CN110433289A - For treating the drug of periodontal ligament cell dysfunction and Alveolar Bone Loss caused by Periodontal Pathogens infect - Google Patents
For treating the drug of periodontal ligament cell dysfunction and Alveolar Bone Loss caused by Periodontal Pathogens infect Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
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- 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
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- 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
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
Abstract
The present invention provides the drugs for treating periodontal ligament cell dysfunction and Alveolar Bone Loss caused by Periodontal Pathogens infect, and the drug includes IL-18 antibody and/or IL-18R receptor siRNA excretion body.Drug of the invention is using IL-18 antibody and/or loads IL-18R receptor siRNA excretion body as functional component, by in conjunction with IL-18 in periodontium, inhibit periodontal ligament cell production of pro-inflammatory cytokines, improve the forfeiture of Periodontal Supporting Tissue caused by periodontal mesangial cell function disorder and the effect of inflammatory cell infiltration under inflammatory conditions, to prevent the occurrence and development of periodontal disease, there are potential huge applications to be worth the clinical prevention treatment of periodontal disease.
Description
Technical field
The invention belongs to pharmaceutical technology field, it is related to for treating periodontal ligament cell function caused by Periodontal Pathogens infect
The drug of disorder and Alveolar Bone Loss.
Background technique
Periodontitis is common mouth disease, mainly by Dental plaque biofilm pathogenic microorganisms starting inherent immunity,
Adaptive immunity and inflammatory response cause, and show as the inflammation syndrome of a series of complex, main to influence parodontium and wrapping
With the tissue for supporting tooth, it can lead to the forfeiture of periodontal alveolar bone progressive, in turn result in loss of tooth.
Pathogenic microorganisms in Dental plaque biofilm is the main reason for causing periodontal disease, and gum micropopulation falls in tooth
It is transplanted in all bags, makes to account for leading microbiologic population in oral pocket and be changed into Gram-negative anaerobism from gram-positive aerobic bacteria
Bacterium, so that periodontal disease occurs for the periodontium of health.Porphyromonas gingivalis is considered as the main pathogenic bacteria of periodontitis, In
Negligible amounts in the oral cavity of Healthy People, but significantly increased in the destructive structure bacterial plaque and dental calculus of periodontitis.Porphyromonas list
Born of the same parents bacterium has a variety of virulence factors, comprising: pili, degrading enzyme and lipopolysaccharides.The porphyromonas gingivalis of low transplanting amount can lure
The change of oral cavity symbiotic microorganism group quantity and constituent is sent out, the transformation of adverse factor in bacterial clump is promoted.Porphyromonas
Monad can also aid in inhibition body immune system, create good living environment for other bacteriums.In mouse periodontitis mould
In type, the porphyromonas gingivalis of low concentration passes through the interaction of oral cavity symbiotic microorganism group and complement system, induces inflammation
The generation of property periodontitis.
Host can cause rapidly the important component that the identification of pathogenic microorganism is in periodontal disease pathomechanism
Immune response, and then promote by secrete cytokines and chemotactic factor (CF) the activation and recruitment of important immune constituent.Detection
Microbial product can promote the firm and proliferation of microbial colonization group, make host buccal microbiologic population and immunity of organism system
Blanket insurance holds homeostasis.Currently, although the feature of chronic periodontitis progression of disease damage has defined, to host's periodontal
The mechanism of organization protection and damage does not illustrate completely yet.
The clinical manifestation of periodontal disease is ever-changing, at the position that disease is active, mast cell, Monocytes/Macrophages and
The quantity of thick liquid cell is significantly raised, these cells pass through further activated mononuclear/macrophage, lymphocyte, fibroblast
With other cell components, cytokine profiles and inflammatory mediator including IL-1, IL-6 and TNF-α, these cells are secreted
The factor and inflammatory mediator can be used alone or combine and play a role, and promote the absorption and decomposition of collagen of alveolar bone.
Natural kill sample B cell (Natural killer-like B cells, NKB cells) is the spleen in mouse
With a new class of immunocyte subgroup found in lymphonodi mesenterici, Major Secretory IL-18 and IL-12 are played antimicrobial
The effect of infection.IL-18 is the major cytokine of NKB cell secretion, can generate IFN- with induction of lymphocyte and splenocyte
γ.The dual-gene defect of IL-18 and IL-12 can reduce NK cell activity and cell response, and IL-18 and IL-12 are answered in local inflammation
Has the function of activated NK in answering.Since the morbidity of periodontitis is that oral cavity bacterial parasite and body immune system interact
As a result, and NKB cell plays a significant role in bacterium infection, therefore NKB cell and IL-18 may be sent out in the onset of periodontitis
The effect of waving.
