JP5827704B2 - Periodontal disease marker - Google Patents
Periodontal disease marker Download PDFInfo
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- JP5827704B2 JP5827704B2 JP2014011496A JP2014011496A JP5827704B2 JP 5827704 B2 JP5827704 B2 JP 5827704B2 JP 2014011496 A JP2014011496 A JP 2014011496A JP 2014011496 A JP2014011496 A JP 2014011496A JP 5827704 B2 JP5827704 B2 JP 5827704B2
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- periodontal disease
- bacteria
- autoinducer
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Description
本発明は、歯周病マーカーに関する。 The present invention relates to a periodontal disease marker.
歯周組織とは、歯の周囲にあって、歯を支持する役割を担っている組織をいい、歯肉、歯根膜、セメント質、歯槽骨から構成されている。その歯周組織の炎症のうち、炎症が歯肉に限局したものを「歯肉炎」といい、炎症部位が歯肉を超えて広がり歯根膜や歯槽骨が損傷、破壊した場合を「歯周炎」という。これらを総称して「歯周病」という。 Periodontal tissue refers to tissue surrounding the teeth and supporting the teeth, and is composed of gingiva, periodontal ligament, cementum, and alveolar bone. Among the inflammation of the periodontal tissue, the one where the inflammation is confined to the gingiva is called “gingivitis”, and the case where the inflamed part extends beyond the gum and the periodontal ligament and alveolar bone are damaged or destroyed is called “periodontitis” . These are collectively called “periodontal disease”.
歯肉炎及び歯周炎の歯周病は、ともに、歯垢(口腔内の食物残渣等に細菌などが付着して繁殖した塊)の中の細菌が増殖し、他の要因も影響して、歯周組織が炎症を起こすことによって生じる内因性感染症である。歯周病は虫歯と異なりほとんど痛みがなく、気づかないうちに進行することが多い。しかし、歯周病を放置すると症状は進行し、やがて歯が抜けることとなる恐れが大きく、循環器系疾患へのリスク増加も懸念される。 Gingivitis and periodontitis of periodontitis are both affected by the growth of bacteria in plaque (the mass that grew by attaching bacteria etc. to food residues in the oral cavity) Endogenous infection caused by inflammation of periodontal tissue. Periodontal disease, unlike caries, is almost painless and often progresses without being noticed. However, if the periodontal disease is left untreated, the symptom progresses, and there is a great risk that the tooth will eventually fall out, and there is also a concern that the risk of circulatory disease increases.
歯周病対策としては、正しい歯磨きや、定期検診によるプラークコントロール、食生活の改善等の予防行為が挙げられる。しかし、現実的には、歯周病診断により、進行しつつある歯周病をいかに的確に発見し、その症状に適した治療を行うか、というところが非常に重要である。
歯周病の診断は、一般には、歯周ポケットの測定、アタッチメントレベル、X線画像診断等によって行われている。しかし、これらの診断方法は被験者への負担が大きい。さらに、これらの歯周病診断方法は操作手順が複雑であり、歯科医師の経験や技能に基づくため判定基準に個人差があるなどの問題がある。
Periodontal disease measures include preventive actions such as correct tooth brushing, plaque control by regular checkups, and improvement of dietary habits. However, in reality, it is very important how to accurately detect a periodontal disease that is progressing by periodontal disease diagnosis and to perform treatment suitable for the symptom.
Diagnosis of periodontal disease is generally performed by measuring periodontal pockets, attachment level, X-ray image diagnosis, and the like. However, these diagnostic methods place a heavy burden on the subject. Further, these periodontal disease diagnosis methods have a complicated operation procedure and have problems such as individual differences in judgment criteria because they are based on experience and skills of dentists.
歯周病マーカーを用いた歯周病の診断方法として、ブラシ状の採取器具で歯肉溝滲出液を採取し、歯肉溝滲出液に含まれるラクトフェリン、α1−アンチトリプシン、ヘモグロビンなどのバイオマーカーを検出して、歯周病を診断する方法が知られている(例えば、特許文献1、2参照)。しかし、歯肉溝滲出液には歯肉溝滲出液以外の成分も含まれており、唾液が混入した場合に正確性に欠ける場合がある。さらに、唾液中や洗口吐出液中の潜血(ヘモグロビン)を検出し、歯周病を診断する方法も知られている(例えば、非特許文献1参照)。しかし、唾液中の潜血には、歯周病由来以外のものも混入している可能性がある。さらに。唾液量は個人差や日内変動も大きく、ヘモグロビンの定量性がないため、歯周病の進行度までを診断することはできない。 As a method for diagnosing periodontal disease using a periodontal disease marker, gingival crevicular fluid is collected with a brush-like sampling device, and biomarkers such as lactoferrin, α1-antitrypsin, and hemoglobin contained in the gingival crevicular fluid are detected. And the method of diagnosing periodontal disease is known (for example, refer patent documents 1 and 2). However, the gingival crevicular fluid contains components other than the gingival crevicular fluid, and accuracy may be lost when saliva is mixed. Furthermore, a method of diagnosing periodontal disease by detecting occult blood (hemoglobin) in saliva or mouthwash discharge liquid is also known (see Non-Patent Document 1, for example). However, occult blood in saliva may contain other substances than those derived from periodontal disease. further. Saliva volume varies greatly between individuals and diurnal variation, and since there is no hemoglobin quantitativeness, it is impossible to diagnose the degree of progression of periodontal disease.
本発明は、簡便にかつ正確に歯周病の発症の有無を判定することができる歯周病発症判定用マーカーを提供することを課題とする。
また、本発明は、簡便にかつ正確に歯周病の進行の程度を判定することができる歯周病の進行度判定用マーカーを提供することを課題とする。
また、本発明は、簡便にかつ正確に歯周病の発症の有無を判定することができる歯周病発症判定方法を提供することを課題とする。
また、本発明は、簡便にかつ正確に歯周病の進行の程度を判定することができる歯周病進行度判定方法を提供することを課題とする。
さらに、本発明は、歯周病の予防及び改善に有効な歯周病改善剤又は予防剤の簡便かつ正確なスクリーニングに有効な、歯周病改善剤又は予防剤のスクリーニング方法を提供することを課題とする。
This invention makes it a subject to provide the marker for periodontal disease onset determination which can determine the presence or absence of onset of periodontal disease simply and correctly.
Another object of the present invention is to provide a marker for determining the degree of progression of periodontal disease that can easily and accurately determine the degree of progression of periodontal disease.
Moreover, this invention makes it a subject to provide the periodontal disease onset determination method which can determine the presence or absence of onset of a periodontal disease simply and correctly.
Moreover, this invention makes it a subject to provide the periodontal disease progress determination method which can determine the extent of a periodontal disease progress simply and correctly.
