JP4662043B2 - Periodontal disease evaluation system - Google Patents
Periodontal disease evaluation system Download PDFInfo
- Publication number
- JP4662043B2 JP4662043B2 JP2005249742A JP2005249742A JP4662043B2 JP 4662043 B2 JP4662043 B2 JP 4662043B2 JP 2005249742 A JP2005249742 A JP 2005249742A JP 2005249742 A JP2005249742 A JP 2005249742A JP 4662043 B2 JP4662043 B2 JP 4662043B2
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- Prior art keywords
- periodontal disease
- ligand
- alveolar bone
- therapeutic agent
- screening
- Prior art date
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Description
本発明は、非ヒト動物の歯槽骨とTLR4リガンド及び/又はTLR2リガンドとを備えた歯周病発症の評価システム、より詳しくは、歯周病の予防・治療剤のスクリーニング方法、歯周病モデル器官培養物、歯周病の予防・治療剤のスクリーニングセット、歯周病発症モデル動物等に関する。 The present invention relates to an evaluation system for periodontal disease onset comprising an alveolar bone of a non-human animal and TLR4 ligand and / or TLR2 ligand, and more specifically, a screening method for a prophylactic / therapeutic agent for periodontal disease, periodontal disease model The present invention relates to organ cultures, periodontal disease preventive / therapeutic agent screening sets, periodontal disease onset animals, and the like.
歯周病は、歯の表面についた汚れ(プラーク)により、歯を支えている歯ぐきや骨が、化膿したり破壊されたりする病気であり、日本国民の8割が罹患する国民病疾患である。歯周病の治療方法としては外科的治療方法や、ペリオクリンやファンキゾンという抗生物質による薬物治療法や、チアゾール化合物もしくはその薬学的に許容される塩からなる骨粗鬆症、骨折、歯周病等、種々の骨疾病・障害の治療薬(例えば、特許文献1参照)や、抗CD14抗体を有効成分として含有する歯周病治療薬(例えば、特許文献2参照)や、血管内皮細胞増殖因子/血管透過性因子(VEGF/VPF)に対する抗体を含む製剤からなることを特徴とする歯周病治療薬(例えば、特許文献3参照)等が知られている。 Periodontal disease is a disease in which the gums and bones supporting the teeth are suppurated or destroyed by dirt (plaque) on the tooth surface, and is a national disease that affects 80% of the Japanese population. . Various treatment methods for periodontal diseases include surgical treatment methods, drug treatment methods using antibiotics such as periocrine and funkisone, osteoporosis, fractures and periodontal diseases consisting of thiazole compounds or pharmaceutically acceptable salts thereof. A therapeutic agent for bone diseases / disorders (for example, see Patent Document 1), a therapeutic agent for periodontal disease containing an anti-CD14 antibody as an active ingredient (for example, see Patent Document 2), vascular endothelial growth factor / vascular permeability A therapeutic agent for periodontal disease (for example, see Patent Document 3) characterized by comprising a preparation containing an antibody against a factor (VEGF / VPF) is known.
また、歯周病の予防や治療に有用な物質を評価する方法としては、歯垢が付着しやすくなるように、動物の歯の周りに糸を巻いて装着し、数ヶ月放置して歯周病を発症させ、その後、歯周組織を摘出して切片を作製し、形態学的に観察して評価する、自然発症モデルを用いる方法(例えば、非特許文献1参照)や、ヒト歯周病菌の生きた菌を動物に投与して感染させて発症させる歯周病菌を投与する方法(例えば、非特許文献2参照)等が報告されている。 In addition, as a method for evaluating substances useful for the prevention and treatment of periodontal disease, a thread is wound around an animal's tooth so that plaque easily adheres to it, and it is left for several months. A method using a spontaneous onset model (see, for example, Non-Patent Document 1) or a human periodontal disease bacteria that develops a disease, then extracts a periodontal tissue to prepare a section, and observes and evaluates morphologically A method of administering periodontal disease bacteria that develop by infecting animals by infecting them with live bacteria (for example, see Non-patent Document 2) has been reported.
しかし、上記自然発症モデルを用いる評価方法の場合、その評価は切片を用いた顕微鏡観察による形態学的手法を用いて行われるが、歯周組織は石灰度の高い歯を有しているため、脱灰する必要があり、その切片作製の過程は固定した組織の包埋や薄切を含み、手間と時間がかかる上に、熟練した手技をも必要とする。また、歯周病菌を投与する方法の場合、動物を無菌環境で飼育する必要性があり、実施環境の整備が求められるため、スクリーニング系としては汎用性に欠ける上に、この場合の評価方法も形態学的手法によることから、動物の感染に時間がかかり、切片作製と形態学的評価もあわせると数ヶ月の時間を要するという問題があった。 However, in the case of the evaluation method using the spontaneous development model, the evaluation is performed using a morphological technique by microscopic observation using a section, but the periodontal tissue has teeth with high calcification, It is necessary to decalcify, and the process of preparing the slice includes embedding and slicing a fixed tissue, which requires labor and time, and requires a skillful technique. In addition, in the case of the method of administering periodontal disease bacteria, it is necessary to keep the animals in a sterile environment and it is necessary to improve the implementation environment, so the screening system lacks versatility and the evaluation method in this case is also Due to the morphological technique, there is a problem that it takes time for infection of animals, and it takes several months when section preparation and morphological evaluation are combined.
他方、トール(Toll)遺伝子は、ショウジョウバエの胚発生中の背腹軸の決定(例えば、非特許文献3、4参照)、また成体における侵入病原体を検出する自然免疫に関与しており(例えば、非特許文献5〜7参照)、近年、トール遺伝子の哺乳類のホモログが同定され(例えば、非特許文献8〜12参照)、ヒトのトール様受容体(TLR)ファミリーについては、TLR2やTLR4等これまでに10種が報告されている。TLRファミリーの役割は、細菌の共通構造を認識するパターン認識受容体として、別々の病原体会合分子パターンを識別し、転写因子であるNF−κBの核内への移行を導く同様の細胞内シグナル伝達経路の活性化を引き起こす。かかるシグナル伝達経路は、最終的には炎症性サイトカインを産生させ、宿主防衛反応を誘起し、さらに獲得免疫に対しても宿主防衛反応を誘起させる。また、近年多くのTLRリガンドが報告されている。 On the other hand, the Toll gene is involved in the determination of the dorsoventral axis during Drosophila embryogenesis (see, for example, Non-Patent Documents 3 and 4) and innate immunity to detect invading pathogens in adults (for example, In recent years, mammalian homologues of the Toll gene have been identified (see, for example, Non-Patent Documents 8 to 12), and the human Toll-like receptor (TLR) family includes TLR2 and TLR4. Ten species have been reported so far. The role of the TLR family is as a pattern recognition receptor that recognizes bacterial common structures, distinguishing different pathogen-associated molecular patterns, and similar intracellular signaling leading to translocation of the transcription factor NF-κB into the nucleus. Causes activation of the pathway. Such signal transduction pathways ultimately produce inflammatory cytokines and induce host defense responses, as well as host defense responses against acquired immunity. In recent years, many TLR ligands have been reported.