IL-18 in conjunction with its receptor IL-18R by playing a role, and the receptor IL-18R of IL-18 is universally present in more
In kind cell, it is made of heterodimer α chain (IL-18R α) and β chain (IL-18R β), wherein IL-18R α is ligand binding chain,
IL-18R β is used to mediate the signal transduction of IL-18.It is proinflammatory can to mitigate periodontal ligament cell secretion for the expression for reducing IL-18 receptor
Cell factor, and then inhibit the occurrence and development of periodontitis.
In recent years, RNA interferes (RNA interfering, RNAi) to obtain extensive concern, as a kind of new gene therapy
Technology, RNAi refers to through degradation or Translational repression target mRNA, so as to cause posttranscriptional gene silencing, in anti-inflammatory, disease-resistant
The field of medicaments such as poison have broad application prospects.SiRNA efficiently, specifically blocks internal homologous gene by RNAi technology
Expression, promote degradation of homologous mRNA, inducing cell shows the particular phenotype of gene delection.But siRNA is in vivo
Be easy to by ribozyme (RNase) degrade, half-life short, transfect cell low efficiency, therefore, how do not damage normal tissue and
Under the premise of siRNA, efficiently specific silencing siRNA is imported into target organ, becomes that gene therapy is urgently to be resolved to ask
Topic.
Currently, the study on the carrier of therapeutic siRNA be concentrated mainly on polycation polyethyleneimine (PEI) nano particle and
On liposome, however, PEI-RNA compound is easy to accumulate in liver, lungs, spleen and kidney, it is limited in other groups
Application in knitting is easy to cause inflammation corpusculum relevant to IL-1 induction and Apoptosis to activate, promotes inflammation；Liposome or rouge
Matter nano particle not only has hepatotoxicity wind agitation, but also can adsorb opsonin, activating complement and coagulation factor, and since it is intrinsic
Net charge and size are easy to cause the phagocytosis of mononuclear phagocyte system, and as PEI, this process can lead to cell and answer
Swash, inflammation corpusculum activates and Apoptosis.
Excretion body (exosomes) is a kind of typical bimolecular lamellar lipid membrane structure, diameter about 30~150nm, lipid
Bilayer structure and surface membrane protein make excretion body transfection efficiency with higher in vivo, are that cell-tocell transmits
Important carrier is developed to the iuntercellular transmission of each substance (including siRNA).With traditional pharmaceutical carrier such as liposome
Transfection reagent is compared, and excretion body has good biocompatibility, degradable, immunogenicity is small etc. as a kind of natural drug carrier
Advantage.
102488654 A of CN discloses a kind of drug for treating periodontitis, in parts by weight by 5-250 parts of how western ring
Element, 25-1000 parts of metronidazole and 10000 parts of glycerol are made.CN 104688998 A discloses a kind of periodontitis for the treatment of
Chinese medicinal tablet and preparation method thereof takes 8-14 parts of Fructus Forsythiae water extract, 3-7 parts of coptis water extract, chingma abutilon seed ethyl alcohol respectively by weight
5-11 parts of extract, 4-8 parts of cymbopogon distans ethanol extract, 4-9 parts of radix paeoniae rubra ethyl acetate extract, crush 2-5 parts of the rhizome of nutgrass flatsedge,
7-16 parts of 2-6 parts of 15-20 parts of 3-6 parts of fiveleaf akebia fruit, magnesium stearate, glycerol, the modified starch of crushing mix above-mentioned ingredient equal
Tabletting machine is used after even, for the Chinese medicinal tablet for treating periodontitis.CN 103638408 A discloses a kind of periodontitis for the treatment of
External-applied ointment is prepared by following components according to shown weight ratio range: 20-30 parts of cortex periplocae, 15-20 parts of Prunella vulgaris, life
8-15 parts of glutinous rehmannia, 6-12 parts of wild chrysanthemum, 5-10 parts of smilax, 7-10 parts of Fructus Forsythiae, 8-12 parts of the tuber of stemona, 5-10 parts of eupatorium, indian trumpetflower bark
8-12 parts, 6-10 parts of semen euphorbiae, 1-3 parts of Chinese gall.Prior art treatment periodontitis is all made of antibiotic or Chinese medicine, and ingredient is multiple
Miscellaneous, side effect is big, and therapeutic effect is not significant, is unable to reach the purpose of radical cure.