Furthermore, the present invention provides a screening method for a periodontal disease ameliorating agent or preventive agent that is effective for simple and accurate screening of a periodontal disease ameliorating agent or prophylactic agent effective in preventing and improving periodontal disease. Let it be an issue.
細菌は自らの存在環境の変化を敏感に感知する機構を獲得してきた。このような機構の1つとして、特異的な情報伝達物質を介して環境における自らの密度を感知し、その密度に応じて自らのさまざまな生物活性を巧妙に制御していることが明らかとなっている。このような細菌間の情報伝達機構は、クオラムセンシングシステムと称される。 Bacteria have acquired a mechanism for sensitively sensing changes in their environment. As one of such mechanisms, it is clear that it senses its own density in the environment through a specific information transmission substance and skillfully controls its various biological activities according to the density. ing. Such an information transmission mechanism between bacteria is called a quorum sensing system.
クオラムセンシングシステムを有する細菌は、オートインデューサーと呼ばれるシグナル伝達分子を合成し、放出し、そのシグナル伝達分子に応答して、遺伝子発現を細菌密度の関数として制御する。これまでに、異なる細菌種間連絡にはオートインデューサー−2を利用することが報告されている(例えば、Bassler et al.,Bacteriol.179,pp.4043-4045,1997参照)。 Bacteria with a quorum sensing system synthesize and release a signaling molecule called an autoinducer and respond to the signaling molecule to control gene expression as a function of bacterial density. To date, it has been reported that autoinducer-2 is used for communication between different bacterial species (see, for example, Bassler et al., Bacteriol. 179, pp. 4043-4045, 1997).
口腔常在菌として約600種の細菌が存在し、このうち、ポリフィロモナス(Porphyromonas)属細菌、ストレプトコッカス(Streptococcus)属細菌、エイケネラ(Eikenella)属細菌、ラクトバチルス(Lactobacillus)属細菌、アクチノバチルス(Actinobacillus)属細菌、アクチノマイセス(Actinomyces)属細菌、フゾバクテリウム(Fusobacterium)属細菌、ベイロネラ(Veillonella)属細菌、カプノサイトファガ(Capnocytophaga)属細菌及びプレボテラ(Prevotella)属細菌等多数の歯周病原菌も存在することが知られている。これらの歯周病原菌が増殖し、菌体外多糖を分泌し、バイオフィルムを形成することで、病原性が上昇し、歯周病が発症する。
特開2008−214296号公報では、歯周病原菌の1つである、Porphyromonas gingivalis株を用いてオートインデューサー−2受容体のアンタゴニストを添加した場合のバイオフィルム形成抑制効果について検討されている。しかし、歯周病原菌はPorphyromonas gingivalis以外にも多菌種存在する。また、歯周病原菌以外の口腔常在菌の歯周病原菌への作用も存在する。従って、Porphyromonas gingivalis株のみを用いてバイオフィルム形成抑制効果を検討しても、ヒト等の哺乳動物の歯周病のレベル、重篤度を臨床的に説明することはできない。
There are about 600 species of bacteria as oral flora, these, poly Philo MONAS (Porphyromonas) bacteria, Streptococcus (Streptococcus) bacteria, Eikenera (Eikenella) bacteria, Lactobacillus (Lactobacillus) bacteria, Actinobacillus (Actinobacillus) bacteria, Actinomyces (Actinomyces) bacteria, Fusobacterium (Fusobacterium) bacteria, Beironera (Veillonella) bacteria, capsules Roh site file moth (Capnocytophaga) bacteria and Prevotella (Prevotella) bacteria such as multiple teeth It is known that peripathogenic bacteria also exist. When these periodontal pathogens proliferate, secrete exopolysaccharides, and form a biofilm, pathogenicity increases and periodontal disease develops.
Japanese Patent Application Laid-Open No. 2008-214296 discusses the biofilm formation inhibitory effect when an antagonist of an autoinducer-2 receptor is added using a Porphyromonas gingivalis strain, which is one of periodontal pathogens. However, there are many other bacterial species other than Porphyromonas gingivalis . Moreover, there exists an effect | action with respect to periodontal pathogens of oral resident bacteria other than periodontal pathogens. Therefore, even if the biofilm formation inhibitory effect is examined using only the Porphyromonas gingivalis strain, the level and severity of periodontal disease in mammals such as humans cannot be clinically explained.
本発明者等は上記課題に鑑み、鋭意検討を行った。その結果、オートインデューサー−2がin vivoで歯周病に及ぼす影響を明らかにし、口腔内に存在する常在菌が増殖する際に放出する異なる細菌種間の連絡に用いるオートインデューサー−2の量と歯周病との間に相関関係があることを見出した。具体的には、歯周病の発症の有無及び歯周病の進行度が、口腔内におけるオートインデューサー−2量に関係することを見出した。さらに、オートインデューサー−2活性阻害効果を有する物質が歯周病改善・予防に有効であることを見出した。本発明はこれらの知見に基づいて完成させたものである。 In view of the above problems, the present inventors have conducted intensive studies. As a result, the influence of autoinducer-2 on periodontal disease in vivo is clarified, and autoinducer-2 used for communication between different bacterial species released when resident bacteria present in the oral cavity grow. It was found that there is a correlation between the amount of cancer and periodontal disease. Specifically, it was found that the presence or absence of periodontal disease and the degree of progression of periodontal disease are related to the amount of autoinducer-2 in the oral cavity. Furthermore, it discovered that the substance which has an autoinducer-2 activity inhibitory effect was effective in periodontal disease improvement and prevention. The present invention has been completed based on these findings.
本発明は、オートインデューサー−2からなる歯周病発症判定用マーカーに関する。
また、本発明は、オートインデューサー−2からなる歯周病の進行度判定用マーカーに関する。
また、本発明は、被験体の口腔内から検体を採取し、採取した検体に含まれるオートインデューサー−2を定量し、定量したオートインデューサー−2量が多い場合歯周病が発症していると判定する、歯周病発症判定方法に関する。
また、本発明は、被験体の口腔内から検体を採取し、採取した検体に含まれるオートインデューサー−2を定量し、定量したオートインデューサー−2量が多ければ多いほど歯周病が進行していると判定する、歯周病進行度判定方法に関する。
さらに、本発明は、オートインデューサー−2を介するクオラムセンシングシステムを有する細菌と被験剤とを混合し、オートインデューサー−2活性阻害効果を有する被験剤を歯周病改善剤又は予防剤として選択する、歯周病改善剤又は予防剤のスクリーニング方法に関する。
The present invention relates to a periodontal disease onset marker comprising autoinducer-2.
The present invention also relates to a marker for determining the degree of progression of periodontal disease comprising autoinducer-2.
