WO2021047137A1 - 用于治疗牙周致病菌感染引起的牙周疾病的药物 - Google Patents

用于治疗牙周致病菌感染引起的牙周疾病的药物 Download PDF

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WO2021047137A1
WO2021047137A1 PCT/CN2020/071939 CN2020071939W WO2021047137A1 WO 2021047137 A1 WO2021047137 A1 WO 2021047137A1 CN 2020071939 W CN2020071939 W CN 2020071939W WO 2021047137 A1 WO2021047137 A1 WO 2021047137A1
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periodontal
exosomes
periodontitis
pharmaceutical composition
sirna
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French (fr)
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谭家莉
李丹枫
匡威
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中山大学附属口腔医院
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/30—Animals modified by surgical methods
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the invention belongs to the technical field of medicine, and relates to a medicine for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogen infection.
  • Periodontitis is a common oral disease, which is mainly caused by the innate immunity, adaptive immunity and inflammatory response initiated by pathogenic microorganisms in dental plaque biofilm. It manifests as a series of complex inflammatory syndromes, which mainly affect the periodontal membrane and its surroundings. And the tissues that support the teeth can cause the progressive loss of periodontal alveolar bone, which can lead to tooth loss.
  • the pathogenic microorganisms in the dental plaque biofilm are the main cause of periodontal disease.
  • the gingival microbial community is transplanted in the periodontal pocket, so that the dominant microbial community in the periodontal pocket changes from Gram-positive aerobic bacteria to leather.
  • Lan's negative anaerobes cause periodontal disease in healthy periodontal tissues.
  • Porphyromonas gingivalis is considered to be the main pathogen of periodontitis. It is less abundant in the oral cavity of healthy people, but it is significantly increased in the destructive structure of periodontitis, plaque and calculus.
  • Porphyromonas gingivalis has a variety of virulence factors, including: fimbriae, degrading enzymes and lipopolysaccharides.
  • a low transplanted amount of Porphyromonas gingivalis can induce changes in the number and composition of oral symbiotic microorganisms, and promote the transformation of harmful factors in bacterial colonies. Porphyromonas gingivalis can also help suppress the body's immune system and create a good living environment for other bacteria. In the mouse periodontitis model, low concentrations of Porphyromonas gingivalis can induce inflammatory periodontitis through the interaction of oral symbiotic microbiota and the complement system.
  • the host's recognition of pathogenic microorganisms is an important part of the pathological mechanism of periodontal disease, which can quickly trigger an immune response, and then promote the activation and recruitment of important immune components through the secretion of cytokines and chemokines.
  • the detection of microbial products can promote the stability and proliferation of the microbial transplantation community, so that the host oral microbial community and the body's immune system can maintain a stable balance.
  • the characteristics of disease progression and damage in patients with chronic periodontitis have been clear, the mechanism of protection and damage to the host periodontal tissue has not been fully elucidated.
  • cytokines and inflammatory mediators include IL-1, IL-6 and TNF- ⁇ . These cytokines and inflammatory mediators can be used alone or in combination to promote the absorption and absorption of alveolar bone. Collagen breaks down.
  • Natural killer-like B cells are a new type of immune cell subgroup found in the spleen and mesenteric lymph nodes of mice. They mainly secrete IL-18 and IL-12 and exert anti-microbial activity. The role of infection. IL-18 is the main cytokine secreted by NKB cells, which can induce lymphocytes and splenocytes to produce IFN- ⁇ . IL-18 and IL-12 double gene defects can reduce NK cell activity and cell response. IL-18 and IL-12 have the effect of activating NK cells in local inflammatory response. Since the onset of periodontitis is the result of the interaction between oral parasites and the body's immune system, and NKB cells play an important role in bacterial infections, NKB cells and IL-18 may play a role in the onset of periodontitis.
  • IL-18 plays a role by binding to its receptor IL-18R.
  • IL-18's receptor IL-18R is widely present in a variety of cells. It is composed of heterodimer ⁇ chain (IL-18R ⁇ ) and ⁇ chain ( IL-18R ⁇ ), where IL-18R ⁇ is the ligand binding chain, and IL-18R ⁇ is used to mediate IL-18 signal transduction. Reducing the expression of IL-18 receptor can reduce the secretion of pro-inflammatory cytokines by periodontal ligament cells, thereby inhibiting the occurrence and development of periodontitis.
  • RNA interference RNA interfering, RNAi
  • RNAi refers to the inhibition of target mRNA by degradation or translation, thereby causing post-transcriptional gene silencing. It is used in anti-inflammatory, antiviral and other medicines.
  • the field has broad application prospects.
  • siRNA efficiently and specifically blocks the expression of homologous genes in the body, promotes the degradation of homologous mRNA, and induces cells to show a specific phenotype of gene deletion.
  • siRNA is extremely easy to be degraded by ribozyme (RNase) in vivo, has a short half-life and low transfection efficiency of cells. Therefore, how to efficiently introduce specific silencing siRNA into target organs without damaging normal tissues and siRNA China has become an urgent problem in gene therapy.
  • RNase ribozyme
  • PEI-RNA complexes tend to accumulate in the liver, lungs, spleen, and kidneys, which limits them.
  • Application in other tissues can easily lead to the activation of inflammasomes related to IL-1 induction and apoptosis, and promote inflammation; liposomes or lipid nanoparticles not only have hepatotoxicity, but also can adsorb opsonins, activate complement and Coagulation factors, and due to their inherent net charge and size, easily lead to the phagocytosis of the mononuclear phagocyte system.
  • this process can lead to cellular stress, inflammasome activation and cell apoptosis.
  • Exosomes are a typical lipid bilayer membrane structure with a diameter of about 30-150nm. Its lipid bilayer structure and surface membrane proteins make exosomes have high transfection efficiency in vivo. It is an important carrier of information transmission between cells and has been developed for the transmission of various substances (including siRNA) between cells. Compared with traditional drug carriers such as liposome transfection reagents, exosomes, as a natural drug carrier, have the advantages of good biocompatibility, degradability, and low immunogenicity.
  • CN 102488654 A discloses a medicine for treating periodontitis, which is prepared by weight parts of 5-250 parts of doxycycline, 25-1000 parts of metronidazole and 10000 parts of glycerin.
  • CN 104688998 A discloses a traditional Chinese medicine tablet for treating periodontitis and a preparation method thereof, respectively taking 8-14 parts of forsythia water extract, 3-7 parts of coptis water extract, and ethanol extract of mallow seeds 5 by weight -11 parts, 4-8 parts of Russula Ethanol Extract, 4-9 parts of Radix Paeoniae Ethyl Acetate Extract, 2-5 Parts of Fragrant Radix Aconiti, 3-6 Parts of Fragmented August Leaf, 15 Magnesium Stearate -20 parts, 2-6 parts of glycerin, 7-16 parts of modified starch, the above-mentioned ingredients are mixed uniformly and then compressed with a tablet machine, which is a Chinese medicine tablet for treating periodontitis.