The morbidity of periodontitis is related to the interaction of microorganism and body immune system, and pathogenesis not yet illustrates completely,
And the immunopathogenesis of periodontitis is furtherd investigate to find the novel targets of periodontitis treatment and providing theoretical and experimental basis, mesh
Before, there is an urgent need to research and develop the drug that can effectively inhibit periodontitis inflammation, mitigate the onset of periodontitis degree from basic source,
Prevent the generation of periodontitis.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides for treating periodontal ligament cell caused by Periodontal Pathogens infect
The drug of dysfunction and Alveolar Bone Loss, the drug have the excretion body of IL-18R receptor siRNA with IL-18 antibody and load
For functional component, IL-18 antibody and IL-18R receptor siRNA excretion body play synergistic effect, are able to suppress periodontitis alveolar bone
The expression with periodontitis related inflammation cell factor and chemotactic factor (CF) is absorbed, mitigates the morbidity journey of periodontal disease from basic source
Degree.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the drug includes IL-18 antibody and/or IL-18R the present invention provides a kind of pharmaceutical composition
Receptor siRNA excretion body.
In the present invention, inventor is had been surprisingly found that on the basis of the immune response and periodontal disease relationship of research in periodontal disease
In patient, the cell factor IL-18 of natural kill sample B cell (Natural killer-like B cells, NKB) secretion
It is significantly risen in expression quantity in peripheral blood and level in gingival sulcus fluid, moreover, IL-18 and AL in Gingival Crevicular Fluid in Patients with Periodontitis
(Attachment loss, attachment loss) is positively correlated in significant, thus illustrates that IL-18 is NKB cell in the onset of periodontitis
Main effects molecule；On this basis, inventor is it has furthermore been found that the level of IL-18 is aobvious in level in gingival sulcus fluid after periodental non-surgical treatment
Writing reduces, and after removing the microorganisms pathogenic factors such as bacterial plaque, micro organism quantity and antigen levels are remarkably decreased, and reduce part
Inflammatory response reduces the stimulation of NKB cell, and the level of secretion IL-18 also reduces, and further confirms that IL-18 takes part in tooth
The morbidity of Zhou Yan, and mainly locally playing a role in periodontium, thus inventor using drug prepared by IL-18 antibody come
Inhibit the symptom of periodontitis, effectively inhibits periodontitis inflammation.
In the present invention, inventor also found that IL-18 can promote periodontal ligament cell and secrete a variety of cells for promoting inflammatory response
The factor, and then promote the disease process of periodontitis.
In the present invention, the drug using IL-18 antibody and load have the excretion body of IL-18R receptor siRNA as effect at
Point, IL-18 antibody reduces the expression of IL-18 by targeting IL-18, and IL-18R receptor siRNA excretion body passes through translation suppression
The expression of IL-18mRNA processed reduces the expression of IL-18, and the two plays synergistic effect, it is suppressed that inflammation is thin in periodontium
The expression of intracellular cytokine and chemotactic factor (CF), reduce inflammatory cytokine and chemotactic factor (CF) to periodontal ligament cell secrete proinflammatory cytokines because
The effect of son, fundamentally alleviates inflammatory infiltration, improves periodontal ligament cell dysfunction, prevent the generation of periodontal disease
Development.
Preferably, the concentration of the IL-18 antibody is 2~10 μ g/mL, such as can be 2 μ g/mL, 3 μ g/mL, 4 μ g/
ML, 5 μ g/mL, 6 μ g/mL, 7 μ g/mL, 8 μ g/mL, 9 μ g/mL or 10 μ g/mL, preferably 5~8 μ g/mL.
Preferably, the concentration of the IL-18R receptor siRNA excretion body is 0.5~2 μ g/1 μ L, such as can be 0.5 μ g/
1μL、0.6μg/1μL、0.7μg/1μL、0.8μg/1μL、0.9μg/1μL、1.0μg/1μL、1.1μg/1μL、1.2μg/1μL、1.3
μ g/1 μ L, 1.4 μ g/1 μ L, 1.5 μ g/1 μ L, 1.6 μ g/1 μ L, 1.7 μ g/1 μ L, 1.8 μ g/1 μ L, 1.9 μ g/1 μ L or 2.0 μ g/1 μ
L, preferably 1.0 μ g/1 μ L.