In addition, the present invention collects a sample from the oral cavity of a subject, quantifies autoinducer-2 contained in the collected sample, and periodontal disease develops when the amount of quantified autoinducer-2 is large. The present invention relates to a method for determining periodontal disease onset.
Further, the present invention collects a sample from the oral cavity of a subject, quantifies autoinducer-2 contained in the collected sample, and periodontal disease progresses as the amount of quantified autoinducer-2 increases. The present invention relates to a periodontal disease progress determination method.
Furthermore, this invention mixes the bacterium which has a quorum sensing system via an autoinducer-2, and a test agent, and uses the test agent which has an autoinducer-2 activity inhibitory effect as a periodontal disease improving agent or a preventive agent. The present invention relates to a screening method for a periodontal disease improving agent or a preventive agent to be selected.
本発明の歯周病発症判定用マーカーによれば、簡便にかつ正確に歯周病の発症の有無を判定することができる。
また、本発明の歯周病進行度判定用マーカーによれば、簡便にかつ正確に歯周病の進行の程度を判定することができる。
また、本発明の歯周病発症判定方法によれば、簡便にかつ正確に歯周病の発症の有無を判定することができる。
また、本発明の歯周病進行度判定方法によれば、簡便にかつ正確に歯周病の進行の程度を判定することができる。
さらに、本発明の歯周病改善剤又は予防剤のスクリーニング方法は、歯周病の予防及び改善に有効な歯周病改善剤又は予防剤を簡便かつ正確にスクリーニングすることができる。
According to the marker for the onset of periodontal disease of the present invention, the presence or absence of the onset of periodontal disease can be determined easily and accurately.
Moreover, according to the marker for periodontal disease progression degree determination of the present invention, the degree of progression of periodontal disease can be easily and accurately determined.
Moreover, according to the method for determining the onset of periodontal disease of the present invention, the presence or absence of the onset of periodontal disease can be determined easily and accurately.
Moreover, according to the periodontal disease progression degree determination method of the present invention, the degree of progression of periodontal disease can be easily and accurately determined.
Furthermore, the method for screening for a periodontal disease improving agent or preventive agent of the present invention can conveniently and accurately screen for a periodontal disease improving agent or prophylactic agent effective in preventing and improving periodontal disease.
以下、本発明について、その好ましい実施態様に基づき詳細に説明する。
本発明の歯周病発症判定用マーカー(単に、歯周病判定用マーカーともいう)及び歯周病進行度判定用マーカーは、ヒト等の被験体の口腔内から採取したオートインデューサー−2からなる。本発明におけるオートインデューサー−2(以下、「AI−2」ともいう)としては細菌が産生するものでAI−2活性を有するものであれば特に制限はない。
Hereinafter, the present invention will be described in detail based on preferred embodiments thereof.
The marker for determining the onset of periodontal disease of the present invention (also simply referred to as a marker for determining periodontal disease) and the marker for determining the progress of periodontal disease are from autoinducer-2 collected from the oral cavity of a subject such as a human. Become. Autoinducer-2 (hereinafter also referred to as “AI-2”) in the present invention is not particularly limited as long as it is produced by bacteria and has AI-2 activity.
本発明においてAI−2活性は、AI−2がクオラムセンシングシステムを有する細菌に影響を及ぼす活性、すなわち、AI−2を介するクオラムセンシングによりもたらされる細菌の機能を促進する活性をさす。細菌は、AI−2を介するクオラムセンシングにより、発光、スウォーミング、バイオフィルム形成、タンパク質分解酵素の産生、抗生物質の合成、遺伝子受容能の発達、プラスミド接合伝達、病原因子産生および胞子形成等を行うことが知られている。従って、AI−2活性は、換言すれば、AI−2を認識する細菌、すなわちAI−2受容体を有する細菌による生物発光、スウォーミング、バイオフィルム形成、タンパク質分解酵素の産生、抗生物質の合成、遺伝子受容能の発達、プラスミド接合伝達、病原因子産生および胞子形成等の活性ということができる。前記病原因子としては、例えば、エンテロトキシン、アデニル酸シクラーゼ毒素、アドヘシン、アルカリプロテアーゼ、溶血毒、炭疽毒素、APX毒素、α毒素、β毒素、δ毒素、C2毒素、C3毒素、ボツリヌス毒素、束状線毛構造サブユニット、C5Aペプチダーゼ、心臓毒、走化性、コレラ毒素、毛様体毒素、クロストリジウム細胞毒、クロストリジウム神経毒、コラーゲン接着遺伝子、細胞溶解素、嘔吐毒素、内毒素、表皮剥脱毒素、外毒素、細胞外エラスターゼ、フィブリノゲン、フィブロネクチン結合タンパク質、線維状赤血球凝集素、フィンブリア、ゼラチナーゼ、赤血球凝集素、ロイコトキシン、リポタンパク質シグナルペプチダーゼ、リステリオリシンO、Mタンパク質、神経毒、非フィンブリアアドヘシン類、浮腫因子、透過酵素、百日咳毒素、ホスホリパーゼ、線毛、孔形成毒素、プロリンパーミアーゼ、セリンプロテアーゼ、志賀毒素、破傷風毒素、チオール活性化細胞溶解素、気管細胞溶解素、ウレアーゼなどが挙げられるがこれに制限されない。 In the present invention, AI-2 activity refers to an activity in which AI-2 affects bacteria having a quorum sensing system, that is, an activity that promotes the function of bacteria brought about by quorum sensing via AI-2. Bacteria are luminescent, swarming, biofilm formation, proteolytic enzyme production, antibiotic synthesis, gene acceptability development, plasmid conjugation transmission, virulence factor production and spore formation by quorum sensing via AI-2 Etc. are known to perform. Thus, AI-2 activity is, in other words, bioluminescence, swarming, biofilm formation, production of proteolytic enzymes, antibiotics by bacteria that recognize AI-2, ie bacteria having the AI-2 receptor. It can be referred to as activities such as synthesis, development of gene acceptability, plasmid conjugation transmission, pathogenic factor production and sporulation. Examples of the pathogenic factor include enterotoxin, adenylate cyclase toxin, adhesin, alkaline protease, hemolytic toxin, anthrax toxin, APX toxin, α toxin, β toxin, δ toxin, C2 toxin, C3 toxin, botulinum toxin, bundled line Hair structure subunit, C5A peptidase, cardiotoxin, chemotaxis, cholera toxin, ciliary toxin, clostridial cytotoxin, clostridial neurotoxin, collagen adhesion gene, cytolysin, vomiting toxin, endotoxin, exfoliating toxin, external Toxin, extracellular elastase, fibrinogen, fibronectin binding protein, fibrillar hemagglutinin, fimbria, gelatinase, hemagglutinin, leukotoxin, lipoprotein signal peptidase, listeriolysin O, M protein, neurotoxin, non-fimbria adhesin Edema factor, Examples include, but are not limited to, hyperenzymes, pertussis toxin, phospholipase, pili, pore forming toxin, proline permease, serine protease, Shiga toxin, tetanus toxin, thiol activated cytolysin, tracheal cell lysin, urease .
