JP2014215286A - メラニン生成又は色素沈着を調節するための候補化合物のスクリーニング方法 - Google Patents
メラニン生成又は色素沈着を調節するための候補化合物のスクリーニング方法 Download PDFInfo
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- JP2014215286A JP2014215286A JP2013095829A JP2013095829A JP2014215286A JP 2014215286 A JP2014215286 A JP 2014215286A JP 2013095829 A JP2013095829 A JP 2013095829A JP 2013095829 A JP2013095829 A JP 2013095829A JP 2014215286 A JP2014215286 A JP 2014215286A
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- mortalin
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- pigmentation
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- 235000010374 vitamin B1 Nutrition 0.000 description 1
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Abstract
【解決手段】メラニン生成又は色素沈着を調節するための候補化合物をスクリーニングする方法であって、被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とを、in vitroで接触させる工程と、被験化合物から、モータリン及び/又はHsp60の発現レベルを変化させる化合物を選択する工程とを含む。
【選択図】なし
Description
2. Kimura Y, Sumiyoshi M. French maritime pine bark (Pinus maritima Lam.) extract (Flavangenol) prevents chronic UVB radiation-induced skin damage and carcinogenesis in melanin-possessing hairless mice. Photochem Photobiol 2010; 86: 955-63.
3. Gidanian S, Mentelle M, Meyskens FL, Jr., Farmer PJ. Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma. Photochem Photobiol 2008; 84: 556-64.
4. Costin GE, Hearing VJ. Human skin pigmentation: melanocytes modulate skin color in response to stress. Faseb J 2007; 21: 976-94
5. Seo YK, Kim SJ, Boo YC, Baek JH, Lee SH, Koh JS. Effects of p-coumaric acid on erythema and pigmentation of human skin exposed to ultraviolet radiation. Clin Exp Dermatol 2011; 36: 260-6.
[1]メラニン生成又は色素沈着を調節するための候補化合物をスクリーニングする方法であって、
被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とを、in vitroで接触させる工程と、
被験化合物から、モータリン及び/又はHsp60の発現レベルを変化させる化合物を選択する工程とを含む方法。
[2]脱色素、美白、又はメラニン生成の抑制のための候補化合物をスクリーニングする方法であって、
被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とを接触させる工程と、
被験化合物から、モータリン及び/又はHsp60の発現レベルを減少させる化合物を選択する工程とを含む方法。
[3]メラニン生成又は色素沈着を誘導するための候補化合物をスクリーニングする方法であって、
被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とを接触させる工程と、
被験化合物から、モータリン及び/又はHsp60の発現レベルを増加させる化合物を選択する工程とを含む方法。