Preferably, the mass ratio of the IL-18 antibody and the IL-18R receptor siRNA excretion body be (0.01~
0.05): 1, such as 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。
Preferably, in the IL-18R α receptor siRNA excretion body siRNA the nucleic acid sequence such as institute of SEQ ID NO:1~2
Show:
SEQ ID NO:1 (positive-sense strand): 5 '-AAACUCGGCAUCCUUCAGGUUTT-3 '；
SEQ ID NO:2 (antisense strand): 5 '-AACCUGAAGGAUGCCGAGUUUTT-3 '
Preferably, described pharmaceutical composition further includes any in pharmaceutically acceptable carrier, excipient or diluent
It is a kind of or at least two combination.
Second aspect, the present invention provides a kind of evaluating drug effect model of pharmaceutical composition as described in relation to the first aspect, institutes
Stating model is periodontal disease model mice.
The third aspect, the preparation method for the evaluating drug effect model that the present invention provides a kind of as described in second aspect are described
Method carries out model construction using silk thread ligation joint porphyromonas gingivalis induction.
Preferably, it the described method comprises the following steps:
Silk thread is placed in the healthy mice upper jaw of no periodontal disease, and porphyromonas gingivalis is inoculated in gum position
And/or it is applied to oral cavity partial, inducing mouse periodontal disease obtains the periodontal disease model mice.
Preferably, the porphyromonas gingivalis is P.gingivalis bacterial strain.
Preferably, the concentration of the porphyromonas gingivalis is 1 × 108~1 × 1010CFU/mL, such as can be 1 ×
108CFU/mL、1×109CFU/mL or 1 × 1010CFU/mL。
As optimal technical scheme, the preparation side for the evaluating drug effect model that the present invention provides a kind of as described in second aspect
Method, the method carry out model construction using silk thread ligation joint porphyromonas gingivalis induction, include the following steps:
(1) Bacteria Culture:
0.5% hemin vitamin solution and cattle heart brain leaching juice culture medium (BHI culture medium) is mixed according to 1:100
It closes uniformly, by P.gingivalis strain inoculated in culture medium；
Porphyromonas gingivalis (P.gingivalis ATCC 33277) is inoculated in above-mentioned configured culture medium,
It cultivates under the conditions of 85% nitrogen, 10% hydrogen, 5% carbon dioxide, 37 DEG C of strictly anaerobics, after passage 2~3 times, cultivates 2 days；
Up to concentration 1 × 109When CFU/mL, the PBS buffer solution containing 2% sodium carboxymethylcellulose is used to be resuspended thallus stand-by；
(2) foundation of silk thread ligation mouse Periodontitis Model and experimental group:
Filter out that denture is complete, the mouse without dental caries and periodontal disease, the 1% yellow Jackets abdominal cavity (0.15mL/20g) note
Anesthesia, dorsal position fixing limbs are penetrated, 1% iodine tincture disinfection oral cavity and mouth week, 75% alcohol take off iodine, pull the fixed upper jaw with silk thread；
The aseptic suture silk thread of 100 μ m diameters is placed in maxillary second molar gingival sulcus at 1mm, it is near and far across neighbour from its
Gap knots in cheek side or palate side；
Whether inspection silk thread falls off within every 4 days, has the person of falling off to ligature again in time；IL-18 antibody handles experimental group and uses mouth
Chamber partial smearing simultaneously uses micro syringe locally injecting porphyromonas gingivalis suspension in maxillary molar cheek, palate side periodontal group
In knitting, while 50 μ L concentration of locally injecting is the anti-IL-18 antibody of 5 μ g/mL in maxillary molar cheek, palate side periodontium, often
Injection in 2~4 days is primary；
Extra bacterium is embrocated in maxillary molar region in oral cavity using cotton swab；It compares periodontitis group and uses micro-injection general ability
Porphyromonas gingivalis suspension is injected in maxillary molar cheek, palate side periodontium in portion；Normal group is then fine using 2% carboxymethyl
Partial smearing in plain sodium-PBS solution oral cavity is tieed up, and using micro syringe injection equivalent PBS in periodontium；
After 4 weeks, cervical dislocation puts to death mouse, and materials are fixed.
Fourth aspect, the present invention provides a kind of pharmaceutical composition as described in relation to the first aspect and/or such as second aspect institute
Application of the evaluating drug effect model stated in preparation periodontal disease therapeutic agent.
Preferably, it includes parodontium forfeiture and/or Alveolar Bone Loss that the Periodontal Supporting Tissue, which is lost,.
Preferably, the Alveolar Bone Loss is caused by Periodontal Pathogens infection.
Preferably, the Periodontal Pathogens include porphyromonas gingivalis.