本発明におけるAI−2の具体例としては、下記式で表される4,5−ジヒドロキシ−2,3−ペンタンジオン(DPD)が挙げられる。 Specific examples of AI-2 in the present invention include 4,5-dihydroxy-2,3-pentanedione (DPD) represented by the following formula.
前記DPDは、細菌のAI−2受容体と結合するときにボロンを取り込んで、フラノシルボレートジエステルに変換される。本発明におけるAI−2の別の具体例としては、前記フラノシルボレートジエステルが挙げられる。
前記フラノシルボレートジエステルの具体例を以下に示す。しかし、本発明はこれらに制限するものではない。
When DPD binds to the bacterial AI-2 receptor, it incorporates boron and is converted to furanosyl borate diester. Another specific example of AI-2 in the present invention is the furanosyl borate diester.
Specific examples of the furanosyl borate diester are shown below. However, the present invention is not limited to these.
AI−2受容体を有する細菌としては、例えば、ポルフィロモナス(Porphyromonas)属細菌、ヘモフィルス(Haemophilus)属細菌、バシルス(Bacillus)属細菌、ナイセリア(Neisseria)属細菌、ストレプトコッカス(Streptococcus)属細菌、エイケネラ(Eikenella)属細菌、ラクトバチルス(Lactobacillus)属細菌、アクチノバチルス(Actinobacillus)属細菌、アクチノマイセス(Actinomyces)属細菌、バクテロイデス(Bacteroides)属細菌、カプノサイトファガ(Capnocytophaga)属細菌、フゾバクテリウム(Fusobacterium)属細菌、ペプトコッカス(Peptococcus)属細菌、プレボテラ(Prevotella)属細菌、セレモナス(Selemonas)属細菌、ユーバクテリウム(Eubacterium)属細菌およびベイロネラ(Veillonella)属細菌などが挙げられる。より具体的には、ポリフィロモナス・ジンジバリス(Porphyromonas gingivalis)、化膿レンサ球菌(Streptococcus pyogenes)、ストレプトコッカス・ミュータンス(Streptococcus mutans)、エイケネラ・コロデンス(Eikenella corrodens)、ラクトバチルス・サリバリウス(Lactobacillus salivarius)、ストレプトコッカス・サンギニス(Streptococcus sanguinis)、ストレプトコッカス・アンギノーサス(Streptococcus anginosus)、ストレプトコッカス・オラリス(Streptococcus oralis)、ストレプトコッカス・ゴルドニ(Streptococcus gordonii)、ストレプトコッカス・ミティス(Streptococcus mitis)、アクチノバチルス・アクチノマイセテムコミタンス(Actinobacillus actinomycetemcomitans)、ラクトバチルス・ラムノサス(Lactobacillus rhamnosus)、アクチノマイセス・ナエスランディ(Actinomyces naeslundii)、フゾバクテリウム・ヌクレアタム(Fusobacterium nucleatum)、ベイロネラ・パルラ(Veillonella parvula)、カプノサイトファガ・スプティゲナ(Capnocytophaga sputigena)及びプレボテラ・インタメディア(Prevotella intermedia)などが挙げられる。 The bacterium having an AI-2 receptor, e.g., Porphyromonas (Porphyromonas) bacteria, Haemophilus (Haemophilus) bacteria, Bacillus (Bacillus) bacteria, Neisseria (Neisseria) bacteria, Streptococcus (Streptococcus) bacteria, Eikenera (Eikenella) bacteria belonging to the genus Lactobacillus (Lactobacillus) bacteria belonging to the genus Actinobacillus (Actinobacillus) bacteria belonging to the genus Actinomyces (Actinomyces) bacteria belonging to the genus Bacteroides (Bacteroides) bacteria belonging to the genus, Cap Roh site file moth (Capnocytophaga) bacteria belonging to the genus, Fusobacterium (Fusobacterium) bacteria, Peputokokkasu (Peptococcus) bacteria, Prevotella (Prevotella) bacteria, Seremonasu (Selemonas) bacteria, such as Eubacterium (Eubacterium) bacteria and Beironera (Veillonella) bacteria and the like. More specifically, poly Philo Monas gingivalis (Porphyromonas gingivalis), Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus mutans (Streptococcus mutans), Eikenera-Korodensu (Eikenella corrodens), Lactobacillus salivarius (Lactobacillus salivarius), Streptococcus Sanginisu (Streptococcus sanguinis), Streptococcus anginosus (Streptococcus anginosus), Streptococcus oralis (Streptococcus oralis), Streptococcus Gorudoni (Streptococcus gordonii), Streptococcus mitis (Streptococcus mitis), Actinobacillus actinomycetemcomitans (Actinobacillus actinomycetemcomitans), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Actinomyces Naesurandi Actinomyces naeslundii), Fusobacterium nucleatum (Fusobacterium nucleatum), Beironera-Parla (Veillonella parvula), Cap Roh site file moth-Suputigena (Capnocytophaga sputigena) and Prevotella interface media (Prevotella intermedia) and the like.
本発明において、AI−2はヒト等の被験体の口腔内から採取して使用する。ここで、被験体としては、ヒト、サル、チンパンジー、犬、猫、牛、豚、ラット及びマウス等の哺乳動物を挙げることができる。前記AI−2は口腔内から採取したものであれば特に制限はなく、口腔内から採取した唾液、歯垢、舌苔、歯肉溝滲出液などの検体に含まれるAI−2を使用することができる。本発明においては、歯垢に含まれるAI−2を使用することが好ましい。唾液は、無刺激時、刺激時を問わず、そのまま吐出したもの、又は水などで含嗽したものとして得ることができる。歯垢は、歯科用スケーラー、綿棒、ブラシなどで歯面刷掃、またはペーパーポイントの挿入などで採取できる。舌苔は、ブラシ、綿棒、ガーゼなどで刷掃して得る。歯肉溝滲出液は、ペーパーポイントなどを歯肉溝に挿入して得ることができる。ただし、AI−2の採取方法はこの限りではない。 In the present invention, AI-2 is collected from the oral cavity of a subject such as a human and used. Here, examples of the subject include mammals such as humans, monkeys, chimpanzees, dogs, cats, cows, pigs, rats, and mice. The AI-2 is not particularly limited as long as it is collected from the oral cavity, and AI-2 contained in specimens such as saliva, plaque, tongue coating, and gingival crevicular fluid collected from the oral cavity can be used. . In the present invention, it is preferable to use AI-2 contained in dental plaque. The saliva can be obtained as it is discharged as it is or whether it is impregnated with water, regardless of whether it is unstimulated or stimulated. Plaque can be collected by cleaning the tooth surface with a dental scaler, swab, brush, or inserting a paper point. Tongue moss is obtained by brushing with a brush, cotton swab or gauze. The gingival crevicular fluid can be obtained by inserting a paper point or the like into the gingival sulcus. However, the method for collecting AI-2 is not limited to this.
