS, completely lyse the cells with 1mL trizol (a new total RNA extraction reagent), add 200μL chloroform, shake for 15s, let stand for 2 minutes, centrifuge at 12000g for 15 minutes, aspirate the supernatant into a new Eppendorf (EP) tube, add an equal volume of isopropanol, let stand at room temperature for 10 minutes, centrifuge at 12000g for 10 minutes, remove the supernatant, resuspend with 75v / v% alcohol, centrifuge at 8000g for 5 minutes, repeat the above process once, remove the alcohol, dry, add an appropriate amount of ultrapure water, and measure the concentration. The expression of SAA1, SAA2, MMP13 and CSF3, the hallmark genes of the disease-related epithelial cell subpopulation SAA1+Epi, was detected. The expression of the key transcription factor PITX2 was also detected.

[0085] like Fig.13 As shown in P.gingivalis The emergence of the SAA1+Epi epithelial subtype could be detected 24 hours after LPS stimulation, with a significant increase in the marker genes SAA1, SAA2, MMP13 and CSF3, and an increase in the expression of the transcription factor PITX2.

[0086] Example 4

[0087] Verification that the TLR2-ERK1 / 2-Smad2 / 3-PITX2 axis activates SAA1+Epi.

[0088] Protein extraction: remove the culture medium, wash twice with PBS, add RIPA lysis buffer (traditional cell tissue rapid lysis buffer) (containing protease inhibitors), lyse at 4 degrees for 20 minutes, shake several times during the period, after ultrasonic disruption, centrifuge at 12000rpm for 15 minutes, aspirate the supernatant into a new EP tube, and heat at 99 degrees for 5 to 10 minutes.

[0089] Western blot: use 10v / v% protein gel, 150V electrophoresis for 40min, 400mA transfer for 30min, 5m / v% BSA blocking for 1h, TBST (Tris-buffered saline containing Tween 20) washing membrane 3 times, 5min each time, incubate with primary antibody at 4°C overnight, wash membrane 3 times with TBST, 5min each time, incubate with secondary antibody for 1h, wash membrane 3 times with TBST, 5min each time, incubate with ECL (enhanced chemiluminescence) kit, and expose.

[0090] The results show that: P.gingivalis After LPS stimulation of gingival epithelial cells, TLR2 expression increased significantly, and ERK1 / 2 phosphorylation and Smad2 / 3 phosphorylation were significantly activated (see Fig.14 and Fig.15 ).

[0091] Example 5

[0092] Blocking PITX2 validated the expression of the inflammation-associated epithelial cell subtype SAA1+Epi.

[0093] Construction of siPITX2 gingival epithelial cells:

[0094] Design siRNA modified primers: sense: CAGGCUAUUCCUACAACAA(dT)(dT), antisense: UUGUUGUAGGAAUAGCCUG(dT)(dT).

[0095] The cells were seeded in a 12-well plate and the cell aggregation reached 50%. A transfection mixture was prepared, 5 μL of lipo2000 + siRNA (final concentration 100 nM) + DMEM was prepared into a 100 μL mixture, mixed and allowed to stand for 15 min, the original culture medium was removed, PBS was washed twice, 900 μL of DMEM and 100 μL of transfection mixture were added, and the complete culture medium / complete culture medium + stimulation was replaced after 8 hours.

[0096] The experiment was divided into control group, P.gingivalis LPS stimulation group, control group + siPITX2 group, P.gingivalis LPS stimulation group + siPITX2 group.

[0097] The expression of SAA1, SAA2, MMP13 and CSF3, the hallmark genes of the disease-related epithelial cell subpopulation SAA1+Epi, was detected. The expression of the key transcription factor PITX2 was also detected.

[0098] The results showed that after siRNA transfection into gingival epithelial cells, the expression of PITX2 in the cells was significantly reduced. After the expression of PITX2 transcriptional gene was reduced, the expression of SAA1+Epi marker gene was also significantly reduced. This proves that PITX2 plays an important role in inducing the appearance of inflammation-related epithelial cell subtype SAA1+Epi (see Fig.16 ).

[0099] According to the present invention, by targeting the PITX2 gene and effectively inhibiting its expression, the production of inflammatory epithelial cells SAA1+Epi is regulated, thereby inhibiting the progression of periodontitis. The present invention can be designed as an injectable small molecule drug to curb the progression of periodontitis. Compared with basic periodontal treatment, small molecule drugs consume less manpower, have a short single treatment time, a simple and convenient treatment method, a simple configuration, and a significant effect. And the local injection method can accurately act on the part that needs to be treated for periodontitis, and has little effect on the whole body. And due to the effect after siRNA modification, it can remain locally for a long time and play a role continuously. Compared with the group without injection, the injection of siPITX2 can significantly reduce the absorption degree of alveolar bone and delay the progression of periodontitis.

[0100] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A drug for preventing and treating periodontitis, characterized in that: The drug comprises siRNA targeting the PITX2 gene, wherein the siRNA specifically binds to and targets the mRNA of the PITX2 gene, thereby blocking the expression of the PITX2 gene. The siRNA specifically binds to and targets the mRNA of the PITX2 gene, and the siRNA consists of the following sequence: Sense strand: CAGGCUAUUCCUACAACAA(dT)(dT); and Antisense strand: UUGUUGUAGGAAUAGCCUG(dT)(dT).

2. The drug according to claim 1, characterized in that The drug is designed as an injectable small molecule drug, which can act on the area where periodontitis needs to be treated by local injection.

3. A siRNA, characterized in that The siRNA specifically binds to and targets the mRNA of the PITX2 gene, and is used to inhibit the expression of the PITX2 gene. The siRNA consists of the following sequence: Sense strand: CAGGCUAUUCCUACAACAA(dT)(dT); and Antisense strand: UUGUUGUAGGAAUAGCCUG(dT)(dT).

4. Use of the siRNA according to claim 3 in the preparation of periodontitis drugs.

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