TLR2リガンドとしては、グラム陽性菌のペプチドグリカン(PGN)、細菌由来トリアシル化リポタンパク質、マイコプラズマ由来ジアシル化リポタンパク質等の種々の細菌由来のリポタンパク質、結核菌のリポアラビノマンナン、酵母のザイモザン、クルーズトリパノソーマ(Trypanosoma cruzi)のGPIアンカーなどのさまざまな細菌成分が報
告されている(例えば、非特許文献13〜17参照)。また、TLR4リガンドとしては、グラム陰性菌の細胞壁に特異的な糖脂質であるリポポリサッカライド(LPS)が知られており、LPSは、宿主に対して強力な自然免疫誘導活性を有することも知られている(例えば、非特許文献18、19参照)。LPSは、コア領域とO−抗原からなる親水性の多糖部分と、リピドAと総称される疎水性の糖脂質から構成される両親媒性物質であり、O−抗原は、細菌種・菌株に特異的なオリゴ糖による繰り返し構造を持っており、グラム陰性細菌の血清学的な分類に用いられており、また、コア領域は、Kdoやヘプトースといった特徴的な糖成分を含むオリゴ糖であり、その構造はO−抗原多糖よりも多様性が少ない。リピドAは、リン酸化されたグルコサミン二糖に複数のアシル基が結合した構造をもっており、LPSの免疫誘導活性の本体として認識されている。
Examples of TLR2 ligands include gram-positive peptidoglycan (PGN), bacteria-derived triacylated lipoprotein, mycoplasma-derived diacylated lipoprotein, and other lipoproteins derived from various bacteria, tuberculosis lipoarabinomannan, yeast zymosan, and cruise. Various bacterial components such as the GPI anchor of Trypanosoma cruzi have been reported (for example, see Non-Patent Documents 13 to 17). As TLR4 ligand, lipopolysaccharide (LPS), a glycolipid specific to the cell wall of Gram-negative bacteria, is known, and LPS is also known to have a strong innate immunity-inducing activity against the host. (See, for example, Non-Patent Documents 18 and 19). LPS is an amphiphile composed of a hydrophilic polysaccharide part composed of a core region and an O-antigen, and a hydrophobic glycolipid collectively referred to as lipid A. It has a repeating structure with specific oligosaccharides and is used for serological classification of Gram-negative bacteria, and the core region is an oligosaccharide containing characteristic sugar components such as Kdo and heptose. Its structure is less diverse than O-antigen polysaccharides. Lipid A has a structure in which a plurality of acyl groups are bonded to phosphorylated glucosamine disaccharide, and is recognized as the main body of LPS immunity-inducing activity.
前記のように、従来、動物の歯周組織を摘出して切片を作製し、形態学的に観察して評価する方法が用いられてきたが、これらの方法は簡便でなく、評価までの時間もかかり、数ヶ月単位の時間を要する評価系であった。従って、数多くの候補物質のスクリーニングは困難である上に、形態学的評価は作製した切片の作製状態や評価する人によってバラツキが生じやすく、実施者や施設間における実験結果に差が生じやすいことから、客観的評価が求められるスクリーニング系としては適さない。本発明の課題は、簡便かつ迅速で客観性に富む歯周病発症の評価システム、歯周病の予防・治療剤のスクリーニング方法、歯周病モデル器官培養物、歯周病の予防・治療剤のスクリーニングセット、歯周病発症モデル動物等を提供することにある。 As described above, conventionally, methods have been used in which an animal's periodontal tissue is excised and sliced, and morphologically observed and evaluated. However, these methods are not simple and time until evaluation is used. It was an evaluation system that took several months. Therefore, it is difficult to screen a large number of candidate substances, and morphological evaluation is likely to vary depending on the preparation state of the slices prepared and the person evaluating them, and differences in experimental results between the practitioner and the facility are likely to occur. Therefore, it is not suitable as a screening system that requires objective evaluation. An object of the present invention is to provide a simple, rapid and objective evaluation system for the development of periodontal disease, a screening method for a prophylactic / therapeutic agent for periodontal disease, a periodontal disease model organ culture, and a prophylactic / therapeutic agent for periodontal disease It is to provide a screening set, a periodontal disease onset model animal, and the like.
本発明者は、マウスより下顎を摘出し、実体顕微鏡下において臼歯および切歯を抜歯し、大きさを揃えた歯槽骨を採取し、歯槽骨の器官培養系に、LPSまたはTLR2リガンドを添加して6日間培養し、培養後、培養上清中のカルシウム濃度の定量により骨吸収活性を測定したところ、LPSまたはTLR2リガンドを添加すると濃度依存的に骨吸収活性が上昇することを見い出し、次に、インドメタシン(Indomethacin)を併用添加して、歯槽骨の骨吸収活性を検討した結果、インドメタシンを併用添加すると、LPSまたはTLR2リガンドによる骨吸収活性が完全に抑制されることを見い出した。また、本発明者は、実体顕微鏡下において、マウスの下顎臼歯外側の歯肉に、LPSを投与することにより歯槽骨の骨密度が低下することを見い出し、次に、インドメタシンを併用投与すると、LPSによる歯槽骨の骨密度低下が完全に抑制されることを見い出した。本発明は上記の知見に基づいて完成するに至ったものである。 The present inventor removes the mandible from the mouse, extracts the molar teeth and incisors under a stereomicroscope, collects the alveolar bone of the same size, and adds LPS or TLR2 ligand to the alveolar bone organ culture system. When the bone resorption activity was measured by quantifying the calcium concentration in the culture supernatant after the culture, it was found that the addition of LPS or TLR2 ligand increased the bone resorption activity in a concentration-dependent manner. As a result of examining the bone resorption activity of alveolar bone by adding indomethacin in combination, it was found that bone resorption activity by LPS or TLR2 ligand is completely suppressed when indomethacin is added in combination. In addition, the present inventor found that the bone density of the alveolar bone is reduced by administering LPS to the gingiva outside the lower molars of the mouse under a stereomicroscope. It has been found that the bone density decrease of the alveolar bone is completely suppressed. The present invention has been completed based on the above findings.