Preferably, the porphyromonas gingivalis includes P.gingivalis bacterial strain.
In the present invention, periodontitis main pathogenic bacteria porphyromonas gingivalis secretes proinflammatory cytokines by stimulation periodontal ligament cell
On the one hand the factor causes parodontium dysfunction that parodontium is caused to be lost to cause periodontium inflammatory infiltration, on the other hand
Alveolar Bone Loss is caused, described pharmaceutical composition passes through suppression of natural killer sample B cell (Natural killer-like B
Cells, NKB) to the immune inflammation pathogenic mechanism of periodontitis occurrence and development, it is proinflammatory to periodontal ligament cell secretion to reduce IL-18
The ability of cell factor inhibits the expression of periodontitis absorption of alveolar bone and periodontitis related inflammation cell factor and chemotactic factor (CF),
Periodontitis inflammation, absorption of alveolar bone are effectively inhibited to realize, and mitigates the onset of periodontitis degree from inflammatory pathogenesis,
And then prevent the generation of periodontitis.
Compared with prior art, the invention has the following beneficial effects:
(1) present invention discover that cell factor IL-18 can promote periodontal ligament cell to secrete the thin of a variety of promotion inflammatory responses
Intracellular cytokine enhances inflammatory reaction, and then Alveolar Bone Loss is caused to aggravate so as to cause the forfeiture of periodontal membrane tissue, promotes periodontitis
Disease process；
(2) in pharmaceutical composition of the invention, IL-18 antibody carrys out the function of prophylactic treatment periodontal ligament cell as targeted drug
Can disorder, IL-18R receptor siRNA excretion body by Translational repression IL-18mRNA expression reduce IL-18 expression, two
Person plays synergistic effect, to inhibit the forfeiture of periodontal membrane tissue, absorption of alveolar bone and periodontitis related inflammation cell factor and become
Change the expression of the factor, and mitigate the onset of periodontitis degree from basic source, and then prevent the occurrence and development of periodontitis, to periodontal
Scorching clinical prevention treatment has potential huge applications to be worth, and is further research IL-18 antibody and IL-18R receptor
The biological function of siRNA is laid a good foundation；
(3) present invention provides periodontitis mouse model, is generated, is led to using silk thread ligation joint porphyromonas gingivalis induction
The technique study of periodontal locally injecting IL-18 antibody and IL-18R receptor siRNA is crossed to act on the treatment and prevention of periodontitis.
Detailed description of the invention
Fig. 1 is influence research of the IL-18 to periodontal ligament cell proinflammatory secretion, wherein Fig. 1 (A) is IFN-γ
Expression variation, Fig. 1 (B) be GM-CSF expression change, Fig. 1 (C) be IL-2 expression change, Fig. 1 (D)
Change for the expression of TNF-α；
Fig. 2 is that the CT of periodontitis Alveolar Bone Loss schemes, wherein Fig. 2 (A) is Normal group, and Fig. 2 (B) is simple silk thread
Control group is ligatured, Fig. 2 (C) is periodontitis group, and Fig. 2 (D) is periodontitis+IL-18 Antybody therapy group, and Fig. 2 (E) is periodontitis+IL-
18 α siRNA excretion body groups, Fig. 2 (F) are periodontitis+IL-18 antibody+IL-18 α siRNA excretion body group；
Fig. 3 is the histogram of the periodontitis Alveolar Bone Loss distance (mm) obtained according to micro-CT result statistics；
Fig. 4 (A) is the expression of periodontal tissue's factor IL-1 β, and Fig. 4 (B) is the expression feelings of cell factor IL-6
Condition, Fig. 4 (C) are the expression of cell factor IL-8, and Fig. 4 (D) is the expression of IL-18R α；
Fig. 5 is that maxillary molar periodontium region carries out histological stain result figure.
Specific embodiment
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
Material and reagent
IL-18 antibody is bought in Med & Biological Lab Co., Ltd. (Medical & Biological
Laboratories Co.,LTD.Code No:D 048-3)；
P.gingivalis bacterial strain is purchased from U.S. ATCC bacterium library (ATCC 33277)；
C57BL/6 mouse is purchased from Zhongshan University's animal experimental center.