  • CN 103638408 A discloses a topical ointment for the treatment of periodontitis, which is prepared from the following components according to the weight ratio range as shown: 20-30 parts of Xiangjia Bark, 15-20 parts of Prunella vulgaris, 8-15 parts of Radix Rehmanniae, 6-12 parts of wild chrysanthemum, 5-10 parts of Smilax chinensis, 7-10 parts of Forsythia, 8-12 parts of Stemona, 5-10 parts of Peiran, 8-12 parts of Phellodendron amurense, 6-10 parts of Lyceum chinensis, 6-10 parts of Galla Chinensis 1 -3 copies.
  • the prior art treats periodontitis using antibiotics or traditional Chinese medicines, which have complex ingredients, large side effects, and insignificant therapeutic effects, and cannot achieve the purpose of radical cure.
  • periodontitis The pathogenesis of periodontitis involves the interaction between microorganisms and the body's immune system. The pathogenesis has not been fully elucidated. In-depth exploration of the immune pathogenesis of periodontitis provides theoretical and experimental basis for finding new targets for the treatment of periodontitis. At present, it is urgent It is necessary to develop drugs that can effectively inhibit periodontitis inflammation, reduce the incidence of periodontitis from the fundamental source, and prevent the occurrence of periodontitis.
  • the present invention provides a medicine for treating periodontal ligament cell dysfunction and alveolar bone loss caused by periodontal pathogen infection.
  • the medicine is based on IL-18 antibody and loaded with IL-18R.
  • Receptor siRNA exosomes are functional components.
  • IL-18 antibody and IL-18R receptor siRNA exosomes play a synergistic effect to inhibit periodontitis alveolar bone resorption and the expression of periodontitis-related inflammatory cytokines and chemokines , To reduce the incidence of periodontal disease from the fundamental source.
  • the present invention adopts the following technical solutions:
  • the present invention provides a pharmaceutical composition, which comprises IL-18 antibody and/or IL-18R receptor siRNA exosomes.
  • IL-18 can promote periodontal ligament cells to secrete a variety of cytokines that promote inflammatory response, thereby promoting the disease process of periodontitis.
  • the drug uses IL-18 antibody and exosomes loaded with IL-18R receptor siRNA as its effective components.
  • IL-18 antibody reduces the expression level of IL-18 by targeting IL-18
  • IL-18R Receptor siRNA exosomes inhibit the expression of IL-18mRNA by translation and reduce the expression level of IL-18.
  • the two play a synergistic effect to inhibit the expression of inflammatory cytokines and chemokines in periodontal tissues, and reduce the expression of inflammatory cytokines and chemokines.
  • the effect of chemokines on the secretion of pro-inflammatory cytokines by periodontal ligament cells fundamentally reduces inflammatory infiltration, improves periodontal ligament cell dysfunction, and prevents the occurrence and development of periodontal diseases.
  • the concentration of the IL-18 antibody is 2-10 ⁇ g/mL, for example, 2 ⁇ g/mL, 3 ⁇ g/mL, 4 ⁇ g/mL, 5 ⁇ g/mL, 6 ⁇ g/mL, 7 ⁇ g/mL, 8 ⁇ g/mL, 9 ⁇ g /mL or 10 ⁇ g/mL, preferably 5 to 8 ⁇ g/mL.
  • the concentration of the IL-18R receptor siRNA exosomes is 0.5-2 ⁇ g/1 ⁇ L, for example 0.5 ⁇ g/1 ⁇ L, 0.6 ⁇ g/1 ⁇ L, 0.7 ⁇ g/1 ⁇ L, 0.8 ⁇ g/1 ⁇ L, 0.9 ⁇ g/1 ⁇ L , 1.0 ⁇ g/1 ⁇ L, 1.1 ⁇ g/1 ⁇ L, 1.2 ⁇ g/1 ⁇ L, 1.3 ⁇ g/1 ⁇ L, 1.4 ⁇ g/1 ⁇ L, 1.5 ⁇ g/1 ⁇ L, 1.6 ⁇ g/1 ⁇ L, 1.7 ⁇ g/1 ⁇ L, 1.8 ⁇ g/1 ⁇ L, 1.9 ⁇ g/1 ⁇ L Or 2.0 ⁇ g/1 ⁇ L, preferably 1.0 ⁇ g/1 ⁇ L.
  • the mass ratio of the IL-18 antibody to the IL-18R receptor siRNA exosomes is (0.01-0.05):1, for example, it can be 0.01:1, 0.02:1, 0.025:1, 0.03: 1. 0.04:1 or 0.05:1, preferably (0.02-0.025):1.
  • nucleic acid sequence of the siRNA in the IL-18R ⁇ receptor siRNA exosomes is shown in SEQ ID NO: 1 to 2:
  • SEQ ID NO:1 sense chain: 5′-AAACUCGGCAUCCUUCAGGUUTT-3′;
  • SEQ ID NO: 2 antisense strand: 5′-AACCUGAAGGAUGCCGAGUUUTT-3′.
  • the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention provides a pharmacodynamic evaluation model of the pharmaceutical composition as described in the first aspect, and the model is a periodontal disease model mouse.
  • the present invention provides a method for preparing the drug efficacy evaluation model as described in the second aspect, which adopts silk thread ligation combined with Porphyromonas gingivalis induction for model construction.
  • the method includes the following steps:
  • the silk thread is placed in the upper jaw of healthy mice without periodontal disease, and Porphyromonas gingivalis is inoculated on the gingival part and/or smeared on the oral cavity to induce periodontal disease in the mice, and the periodontal disease model mice are obtained .
  • the Porphyromonas gingivalis is a strain of P.gingivalis.
  • the concentration of the Porphyromonas gingivalis is 1 ⁇ 10 8 to 1 ⁇ 10 10 CFU/mL, for example, 1 ⁇ 10 8 CFU/mL, 1 ⁇ 10 9 CFU/mL or 1 ⁇ 10 10 CFU/mL.
  • the present invention provides a method for preparing a pharmacodynamic evaluation model as described in the second aspect.