すなわち本発明は、(1)非ヒト動物の歯槽骨に、TLR4リガンド及び/又はTLR2リガンドを接触せしめることを特徴とする歯周病発症の評価方法や、(2)非ヒト動物の歯槽骨を器官培養した後、培養液にTLR4リガンド及び/又はTLR2リガンドと被検物質とを添加してさらに培養し、培養上清中のカルシウム濃度を測定・評価することを特徴とする歯周病の予防・治療剤のスクリーニング方法や、(3)非ヒト動物の歯槽骨近傍の歯肉に、TLR4リガンド及び/又はTLR2リガンドと被検物質とを投与し、歯槽骨の骨密度を測定・評価することを特徴とする歯周病の予防・治療剤のスクリーニング方法や、(4)TLR4リガンドとして、リポポリサッカライド(LPS)を用いることを特徴とする上記(2)又は(3)記載の歯周病の予防・治療剤のスクリーニング方法や、(5)TLR2リガンドとして、ペプチドグリカン、リポタンパク質、リポアラビノマンナン、ザイモザンの1種又は2種以上を用いることを特徴とする上記(2)又は(3)記載の歯周病の予防・治療剤のスクリーニング方法や、(6)非ヒト動物がマウスであることを特徴とする上記(2)〜(5)のいずれか記載の歯周病の予防・治療剤のスクリーニング方法や、(7)非ヒト動物の歯槽骨を器官培養した後、培養液にTLR4リガンド及び/又はTLR2リガンドを添加してさらに培養した歯周病モデル器官培養物に関する。
That is, the present invention provides (1) a method for evaluating the onset of periodontal disease characterized by contacting a TLR4 ligand and / or a TLR2 ligand with an alveolar bone of a non-human animal, and (2) an alveolar bone of a non-human animal. Periodontal disease prevention characterized by organ culture, adding TLR4 ligand and / or TLR2 ligand and test substance to culture medium, further culturing, and measuring and evaluating calcium concentration in culture supernatant therapeutic agent or screening how the (3) in the alveolar bone near the non-human animal gingiva, administered and a test substance TLR4 ligand and / or TLR2 ligand, measuring and evaluating the bone density of alveolar bone and periodontal disease screening how the agent for the prophylaxis or treatment of characterized by, (4) a TLR4 ligand, above, wherein the use of lipopolysaccharide (LPS) (2) or 3) and the screening how periodontal disease preventive and therapeutic agent according, (5) as a TLR2 ligand to peptidoglycan, lipoproteins, lipoarabinomannan, characterized by using one or two or more of zymosan (2) or (3) and screening how periodontal disease preventive and therapeutic agent according to any of the above (6) a non-human animal characterized in that it is a mouse (2) to (5) and screening how periodontal disease preventive and therapeutic agent according, (7) periodontal which the alveolar bone of a non-human animal after organ culture, and further cultured with the addition of TLR4 ligand and / or TLR2 ligand to the culture medium about the disease model organ cultures.
また本発明は、(8)TLR4リガンドが、リポポリサッカライド(LPS)であることを特徴とする上記(7)記載の歯周病モデル器官培養物や、(9)TLR2リガンドが、ペプチドグリカン、リポタンパク質、リポアラビノマンナン、ザイモザンの1種又は2種以上であることを特徴とする上記(7)記載の歯周病モデル器官培養物や、(10)非ヒト動物がマウスであることを特徴とする上記(7)〜(9)のいずれか記載の歯周病モデル器官培養物や、(11)非ヒト動物の歯槽骨を器官培養用培地、TLR4リガンド及び/又はTLR2リガンド、及び培養上清中のカルシウム濃度の測定キットを有することを特徴とする歯周病の予防・治療剤のスクリーニングセットや、(12)TLR4リガンドが、リポポリサッカライド(LPS)であることを特徴とする上記(11)記載の歯周病の予防・治療剤のスクリーニングセットや、(13)TLR2リガンドが、ペプチドグリカン、リポタンパク質、リポアラビノマンナン、ザイモザンの1種又は2種以上であることを特徴とする上記(11)記載の歯周病の予防・治療剤のスクリーニングセットや、(14)非ヒト動物がマウスであることを特徴とする上記(11)〜(13)のいずれか記載の歯周病の予防・治療剤のスクリーニングセットに関する。
The present invention also relates to (8) a periodontal disease model organ culture according to (7) above , wherein the TLR4 ligand is lipopolysaccharide (LPS), and (9) the TLR2 ligand is a peptidoglycan, lipopolysaccharide (LPS) One or more of protein, lipoarabinomannan and zymosan, or the periodontal disease model organ culture according to (7) above , or (10) the non-human animal is a mouse above and (7) - or periodontal disease model organ culture according to any one of (9), (11) the alveolar bone medium for organ culture of non-human animals, TLR4 ligand and / or TLR2 ligands, and the culture and screening set of an agent for the prophylaxis or treatment of periodontal disease characterized by having a measurement kit calcium concentration in supernatant, (12) TLR4 ligands, Ripoporisakkara And screening set of (11), wherein the periodontal disease preventive and therapeutic agent, characterized in that the id (LPS), (13) TLR2 ligand peptidoglycan, lipoproteins, lipoarabinomannan, the zymosan (11) screening and set of an agent for preventing or treating periodontal disease, wherein a is one or more, above, wherein the (14) non-human animal is a mouse ( 11) - (13) about the screening set of periodontal disease preventive and therapeutic agent of any one.
さらに本発明は、(15)非ヒト動物の歯槽骨近傍の歯肉に、TLR4リガンド及び/又はTLR2リガンドを投与することにより得られる歯周病発症モデル動物や、(16)TLR4リガンドが、リポポリサッカライド(LPS)であることを特徴とする上記(15)記載の歯周病発症モデル動物や、(17)TLR2リガンドが、ペプチドグリカン、リポタンパク質、リポアラビノマンナン、ザイモザンから選ばれる1種又は2種以上であることを特徴とする上記(15)記載の歯周病発症モデル動物や、(18)非ヒト動物がマウスであることを特徴とする上記(15)〜(17)のいずれか記載の歯周病発症モデル動物に関する。 Furthermore, the present invention relates to (15) a periodontal disease onset model animal obtained by administering TLR4 ligand and / or TLR2 ligand to the gingiva near the alveolar bone of a non-human animal, and (16) TLR4 ligand is The periodontal disease onset model animal according to (15) above, which is a saccharide (LPS), and (17) the TLR2 ligand is selected from peptidoglycan, lipoprotein, lipoarabinomannan and zymosan Periodontal disease onset model animal according to (15) above, characterized in that it is a species or more, or (18) any one of (15) to (17) above, wherein the non-human animal is a mouse about the periodontal disease onset model animal.