1 silk thread of embodiment ligatures joint porphyromonas gingivalis periodontitis molding
(1) Bacteria Culture
0.5% hemin vitamin solution and cattle heart brain leaching juice culture medium (BHI culture medium) is mixed according to 1:100
It closes uniformly, by P.gingivalis strain inoculated in culture medium, in 85% nitrogen, 10% hydrogen, 5% carbon dioxide, 37 DEG C
It is cultivated under the conditions of strictly anaerobic；
After cultivating for two generations, bacterial concentration is up to 1 × 108When CFU/mL, by PBS of the thallus containing 2% sodium carboxymethylcellulose
Buffer is resuspended stand-by；
It is reflected according to the bacterium of colonial morphology, Grain stain, thalli morphology and biochemical reaction result to culture 48h
It is fixed；
(2) modeling
Select that 8 weeks dentures are complete, the C57BL/6 mouse without dental caries and periodontal disease, it is good in indoor lighting condition, clearly
Clean, quiet, well-ventilated, under conditions of 22 DEG C of room temperature, give aseptic feed in the environment without special pathogenic bacteria cage divided to feed, light
It is 12h:12h according to the period；
Using 4% hydration chloric acid intraperitoneal injection of anesthesia (0.35mg/kg), mouse takes dorsal position after anaesthetizing, and adhesive plaster is solid
Due on operating table, using 1% iodine tincture disinfection oral cavity and mouth week, and iodine is taken off with 75% alcohol, fixed upper jaw door is pulled with cotton thread
Tooth is allowed in big gaping；Left side is separated with pointed probe, maxillary first molar gingiva tissue is ground one's teeth in sleep with silk thread insertion first and second
Neck portion between neighbour in the nearly dens in dente of first molar with ligaturing fixed placement at nipple at first molar gingival margin position, and is set as far as possible
In in gingival sulcus；
It checks whether needle and silk thread fall off once a week, there is the person of falling off to ligature again in time；
Inoculation 100 μ L right maxillary the 2nd of porphyromonas gingivalis grind one's teeth in sleep palate side center gum bottom of trench, weekly inject 2 times.
The preparation of embodiment 2IL-18 receptor siRNA excretion body
(1) design and synthesis IL-18R α siRNA sequence:
The nucleic acid sequence of IL-18R α siRNA is as shown in NO:1~2 SEQ ID:
SEQ ID NO:1 (positive-sense strand): 5 '-AAACUCGGCAUCCUUCAGGUU-3 '；
SEQ ID NO:2 (antisense strand): 5 '-AACCUGAAGGAUGCCGAGUUUTT-3 '；
(2) separating-purifying of mice serum source excretion body
1. mice serum 300g at 4 DEG C is taken to be centrifuged 10min, precipitating is abandoned, cell is removed；
2. taking supernatant, 2000g is centrifuged 10min, abandons precipitating, removes dead cell；
3. taking supernatant, 10000g is centrifuged 30min, abandons precipitating, removes cell fragment；
4. taking supernatant, 100000g is centrifuged 70min, and gained precipitating is excretion body；
5. abandoning supernatant, suspended precipitating again with PBS, and 100000g is centrifuged 70min；
6. abandoning supernatant, with suitable PBS suspended sediment, it is placed in -80 DEG C of refrigerators and saves backup.
(3) the excretion body building of siRNA is loaded:
SiRNA is loaded into serum excretion body by electroporation, the specific steps are as follows:
10 μ g siRNA and 100 μ g excretion weight are suspended from 400 μ L PBS, Bio-RAD Gene Pulser is used
XCell TM instrument carries out electroporation, voltage 350V, and capacitor is 150 μ F, is selected Pulse 2 times, obtains loading the outer of siRNA
Body is secreted, it is stand-by to be placed in 20min on ice.
Influence of the embodiment 3IL-18 to periodontal ligament cell proinflammatory secretion
The present embodiment application IL-18 is to cell conditioned medium is collected after periodontal ligament cell stimulation culture 96 hours, using ELISA method
The expression of IFN-γ, GM-CSF, IL-2 and TNF-α is detected.
As a result above-mentioned in culture supernatant after IL-18 stimulation as shown in Fig. 1 (A), Fig. 1 (B), Fig. 1 (C), Fig. 1 (D) and table 1
The expression of cell factor is significantly raised (P < 0.05 *).
The level (pg/mL) of periodontal ligament cell secrete cytokines after table 1IL-18 stimulation
| Cell factor | Control group (pg/mL) | IL-18 stimulates (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* |
The verifying of embodiment 4IL-18 antibody drug
The Periodontitis Model mouse being prepared to embodiment 1 provides cell factor IL-18 antibody, the specific steps are as follows:
Apply drug: to the bilateral upper jaw the 1st, 2 of Periodontitis Model mouse grind one's teeth in sleep palate side center gingival sulcus rising pouring penetrate anti-IL-18
50 μ L of antibody, concentration are 5 μ g/mL, are injected 1 time weekly, and continuous injection 4 weeks amounts to 4 times.