本発明者等は、被験体の口腔内に存在するAI−2量が多い場合、被験体は歯周病を発症していることを見出した。さらに、AI−2量が多ければ多いほど、歯周病が進行していることを見出した。従って、口腔内から採取したAI−2量を定量することにより、被験体の歯周病の発症の有無および歯周病の進行度(重篤度)を判定することができる。一例として、DPD10μMの場合のAI−2活性を100とした場合、AI−2活性が0以上3未満の場合は健常・軽度歯周病、3以上10未満の場合は中等度歯周病、10以上は重度歯周病であると判定することができる。また、歯周病治療開始前のAI−2量と治療後のAI−2量を比較することにより治療効果の判定を行うことができる。 The present inventors have found that when the amount of AI-2 present in the oral cavity of the subject is large, the subject has developed periodontal disease. Furthermore, it discovered that periodontal disease progressed, so that there were many amounts of AI-2. Therefore, by quantifying the amount of AI-2 collected from the oral cavity, it is possible to determine the presence or absence of the onset of periodontal disease and the degree of progression (severity) of the periodontal disease. As an example, when the AI-2 activity in the case of 10 μM DPD is 100, when the AI-2 activity is 0 or more and less than 3, normal / mild periodontal disease, and when it is 3 or more and less than 10, moderate periodontal disease, 10 The above can be determined to be severe periodontal disease. Further, the therapeutic effect can be determined by comparing the amount of AI-2 before the start of periodontal disease treatment and the amount of AI-2 after treatment.
AI−2の定量方法としては特に制限はなく、通常の方法により行うことができる(例えば、Chen X.et al.,Nature,vol.415,p.545-549,2002;及びThiel V.et al.,Chembiochem.,vol.10(3),p.479-485,2008参照)。
例えば、AI−2を認識することにより発光するレポーター細菌、好ましくはAI-2受容体およびルシフェラーゼ活性を有する細菌などを用い、その発光強度を測定するバイオアッセイにより、AI−2を定量することができる。より詳しくは、ビブリオ・ハーベイBB170株をAI−2レポーター細菌とし、AI−2の標準試料としての4−ヒドロキシ−5−メチル−3(2H)−フラノン(HMF)標品及び/又はDPD標品による検量線を作成し、被検試料を添加した場合の発光強度からAI−2を定量することができる(例えば、Chen X.et al.,Nature,vol.415,p.545-549,2002参照)。また、被検試料に含まれるAI−2をフェニレンジアミン及びN−メチル−N−(トリメチルシリル)トリフルオロ−アセトアミドと反応させた後、GC−MSによってAI−2を定量することができる(例えば、Thiel V.et al.,Chembiochem.,vol.10(3),p.479-485,2008参照)。しかし、本発明はこれらに限定するものではない。
AI−2を定量するための被験試料は、口腔内から採取した検体を破砕/抽出処理後にカットオフ分子量が3000以下のフィルターで限外ろ過し、夾雑物を除去することが好ましい。
本発明において、被験試料の調製、保存をガラス容器などAI−2の吸着性が低い容器を用いることが好ましい。ガラス容器を用いることで被験試料に含まれるAI−2を正確に定量することができる。
There is no restriction | limiting in particular as a quantification method of AI-2, It can carry out by a normal method (For example, Chen X. et al., Nature, vol. 415, p. 545-549, 2002; and Thiel V. et. al., Chembiochem., vol. 10 (3), p. 479-485, 2008).
For example, AI-2 can be quantified by a bioassay that measures luminescence intensity using a reporter bacterium that emits light by recognizing AI-2, preferably a bacterium having AI-2 receptor and luciferase activity. it can. More specifically, Vibrio Harvey BB170 strain is used as an AI-2 reporter bacterium, and 4-hydroxy-5-methyl-3 (2H) -furanone (HMF) standard and / or DPD standard as a standard sample for AI-2. A calibration curve can be prepared and AI-2 can be quantified from the luminescence intensity when a test sample is added (for example, Chen X. et al., Nature, vol. 415, p. 545-549, 2002). reference). Moreover, after reacting AI-2 contained in a test sample with phenylenediamine and N-methyl-N- (trimethylsilyl) trifluoro-acetamide, AI-2 can be quantified by GC-MS (for example, Thiel V. et al., Chembiochem., Vol. 10 (3), p. 479-485, 2008). However, the present invention is not limited to these.
A test sample for quantifying AI-2 is preferably subjected to ultrafiltration of a specimen collected from the oral cavity with a filter having a cutoff molecular weight of 3000 or less after the crushing / extraction process to remove impurities.
In the present invention, it is preferable to use a container having low AI-2 adsorptivity, such as a glass container, for preparing and storing a test sample. By using a glass container, AI-2 contained in the test sample can be accurately quantified.
本発明の歯周病改善剤又は予防剤のスクリーニング方法において、オートインデューサー−2を介するクオラムセンシングシステムを有する細菌と被験剤とを混合し、オートインデューサー−2活性阻害効果を有する被験剤を歯周病改善剤又は予防剤として選択する。オートインデューサー−2を介するクオラムセンシングシステムを有する細菌としては特に制限はなく、オートインデューサー−2を認識して発光するレポーター細菌が好ましく、口腔内細菌や歯周病原菌を用いることもできる。また、被験体の口腔内から採取したオートインデューサー−2を介するクオラムセンシングシステムを有する細菌を検体として用いることもできる。
本発明における「オートインデューサー−2活性阻害効果」とは、AI−2活性に対する阻害効果をいう。具体的には、前述の、AI−2受容体を有する細菌による生物発光、スウォーミング、バイオフィルム形成、タンパク質分解酵素の産生、抗生物質の合成、遺伝子受容能の発達、プラスミド接合伝達、病原因子産生および胞子形成等のAI−2活性に対する阻害効果をいう。
In the screening method for periodontal disease ameliorating agent or preventive agent of the present invention, a test agent having an autoinducer-2 activity inhibitory effect by mixing a bacterium having a quorum sensing system via autoinducer-2 with a test agent Is selected as a periodontal disease improving agent or preventive agent. The bacterium having a quorum sensing system via autoinducer-2 is not particularly limited, and a reporter bacterium that recognizes autoinducer-2 and emits light is preferable, and oral bacteria and periodontal pathogens can also be used. In addition, bacteria having a quorum sensing system via autoinducer-2 collected from the oral cavity of the subject can also be used as a specimen.