本発明の歯周病の予防・治療剤のスクリーニング方法、歯周病モデル器官培養物、歯周病の予防・治療剤のスクリーニングセット、歯周病発症モデル動物を用いることにより、歯周病の予防あるいは治療に有用な候補物質を1週間程度の短期間に評価することができる。マウス歯槽骨の器官培養系を用いたインビトロ(in vitro)スクリーニング系においては、歯周病に対する数多くの候補物質の有効性の有無を簡便に調べることが可能となり、一次スクリーニング系として特に適している。また、歯周病発症モデル動物を用いたインビボ(in vivo)スクリーニング系においては、歯周病に対する候補物質の有効性の有
無を、生体を用いて極めて短期間で評価することが可能となり、生体を用いる系であることから、ヒトにおける有効性の有無を判断する材料として特に適している。これら2つの評価システムは、歯磨剤などのオーラルケア製品、ガムや飴などの食品やサプリメント、及び歯周病予防・治療薬などを対象として、有効成分の添加を検討する際に用いるスクリーニング系としても適している。
Periodontal disease preventive / therapeutic agent screening method, periodontal disease model organ culture, periodontal disease preventive / therapeutic agent screening set, periodontal disease onset model animal, Candidate substances useful for prevention or treatment can be evaluated in a short time of about one week. The in vitro screening system using mouse alveolar bone organ culture system makes it possible to easily check the effectiveness of many candidate substances for periodontal disease and is particularly suitable as a primary screening system. . In addition, in an in vivo screening system using a periodontal disease onset model animal, it is possible to evaluate the effectiveness of a candidate substance for periodontal disease in a very short time using a living body. Therefore, it is particularly suitable as a material for judging the presence or absence of effectiveness in humans. These two evaluation systems are used as screening systems when examining the addition of active ingredients for oral care products such as dentifrices, foods and supplements such as gums and salmon, and preventive and therapeutic agents for periodontal diseases. Is also suitable.
本発明の歯周病発症の評価システムとしては、非ヒト動物の歯槽骨に、TLR4リガンド及び/又はTLR2リガンドを接触せしめることを特徴とし、かかる評価システムの具体的な実施の態様としては、かかる評価システムを利用した歯周病の予防・治療剤のスクリーニング方法、歯周病モデル器官培養物、歯周病の予防・治療剤のスクリーニングセット、歯周病発症モデル動物等を挙げることができる。また、本発明における非ヒト動物としては、マウス、ラット、ウサギ、イヌ、ネコ、ヒツジ、ウマ、ウシ、サル等を挙げることができるが、短期間に歯槽骨に歯周病を発症させることができ、かつ取扱いが簡便である点で、特にマウスが好ましい。 The evaluation system for the onset of periodontal disease according to the present invention is characterized in that a TLR4 ligand and / or a TLR2 ligand is brought into contact with the alveolar bone of a non-human animal. Examples include a screening method for a prophylactic / therapeutic agent for periodontal disease using an evaluation system, a periodontal disease model organ culture, a screening set for a prophylactic / therapeutic agent for periodontal disease, and a periodontal disease onset model animal. In addition, examples of the non-human animal in the present invention include mice, rats, rabbits, dogs, cats, sheep, horses, cows, monkeys, etc., but it is possible to develop periodontal disease in the alveolar bone in a short period of time. A mouse is particularly preferable because it can be easily handled.
本発明の歯周病の予防・治療剤のスクリーニング方法としては、インビトロ系とインビボ系に大別することができ、インビトロ系としては、非ヒト動物の歯槽骨を器官培養した後、培養液にTLR4リガンド及び/又はTLR2リガンドと被検物質とを添加してさらに培養し、培養上清中のカルシウム濃度を測定・評価するスクリーニング方法であれば特に制限されるものではないが、被検物質を添加しない対照におけるカルシウム濃度を測定し、比較評価することが好ましい。非ヒト動物がマウスの場合、インビトロ系における器官培養する歯槽骨として、臼歯および切歯を抜歯したものを有利に用いることができ、ここで、器官培養とは、器官片を生体より取り出し、もとの三次元的な構築を失わせることなく、有機的な成長を行わせる培養を云う。また、インビボ系としては、非ヒト動物の歯槽骨近傍の歯肉に、TLR4リガンド及び/又はTLR2リガンドと被検物質とを投与し、歯槽骨の骨密度を測定・評価するスクリーニング方法であれば特に制限されるものではないが、被検物質を投与しない対照における歯槽骨の骨密度を測定し、比較評価することが好ましく、特に同一動物における被検物質を併用投与した以外の左右どちらか一方の歯槽骨の骨密度を測定し、比較評価することが好ましい。また、非ヒト動物がマウスの場合、インビボ系における歯槽骨近傍の歯肉として、下顎臼歯外側の歯肉を好適に例示することができる。 The screening method for the preventive / therapeutic agent for periodontal disease of the present invention can be broadly classified into in vitro system and in vivo system. As an in vitro system, an alveolar bone of a non-human animal is organ-cultured and then used as a culture solution. The screening method is not particularly limited as long as it is a screening method in which TLR4 ligand and / or TLR2 ligand and a test substance are added and further cultured, and the calcium concentration in the culture supernatant is measured and evaluated. It is preferable to measure and compare the calcium concentration in the control without addition. When the non-human animal is a mouse, as an alveolar bone for organ culture in an in vitro system, those extracted from molars and incisors can be advantageously used. Here, organ culture refers to removing organ pieces from a living body. This culture means organic growth without losing the three-dimensional structure. The in vivo system is particularly a screening method in which TLR4 ligand and / or TLR2 ligand and a test substance are administered to the gingiva near the alveolar bone of a non-human animal, and the bone density of the alveolar bone is measured and evaluated. Although not limited, it is preferable to measure and compare the bone mineral density of the alveolar bone in the control to which the test substance is not administered, and in particular, either the left or right other than the test substance in combination in the same animal. It is preferable to measure and compare the bone density of the alveolar bone. When the non-human animal is a mouse, the gingiva on the outer side of the mandibular molar can be suitably exemplified as the gingiva near the alveolar bone in the in vivo system.
上記TLR4リガンドとしては、LPSを好適に例示することができる。また、TLR2リガンドとしては、グラム陽性菌のPGN、細菌由来トリアシル化リポタンパク質、マイコプラズマ由来ジアシル化リポタンパク質等の種々の細菌由来のリポタンパク質、結核菌のリポアラビノマンナン、酵母のザイモザン、クルーズトリパノソーマ(Trypanosoma cruzi)のGPIアンカー等を具体的に挙げることができる。これらTLR4リガンドや
TLR2リガンドの使用濃度や使用量は、リガンドの種類、動物種やその体重によって適宜変更しうるが、短期間で歯槽骨に歯周病が発症するように使用濃度や使用量を決定する
ことが好ましく、例えばLPSを使用するマウスの場合、インビトロ・スクリーニング系では、培養液中の濃度が0.1〜10μg/ml、好ましくは1〜10μg/mlとなる
ように添加すればよく、インビボ・スクリーニング系では825〜1650μg/kgとなるように投与すればよい。TLR2リガンドを使用するマウスの場合、インビトロ・スクリーニング系では、培養液中の濃度が30〜300μg/ml、好ましくは100〜300μg/mlとなるように添加すればよく、インビボ・スクリーニング系では8.3〜
83mg/kgとなるように投与すればよい。
As the TLR4 ligand, LPS can be preferably exemplified. Examples of TLR2 ligands include gram-positive bacterial PGN, bacterial-derived triacylated lipoproteins, lipoproteins derived from various bacteria such as mycoplasma-derived diacylated lipoproteins, M. tuberculosis lipoarabinomannan, yeast zymosan, and cruising trypanosoma. The GPI anchor of (Trypanosoma cruzi) can be specifically mentioned. The concentration and amount used of these TLR4 ligands and TLR2 ligands can be appropriately changed depending on the type of ligand, animal species and their body weight, but the concentration and amount used are set so that periodontal disease develops in the alveolar bone in a short period of time. For example, in the case of a mouse using LPS, in an in vitro screening system, it may be added so that the concentration in the culture solution is 0.1 to 10 μg / ml, preferably 1 to 10 μg / ml. In an in vivo screening system, the dose may be 825 to 1650 μg / kg. In the case of a mouse using a TLR2 ligand, the in vitro screening system may be added so that the concentration in the culture solution is 30 to 300 μg / ml, preferably 100 to 300 μg / ml. In the in vivo screening system, 8. 3 to
What is necessary is just to administer so that it may become 83 mg / kg.