  • the method adopts silk thread ligation combined with Porphyromonas gingivalis induction for model construction, which includes the following steps:
  • BHI medium bovine heart brain infusion medium
  • Porphyromonas gingivalis (P. gingivalis ATCC 33277) was inoculated into the prepared medium, cultured under 85% nitrogen, 10% hydrogen, 5% carbon dioxide, 37°C under strict anaerobic conditions, and passaged 2 to 3 After the second, incubate for 2 days; when the concentration reaches 1 ⁇ 10 9 CFU/mL, resuspend the bacteria in PBS buffer containing 2% sodium carboxymethyl cellulose for later use;
  • mice with intact dentition, no caries and periodontal disease 1% sodium pentobarbital (0.15mL/20g) intraperitoneal injection anesthesia, fixed limbs in supine position, 1% iodine tincture disinfects the oral cavity and perioral area, 75% Alcohol is deiodized, and the upper jaw is fixed with silk thread;
  • the IL-18 antibody treatment experimental group uses local oral smear and uses a micro syringe to locally inject the Porphyromonas gingivalis suspension into the cheek and palatal periodontal of the maxillary molars
  • 50 ⁇ L of anti-IL-18 antibody with a concentration of 5 ⁇ g/mL was injected locally into the periodontal tissues of the cheek and palatal sides of the maxillary molars at the same time, and injected once every 2 to 4 days;
  • the control periodontitis group uses a micro syringe to locally inject the Porphyromonas gingivalis suspension into the maxillary molar cheek and palatal periodontal tissue;
  • the normal group uses 2% Apply the sodium carboxymethyl cellulose-PBS solution locally in the oral cavity, and use a micro syringe to inject an equal amount of PBS into the periodontal tissue;
  • mice Four weeks later, the mice were sacrificed by cervical dislocation and fixed.
  • the present invention provides an application of the pharmaceutical composition as described in the first aspect and/or the pharmacodynamic evaluation model as described in the second aspect in the preparation of periodontal disease treatment drugs.
  • the periodontal support tissue loss includes periodontal ligament loss and/or alveolar bone loss.
  • the alveolar bone loss is caused by periodontal pathogen infection.
  • the periodontal pathogens include Porphyromonas gingivalis.
  • the Porphyromonas gingivalis includes P. gingivalis strain.
  • the main pathogen of periodontitis Porphyromonas gingivalis
  • periodontal ligament dysfunction leads to periodontal ligament loss.
  • the pharmaceutical composition reduces IL-18 by inhibiting natural killer-like B cells (NKB) from the immune-inflammatory pathogenesis of periodontitis.
  • periodontal ligament cells to secrete pro-inflammatory cytokines, inhibit periodontitis alveolar bone resorption, and the expression of periodontitis-related inflammatory cytokines and chemokines, thereby effectively inhibiting periodontitis inflammation and alveolar bone resorption, And reduce the incidence of periodontitis from the pathogenesis of inflammation, thereby preventing the occurrence of periodontitis.
  • the present invention has the following beneficial effects:
  • the present invention found that the cytokine IL-18 can promote periodontal ligament cells to secrete a variety of cytokines that promote inflammatory response, leading to the loss of periodontal ligament tissue, enhancing the inflammatory response, leading to aggravated alveolar bone loss and promoting periodontal The disease process of inflammation;
  • IL-18 antibody is used as a targeted drug to prevent and treat the dysfunction of periodontal ligament cells
  • IL-18R receptor siRNA exosomes can inhibit the expression of IL-18mRNA by translation and reduce IL-
  • the expression level of 18, the two play a synergistic effect, thereby inhibiting the loss of periodontal ligament tissue, alveolar bone resorption and the expression of periodontitis-related inflammatory cytokines and chemokines, and reducing the incidence of periodontitis from the fundamental source.
  • preventing the occurrence and development of periodontitis has great potential application value for the clinical prevention and treatment of periodontitis, and lays the foundation for further research on the biological functions of IL-18 antibody and IL-18R receptor siRNA;
  • the present invention provides a mouse model of periodontitis, which is induced by silk ligation combined with Porphyromonas gingivalis, and the effects of IL-18 antibody and IL-18R receptor siRNA on periodontitis Therapeutic and preventive effects.
  • Figure 1 shows the effect of IL-18 on the secretion of pro-inflammatory cytokines in periodontal ligament cells.
  • Figure 1(A) shows the changes in the expression level of IFN- ⁇
  • Figure 1(B) shows the changes in the expression level of GM-CSF
  • Figure 1(C) shows the changes in the expression level of IL-2
  • Figure 1(D) shows the changes in the expression level of TNF- ⁇ ;
  • Fig. 2 is a CT image of periodontitis alveolar bone loss.
  • Fig. 2(A) is a normal control group
  • Fig. 2(B) is a simple silk ligation control group
  • Fig. 2(C) is a periodontitis group
  • Fig. 2( D) is the periodontitis + IL-18 antibody treatment group
  • Figure 2 (E) is the periodontitis + IL-18 ⁇ siRNA exosomes group
  • Figure 2 (F) is the periodontitis + IL-18 antibody + IL- 18 ⁇ siRNA exosomes group;
  • Figure 3 is a histogram of the periodontitis alveolar bone loss distance (mm) calculated according to the micro-CT results;
  • Figure 4(A) shows the expression of cytokine IL-1 ⁇ in periodontal tissue
  • Figure 4(B) shows the expression of cytokine IL-6
  • Figure 4(C) shows the expression of cytokine IL-8
  • Figure 4 (D) is the expression of IL-18R ⁇ ;
  • Figure 5 shows the results of histological staining of the periodontal tissue area of the maxillary molars.
  • the IL-18 antibody was purchased from the Medical & Biological Laboratories Co., LTD. Code No: D 048-3;
  • the P.gingivalis strain was purchased from the American ATCC Bacteria Bank (ATCC 33277);
  • C57BL/6 mice were purchased from the Animal Experiment Center of Sun Yat-Sen University.
  • Example 1 Silk thread ligation combined with Porphyromonas gingivalis periodontitis model
  • the 0.5% hemin vitamin solution and the bovine heart brain infusion medium (BHI medium) were mixed uniformly at a ratio of 1:100, and the P.gingivalis strain was inoculated into the medium.
  • the medium was inoculated with 85% nitrogen, 10% hydrogen, and 5%. Cultivate under strict anaerobic conditions under carbon dioxide and 37°C;
  • mice Anesthetized by intraperitoneal injection of 4% chloric acid hydrate (0.35 mg/kg). After anesthesia, the mice were placed in a supine position and fixed on the operating table with tape. The oral cavity and perioral area were disinfected with 1% iodine tincture, and 75% alcohol was used to deiodize. Pull and fix the upper incisor teeth with cotton thread to make them look like a wide mouth; separate the left side, the gingival tissue of the maxillary first molar with a pointed probe, and insert silk thread into the neck between the first and second molars, in the middle of the first molar. Ligate the nipple and place it on the gingival margin of the first molar, and place it in the gingival sulcus as much as possible;
  • Porphyromonas gingivalis 100 ⁇ L was inoculated on the palatal median gingival sulcus floor of the right maxillary second molar, and injected twice a week.
  • the nucleic acid sequence of IL-18R ⁇ siRNA is shown in SEQ ID NO: 1 to 2:
  • SEQ ID NO:1 sense chain: 5′-AAACUCGGCAUCCUUCAGGUU-3′;
  • SEQ ID NO: 2 antisense strand: 5′-AACCUGAAGGAUGCCGAGUUUTT-3′;
  • Example 3 The effect of IL-18 on the secretion of pro-inflammatory cytokines in periodontal ligament cells
  • IL-18 was used to stimulate and culture periodontal ligament cells for 96 hours and then the cell supernatant was collected, and the expression levels of IFN- ⁇ , GM-CSF, IL-2 and TNF- ⁇ were detected by ELISA.