The “autoinducer-2 activity inhibitory effect” in the present invention refers to an inhibitory effect on AI-2 activity. Specifically, bioluminescence, swarming, biofilm formation, production of proteolytic enzymes, synthesis of antibiotics, development of gene acceptability, plasmid conjugation transmission, pathogenesis by the aforementioned bacteria having AI-2 receptor It refers to inhibitory effects on AI-2 activity such as factor production and sporulation.
以下、本発明を実施例に基づきさらに詳細に説明するが、本発明はこれに限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
実施例1 歯周病発症及び歯周病進行度の判定
(1) 歯垢中AI−2の定量
ビブリオ・ハーベイBB170株(ATCCより分譲、AB(Autoinducer Bioassay)培地(10mMリン酸カリウムbuffer[pH7.0]、0.3M NaCL、0.05M MgSO4、0.2%カザミノ酸、2%グリセロール、1mM L−アルギニン、1μg/mLチアミン、0.01μg/mLリボフラビン)で30℃にて一晩培養)をAI−2レポーター細菌とし、AB培地にて5000倍に希釈し、レポーター細菌液を調製した。レポーター細菌液に様々な濃度のHMF標品(シグマ製)又はDPD標品(OMM Scientific製)をレポーター細菌液:標品の容量比が9:1となるように混合し、30℃にて好気振盪培養を行った。4時間後の発光強度をケミルミネッセンス計(LB940型、ベルトールドジャパン製)で測定し、発光強度とAI−2量とに関する検量線を作成した。
Example 1 Determination of Periodontal Disease Onset and Periodontal Disease Progression (1) Determination of AI-2 in plaque Vibrio Harvey BB170 strain (distributed from ATCC, AB (Autoinducer Bioassay) medium (10 mM potassium phosphate buffer [pH 7] 0.0], 0.3 M NaCl, 0.05 M MgSO 4 , 0.2% casamino acid, 2% glycerol, 1 mM L-arginine, 1 μg / mL thiamine, 0.01 μg / mL riboflavin) at 30 ° C. overnight. Culture) was used as AI-2 reporter bacteria and diluted 5000 times in AB medium to prepare a reporter bacteria solution. Various concentrations of HMF preparation (manufactured by Sigma) or DPD preparation (manufactured by OMM Scientific) were mixed with the reporter bacterial solution so that the volume ratio of the reporter bacterial solution: preparation was 9: 1, and the mixture was favorably mixed at 30 ° C. Air shaking culture was performed. The luminescence intensity after 4 hours was measured with a chemiluminescence meter (LB940 type, manufactured by Bertrud Japan), and a calibration curve regarding the luminescence intensity and the amount of AI-2 was prepared.
20〜90歳代の男女94名の被験者のうち、午前に試料を採取する被験者は、試験前日就寝前の歯磨き後から、午後の被験者は試験日朝の歯磨き後から、歯磨きを停止させた。さらに、試料採取30分前から飲食・喫煙を禁止した。 Of the 94 male and female subjects in their 20s to 90s, subjects who collected samples in the morning stopped brushing after brushing before sleeping on the day before the test, and those in the afternoon after brushing on the morning of the test day. In addition, eating, drinking and smoking were prohibited 30 minutes before sampling.
1歯の歯面に付着している歯肉縁下歯垢の全量0.001〜100mgを、歯科用スケーラーで採取した。採取した歯垢は1mLのPBSで分散させ、90℃で10分間インキュベート後、0.3gのジルコニア/シリカビーズ(粒径0.1mm、トミー精工社製)で破砕した。14000rpmで5分間遠心分離後、上清を0.22μmのフィルター(ウルトラフリーMC、ミリポア製)、さらに分子量3000カットのフィルター(マイクロコンYM−3、ミリポア製)に通すことで、サンプルを調製した。このようにして調製したサンプルを前記と同様に調製したレポーター細菌液に、レポーター細菌液:標品の容量比が9:1の割合で混合し、30℃にて好気振盪培養し、4時間後の発光強度をケミルミネッセンス計で測定した。測定した発光強度と、前記検量線から、歯垢中に含まれるAI−2を定量した。 A total amount of 0.001 to 100 mg of subgingival plaque adhering to the tooth surface of one tooth was collected with a dental scaler. The collected plaque was dispersed in 1 mL of PBS, incubated at 90 ° C. for 10 minutes, and then crushed with 0.3 g of zirconia / silica beads (particle size: 0.1 mm, manufactured by Tommy Seiko Co., Ltd.). After centrifuging at 14,000 rpm for 5 minutes, the supernatant was passed through a 0.22 μm filter (Ultra Free MC, manufactured by Millipore) and a filter with a molecular weight of 3000 cut (Microcon YM-3, manufactured by Millipore) to prepare a sample. . The sample prepared in this manner was mixed with the reporter bacterial solution prepared in the same manner as described above at a reporter bacterial solution: standard volume ratio of 9: 1, cultured at 30 ° C. under aerobic shaking, and cultured for 4 hours. The later emission intensity was measured with a chemiluminescence meter. AI-2 contained in dental plaque was quantified from the measured luminescence intensity and the calibration curve.
(2) 歯垢中AI−2量と歯周ポケットの深さ(PD)との関係
被験者の歯周ポケットに歯科用プローブを挿入し、歯周ポケットの深さ(PD)を測定し、3段階で評価した。なお、歯周ポケットの深さ(PD)は1mmごとに測定した。
(PD)
3mm以下:健常・軽度歯周病(歯肉炎)
4〜5mm:中等度歯周病(歯周炎)
6mm以上:重度歯周病(歯周炎)
(2) Relationship between the amount of AI-2 in dental plaque and the periodontal pocket depth (PD) A dental probe was inserted into the subject's periodontal pocket, and the periodontal pocket depth (PD) was measured. Rated by stage. The depth of the periodontal pocket (PD) was measured every 1 mm.
(PD)
3 mm or less: Healthy / mild periodontal disease (gingivitis)
4-5 mm: Moderate periodontal disease (periodontitis)
6 mm or more: Severe periodontal disease (periodontitis)
上記方法に従い測定したPDと、前記(1)で定量したAI−2量の関係を表1に示す。 Table 1 shows the relationship between the PD measured according to the above method and the amount of AI-2 quantified in (1).