上記被検物質としては特に制限されないが、鎮痛・消炎・抗炎症薬(NSAID、ステロイ
ドなど)、抗生物質、動植物からの抽出物、生薬、核酸、糖類、糖アルコール、ポリペプチドなどを挙げることができるが、動植物からの抽出物、生薬、糖類、糖アルコール等の歯磨剤などのオーラルケア製品、ガムや飴などの食品やサプリメントに適用可能な物が好ましい。
The test substance is not particularly limited, but examples include analgesic / anti-inflammatory / anti-inflammatory drugs (NSAID, steroids, etc.), antibiotics, extracts from animals and plants, crude drugs, nucleic acids, saccharides, sugar alcohols, polypeptides, etc. However, it is preferably an extract from animals and plants, oral care products such as dentifrices such as herbal medicines, sugars and sugar alcohols, and products applicable to foods and supplements such as gums and candy.
候補薬剤としての上記鎮痛・消炎・抗炎症薬としては、イブプロフェンピコノール、インドメタシン、ウフェナマート、ケトプロフェン、グリチルレチン酸、ジクロフェナクナトリウム、スプロフェン、ピロキシカム、フェルビナク、ブフェキサマク、フルルビプロフェン、ペンダザック、ジフェンヒドラミン、ラウリル硫酸ジフェンヒドラミン、アムシノニド、吉草酸酢酸プレドニゾロン、吉草酸ジフルコルトロン、吉草酸デキサメタゾン、吉草酸ベタメタゾン、酢酸ジフロラゾン、酢酸ヒドロコルチゾン、ジフルプレドナート、ジプロピオン酸ベタメタゾン、デキサメタゾン、トリアムシノロンアセトニド、ハルシノニド、ピバル酸フルメタゾン、フランカルボン酸モメタゾン、フルオシノニド、フルオシノロンアセトニド、フルドロキシコルチド、プレドニゾロン、プロピオン酸アルクロメタゾン、プロピオン酸クロベタゾール、プロピオン酸デキサメタゾン、プロピオン酸デプロドン、プロピオン酸ベクロメタゾン、酪酸クロベタゾン、酪酸ヒドロコルチゾン、酪酸プロピオン酸ヒドロコルチゾン、酪酸プロピオン酸ベタメタゾン、酢酸プレドニゾロンなどを挙げることができる。また、候補薬剤としての上記抗生物質としては、塩酸オキシテトラサイクリン、塩酸テトラサイクリン、ゲンタマイシン、フラジオマイシン、エリスロマイシン、ピマリシン、硫酸ブレオマイシン、合成ペニシリン、合成セファロスポリン、バンコマイシンなどを挙げることができる。さらに、植物からの抽出物としては、柑橘類由来のポリメトキシフラボノイド類であるノビレチン、タンゲレチン、ナリルチン、シネンセチンなどを挙げることができ、またこれらのポリメトキシフラボノイドに代えて、シークヮーサーエキスなどのポリメトキシフラボノイド類を含む柑橘類由来のエキス類を候補薬剤として用いることもできる。 The analgesic / anti-inflammatory / anti-inflammatory drugs as candidate drugs include ibuprofen piconol, indomethacin, ufenamate, ketoprofen, glycyrrhetinic acid, diclofenac sodium, suprofen, piroxicam, felbinac, bufexamac, flurbiprofen, pendazac, diphenhydramine, lauryl sulfate Diphenhydramine, Amsinonide, Prednisolone valerate acetate, Diflucortron valerate, Dexamethasone valerate, Betamethasone valerate, Diflorazone acetate, Hydrocortisone acetate, Difluprednate, Betamethasone dipropionate, Dexamethasone, Triamcinolone acetonide, Halsinonide, Pivalate Mometasone furoate, fluocinonide, fluocinolone acetonide, flurdoxy Koruchido, prednisolone, alclometasone propionate, clobetasol propionate, can be given dexamethasone propionate, deprodone propionate, beclomethasone propionate, clobetasone butyrate, hydrocortisone butyrate, butyrate propionate, hydrocortisone, betamethasone butyrate propionate, acetate and the like prednisolone. Examples of the antibiotics as candidate drugs include oxytetracycline hydrochloride, tetracycline hydrochloride, gentamicin, fradiomycin, erythromycin, pimaricin, bleomycin sulfate, synthetic penicillin, synthetic cephalosporin, vancomycin and the like. Furthermore, examples of extracts from plants include citrus-derived polymethoxyflavonoids such as nobiletin, tangeretin, nariltin, and synencetin. In addition to these polymethoxyflavonoids, polymethoxyflavonoids such as seeker extract Extracts derived from citrus fruits including citrus can also be used as candidate drugs.