  • Cytokines Control group pg/mL
  • IL-18 stimulation pg/mL
  • IFN- ⁇ 82.11 ⁇ 17.24 687.1 ⁇ 109.3*
  • GM-CSF 32.90 ⁇ 7.29 89.27 ⁇ 19.47*
  • IL-2 114.2 ⁇ 28.39 197.6 ⁇ 54.28*
  • Drug application 50 ⁇ L of anti-IL-18 antibody was injected into the palatal median gingival sulcus floor of the bilateral maxillary first and second molars of periodontitis model mice, at a concentration of 5 ⁇ g/mL, injected once a week for 4 consecutive weeks. A total of 4 times.
  • the periodontitis model mice prepared in Example 1 were provided with IL-18R ⁇ siRNA exosomes.
  • the nucleic acid sequence of the siRNA is shown in SEQ ID NO: 1 to 3. The specific steps are as follows:
  • IL-18R ⁇ siRNA exosomes were injected into the palatal median gingival sulcus floor of the bilateral maxillary first and second molars of periodontitis model mice, injected once a week for 4 consecutive weeks, a total of 4 times.
  • the PBS mixture of anti-IL-18 antibody (5 ⁇ g/mL) and siRNA exosomes (10 ⁇ g per injection per side) was injected into the palatal median sulcus floor of the bilateral maxillary first and second molars of periodontitis model mice , Inject once a week for 4 consecutive weeks, a total of 4 times.
  • Example 7 The therapeutic effect of IL-18 antibody synergistically with siRNA exosomes
  • siRNA exosomes group was irradiated with micro-CT.
  • Figure 2 (A) is the normal control group
  • Figure 2 (B) is the simple silk ligation control group
  • Figure 2 (C) is the periodontitis group
  • Figure 2 (D) is the periodontitis + IL-18 antibody treatment group
  • Figure 2 (E) is the periodontitis + IL-18R ⁇ siRNA exosomes group
  • Figure 2 (F) is the periodontitis + IL-18 antibody + IL-18R ⁇ siRNA exosomes group of micro-CT detection results.
  • the amount of alveolar bone loss (the distance between the enamel-cementum boundary and the alveolar crest) between the first molar and the second molar in the periodontitis group was higher than that of the normal control group and the simple silk thread group;
  • Treatment with IL-18 antibody or IL-18R ⁇ siRNA exosomes can reduce the amount of alveolar bone loss; the combined treatment of IL-18 antibody and IL-18R ⁇ siRNA exosomes has a more significant effect on reducing alveolar bone loss.
  • Figure 3 shows the periodontitis alveolar bone loss distance (mm) calculated according to the micro-CT results. After treatment with IL-18 antibody and siRNA exosomes, the periodontitis alveolar bone loss distance is higher than that of bacteria-induced periodontitis The group distance is significantly reduced.
  • Figure 4 (A) is the expression of periodontal tissue cytokine IL-1 ⁇
  • Figure 4 (B) is the expression of periodontal tissue cytokine IL-6
  • Figure 4 (C) is the periodontal tissue cytokine IL-8
  • Figure 4(D) shows the expression of IL-18R ⁇ . It can be seen that after the treatment of IL-18 antibody and siRNA exosomes, IL-1 ⁇ , IL-6, IL-8 and IL in periodontal tissue The expression of -18R ⁇ decreased significantly, indicating that IL-18 antibody and IL-18R ⁇ siRNA exosomes have a synergistic effect and have a significant intervention efficiency on gene mRNA.
  • Figure 5 is the results of histological staining of the periodontal tissue area of the maxillary molars. It can be seen that the inflammatory cells in the periodontal tissue between the first molar and the second molar in the bacteria-induced periodontitis group are more infiltrated than the normal control group and simple The silk thread group was significantly increased; while the IL-18 antibody or IL-18R ⁇ siRNA exosome treatment group alone alleviated inflammatory cell infiltration; the combination of IL-18 antibody and IL-18R ⁇ siRNA exosomes significantly reduced inflammation Cell infiltration.
  • the present invention finds that the cytokine IL-18 can promote periodontal ligament cells to secrete a variety of cytokines that promote inflammatory response, which leads to the loss of periodontal ligament tissue, enhances the inflammatory response, and then leads to increased alveolar bone loss and promote The disease process of periodontitis; the present invention uses IL-18 antibody to cooperate with IL-18R ⁇ receptor siRNA exosomes to prevent and treat the dysfunction of periodontal ligament cells, inhibit periodontal ligament tissue loss, alveolar bone resorption and The expression of inflammatory cytokines and chemokines related to periodontitis can reduce the incidence of periodontitis from the fundamental source, thereby preventing the occurrence and development of periodontitis. It has potential great application value for the clinical prevention and treatment of periodontitis. And it laid the foundation for further research on the biological functions of IL-18 antibody and IL-18R ⁇ receptor siRNA.