表1から明らかなように、歯垢中AI−2量とPDとの間に相関関係が有り、PDが深くなるほど、歯肉縁下歯垢に含まれるAI−2量が増加している。従って、被験者の口腔内のAI−2量を定量することにより、歯周病の進行度を簡便にかつ正確に判定することができる。 As is apparent from Table 1, there is a correlation between the amount of AI-2 in plaque and PD, and the amount of AI-2 contained in subgingival plaque increases as PD becomes deeper. Therefore, by quantifying the amount of AI-2 in the oral cavity of the subject, the progress of periodontal disease can be determined easily and accurately.
(3) 歯垢中AI−2量と歯肉炎症指数(GI)との関係
被験者の歯周ポケットに歯科用プローブを挿入し、歯肉の炎症度合いを4段階で評価した。Gingival Index(GI)スコアの判定は、以下の通りとした。
(GI)
0: 正常歯肉
1: 歯肉に炎症あり。プロービングで出血なし。
2: 歯肉に炎症あり。プロービングで出血あり。
3: 自然出血・腫瘍形成
(3) Relationship between the amount of AI-2 in dental plaque and the gingival inflammation index (GI) A dental probe was inserted into the periodontal pocket of the subject, and the degree of inflammation of the gingiva was evaluated in four stages. The determination of Gingival Index (GI) score was as follows.
(GI)
0: Normal gingiva 1: Inflamed gingiva. No bleeding with probing.
2: The gums are inflamed. Probing and bleeding.
3: Spontaneous bleeding / tumor formation
上記方法に従い測定したGIと、前記(1)で定量したAI−2量の関係を表2に示す。 Table 2 shows the relationship between the GI measured according to the above method and the amount of AI-2 quantified in (1).
表2から明らかなように、歯垢中AI−2量とGIの間に相関関係が有り、歯肉に炎症が生じると、歯肉縁下歯垢に含まれるAI−2量が増加している。従って、被験者の口腔内のAI−2量を定量することにより、歯周病発症の有無を簡便にかつ正確に判定することができる。 As is clear from Table 2, there is a correlation between the amount of AI-2 in plaque and GI, and when inflammation occurs in the gingiva, the amount of AI-2 contained in subgingival plaque increases. Therefore, by determining the amount of AI-2 in the oral cavity of the subject, the presence or absence of periodontal disease can be determined easily and accurately.
(4) 歯垢中AI−2量と出血指数(BOP)との関係
被験者の歯周ポケットに歯科用プローブを挿入し、歯周ポケットからの出血の有無を4段階で評価した。出血指数(Bleeding on Probing、BOP)スコアの判定は、以下の通りとした。
(BOP)
0: 出血なし
1: 点状出血あり
2: 線状出血あり
3: 自然出血あり
(4) Relationship between the amount of AI-2 in dental plaque and the bleeding index (BOP) A dental probe was inserted into the periodontal pocket of the subject, and the presence or absence of bleeding from the periodontal pocket was evaluated in four stages. Bleeding index (Bleeding on Probing, BOP) score was determined as follows.
(BOP)
0: No bleeding 1: With punctate bleeding 2: With linear bleeding 3: With natural bleeding
上記方法に従い測定したBOPと、前記(1)で定量したAI−2量の関係を表3に示す。 Table 3 shows the relationship between BOP measured according to the above method and the amount of AI-2 quantified in (1) above.
表3から明らかなように、歯垢中AI−2量とBOPの間に相関関係が有り、プロービングにより歯肉に出血が生じると、歯肉縁下歯垢に含まれるAI−2量が増加している。従って、被験者の口腔内のAI−2量を定量することにより、歯周病発症の有無を簡便にかつ正確に判定することができる。 As is apparent from Table 3, there is a correlation between the amount of AI-2 in plaque and BOP, and when bleeding occurs in the gingiva due to probing, the amount of AI-2 contained in subgingival plaque increases. Yes. Therefore, by determining the amount of AI-2 in the oral cavity of the subject, the presence or absence of periodontal disease can be determined easily and accurately.
以上の結果から、口腔内のAI−2量と歯周病との間に相関関係を有することが臨床的に示された。したがって、口腔内に存在するAI−2は、歯周病発症判定用マーカー及び歯周病の進行度判定用マーカーに用いることができる。 From the above results, it was clinically shown that there is a correlation between the amount of AI-2 in the oral cavity and periodontal disease. Therefore, AI-2 present in the oral cavity can be used as a periodontal disease onset determination marker and a periodontal disease progression degree determination marker.
実施例2 歯周病改善剤又は予防剤のスクリーニング
ビブリオ・ハーベイBB170株(AB培地で30℃にて一晩培養)をAI−2レポーター細菌とし、AB培地にて4500倍に希釈し、レポーター細菌液を調製した。レポーター細菌液及び各被験剤を混合し室温で10分プレインキュベートした後、DPDを添加し、30℃にて好気振盪培養を行い、4時間後の発光強度をケミルミネッセンス計(LB940型、ベルトールドジャパン製)で測定した。測定した発光強度から、実施例1と同様の方法でAI−2を定量した。
Example 2 Screening for Periodontal Disease Improvement Agent or Preventive Agent Vibrio Harvey BB170 strain (cultured overnight at 30 ° C. in AB medium) as AI-2 reporter bacteria, diluted 4500 times in AB medium, reporter bacteria A liquid was prepared. After mixing the reporter bacterial solution and each test agent and preincubating at room temperature for 10 minutes, DPD was added, aerobic shaking culture was performed at 30 ° C., and the luminescence intensity after 4 hours was measured with a chemiluminescence meter (LB940 type, belt-type). Manufactured by Ludo Japan). From the measured emission intensity, AI-2 was quantified in the same manner as in Example 1.
被験剤としては、化合物1:4−ブロモ−5−(4−メトキシフェニル)−2(5H)−フラノン(シグマ社製)、化合物2:3,4−ジブロモ−5−ヒドロキシ−2(5H)−フラノン(シグマ社製)、化合物3:4−ブロモ−5−メトキシ−5−(4−メトキシフェニル)−2(5H)−フラノン(シグマ社製)、及び化合物4:4−{[3−ブロモ2−(4−メトキシフェニル)−5−オキソ−2,5−ジヒドロ−2−フラニル]オキシ}安息香酸(シグマ社製)を用いた。また、陽性対照として、AI−2阻害化合物として公知の3種の化合物(化合物5:4−ヒドロキシ−2,5−ジメチル−3(2H)−フラノン(シグマ製)、化合物6:2−メトキシ−2,4−ジフェニル−3(2H)−フラノン(シグマ製)、化合物7:2−ペンチル−2−シクロペンテン−1−オン(シグマ製))を用いた(Bassseler et al.、Bacteriol.179、p.4043-4045、1997年;Yoshida A.et al.、Appl.Environ.Microbiol.、71(5)、p.2372-2380、2005年;及びWen Z.T.et al.、J.Bacteriol.、189(9)、p.2682-2691、2004年)。 As test agents, compound 1: 4-bromo-5- (4-methoxyphenyl) -2 (5H) -furanone (manufactured by Sigma), compound 2: 3,4-dibromo-5-hydroxy-2 (5H) -Furanone (manufactured by Sigma), compound 3: 4-bromo-5-methoxy-5- (4-methoxyphenyl) -2 (5H) -furanone (manufactured by Sigma), and compound 4: 4-{[3- Bromo 2- (4-methoxyphenyl) -5-oxo-2,5-dihydro-2-furanyl] oxy} benzoic acid (manufactured by Sigma) was used. As positive controls, three compounds known as AI-2 inhibitor compounds (compound 5: 4-hydroxy-2,5-dimethyl-3 (2H) -furanone (manufactured by Sigma), compound 6: 2-methoxy- 2,4-diphenyl-3 (2H) -furanone (Sigma), compound 7: 2-pentyl-2-cyclopenten-1-one (Sigma)) (Bassseler et al., Bacteriol. 179, p. 4043-4045, 1997; Yoshida A. et al., Appl. Environ. Microbiol., 71 (5), p. 2372-2380, 2005; and Wen Z. T. et al., J. Bacteriol. 189 (9), p.2682-2691, 2004).