本発明の歯周病予防・治療剤としては、上記本発明の歯周病の予防・治療剤のスクリーニング方法により得られるものであれば特に制限されず、具体的には、インドメタシンや、ノビレチン、タンゲレチン、ナリルチン、シネンセチン等のポリメトキシフラボノイド類などを挙げることができる。これらは1種単独若しくは2種以上を混合して用いてもよく、又は、これらの混合物であるシークヮーサーエキスなどのポリメトキシフラボノイド類を含む柑橘類由来のエキス類を用いることもできる。これらの歯周病予防・治療剤を医薬用の治療剤として用いる場合は、薬学的に許容される通常の担体、結合剤、安定化剤、賦形剤、希釈剤、pH緩衝剤、崩壊剤、可溶化剤、溶解補助剤、等張剤などの各種調剤用配合成分を添加することができる。これら治療剤は、経口的又は非経口的に投与することができ、例えば、粉末、顆粒、錠剤、カプセル剤、シロップ剤、懸濁液等の剤型で経口的に投与することができ、あるいは、例えば、練り歯磨、口腔内軟膏剤、含嗽剤溶液等の口腔内使用剤や、乳剤、懸濁液等の剤型にしたものを注射剤として非経口投与することができる。また、本発明の歯周病予防・治療剤を食品又は食品素材に添加・配合して歯周病予防・治療用の食品又は食品素材とすることもできる。上記食品又は食品素材としては、ヨーグルト、ドリンクヨーグルト、ジュース、牛乳、豆乳、酒類、コーヒー、紅茶、煎茶、ウーロン茶、スポーツ飲料等の各種飲料や、プリン、クッキー、パン、ケーキ、ゼリー、煎餅などの焼き菓子、羊羹などの和菓子、冷菓、チューインガム等のパン・菓子類や、うどん、そば等の麺類や、かまぼこ、ハム、魚肉ソーセージ等の魚肉練り製品や、みそ、しょう油、ドレッシング、マヨネーズ、甘味料等の調味類や、チーズ、バター等の乳製品や、豆腐、こんにゃく、その他佃煮、餃子、コロッケ、サラダ等の各種総菜へ配合して食品として使用することができる。なお、歯周病予防・治療用とは、包装容器や説明書に、歯周病の予防や治療に有効である旨表示されている場合などを意味する。 The periodontal disease preventive / therapeutic agent of the present invention is not particularly limited as long as it is obtained by the above-described method for screening for the preventive / therapeutic agent of periodontal disease of the present invention. Specifically, indomethacin, nobiletin, Examples thereof include polymethoxyflavonoids such as tangeretin, nariltin, and synencetin. These may be used singly or in combination of two or more, or extracts derived from citrus fruits containing polymethoxyflavonoids such as seeker extract which is a mixture thereof. When these periodontal disease preventive / therapeutic agents are used as pharmaceutical therapeutic agents, pharmaceutically acceptable ordinary carriers, binders, stabilizers, excipients, diluents, pH buffering agents, disintegrating agents In addition, various blending ingredients such as a solubilizer, a solubilizer, and an isotonic agent can be added. These therapeutic agents can be administered orally or parenterally, for example, can be administered orally in a dosage form such as powder, granules, tablets, capsules, syrups, suspensions, etc., or For example, oral preparations such as toothpastes, oral ointments, gargle solutions, and preparations such as emulsions and suspensions can be administered parenterally as injections. Moreover, the periodontal disease preventive / therapeutic agent of this invention can also be added and mix | blended with a foodstuff or a foodstuff material, and it can also be set as the foodstuff or foodstuff material for a periodontal disease prevention / treatment. Examples of the food or food material include yogurt, drink yogurt, juice, milk, soy milk, liquor, coffee, tea, sencha, oolong tea, sports drinks, pudding, cookies, bread, cakes, jelly, rice crackers, etc. Japanese confectionery such as baked confectionery, sheep candy, frozen confectionery, bread and confectionery such as chewing gum, noodles such as udon and soba, fish paste products such as kamaboko, ham and fish sausage, miso, soy sauce, dressing, mayonnaise, sweeteners, etc. It can be used as a food by blending it with dairy products such as cheese, butter and other dairy products, tofu, konnyaku, other boiled rice, dumplings, croquettes and salads. The term “for periodontal disease prevention / treatment” refers to a case where it is indicated on the packaging container or the instructions that it is effective for the prevention or treatment of periodontal disease.
本発明の歯周病モデル器官培養物としては、マウス等の非ヒト動物の歯槽骨を器官培養した後、培養液にTLR4リガンド及び/又はTLR2リガンドを添加してさらに培養したものであれば特に制限されるものではなく、かかる歯周病モデル器官培養物は、インビトロにおける歯周病の予防・治療剤のスクリーニングに有利に用いることができる他、歯周病発症のメカニズムの解明に有利に用いることができる。歯槽骨の器官培養は、歯槽骨を生体より取り出し、もとの三次元的な構築を失わせることなく、有機的な成長を行わせる培養であれば公知の方法を含めどのような方法であってもよい。 The periodontal disease model organ culture of the present invention is particularly suitable if it is obtained by organ-culturing the alveolar bone of a non-human animal such as a mouse and then further adding TLR4 ligand and / or TLR2 ligand to the culture solution. The periodontal disease model organ culture is not limited, and can be advantageously used for in vitro screening of preventive / therapeutic agents for periodontal disease, as well as for elucidating the mechanism of periodontal disease onset. be able to. Organ culture of alveolar bone, the alveolar bone is taken out from a living body, without loss of three-dimensional construction of the original, there in any way, including the known methods as long as the culture to perform organic growth May be.
本発明の歯周病の予防・治療剤のスクリーニングセットとしては、マウス等の非ヒト動物の歯槽骨の器官培養用培地、TLR4リガンド及び/又はTLR2リガンド、及び培養上清中のカルシウム濃度の測定キットを有するセットであれば特に制限されず、歯槽骨の器官培養用培地としては、1mg/mlBSA, Penicillin(P)(63.2mg/l),Streptomycin(S)(100mg/l)を含むBGJb液体培地、アルファMEM液体培地等を挙げることができる。 As a screening set for a prophylactic / therapeutic agent for periodontal disease of the present invention, medium for culture of alveolar bone of non-human animals such as mice, TLR4 ligand and / or TLR2 ligand, and measurement of calcium concentration in the culture supernatant If it is a set which has a kit, it will not restrict | limit in particular, As a culture medium for an alveolar bone organ culture, BGJb containing 1 mg / ml BSA, Penicillin (P) (63.2 mg / l), Streptomycin (S) (100 mg / l) A liquid culture medium, an alpha MEM liquid culture medium, etc. can be mentioned.
