JP6643314B2 - 骨分化促進能及び歯周靭帯線維母細胞活性促進能を有するペプチド及びその用途 - Google Patents
骨分化促進能及び歯周靭帯線維母細胞活性促進能を有するペプチド及びその用途 Download PDFInfo
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Description
(i)本発明の配列番号1または配列番号2のアミノ酸配列からなるペプチドは、造骨細胞の増殖と分化促進、及び歯周靭帯線維母細胞の増殖と活性促進効能を示す。
(ii)本発明のペプチドは、smad1/5/8リン酸化のようなBMPシグナリングを増加させ、造骨細胞の成長及びCOL1A1、BSP、ALPのような分化マーカーの発現を増加させることにより、究極的に造骨活性を示す。
(iii)本発明のペプチドは、PI3K及びAktリン酸化を通じて、歯周靭帯線維母細胞の成長を促進し、COL1A1及びDSPPのような活性マーカーの発現を増加させることにより、究極的に歯周組織再生活性を示す。
(iv)本発明は、上述のペプチドを含む骨疾患の予防または治療用組成物及び歯周疾患の予防または治療用組成物を提供する。
クロロトリチルクロライドレジン(Chloro trityl chloride resin; CTL resin, Nova biochem Cat No. 01−64−0021) 700mgを反応容器に入れて、メチレンクロライド(MC) 10mlを加えて3分間攪拌した。溶液を除去してジメチルホルムアミド(DMF) 10mlを入れて3分間攪拌した後、再び溶媒を除去した。反応器に10mlのジクロロメタン(DCM)溶液を入れて、Fmoc−Ser(tBu)−OH(Bachem, Swiss) 200mmole及びジイソプロピルエチルアミン(DIEA) 400mmoleを入れた後、攪拌してよく溶かし、1時間攪拌しながら反応した。反応後、洗浄して、メタノールとDIEA(2:1)をDCMに溶かして10分間反応して、過量のDCM/DMF(1:1)で洗浄した。溶液を除去して、DMFを10ml入れて、3分間攪拌した後、再び溶媒を除去した。脱保護溶液(20%のピぺリジン(Piperidine)/DMF) 10mlを反応容器に入れて、10分間常温で攪拌した後、溶液を除去した。同量の脱保護溶液を入れて、再び10分間反応を維持した後、溶液を除去し、それぞれ3分間ずつDMFで2回、MCで1回、DMFで1回洗浄し、Ser(tBu)−CTL Resinを製造した。
合成例1で合成された配列ペプチドに対するBMP2類似効能を分析するために、筋芽細胞株(myoblast cell line)のC2C12細胞を利用したMTTアッセイを進行し、増殖促進効能があるかどうかを確認した。
合成例1で合成された配列ペプチドに対するBMP2類似効能を分析するために、前駆−骨芽細胞株(pre−osteoblast cell line)のMC3T3−E1細胞を利用して、ALP(Alkaline Phosphatase)染色を通じて、造骨細胞分化促進効能を示すかどうか確認した。MC3T3−E1細胞を24−ウェルプレートに3×104細胞になるように入れて、37℃、5% CO2条件下で24時間培養した。24時間後、10% FBS, 50ug/ml アスコルビン酸+100mM b−グリセロホスフェートが含有されたアルファ−MEM培地に、合成したペプチドを10ug/ml, 50ug/ml濃度で処理して、培地を3日に1回ずつ入れ替えながら13日間培養した。培養完了されたプレートウェルをPBSで2回洗浄後、アセトン、37%ホルムアルデヒド、クエン酸溶液を含む固定バッファーで30秒間細胞固定した。白血球アルカリホスファターゼ染色キット(SIGMA)を利用して、下記の染色を進行した。
合成例1で合成された配列ペプチドによる骨分化関連遺伝子BSP(bone sialoprotin)及びCOL1A1(collagen type I alpha 1)のmRNA発現程度を確認するために、MC3T3−E1細胞を6−ウェルプレートに2×105細胞になるように入れて、24時間37℃, 5% CO2条件下で培養した。24時間後、FBS 10%含まれた培地に、それぞれのペプチドを10ug/ml、50ug/ml濃度で処理し、陽性対照群として使用されたBMP2は、100ng/ml濃度で処理した。2日後、PBSで洗浄し、Easy blue(Intron)を利用してRNAを分離した。1ugのRNAとRT premixture(Intron)を利用してcDNAを合成し、PCR premixture(Bioneer)を利用してPCRを進行した後、アガロースゲルにかけて各遺伝子のmRNA発現程度を確認した。