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Abstract

本发明提供了用于治疗牙周致病菌感染引起的牙周膜细胞功能紊乱与牙槽骨丧失的药物，所述药物包括IL-18抗体和/或IL-18R受体siRNA外泌体。本发明的药物以IL-18抗体和/或装载IL-18R受体siRNA外泌体为功效成分，通过与牙周组织中IL-18结合，抑制牙周膜细胞分泌促炎细胞因子，改善炎症状态下牙周膜细胞功能紊乱导致的牙周支持组织丧失及炎症细胞浸润的作用，从而阻止牙周疾病的发生发展，对牙周疾病的临床预防治疗有着潜在的巨大应用价值。

Description

用于治疗牙周致病菌感染引起的牙周疾病的药物 技术领域
本发明属于医药技术领域，涉及用于治疗牙周致病菌感染引起的牙周膜细胞功能紊乱与牙槽骨丧失的药物。
背景技术
牙周炎是常见的口腔疾病，主要由牙菌斑生物膜中的致病微生物启动固有免疫、适应性免疫和炎症应答引起，表现为一系列复杂的炎症症候群，主要影响牙周膜以及包绕和支持牙齿的组织，可导致牙周牙槽骨进行性丧失，进而造成牙齿脱落。
牙菌斑生物膜中的致病微生物是引起牙周疾病的主要原因，牙龈微生物群落在牙周袋中移植，使牙周袋中占主导的微生物群落从革兰氏阳性需氧菌转变为革兰氏阴性厌氧菌，使得健康的牙周组织发生牙周疾病。牙龈卟啉单胞菌被认为是牙周炎的主要致病菌，在健康人的口腔中数量较少，但在牙周炎的破坏性结构菌斑和牙石中显著升高。牙龈卟啉单胞菌具有多种毒力因素，包括：菌毛、降解酶和脂多糖。低移植量的牙龈卟啉单胞菌即可诱发口腔共生微生物群数量和组成成分的改变，促进细菌菌落中有害因素的转变。牙龈卟啉单胞菌还可以协助抑制机体免疫系统，为其他细菌创造良好的生存环境。在小鼠牙周炎模型中，低浓度的牙龈卟啉单胞菌通过口腔共生微生物群和补体系统的相互作用，诱导炎症性牙周炎的发生。
宿主对病原微生物的识别是牙周疾病病理机制中的重要组成部分，可以迅速引发免疫应答，进而通过分泌细胞因子和趋化因子促进重要免疫组成成分的活化和募集。检测微生物产物可以促进微生物移植群落的稳固和增殖，使宿主口腔微生物群落与机体免疫系统保持稳态平衡。目前，虽然慢性牙周炎患者疾病进展损伤的特征已明确，但是对宿主牙周组织保护和损伤的机制仍未完全阐明。
牙周疾病的临床表现千变万化，在疾病活跃的部位，肥大细胞、单核/巨噬细胞和浆细胞的数量明显升高，这些细胞通过进一步活化单核/巨噬细胞、淋巴细胞、成纤维细胞和其他细胞成分，分泌包括IL-1、IL-6和TNF-α在内的多种细胞因子和炎症介质，这些细胞因子和炎症介质可以单独使用或联合发挥作用，促进牙槽骨的吸收和胶原分解。
自然杀伤样B细胞(Natural killer-like B cells，NKB cells)是在小鼠的脾脏和肠系膜淋巴结中发现的一类新的免疫细胞亚群，主要分泌IL-18和IL-12，发挥抗微生物感染的作用。IL-18是NKB细胞分泌的主要细胞因子，可以诱导淋巴细胞和脾细胞产生IFN-γ。IL-18和IL-12双基因缺陷可降低NK细胞活性和细胞应答，IL-18和IL-12在局部炎症应答中具有活化NK细胞的作用。由于牙周炎的发病是口腔寄生菌与机体免疫系统相互作用的结果，而NKB细胞在细菌感染中发挥重要作用，因此NKB细胞和IL-18可能在牙周炎发病中发挥作用。
IL-18通过与其受体IL-18R结合来发挥作用，IL-18的受体IL-18R广泛地存在于多种细胞中，由异源二聚体α链(IL-18Rα)和β链(IL-18Rβ)组成，其中，IL-18Rα是配体结合链，IL-18Rβ用于介导IL-18的信号传导。降低IL-18受体的表达，可以减轻牙周膜细胞分泌促炎细胞因子，进而抑制牙周炎的发生发展。
近年来，RNA干扰(RNA interfering，RNAi)获得广泛关注，作为一种新的基因治疗技术，RNAi是指通过降解或翻译抑制目标mRNA，从而引起转录后基因沉默，在抗炎症、抗病毒等医药领域具有广阔的应用前景。siRNA通过RNAi技术，高效、特异地阻断体内同源基因的表达，促使同源mRNA降解，诱导细胞表现出基因缺失的特定表型。但是，siRNA在生物体内极容易被核酶(RNase)降解，半衰期短，转染细胞效率低，因此，如何在不损伤正常组织及siRNA的前提下，高效地将特异性沉默siRNA导入到靶器官中，成为基因治疗亟待解决的问题。
目前，治疗性siRNA的载体研究主要集中在聚阳离子聚乙烯亚胺(PEI)纳米颗粒和脂质体上，然而，PEI-RNA复合物容易在肝脏、肺脏、脾脏和肾脏中积累，限制了其在其他组织中的应用，容易导致与IL-1诱导和细胞凋亡相关的炎症小体激活，促进炎症；脂质体或脂质纳米颗粒不仅具有肝毒性，而且可以吸附调理素，激活补体和凝血因子，并由于其固有的净电荷和尺寸容易导致单核吞噬细胞系统的吞噬作用，与PEI一样，这一过程可导致细胞应激、炎症小体激活和细胞凋亡。
外泌体(exosomes)是一种典型的脂质双分子层膜结构，直径约30～150nm，其脂质双分子层结构和表面膜蛋白使得外泌体在体内具有较高的转染效率，是细胞间信息传递的重要载体，已被开发用于各类物质(包括siRNA)的细胞间传输。与传统的药物载体如脂质体转染试剂相比，外泌体作为一种天然药物载体，具有生物相容性好、可降解、免疫原性小等优点。
CN 102488654 A公开了一种治疗牙周炎的药物，按重量份数由5-250份的多西环素、25-1000份的甲硝唑和10000份的甘油制成。CN 104688998 A公开了一种治疗牙周炎的中药片剂及其制备方法，按重量分别取连翘水提物8-14份、黄连水提物3-7份、冬葵子乙醇提取物5-11份、芸香草乙醇提取物4-8份、赤芍乙酸乙酯提取物4-9份、粉碎的香附子2-5份、粉碎的八月札3-6份、硬脂酸镁15-20份、甘油2-6份、改性淀粉7-16份，将上述的成分混合均匀后用压片机压片，为治疗牙周炎的中药片剂。CN 103638408 A公开了一种治疗牙周炎的外用药膏，由以下组分按照所示重量份比例范围配制：香加皮20-30份、夏枯草15-20份、生地黄8-15份、野菊花6-12份、土茯苓5-10份、连翘7-10份、百部8-12份、佩兰5-10份、土黄柏8-12份、千金子6-10份、五倍子1-3份。现有技术治疗牙周炎均采用抗生素或中药，成分复杂，副作用大，治疗效果不显著，无法达到根治的目的。
牙周炎的发病涉及微生物与机体免疫系统的相互作用，发病机制尚未完全 阐明，而深入探索牙周炎的免疫发病机制为寻找牙周炎治疗的新靶点提供理论和实验依据，目前，迫切需要研发能够有效地抑制牙周炎炎症的药物，从根本源头上减轻牙周炎发病程度，阻止牙周炎的发生。