本発明の歯周病発症モデル動物としては、マウス、ラット、ウサギ、イヌ、ネコ、ヒツジ、ウマ、ウシ、サル等の非ヒト動物の歯槽骨近傍の歯肉に、TLR4リガンド及び/又はTLR2リガンドを投与することにより得られる非ヒト動物であれば特に制限されず、かかる歯周病発症モデル動物はインビボにおける歯周病の予防・治療剤のスクリーニングに有利に用いることができる他、歯周病発症のメカニズムの解明に有利に用いることができる。 As the periodontal disease onset model animal of the present invention, a TLR4 ligand and / or a TLR2 ligand is added to the gingiva near the alveolar bone of a non-human animal such as a mouse, rat, rabbit, dog, cat, sheep, horse, cow, monkey or the like. It is not particularly limited as long as it is a non-human animal obtained by administration, and such a periodontal disease onset model animal can be advantageously used for screening of preventive / therapeutic agents for periodontal disease in vivo. It can be advantageously used to elucidate the mechanism of
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
[インビトロ・スクリーニング系(マウス歯槽骨の器官培養系:LPSによる骨吸収活性の上昇)]
6週齢のddy系マウスより下顎を採取し、実体顕微鏡下において、臼歯および切歯を抜歯したものを歯槽骨とした。48ウェルプレートを用い、1mg/mlBSA/BGJb medium/Penicillin(P)(63.2mg/l), Streptomycin(S)(100mg/l)培養液500μlにて37℃で24時間培養した。その後、同培養液、すなわち、1mg/mlBSA/BGJb medium/PSの200μlにLPS(1μg/ml;Sigma社製)を添加した培地に変え、さらに6日間培養した。培養後、カルシウムCテストワコー(和光社製)により培養上清中のカルシウム濃度を測定した(n=4)。培養液のみの場合のカルシウム濃度を差引き、カルシウム濃度の上昇分を歯槽骨から遊出したカルシウムであると判断し、骨吸収活性とした。結果を図1(左)に示す。図1(左)中、*はp<0.001を表す。図1(左)に示されるように、LPS添加により骨吸収活性の上昇が認められた。
[In vitro screening system (mouse alveolar bone organ culture system: increase in bone resorption activity by LPS)]
The mandible was collected from a 6-week-old ddy mouse and the molar and incisor extracted under a stereomicroscope were used as alveolar bone. Using a 48-well plate, the cells were cultured at 37 ° C. for 24 hours in 500 μl of 1 mg / ml BSA / BGJb medium / Penicillin (P) (63.2 mg / l), Streptomycin (S) (100 mg / l) culture solution. Thereafter, the medium was changed to the same culture solution, ie, a medium in which LPS (1 μg / ml; manufactured by Sigma) was added to 200 μl of 1 mg / ml BSA / BGJb medium / PS, and further cultured for 6 days. After the culture, the calcium concentration in the culture supernatant was measured with calcium C test Wako (manufactured by Wako) (n = 4). The calcium concentration in the case of only the culture solution was subtracted, and the increase in the calcium concentration was judged to be calcium released from the alveolar bone, and was defined as bone resorption activity. The results are shown in FIG. 1 (left). In FIG. 1 (left), * represents p <0.001. As shown in FIG. 1 (left), an increase in bone resorption activity was observed with the addition of LPS.
[インビボ・スクリーニング系(マウス歯槽骨の骨密度による歯周病評価:下顎へのLPS投与による骨密度低下)]
実体顕微鏡下において、6週齢ddy系マウスの左下顎臼歯外側の歯肉にLPS(0.5mg/ml)を2ヶ所に分けて25μlずつ(total 50μl)を1日置きに3回投与する(LPS(25μg/body/day))。右下顎臼歯外側の歯肉には対象としてPBSを同様に投与した。3回目投与の3日後に左右下顎より歯槽骨を取り出し、抜歯の後、DEXA(dual energy X-ray absorptiometry:Aloka社製)法により歯槽骨の骨密度(BMD)を測定した(n=4)。結果を図1(右)に示す。図1(右)中、*はp<0.001を表す。図1(右)に示されるように、LPS投与により骨密度の減少が認められた。
[In vivo screening system (assessment of periodontal disease by bone mineral density of mouse alveolar bone: reduction of bone density by LPS administration to mandible)]
Under a stereomicroscope, LPS (0.5 mg / ml) was divided into two portions of 6-week-old ddy mice external to the left mandibular molar and 25 μl (total 50 μl) was administered 3 times every other day (LPS (25 μg / body / day)). PBS was similarly administered to the gingiva outside the right lower molar. Three days after the third administration, the alveolar bone was removed from the left and right lower jaws, and after extraction, the bone density (BMD) of the alveolar bone was measured by the DEXA (dual energy X-ray absorptiometry: Aloka) method (n = 4). . The results are shown in FIG. 1 (right). In FIG. 1 (right), * represents p <0.001. As shown in FIG. 1 (right), a decrease in bone density was observed with LPS administration.
[インビトロ・スクリーニング系(マウス歯槽骨の器官培養系:LPS、TLR2リガンドによる骨吸収活性の上昇とインドメタシン併用による骨吸収活性の上昇抑制)]
実施例1と同様に、6週齢のddy系マウスより下顎を採取し、実体顕微鏡下において、臼歯および切歯を抜歯したものを歯槽骨とした。48ウェルプレートを用い、1mg/mlBSA/BGJb medium/PS培養液500μlにて37℃で24時間培養した。その後、同培養液の200μlに、それぞれLPS(1μg/ml;Sigma社製)を添加した培地、LPS(1μg/ml;Sigma社製)とインドメタシン(10−6M;Sigma社製)を添加した培地、TLR2リガンドであるリポペプチド(300μg/ml;BACHEM社製)を添加した培地、リポペプチド(300μg/ml;BACHEM社製)とインドメタシン(10−6M;Sigma社製)を添加した培地に変え、さらに6日間培養した。培養後、カルシウムCテストワコー(和光社製)により培養上清中のカルシウム濃度を測定した(n=各4)。培養液のみの場合のカルシウム濃度を差引き、カルシウム濃度の上昇分を歯槽骨から遊出したカルシウムであると判断し、骨吸収活性とした。結果を図2に示す。図2中、*はp<0.01を、**はp<0.001を表す。図2に示されるように、LPS、TLR2リガンドの添加により骨吸収活性の上昇が認められたが、インドメタシンの併用により、骨吸収活性の上昇が抑制された。
[In vitro screening system (organ culture system of mouse alveolar bone: increase in bone resorption activity by LPS, TLR2 ligand and suppression of increase in bone resorption activity by combined use of indomethacin]]
In the same manner as in Example 1, the mandible was collected from a 6-week-old ddy mouse, and the molar and incisor extracted under a stereomicroscope were used as alveolar bone. Using a 48-well plate, the cells were cultured at 37 ° C. for 24 hours in 500 μl of 1 mg / ml BSA / BGJb medium / PS culture solution. Thereafter, a medium supplemented with LPS (1 μg / ml; manufactured by Sigma) and a medium supplemented with LPS (1 μg / ml; manufactured by Sigma) and indomethacin (10-6M; manufactured by Sigma) were added to 200 μl of the same culture solution. The medium was supplemented with lipopeptide (300 μg / ml; manufactured by BACHEM), which is a TLR2 ligand, and the medium supplemented with lipopeptide (300 μg / ml; manufactured by BACHEM) and indomethacin (10-6M; manufactured by Sigma). The culture was further continued for 6 days. After the cultivation, the calcium concentration in the culture supernatant was measured with calcium C test Wako (manufactured by Wako) (n = 4 for each). The calcium concentration in the case of only the culture solution was subtracted, and the increase in the calcium concentration was judged to be calcium released from the alveolar bone, and was defined as bone resorption activity. The results are shown in FIG. In FIG. 2, * represents p <0.01, and ** represents p <0.001. As shown in FIG. 2, an increase in bone resorption activity was observed by the addition of LPS and TLR2 ligand, but the increase in bone resorption activity was suppressed by the combined use of indomethacin.
