JP6871555B2 - 歯周病菌細胞侵入抑制用組成物 - Google Patents
歯周病菌細胞侵入抑制用組成物 Download PDFInfo
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- JP6871555B2 JP6871555B2 JP2019183374A JP2019183374A JP6871555B2 JP 6871555 B2 JP6871555 B2 JP 6871555B2 JP 2019183374 A JP2019183374 A JP 2019183374A JP 2019183374 A JP2019183374 A JP 2019183374A JP 6871555 B2 JP6871555 B2 JP 6871555B2
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- periodontal disease
- cells
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- cell invasion
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Description
る。侵入した菌は、強力な細胞傷害性を発揮する。侵入した菌の幾分かはリソソームで分解されるが、たとえ分解される運命となってもP. gingivalisはかなり長い時間オートラ
イソソーム内で生き続け、細胞傷害性を発揮するのである。さらには細胞内に侵入した菌の半数近くは初期エンドソームからrecycling pathwayを経由して細胞外に出て、周囲の
細胞に再侵入する。こたのめ、P. gingivalisは細胞間を往来し・生き長らえ・増殖し・
感染を続けるのである(例えば次のウェブページを参照:http://web.dent.osaka-u.ac.jp/~prevent/research01.html)。しかも、歯肉細胞内へ歯周病菌が侵入すると、歯周病菌殺菌又は抑制効果を奏する有効成分を含有する口腔用組成物を口腔内に適用しても、有効成分が歯周病菌まで到達することが難しくなるため、歯周病菌を殺菌又は抑制することが難しい。以上のことから、歯周病菌が細胞に侵入するのを抑制する手段を見出すことは歯周病の予防又は治療に大きく貢献すると考えられるため、本発明者らは検討を行った。
項1.
α−シクロデキストリン、β−シクロデキストリン、銅クロロフィリンナトリウム、フラボン、及びレスベラトロールからなる群より選択される少なくとも1種を含有する、歯周病菌細胞侵入抑制用組成物。
項2.
歯周病菌がP.gingivalisである、項1に記載の組成物。
項3.
細胞が歯肉上皮細胞である、項1又は2に記載の組成物。
項4.
口腔用組成物である、項1〜3のいずれかに記載の組成物。
P.gingivalisは恒常的に、外膜小胞Outer membrane vesicles(OMVs)を分泌していることが知られている。OMVsにはP. gingivalisのほぼ全ての細胞傷害性因子が含まれており、しかもOMVs単独でも
細胞内に侵入する。OMVsを培養細胞に添加するとエンドサイトーシスによって細胞内に取り込まれるのである。このため、P.gingivalisから精製したOMVsを粒子(例えば蛍光ビーズ)にコーティングさせることにより、細胞侵入能を有する擬似P.gingivalisを調製することができる。
止)、遮光して室温でインキュベート後、13000rpmで遠心した。遠心後、上清を除去し、PBS(−)で2回洗浄した。洗浄後、1mlのPBSに懸濁させた。当該懸濁液74μl(8サンプル分)をチューブに移し、12,000rpmで遠心した。遠心後、上清を除去し、450μlのMEMで再懸濁した。これをベシクルビーズ液として、以下検討に用いた。
Ca9−22細胞(ヒト歯肉上皮細胞由来)300μlを各ウェルに継代し(接種量:5x104 cells/300μl)、1日間培養した。1日間培養した古い培地を捨て、各種濃度の被験物質(10mM、1mM、又は0.1mM)を溶解したMEM培地200μlに置換し、予め超音波でベシクルビーズをよく分散させたベシクルビーズ液50μlずつ各ウェルに添加し、37℃、5%CO2で1時間インキュベートした。(したがって、細胞に処理した各被験物質の濃度(処理濃度)は、初期濃度の0.8倍であり、8mM、0.8mM、又は0.08mMである。)
88 Streptavidin (1:1000 in 1% BSA/PBS(−))を添加して室温でインキュベートし、細胞膜を標識したビオチンを染色した。細胞膜染色後、PBS(−)で3回洗浄を行い、共焦点レーザー顕微鏡で観察した(細胞膜染色:励起光488nm、放出光519nm;ベシクルビーズ:励起光580nm、放出光605nm)。被験物質として銅クロロフェリンナトリウム1mMを用いて検討した際の、共焦点レーザー顕微鏡観察像を、図1に示す。図1左側に被験物質無添加のときの結果を、図1右側に被験物質添加のときの結果を、それぞれ示す。
〜40個になる領域をランダムに3箇所選び観察した(N=3)。そして、領域に含まれる細胞内に存在するビーズ数をカウントすることにより、1細胞あたりのベシクルビーズ侵入数(beads/cell)を算出した。また、各被験物質の細胞侵入阻害効果を検討するため、以下の式でベシクルビーズの細胞侵入率を算出した。
被験物質含有培地で処理した1細胞あたりのベシクル侵入数/被験物質非含有培地(MEM培地のみ)で処理した1細胞あたりのベシクル侵入数×100
Claims (4)
- 2,3−ジデヒドロフラバン−4−オンを含有する、歯周病菌細胞侵入抑制用組成物。
- 歯周病菌がP.gingivalisである、請求項1に記載の組成物。
- 細胞が歯肉上皮細胞である、請求項1又は2に記載の組成物。
- 口腔用組成物である、請求項1〜3のいずれかに記載の組成物。
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