AI−2量は、被験剤を添加せずDPDのみを添加したものをコントロールとしたときの、コントロールのDPD量(AI−2量)に対する相対値とした。その結果を表4に示す。 The amount of AI-2 was defined as a relative value to the control DPD amount (AI-2 amount) when the control agent was added with only DPD without adding the test agent. The results are shown in Table 4.
表4に示すように、化合物1〜4は、公知のAI−2阻害化合物と比べて、同等又はそれ以上のAI−2活性阻害効果を有することがわかった。 As shown in Table 4, it was found that Compounds 1 to 4 have an AI-2 activity inhibitory effect equivalent to or higher than that of known AI-2 inhibitory compounds.
シリアンハムスター(7週齢、雄各3匹)の下顎骨を麻酔下で摘出し、実体顕微鏡下で撮影した。評価部位は摘出下顎骨の舌側面とし、得られた写真を画像解析に供し、第一臼歯近心咬頭頂から歯槽骨頂上までの垂直距離を測定した。 The mandible of a Syrian hamster (7 weeks old, 3 males) was removed under anesthesia and photographed under a stereomicroscope. The evaluation site was the lingual side of the extracted mandible, and the obtained photograph was subjected to image analysis, and the vertical distance from the first molar mesial cusp to the alveolar crest was measured.
別のシリアンハムスター(7週齢、雄各3匹)の下顎左右第一臼歯歯頚部に絹糸を結紮し、翌日より同結紮部位に前記化合物1若しくは2の1%DMSO含有PBS溶液(濃度10μM)、又は1%DMSO含有PBS200μLを1日2回、2週間滴下し、飼育した。
飼育終了後、麻酔下にて下顎骨を摘出し、実体顕微鏡下で撮影した。顕微鏡写真を図1(a)〜(c)に示す。評価部位は摘出下顎骨の舌側面とし、得られた写真を画像解析に供した。得られた写真を画像解析に供し、第一臼歯近心咬頭頂から歯槽骨頂上までの垂直距離を測定した。
Another Syrian hamster (7 weeks old, 3 males each) was ligated with silk thread to the lower left and right first molar teeth neck of the lower jaw, and from the next day, 1% DMSO-containing PBS solution (concentration: 10 μM) of the above compound 1 or 2 Alternatively, 200 μL of PBS containing 1% DMSO was dropped twice a day for 2 weeks and reared.
After the rearing, the mandible was removed under anesthesia and photographed under a stereomicroscope. The photomicrographs are shown in FIGS. The evaluation site was the lingual side of the extracted mandible, and the obtained photograph was subjected to image analysis. The obtained photograph was used for image analysis, and the vertical distance from the first molar mesial cusp to the alveolar crest was measured.
被験剤の添加前及び2週間添加後の、第一臼歯近心咬頭頂から歯槽骨頂上までの垂直距離から、歯槽骨吸収深度(2週間添加後の値−添加前の値)を測定した。その結果を表5に示す。 The alveolar bone resorption depth (value after addition for 2 weeks−value before addition) was measured from the vertical distance from the first molar mesial apex to the alveolar crest before addition of the test agent and after addition for 2 weeks. The results are shown in Table 5.
図1及び表5から、AI−2活性阻害効果を有する化合物1及び2を口腔内に滴下することにより歯槽骨の吸収が抑制され、口腔内に存在するAI−2量と歯周病との間に相関関係があり、AI−2活性阻害効果を有する化合物が歯周病の予防及び改善(治療)に有効であることが示された。
従って、AI−2活性阻害効果を有する被験剤を選択することにより、歯周病改善剤又は予防剤のスクリーニングすることができる。
From FIG. 1 and Table 5, by dripping the compounds 1 and 2 having an AI-2 activity inhibitory effect into the oral cavity, the absorption of alveolar bone is suppressed, and the amount of AI-2 present in the oral cavity and periodontal disease It was shown that a compound having an AI-2 activity inhibitory effect is effective for the prevention and improvement (treatment) of periodontal disease.
Therefore, by selecting a test agent having an AI-2 activity inhibitory effect, it is possible to screen for a periodontal disease improving agent or preventive agent.
Claims (3)
被験剤を混合せずに、オートインデューサー−2を介するクオラムセンシングシステムを有する細菌と4,5−ジヒドロキシ−2,3−ペンタンジオンとを混合した場合のオートインデューサー−2活性を100としたとき、この値に対する、前記被験剤を混合したときのオートインデューサー−2活性の定量値が相対値で29.6%以下となる被験剤を、オートインデューサー−2活性阻害効果を有し、歯槽骨の吸収を抑制する歯周病改善剤又は予防剤として選択する、
歯槽骨の吸収を抑制する歯周病改善剤又は予防剤のスクリーニング方法。 Autoinducer-2 activity is quantified by mixing bacteria having a quorum sensing system via autoinducer-2 with a test agent that is a candidate for a periodontal disease ameliorating agent or prophylactic agent that suppresses alveolar bone resorption. Measured
The autoinducer-2 activity when the bacteria having the quorum sensing system via autoinducer-2 and 4,5-dihydroxy-2,3-pentanedione are mixed without mixing the test agent is 100. when, for this value, a test agent quantitative value of autoinducer-2 activity when mixed with the test agent is less than 29.6% as a relative value, it has a autoinducer-2 activity inhibitory effect Select as periodontal disease ameliorating agent or preventive agent to suppress alveolar bone resorption ,
A screening method for an agent for improving or preventing periodontal disease that suppresses alveolar bone resorption .
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