发明内容
针对现有技术的不足，本发明提供了用于治疗牙周致病菌感染引起的牙周膜细胞功能紊乱与牙槽骨丧失的药物，所述药物以IL-18抗体和负载有IL-18R受体siRNA的外泌体为功效成分，IL-18抗体和IL-18R受体siRNA外泌体发挥协同作用，能够抑制牙周炎牙槽骨吸收和牙周炎相关炎症细胞因子和趋化因子的表达，从根本源头上减轻牙周疾病的发病程度。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种药物组合物，所述药物包括IL-18抗体和/或IL-18R受体siRNA外泌体。
本发明中，发明人在研究免疫反应与牙周疾病关系的基础上，意外发现在牙周疾病患者中，自然杀伤样B细胞(Natural killer-like B cells，NKB)分泌的细胞因子IL-18在外周血和龈沟液中的表达量中显著上升，不仅如此，牙周炎患者龈沟液中的IL-18与AL(Attachment loss，附着丧失)呈显著正相关，由此说明IL-18是NKB细胞在牙周炎发病中的主要效应分子；在此基础上，发明人进一步发现，牙周基础治疗后龈沟液中IL-18的水平显著降低，当去除菌斑等微生物致病因素后，微生物数量和抗原水平均显著下降，降低了局部炎症应答，对NKB细胞的刺激降低，其分泌IL-18的水平亦降低，进一步证实IL-18参与了牙周炎的发病，且主要在牙周组织局部发挥作用，因此发明人采用IL-18抗体所制备的药物来抑制牙周炎的症状，有效抑制牙周炎炎症。
本发明中，发明人还发现IL-18可促进牙周膜细胞分泌多种促进炎症应答的细胞因子，进而促进牙周炎的疾病进程。
本发明中，所述药物以IL-18抗体和负载有IL-18R受体siRNA的外泌体为 功效成分，IL-18抗体通过靶向IL-18降低IL-18的表达水平，IL-18R受体siRNA外泌体通过翻译抑制IL-18mRNA的表达降低IL-18的表达水平，两者发挥协同作用，抑制了牙周组织中炎症细胞因子和趋化因子的表达，减少了炎症细胞因子和趋化因子对牙周膜细胞分泌促炎细胞因子的作用，从根本上减轻了炎症浸润，改善了牙周膜细胞功能紊乱，阻止了牙周疾病的发生发展。
优选地，所述IL-18抗体的浓度为2～10μg/mL，例如可以是2μg/mL、3μg/mL、4μg/mL、5μg/mL、6μg/mL、7μg/mL、8μg/mL、9μg/mL或10μg/mL，优选为5～8μg/mL。
优选地，所述IL-18R受体siRNA外泌体的浓度为0.5～2μg/1μL，例如可以是0.5μg/1μL、0.6μg/1μL、0.7μg/1μL、0.8μg/1μL、0.9μg/1μL、1.0μg/1μL、1.1μg/1μL、1.2μg/1μL、1.3μg/1μL、1.4μg/1μL、1.5μg/1μL、1.6μg/1μL、1.7μg/1μL、1.8μg/1μL、1.9μg/1μL或2.0μg/1μL，优选为1.0μg/1μL。
优选地，所述IL-18抗体与所述IL-18R受体siRNA外泌体的质量比为(0.01～0.05):1，例如可以是0.01:1、0.02:1、0.025:1、0.03:1、0.04:1或0.05:1，优选为(0.02～0.025):1。
优选地，所述IL-18Rα受体siRNA外泌体中siRNA的核酸序列如SEQ ID NO:1～2所示：
SEQ ID NO:1(正义链)：5′-AAACUCGGCAUCCUUCAGGUUTT-3′；
SEQ ID NO:2(反义链)：5′-AACCUGAAGGAUGCCGAGUUUTT-3′.
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第二方面，本发明提供了一种如第一方面所述的药物组合物的药效评价模型，所述模型为牙周疾病模型小鼠。
第三方面，本发明提供了一种如第二方面所述的药效评价模型的制备方法，所述方法采用丝线结扎联合牙龈卟啉单胞菌诱导进行模型构建。
优选地，所述方法包括以下步骤：
将丝线放置于无牙周疾病的健康小鼠上颌，并将牙龈卟啉单胞菌接种于牙龈部位和/或涂抹于口腔局部，诱导小鼠牙周疾病，得到所述牙周疾病模型小鼠。
优选地，所述牙龈卟啉单胞菌为P.gingivalis菌株。
优选地，所述牙龈卟啉单胞菌的浓度为1×10 8～1×10 10CFU/mL，例如可以是1×10 8CFU/mL、1×10 9CFU/mL或1×10 10CFU/mL。
作为优选技术方案，本发明提供了一种如第二方面所述的药效评价模型的制备方法，所述方法采用丝线结扎联合牙龈卟啉单胞菌诱导进行模型构建，包括如下步骤：
(1)细菌培养：
将0.5％氯化血红素维生素溶液和牛心脑浸汁液培养基(BHI培养基)按照1:100混合均匀，将P.gingivalis菌株接种于培养基中；
将牙龈卟啉单胞菌(P.gingivalis ATCC 33277)接种于上述配置好的培养基中，在85％氮气、10％氢气、5％二氧化碳、37℃严格厌氧条件下培养，传代2～3次后，培养2天；达浓度1×10 9CFU/mL时，将菌体用含2％羧甲基纤维素钠的PBS缓冲液重悬待用；
(2)丝线结扎小鼠牙周炎模型建立及实验分组：
筛选出牙列完整、无龋病及牙周疾病的小鼠，1％戊巴比妥钠(0.15mL/20g)腹腔注射麻醉，仰卧位固定四肢，1％碘酊消毒口腔及口周，75％酒精脱碘，以丝线牵拉固定上颌；
将100μm直径的无菌缝合丝线置于上颌第二磨牙龈沟上1mm处，从其近远中穿过邻间隙，在颊侧或腭侧打结；
每4天检查丝线是否脱落，有脱落者及时重新结扎；IL-18抗体处理实验组采用口腔局部涂抹并使用微量注射器局部注射牙龈卟啉单胞菌悬浊液于上颌磨牙颊、腭侧牙周组织中，同时局部注射50μL浓度为5μg/mL的抗-IL-18抗体于上颌磨牙颊、腭侧牙周组织中，每2～4天注射一次；
使用棉签涂擦多余细菌于口腔内上颌磨牙区域；对照牙周炎组使用微量注射器局部注射牙龈卟啉单胞菌悬浊液于上颌磨牙颊、腭侧牙周组织中；正常组则使用2％羧甲基纤维素钠-PBS溶液口腔内局部涂抹，并使用微量注射器注射等量PBS于牙周组织中；
4周后，颈椎脱臼处死小鼠，取材固定。
第四方面，本发明提供了一种如第一方面所述的药物组合物和/或如第二方面所述的药效评价模型在制备牙周疾病治疗药物中的应用。
优选地，所述牙周支持组织丧失包括牙周膜丧失和/或牙槽骨丧失。
优选地，所述牙槽骨丧失由牙周致病菌感染引起。
优选地，所述牙周致病菌包括牙龈卟啉单胞菌。