Embodiment 5IL-18R α siRNA excretion body drug verification
The Periodontitis Model mouse being prepared to embodiment 1 provides IL-18R α siRNA excretion body, the nucleic acid of siRNA
Sequence is as shown in NO:1~3 SEQ ID, the specific steps are as follows:
Apply drug: to the bilateral upper jaw the 1st, 2 of Periodontitis Model mouse grind one's teeth in sleep palate side center gingival sulcus rising pouring penetrate IL-18R α
SiRNA excretion body is injected 1 time weekly, and continuous injection 4 weeks amounts to 4 times.
Embodiment 6IL-18 antibody cooperates with the effect of siRNA excretion body to verify
The Periodontitis Model mouse being prepared to embodiment 1 provides cell factor IL-18 antibody and siRNA excretion body,
Specific step is as follows:
To the bilateral upper jaw the 1st, 2 of Periodontitis Model mouse grind one's teeth in sleep palate side center gingival sulcus rising pouring penetrate anti-IL-18 antibody (5 μ g/
ML) and the PBS mixed liquor of siRNA excretion body (every 10 μ g of side per injection), weekly injection 1 time, continuous injection 4 weeks amount to 4
It is secondary.
The therapeutic effect of embodiment 7IL-18 antibody collaboration siRNA excretion body
Normal group is set, simple silk thread ligatures control group, periodontitis group, periodontitis+IL-18 Antybody therapy group, tooth
Zhou Yan+IL-18 Antybody therapy group and periodontitis+IL-18 antibody+IL-18 α siRNA excretion body group carry out micro-CT irradiation.
Fig. 2 (A) is Normal group, and Fig. 2 (B) is that simple silk thread ligatures control group, and Fig. 2 (C) is periodontitis group, Fig. 2 (D)
For periodontitis+IL-18 Antybody therapy group, Fig. 2 (E) is periodontitis+IL-18R α siRNA excretion body group, Fig. 2 (F) be periodontitis+
The micro-CT testing result of IL-18 antibody+IL-18R α siRNA excretion body group.As can be seen that periodontitis group first molar and
Alveolar Bone Loss amount (amelocemental junction-crest of alveolar ridge distance) is compared with Normal group and simple silk group between second molar
It is all higher；And it can reduce Alveolar Bone Loss amount using IL-18 antibody or the treatment of IL-18R α siRNA excretion body merely；IL-18 is anti-
Body and IL-18R α siRNA excretion body combination therapy are more significant to the effect for slowing down Alveolar Bone Loss.
Fig. 3 show the periodontitis Alveolar Bone Loss distance (mm) obtained according to micro-CT result statistics, using IL-18
After antibody collaboration siRNA excretion body is treated, periodontitis Alveolar Bone Loss distance is bright compared with the distance of bacteria-induction periodontitis group
It is aobvious to reduce.
Fig. 4 (A) is the expression of periodontal tissue's factor IL-1 β, and Fig. 4 (B) is periodontal tissue's factor IL-6
Expression, Fig. 4 (C) be periodontal tissue's factor IL-8 expression, Fig. 4 (D) be IL-18R α expression,
As can be seen that after the collaboration siRNA excretion body treatment of IL-18 antibody, IL-1 β in periodontium, IL-6, IL-8 and IL-18R α
Expression quantity is remarkably decreased, and illustrates IL-18 antibody and IL-18R α siRNA excretion body synergistic effect, is had to gene mRNA aobvious
The intervention efficiency of work.
Fig. 5 is that maxillary molar periodontium region carries out histological stain result figure, it can be seen that bacteria-induction periodontitis
Inflammatory cell infiltration is obvious in periodontium between group first molar and second molar, all compared with Normal group and simple silk group
It is significant to increase；And subtracted using IL-18 antibody or IL-18R α siRNA excretion body treatment group inflammatory cell infiltration merely
Gently；Substantially reduced inflammatory cell infiltration after IL-18 antibody and IL-18R α siRNA excretion body combination therapy.