[インビボ・スクリーニング系(マウス歯槽骨の骨密度による歯周病評価:下顎へのLPS投与による骨密度低下とインドメタシン併用による骨密度低下の抑制)]
実施例2と同様に、実体顕微鏡下において、6週齢ddy系マウスの左下顎臼歯外側の歯肉に、それぞれLPS(0.5mg/ml)のみを2ヶ所に分けて25μlずつ(total 50μl)、LPS(0.5mg/ml)を2ヶ所に分けて25μlずつ(total 50μl)とインドメタシン(Sigma社製)1μg、LPS(0.5mg/ml)を2ヶ所に分けて25μlずつ(total 50μl)とインドメタシン(Sigma社製)10μgを投与した。右下顎臼歯外側の歯肉には対象としてPBSを同様に投与した。投与の7日後に左右下顎より歯槽骨を取り出し、抜歯の後、DEXA(dual energy X-ray absorptiometry:Aloka社製)法により歯槽骨の骨密度(BMD)を測定した(n=各4)。結果を図3に示す。図3中、*は対照と比較してp<0.05を、**はp<0.001を表し、##はLPS単独投与と比較してp<0.001を表す。図3に示されるように、LPS投与により骨密度の減少が認められたが、インドメタシンの併用により、濃度依存的に骨密度の減少が抑制された。
[In vivo screening system (periodontal disease assessment by bone mineral density of mouse alveolar bone: bone density reduction by LPS administration to the mandible and suppression of bone density reduction by combined use of indomethacin)]
In the same manner as in Example 2, under a stereomicroscope, 25 μl each of LPS (0.5 mg / ml) alone was divided into two portions on the gingiva outside the left mandibular molar teeth of 6-week-old ddy mice (total 50 μl), LPS (0.5 mg / ml) is divided into two parts, 25 μl each (total 50 μl), indomethacin (Sigma) 1 μg, LPS (0.5 mg / ml) is divided into two parts, 25 μl each (total 50 μl) 10 μg of indomethacin (Sigma) was administered. PBS was similarly administered to the gingiva outside the right lower molar. Seven days after administration, the alveolar bone was removed from the left and right lower jaws, and after extraction, the bone density (BMD) of the alveolar bone was measured by the DEXA (dual energy X-ray absorptiometry: Aloka) method (n = 4 each). The results are shown in FIG. In FIG. 3, * represents p <0.05 compared to the control, ** represents p <0.001, and ## represents p <0.001 compared to administration of LPS alone. As shown in FIG. 3, a decrease in bone density was observed by LPS administration, but the decrease in bone density was suppressed in a concentration-dependent manner by the combined use of indomethacin.
[歯槽骨器官培養系におけるノビレチンおよびタンゲレチンの骨吸収抑制作用]
マウスより下顎を採取し、実体顕微鏡下において、臼歯、および切歯を抜歯したものを歯槽骨とした。1mg/mlBSA/BGJb medium/PSにて24時間培養後、LPS(10μg/ml) および、式(1)に示すノビレチン(30μM)または式(2)に示すタンゲレチン(30μM)を併用処理し、さらに3日間培養後、培養上清中のCa濃度を測定し、骨吸収活性を求めた。その結果を図4に示す。処理後3日目において、骨吸収活性はLPS処理により有意に増加したが(*P<0.001 vs control)、ノビレチンまたはタンゲレチン併用処理により、有意に低下した(###P<0.001 vs LPS)。
[Inhibition of bone resorption by nobiletin and tangeretin in alveolar bone organ culture system]
The mandible was collected from the mouse, and the molar and incisor extracted under the stereomicroscope were used as the alveolar bone. After culturing in 1 mg / ml BSA / BGJb medium / PS for 24 hours, LPS (10 μg / ml) and nobiletin (30 μM) represented by formula (1) or tangeretin (30 μM) represented by formula (2) were further treated. After culturing for 3 days, the Ca concentration in the culture supernatant was measured to determine bone resorption activity. The result is shown in FIG. On the third day after treatment, bone resorption activity was significantly increased by LPS treatment (* P <0.001 vs control), but significantly decreased by combined treatment with nobiletin or tangeretin (#### P <0.001) vs LPS).
[歯周病モデルマウスの歯槽骨の骨密度に及ぼすノビレチンおよびタンゲレチンの投与効果]
6週齢マウスの下顎臼歯外側の歯肉に溶媒 (DMSO/PEG300 1:4) を1日置きに3回投与したマウスを control群、LPS(30μg/body/day)を同様のスケジュールで投与したマウスをLPS群として、歯周病モデルマウスを作製した。さらに、LPSにノビレチン(0.3mg/body/day)またはタンゲレチン(0.5mg/body/day)を混合した液を1日置きに3回併用投与した。4群について、3回目投与の3日後に歯槽骨を摘出し、抜歯の後、Dual Energy X-ray Absorptiometry (DEXA) 法にて歯槽骨の骨密度を測定した。その結果を図5に示す。LPS投与により低下した歯槽骨の骨密度はノビレチン(0.3mg/body/day)併用投与によりコントロールレベルに維持された(LPS投与群:*P<0.001 vs control, ノビレチン群:##P<0.01 vs LPS)。また、LPS投与により低下した歯槽骨の骨密度はタンゲレチン(0.5mg/body/day)併用投与により有意に回復した(タンゲレチン群:#P<0.05 vs LPS)。
[Effects of nobiletin and tangeretin on bone mineral density of alveolar bone in periodontal disease model mice]
Mice in which 6-week-old mice were administered with the solvent (DMSO / PEG300 1: 4) three times every other day on the gingiva of the lower mandibular molar group were mice in the control group and LPS (30 μg / body / day) was administered in the same schedule As an LPS group, periodontal disease model mice were prepared. Furthermore, LPS mixed with nobiletin (0.3 mg / body / day) or tangeretin (0.5 mg / body / day) was co-administered 3 times every other day. For group 4, the alveolar bone was removed 3 days after the third administration, and after tooth extraction, the bone density of the alveolar bone was measured by the Dual Energy X-ray Absorptiometry (DEXA) method. The result is shown in FIG. Alveolar bone density decreased by LPS administration was maintained at a control level by combined administration of nobiletin (0.3 mg / body / day) (LPS administration group: * P <0.001 vs control, nobiletin group: ## P <0.01 vs LPS). In addition, the bone density of the alveolar bone decreased by the LPS administration was significantly recovered by the combined administration of tangeretin (0.5 mg / body / day) (tangeretin group: #P <0.05 vs LPS).
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JP2003528044A (en) * | 1999-12-07 | 2003-09-24 | ラトガーズ, ザ ステイト ユニバーシティ オブ ニュー ジャージー | Therapeutic compositions and methods of treatment |
JP2003529349A (en) * | 2000-02-18 | 2003-10-07 | ロード アイランド ホスピタル, ア ライフスパン パートナー | Treatment of bone disorders |
JP2004501942A (en) * | 2000-06-30 | 2004-01-22 | ザ、プロクター、エンド、ギャンブル、カンパニー | Chlorite-containing oral care composition and method |
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JP2003529349A (en) * | 2000-02-18 | 2003-10-07 | ロード アイランド ホスピタル, ア ライフスパン パートナー | Treatment of bone disorders |
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