骨分化促進に係るシグナルのpSmad1/5/8を、本ペプチドが活性化させるかどうか確認するために、MC3T3−E1細胞を6−ウェルプレートに2×105細胞になるように入れて、37℃、5% CO2条件下で24時間培養した。24時間後、血清の含まれていない培地に入れ替え、24時間培養した後、ペプチドを10μg/ml濃度で処理して、陽性対照群として使用されたBMP2は、50ng/ml濃度で処理して15分間、30分間後にPBSで洗浄し、溶解バッファーを入れて溶解させた後、タンパク質を得てウェスタンブロッティングを進行した。
配列番号1のペプチド100μgに0.5mm×0.5mmのコラーゲンスポンジを浸して、Balb/cマウスの背中に移植して、2週間放置した。その後、コラーゲンスポンジを摘出し、パラフィンブロックに制作して、H&E染色を通じてコラーゲンスポンジ内の骨細胞浸透及び増殖程度を測定した(図5)。
配列番号1及び2のペプチドの歯周組織に対する効果を確認するために、歯周靭帯線維母細胞(Human periodontal ligament fibroblast)に対する成長促進効果を分析した。歯周靭帯線維母細胞を48−ウェルプレートに1×103細胞になるように入れて、37℃、5% CO2条件下で24時間培養した。24時間後、血清の含まれていない培地に入れ替え、6時間培養した後、ペプチドを0.05〜10μg/ml濃度で処理して、陽性対照群として使用されたBMP2及びIGF−1は、0.2μg/ml濃度で処理して、72時間培養した。培養が完了した後、培養上澄み液を除去し、エタノールを利用して細胞を固定化した後、細胞固定が終わった後、PBS(phosphate buffer saline)で3回洗浄した。洗浄溶液を除去した後、比色SRB溶液で処理して、1%アセト酸で十分洗浄した後、顕微鏡で細胞を観察して生存細胞の状態を観察して、20mMトリスで脱染色された溶液に対して紫外線560nmで吸光度を測定し、細胞の生存状態を測定した。
配列番号1及び2のペプチドの歯周靭帯線維母細胞(Human periodontal ligament fibroblast)に対する成長促進効果を確認するために、関連シグナリング分子のリン酸化程度をウェスタンブロッティングで確認した。歯周靭帯線維母細胞を6−ウェルプレートに5×105細胞になるように入れて、37℃、5% CO2条件下で24時間培養した後、ペプチドを10μg/ml濃度で処理して、陽性対照群として使用されたbFGFは、0.2μg/ml濃度で処理した後、5〜15分間培養時間別に細胞を回収して、全体タンパク質を分離し、p−PI3K、p−Aktに対するウェスタンブロッティングを進行した。
配列番号1及び2のペプチドの歯周靭帯線維母細胞活性促進効果を確認するために、関連遺伝子のmRNA発現をRT−PCRで確認した。歯周靭帯線維母細胞を24−ウェルプレートに1.5×104細胞になるように入れて、37℃、5% CO2条件下で24時間培養した。24時間後、血清の含まれていない培地に入れ替え、24時間培養した後、ペプチドを1、10μg/ml濃度で処理して、陽性対照群として使用されたBMP−2及びIGF−1は、0.2μg/ml濃度で処理して、72時間培養した。培養完了後、PBSで洗浄して、Easy blue(Intron)を利用してRNAを分離した。1μgのRNAとRT premixture(Intron)を利用してcDNAを合成して、PCR premixture(Bioneer)を利用してPCRを進行した後、アガロースゲルにかけて各マーカーのmRNAレベルを確認した。
Claims (6)
- 配列番号1で表されるアミノ酸配列及び配列番号2で表されるアミノ酸配列からなる群から選択される1つのアミノ酸配列からなり、
ALP(Alkaline phosphatase)、COL1A1(Collagen type I alpha 1)、BSP(Bone sialoprotein)及びDSPP(Dentin sialophosphoprotein)の少なくとも1つの発現を増加させる、ペプチド。 - 前記ペプチドは、造骨細胞の増殖を促進することを特徴とする、請求項1に記載のペプチド。
- 前記ペプチドは、Smad1、Smad5及びSmad8又は、PI3K及びAktのリン酸化を増加させることを特徴とする、請求項1に記載のペプチド。
- 前記ペプチドは、歯周靭帯線維母細胞の増殖を促進することを特徴とする、請求項1に記載のペプチド。
- 請求項1から4のいずれかに記載のペプチドを有効成分として含む、骨疾患の予防または治療用組成物。
- 請求項1から4のいずれかに記載のペプチドを有効成分として含む、歯周疾患の予防または治療用組成物。
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