In conclusion present invention discover that cell factor IL-18 can promote a variety of promotion inflammation of periodontal ligament cell secretion to answer
The cell factor answered enhances inflammatory reaction so as to cause the forfeiture of periodontal membrane tissue, and then Alveolar Bone Loss is caused to aggravate and then promote
Into the disease process of periodontitis；The present invention cooperates with the therapeutic scheme of IL-18R α receptor siRNA excretion body using IL-18 antibody, in advance
The dysfunction of anti-treatment periodontal ligament cell, inhibits periodontal membrane tissue to lose, absorption of alveolar bone and periodontitis related inflammation cell
The expression of the factor and chemotactic factor (CF), and mitigate the onset of periodontitis degree from basic source, and then periodontitis is prevented to send out
Exhibition has potential huge applications to be worth the treatment of the clinical prevention of periodontitis, and for further research IL-18 antibody and
The biological function of IL-18R α receptor siRNA is laid a good foundation.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
SEQUENCE LISTING
<110>The Stomatologial Hospital of Zhongshan University
<120>for treating the drug of periodontal ligament cell dysfunction and Alveolar Bone Loss caused by Periodontal Pathogens infect
<130> 20190903
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>artificial synthesized
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aaacucggca uccuucaggu utt 23
<210> 2
<211> 23
<212> DNA
<213>artificial synthesized
<400> 2
aaccugaagg augccgaguu utt 23
Claims (10)
1. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes IL-18 antibody and/or IL-18R receptor
SiRNA excretion body.
2. pharmaceutical composition according to claim 1, which is characterized in that the concentration of the IL-18 antibody is 2~10 μ g/
ML, preferably 5~8 μ g/mL；
Preferably, the concentration of the IL-18R receptor siRNA excretion body is 0.5~2 μ g/ μ L；
Preferably, the mass ratio of the IL-18 antibody and the IL-18R receptor siRNA excretion body is (0.01~0.05): 1,
Preferably (0.02~0.025): 1.
3. pharmaceutical composition according to claim 1 or 2, which is characterized in that the IL-18R α receptor siRNA excretion body
The nucleic acid sequence of middle siRNA is as shown in NO:1~2 SEQ ID.
4. pharmaceutical composition according to claim 1-3, which is characterized in that described pharmaceutical composition further includes medicine
On in acceptable carrier, excipient or diluent any one or at least two combination.
5. a kind of evaluating drug effect model of pharmaceutical composition according to any one of claims 1-4, which is characterized in that the mould
Type is periodontal disease model mice.
6. a kind of preparation method of evaluating drug effect model as claimed in claim 5, which is characterized in that the method uses silk thread
It ligatures the induction of joint porphyromonas gingivalis and carries out model construction.
7. according to the method described in claim 6, it is characterized in that, the described method comprises the following steps:
Silk thread is placed in the healthy mice upper jaw of no periodontal disease, and by porphyromonas gingivalis be inoculated in gum position and/
Or it is applied to oral cavity partial, inducing mouse periodontal disease obtains the periodontal disease model mice.
8. method according to claim 6 or 7, which is characterized in that the porphyromonas gingivalis is P.gingivalis
Bacterial strain；
Preferably, the concentration of the porphyromonas gingivalis is 1 × 108~1 × 1010CFU/mL。
9. a kind of pharmaceutical composition according to any one of claims 1-4 and/or evaluating drug effect mould as claimed in claim 5
Application of the type in preparation periodontal disease therapeutic agent.
10. application according to claim 9, which is characterized in that the periodontal disease include Periodontal Supporting Tissue lose and/
Or periodontal disease；
Preferably, it includes parodontium forfeiture and/or Alveolar Bone Loss that the Periodontal Supporting Tissue, which is lost,；
Preferably, the Alveolar Bone Loss is caused by Periodontal Pathogens infection；
Preferably, the Periodontal Pathogens include porphyromonas gingivalis；
Preferably, the porphyromonas gingivalis includes P.gingivalis bacterial strain.
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| PCT/CN2020/071939 WO2021047137A1 (en) | 2019-09-10 | 2020-01-14 | Medicine for treating periodontal disease caused by periodontal pathogen infection |
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| WO2021047137A1 (en) * | 2019-09-10 | 2021-03-18 | 中山大学附属口腔医院 | Medicine for treating periodontal disease caused by periodontal pathogen infection |
| CN115487350A (en) * | 2021-06-17 | 2022-12-20 | 中国科学院苏州纳米技术与纳米仿生研究所 | Hydrogel scaffold for regulating immune inflammation microenvironment as well as preparation method and application thereof |
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| CN115487350A (en) * | 2021-06-17 | 2022-12-20 | 中国科学院苏州纳米技术与纳米仿生研究所 | Hydrogel scaffold for regulating immune inflammation microenvironment as well as preparation method and application thereof |
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