优选地，所述牙龈卟啉单胞菌包括P.gingivalis菌株。
本发明中，牙周炎主要致病菌牙龈卟啉单胞菌通过刺激牙周膜细胞分泌促炎细胞因子，从而引发牙周组织炎症浸润，一方面造成牙周膜功能紊乱导致牙周膜丧失，另一方面造成牙槽骨丧失，所述药物组合物通过抑制自然杀伤样B细胞(Natural killer-like B cells，NKB)对牙周炎发生发展的免疫炎症致病机制，减少了IL-18对牙周膜细胞分泌促炎细胞因子的能力，抑制牙周炎牙槽骨吸收，和牙周炎相关炎症细胞因子和趋化因子的表达，从而实现有效地抑制牙周炎炎症、牙槽骨吸收，并从炎症发病机制上减轻牙周炎发病程度，进而阻止牙周炎的发生。
与现有技术相比，本发明具有如下有益效果：
(1)本发明发现细胞因子IL-18可以促进牙周膜细胞分泌多种促进炎症应答的细胞因子，从而导致牙周膜组织丧失，增强炎症反应，进而导致牙槽骨丧失加剧，促进牙周炎的疾病进程；
(2)本发明的药物组合物中，IL-18抗体作为靶向药物来预防治疗牙周膜细胞的功能紊乱，IL-18R受体siRNA外泌体通过翻译抑制IL-18mRNA的表达降低IL-18的表达水平，两者发挥协同作用，从而抑制牙周膜组织丧失，牙槽骨吸收和牙周炎相关炎症细胞因子和趋化因子的表达，并从根本源头上减轻牙周炎发病程度，进而阻止牙周炎的发生发展，对牙周炎的临床预防治疗有着潜在的巨大应用价值，并且为进一步研究IL-18抗体和IL-18R受体siRNA的生物学功能奠定了基础；
(3)本发明提供牙周炎小鼠模型，采用丝线结扎联合牙龈卟啉单胞菌诱导产生，通过牙周局部注射的方法研究了IL-18抗体和IL-18R受体siRNA对牙周炎的治疗和预防作用。
附图说明
图1为IL-18对牙周膜细胞促炎细胞因子分泌的影响研究，其中，图1(A)为IFN-γ的表达水平变化，图1(B)为GM-CSF的表达水平变化，图1(C)为IL-2的表达水平变化，图1(D)为TNF-α的表达水平变化；
图2为牙周炎牙槽骨丧失的CT图，其中，图2(A)为正常对照组，图2(B)为单纯丝线结扎对照组，图2(C)为牙周炎组，图2(D)为牙周炎+IL-18抗体治疗组，图2(E)为牙周炎+IL-18α siRNA外泌体组，图2(F)为牙周炎+IL-18抗体+IL-18α siRNA外泌体组；
图3为根据micro-CT结果统计得出的牙周炎牙槽骨丧失距离(mm)的柱状图；
图4(A)为牙周组织细胞因子IL-1β的表达情况，图4(B)为细胞因子IL-6的表达情况，图4(C)为细胞因子IL-8的表达情况，图4(D)为IL-18Rα的表达情况；
图5为上颌磨牙牙周组织区域进行组织学染色结果图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
材料和试剂
IL-18抗体购买于株式会社医学生物学研究所(Medical&Biological Laboratories Co.,LTD.Code No:D 048-3)；
P.gingivalis菌株购于美国ATCC细菌库(ATCC 33277)；
C57BL/6小鼠购于中山大学动物实验中心。
实施例1丝线结扎联合牙龈卟啉单胞菌牙周炎造模
(1)细菌培养
将0.5％氯化血红素维生素溶液和牛心脑浸汁液培养基(BHI培养基)按照1:100混合均匀，将P.gingivalis菌株接种于培养基中，在85％氮气、10％氢气、 5％二氧化碳、37℃严格厌氧条件下培养；
培养两代后，细菌浓度达1×10 8CFU/mL时，将菌体用含2％羧甲基纤维素钠的PBS缓冲液重悬待用；
根据菌落形态、革兰染色性、菌体形态及生化反应结果对培养48h的细菌进行鉴定；
(2)造模
选用8周牙列完整、无龋病及牙周疾病的C57BL/6小鼠，于室内采光条件良好、清洁、安静、通风良好、室温22℃的条件下、无特殊致病菌的环境中给予无菌饲料分笼喂养，光照周期为12h:12h；
采用4％水合氯酸腹腔注射麻醉(0.35mg/kg)，待麻醉后小鼠采取仰卧位，胶布固定于手术台上，采用1％碘酊消毒口腔及口周，并用75％酒精脱碘，以棉线牵拉固定上颌门牙，使之呈大张口状；用尖探针分离左侧，上颌第一磨牙牙龈组织，用丝线嵌入第一、二磨牙邻间牙颈部，于第一磨牙近中牙跟乳头处结扎固定放置在第一磨牙牙龈缘部位，并尽量置于牙龈沟内；
每周一次检查针和丝线是否脱落，有脱落者及时重新结扎；
接种牙龈卟啉单胞菌100μL右侧上颌第2磨牙腭侧正中龈沟底，每周注射2次。
实施例2 IL-18受体siRNA外泌体的制备
(1)设计与合成IL-18Rα siRNA序列：
IL-18Rα siRNA的核酸序列如SEQ ID NO:1～2所示：
SEQ ID NO:1(正义链)：5′-AAACUCGGCAUCCUUCAGGUU-3′；
SEQ ID NO:2(反义链)：5′-AACCUGAAGGAUGCCGAGUUUTT-3′；
(2)小鼠血清来源外泌体的分离提纯
①取小鼠血清在4℃下300g离心10min，弃沉淀，去除细胞；
②取上清液，2000g离心10min，弃沉淀，去除死细胞；
③取上清，10000g离心30min，弃沉淀，去除细胞碎片；
④取上清，100000g离心70min，所得沉淀即为外泌体；
⑤弃上清，用PBS重新悬浮沉淀，100000g离心70min；
⑥弃上清，用适量的PBS悬浮沉淀物，置于-80℃冰箱内保存备用。
(3)装载siRNA的外泌体构建：
通过电穿孔法将siRNA加载到血清外泌体中，具体步骤如下：
将10μg siRNA和100μg外泌体重悬于400μL PBS中，使用Bio-RAD Gene Pulser XCell TM仪器进行电穿孔，电压为350V，电容为150μF，选择Pulse 2次，得到装载siRNA的外泌体，置于冰上20min待用。
实施例3 IL-18对牙周膜细胞促炎细胞因子分泌的影响
本实施例应用IL-18对牙周膜细胞刺激培养96小时后收集细胞上清，应用 ELISA法对IFN-γ、GM-CSF、IL-2和TNF-α的表达水平进行检测。
结果如图1(A)、图1(B)、图1(C)、图1(D)和表1所示，IL-18刺激后培养上清中上述细胞因子的表达均明显升高(*P<0.05)。
表1IL-18刺激后牙周膜细胞分泌细胞因子的水平(pg/mL)
细胞因子 对照组(pg/mL) IL-18刺激(pg/mL)
IFN-γ 82.11±17.24 687.1±109.3*
GM-CSF 32.90±7.29 89.27±19.47*
IL-2 114.2±28.39 197.6±54.28*
TNF-α 881.7±263.2 2269±746.1*
实施例4 IL-18抗体药物验证
向实施例1制备得到的牙周炎模型小鼠提供细胞因子IL-18抗体，具体步骤如下：
施加药物：向牙周炎模型小鼠的双侧上颌第1、2磨牙腭侧正中龈沟底注射抗IL-18抗体50μL，浓度为5μg/mL，每周注射1次，连续注射4周，共计4次。
实施例5 IL-18Rα siRNA外泌体药物验证
向实施例1制备得到的牙周炎模型小鼠提供IL-18Rα siRNA外泌体，siRNA的核酸序列如SEQ ID NO:1～3所示，具体步骤如下：
施加药物：向牙周炎模型小鼠的双侧上颌第1、2磨牙腭侧正中龈沟底注射IL-18Rα siRNA外泌体，每周注射1次，连续注射4周，共计4次。
实施例6 IL-18抗体协同siRNA外泌体的功效验证
向实施例1制备得到的牙周炎模型小鼠提供细胞因子IL-18抗体和siRNA外泌体，具体步骤如下：
向牙周炎模型小鼠的双侧上颌第1、2磨牙腭侧正中龈沟底注射抗IL-18抗 体(5μg/mL)和siRNA外泌体(每侧每次注射10μg)的PBS混合液，每周注射1次，连续注射4周，共计4次。
实施例7 IL-18抗体协同siRNA外泌体的治疗效果
设置正常对照组、单纯丝线结扎对照组、牙周炎组、牙周炎+IL-18抗体治疗组、牙周炎+IL-18抗体治疗组和牙周炎+IL-18抗体+IL-18α siRNA外泌体组，进行micro-CT照射。
图2(A)为正常对照组，图2(B)为单纯丝线结扎对照组，图2(C)为牙周炎组，图2(D)为牙周炎+IL-18抗体治疗组，图2(E)为牙周炎+IL-18Rα siRNA外泌体组，图2(F)为牙周炎+IL-18抗体+IL-18Rα siRNA外泌体组的micro-CT检测结果。可以看出，牙周炎组第一磨牙和第二磨牙之间牙槽骨丧失量(釉质牙骨质界-牙槽嵴顶的距离)较正常对照组和单纯丝线组都更高；而单纯使用IL-18抗体或IL-18Rα siRNA外泌体治疗可减轻牙槽骨丧失量；IL-18抗体和IL-18Rα siRNA外泌体联合治疗对减缓牙槽骨丧失的效果更为显著。
图3所示为根据micro-CT结果统计得出的牙周炎牙槽骨丧失距离(mm)，采用IL-18抗体协同siRNA外泌体进行治疗后，牙周炎牙槽骨丧失距离较细菌诱导牙周炎组的距离明显减小。
图4(A)为牙周组织细胞因子IL-1β的表达情况，图4(B)为牙周组织细胞因子IL-6的表达情况，图4(C)为牙周组织细胞因子IL-8的表达情况，图4(D)为IL-18Rα的表达情况，可以看出，IL-18抗体协同siRNA外泌体治疗后，牙周组织中IL-1β、IL-6、IL-8和IL-18Rα的表达量均显著下降，说明IL-18抗体和IL-18Rα siRNA外泌体协同作用，对基因mRNA具有显著的干预效率。
图5为上颌磨牙牙周组织区域进行组织学染色结果图，可以看出，细菌诱导牙周炎组第一磨牙和第二磨牙之间牙周组织中炎症细胞浸润明显，较正常对照组和单纯丝线组都显著升高；而单纯使用IL-18抗体或IL-18Rα siRNA外泌体治疗组炎症细胞浸润均有所减轻；IL-18抗体和IL-18Rα siRNA外泌体联合治疗 后明显减轻炎症细胞浸润。
综上所述，本发明发现细胞因子IL-18可以促进牙周膜细胞分泌多种促进炎症应答的细胞因子，从而导致牙周膜组织丧失，增强炎症反应，进而导致牙槽骨丧失加剧进而促进牙周炎的疾病进程；本发明采用IL-18抗体协同IL-18Rα受体siRNA外泌体的治疗方案，预防治疗牙周膜细胞的功能紊乱，抑制牙周膜组织丧失，牙槽骨吸收和牙周炎相关炎症细胞因子和趋化因子的表达，并从根本源头上减轻牙周炎发病程度，进而阻止牙周炎的发生发展，对牙周炎的临床预防治疗有着潜在的巨大应用价值，并且为进一步研究IL-18抗体和IL-18Rα受体siRNA的生物学功能奠定了基础。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (15)

  1. 一种药物组合物，其包括IL-18抗体和/或IL-18R受体siRNA外泌体。
  2. 根据权利要求1所述的药物组合物，其中，所述IL-18抗体的浓度为2～10μg/mL。
  3. 根据权利要求1或2所述的药物组合物，其中，所述IL-18R受体siRNA外泌体的浓度为0.5～2μg/μL。
  4. 根据权利要求1-3任一项所述的药物组合物，其中，所述IL-18抗体与所述IL-18R受体siRNA外泌体的质量比为(0.01～0.05):1，优选为(0.02～0.025):1。
  5. 根据权利要求1所述的药物组合物，其中，所述IL-18抗体的浓度为5～8μg/mL。
  6. 根据权利要求1-5任一项所述的药物组合物，其中，所述IL-18Rα受体siRNA外泌体中siRNA的核酸序列如SEQ ID NO:1～2所示。
  7. 根据权利要求1-6任一项所述的药物组合物，其还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
  8. 一种牙周疾病模型小鼠的制备方法，其中，所述方法采用丝线结扎联合牙龈卟啉单胞菌诱导进行模型构建。
  9. 根据权利要求8所述的方法，其中，所述方法包括以下步骤：
    将丝线放置于无牙周疾病的健康小鼠上颌，并将牙龈卟啉单胞菌接种于牙龈部位和/或涂抹于口腔局部，诱导小鼠牙周疾病，得到所述牙周疾病模型小鼠。
  10. 根据权利要求8或9所述的方法，其中，所述牙龈卟啉单胞菌为P.gingivalis菌株。
  11. 根据权利要求8-10任一项所述的方法，其中，所述牙龈卟啉单胞菌的浓度为1×10 8～1×10 10CFU/mL。
  12. 一种如权利要求1-7任一项所述的药物组合物在制备用于治疗牙周疾病的药物中的用途。
  13. 根据权利要求12所述的用途，其中，所述牙周疾病包括牙周支持组织丧失和/或牙周炎症；
    优选地，所述牙周支持组织丧失包括牙周膜丧失和/或牙槽骨丧失；
    优选地，所述牙槽骨丧失由牙周致病菌感染引起；
    优选地，所述牙周致病菌包括牙龈卟啉单胞菌；
    优选地，所述牙龈卟啉单胞菌包括P.gingivalis菌株。
  14. 牙周疾病模型小鼠用于如权利要求1-7任一项所述的药物组合物的药效评价的用途。
  15. 根据权利要求14所述的用途，其中，所述牙周疾病模型小鼠通过权利要求8-11任一项所述的方法制得。
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