[4]被験化合物と細胞とを接触させる工程の前に、メラニン生成及び/又は色素沈着を誘導する工程をさらに含む、[1]〜[3]のいずれか1項に記載の方法。
[5]モータリン及び/又はHsp60の発現レベルは、ELISAによって決定される、[1]〜[4]のいずれか1項に記載の方法。
[6]モータリン及び/又はHsp60を発現可能な細胞は、ヒトメラノーマ細胞又はメラノサイトを含む、[1]〜[5]のいずれか1項に記載の方法。
[7]色素沈着又はメラニン生成を誘導するための医薬組成物であって、(i)Hsp60遺伝子及び/又はモータリン遺伝子を含む発現ベクター、又は(ii)Hsp60タンパク質及び/又はモータリンタンパク質を含む、医薬組成物。
[8]組成物が皮膚の障害を治療するために使用される、[7]に記載の医薬組成物。
[9]皮膚の障害が、癌、色素沈着過度、発疹及びケロイドからなる群から選択される、[8]に記載の医薬組成物。
[10]モータリン、HSP60、及びミトコンドリア中の他のタンパク質に基づいてミトコンドリア機能を操作する工程を含む、色素沈着又はメラニン生成に介入する方法。
[11]モータリン、HSP60、及びミトコンドリア中の他のタンパク質に関与する細胞の酸化ストレス反応を操作する工程を含む、色素沈着又はメラニン生成に介入する方法。
[12]皮膚の美白のために、[1]〜[6]のいずれか1項の方法により得られる、モータリン及びHsp60から選択される少なくとも1つの遺伝子の発現抑制剤の化粧品への使用。
[13]培養皮膚細胞のメラニン生成又は色素沈着を操作する方法であって、
幹細胞を皮膚細胞にin vitroで分化させる工程と、
培養皮膚細胞中のモータリン及び/又はHsp60の量を調節する工程とを含む方法。
[0013] 本発明のスクリーニング法は、メラニン生成又は色素沈着を調節する候補化合物をスクリーニングする方法であって、(i)被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とをin vitroで接触させる工程と、(ii)被験化合物からモータリン及び/又はHsp60の発現レベルを変化させる化合物を選択する工程とを含む方法である。工程(ii)では、モータリン及び/又はHsp60の発現レベルを少なくとも20%変化させる任意の化合物を選択することが好ましい。
[0034] 本発明の化粧品は、細胞中のモータリン及び/又はHsp60の発現レベルを低下させる化合物を含む。
[0051] 乳化オルガノシロキサンエラストマーとしては、ポリアルコキシル化シリコーンエラストマー又はポリグリセロール化シリコーンエラストマーなどが挙げられる。
[0052] ポリアルコキシル化シリコーンエラストマーとしては、DOW CORNING社より「DC9010」及び「DC9011」などの名称で市販されているもの、信越化学工業株式会社より「KSG−20」、「KSG−21」、「KSG−30」、「KSG−31」、「KSG−32」、「KSG−33」、「KSG−210」、「KSG−310」、「KSG−320」、「KSG−330」、「KSG−340」及び「X−226146」などの名称で市販されているものなどが挙げられる。
[0053] ポリグリセロール化シリコーンエラストマーとしては、信越化学工業株式会社より「KSG−710」、「KSG−810」、「KSG−820」、「KSG−830」、「KSG−840」、「KSG−31」、「KSG−32」、「KSG−41」、「KSG−42」、「KSG−43」及び「KSG−44」などの名称で市販されているものなどが挙げられる。
[0060] 金属イオン封鎖剤としては、例えば、1−ヒドロキシエタン−1,1−ジフォスホン酸、1−ヒドロキシエタン−1,1−ジフォスホン酸四ナトリウム塩、エデト酸二ナトリウム、エデト酸三ナトリウム、エデト酸四ナトリウム、クエン酸ナトリウム、ポリリン酸ナトリウム、メタリン酸ナトリウム、グルコン酸、リン酸、クエン酸、アスコルビン酸、コハク酸、エデト酸、エチレンジアミンヒドロキシエチル三酢酸3ナトリウム等が挙げられる。
[0062] 多価アルコール又はその誘導体としては、例えば、2価のアルコール(例えば、エチレングリコール、プロピレングリコール、ペンチレングリコール、トリメチレングリコール、1,2−ブチレングリコール、1,3−ブチレングリコール、テトラメチレングリコール、2,3−ブチレングリコール、ペンタメチレングリコール、2−ブテン−1,4−ジオール、ヘキシレングリコール、オクチレングリコール等);3価のアルコール(例えば、グリセリン、トリメチロールプロパン等);4価アルコール(例えば、1,2,6−ヘキサントリオール等のペンタエリスリトール等);5価アルコール(例えば、キシリトール等);6価アルコール(例えば、ソルビトール、マンニトール等);多価アルコール重合体(例えば、ジエチレングリコール、ジプロピレングリコール、トリエチレングリコール、ポリプロピレングリコール、テトラエチレングリコール等);2価アルコールアルキルエーテル類(例えば、ジエチレングリコールモノメチルエーテル、ジエチレングリコールモノエチルエーテル、ジエチレングリコールモノブチルエーテル等);2価アルコールエーテルエステル(例えば、エチレングリコールモノメチルエーテルアセテート、エチレングリコールモノエチルエーテルアセテート等);グリセリンモノアルキルエーテル(例えば、キミルアルコール、セラキルアルコール、バチルアルコール等);糖アルコール(例えば、ソルビトール、マルチトール、マルトトリオース、マンニトール、ショ糖、エリトリトール、グルコース、フルクトース、デンプン分解糖、マルトース、キシリトース、デンプン分解糖還元アルコール等)等が挙げられる。
[0063] 単糖としては、例えば、三炭糖(例えば、D−グリセリルアルデヒド、ジヒドロキシアセトン等);四炭糖(例えば、D−エリトロース、D−エリトルロース、D−トレオース、エリスリトール等);五炭糖(例えば、L−アラビノース、D−キシロース、L−リキソース、D−アラビノース、D−リボース、D−リブロース、D−キシルロース、L−キシルロース等);六炭糖(例えば、D−グルコース、D−タロース、D−プシコース、D−ガラクトース、D−フルクトース、L−ガラクトース、L−マンノース、D−タガトース等);七炭糖(例えば、アルドヘプトース、ヘプロース等);八炭糖(例えば、オクツロース等);デオキシ糖(例えば、2−デオキシ−D−リボース、6−デオキシ−L−ガラクトース、6−デオキシ−L−マンノース等);アミノ糖(例えば、D−グルコサミン、D−ガラクトサミン、シアル酸、アミノウロン酸、ムラミン酸等);ウロン酸(例えば、D−グルクロン酸、D−マンヌロン酸、L−グルロン酸、D−ガラクツロン酸、L−イズロン酸等)等が挙げられる。
[0064] オリゴ糖としては、例えば、ショ糖、ラクトース、マルトース、トレハロース、セロビオース、ゲンチオビオース、ウンビリシン、ラフィノース、ゲンチアノース、マルトトリオース、メレジトース、プランテオース、ウンベリフェロース、スタキオース、ベルバスコース等が挙げられる。
[0065] アミノ酸としては、例えば、中性アミノ酸(例えば、スレオニン、システイン等);塩基性アミノ酸(例えば、ヒドロキシリジン等)等が挙げられる。また、アミノ酸誘導体として、例えば、アシルサルコシンナトリウム(ラウロイルサルコシンナトリウム)、アシルグルタミン酸塩、アシルβ−アラニンナトリウム、グルタチオン、ピロリドンカルボン酸等が挙げられる。
[0066] 有機アミンとしては、例えば、モノエタノールアミン、ジエタノールアミン、トリエタノールアミン、モルホリン、トリイソプロパノールアミン、2−アミノ−2−メチル−1,3−プロパンジオール、2−アミノ−2−メチル−1−プロパノール等が挙げられる。
[0067] 高分子エマルジョンとしては、例えば、アクリル樹脂エマルジョン、ポリアクリル酸エチルエマルジョン、アクリルレジン液、ポリアクリル酸アルキルエマルジョン、ポリ酢酸ビニル樹脂エマルジョン、天然ゴムラテックス等が挙げられる。
[0070] ビタミン類としては、例えば、ビタミンA、B1、B2、B6、C、Eおよびその誘導体、パントテン酸およびその誘導体、ビオチン等が挙げられる。
[0071] 抗酸化剤としては、例えば、パルミチン酸アスコルビル、テトライソパルミチン酸アスコルビル、グルコシドアスコルビル、リン酸アスコルビルマグネシウム、リン酸アスコルビルナトリウム、ソルビン酸アスコルビルなどのアスコルビン酸及びその誘導体;酢酸トコフェロール、ソルビン酸トコフェロール、その他のトコフェロールのエステルなどのトコフェロール及びその誘導体;ジブチルヒドロキシトルエン(BHT)及びブチルヒドロキシアニソール(BHA);没食子酸エステル;リン酸;クエン酸;マレイン酸;マロン酸;コハク酸;フマル酸;ケファリン;ヘキサメタリン酸塩;フィチン酸;エチレンジアミンテトラ酢酸:及びアイリッシュモス(Chondrus crispus)、ロディオラ属(Rhodiola)、高度好熱菌、マテ茶葉、オーク材、カユ・ラペ樹皮(kayu rapet bark)、サクラ葉、イランイラン葉(ylang ylang leaves)などの植物エキスが挙げられる。
[0072] 保湿剤としては、例えば、ポリエチレングリコール;プロピレングリコール;ジプロピレングリコール;グリセリン;1,3−ブチレングリコール;キシリトール;ソルビトール;マルチトール;コンドロイチン硫酸などのムコ多糖類;ヒアルロン酸;ヒアルロン酸ナトリウム;ヒアルロン酸アセチルナトリウム;ムコイチン硫酸;カロニン酸;アテロコラーゲン;コレステリル−12−ヒドロキシステアレート;胆汁酸塩;ピロリドンカルボン酸塩及び乳酸塩などのNMF(自然保湿因子)の主成分;尿素、システイン及びセリンなどのアミノ酸類;短鎖可溶性コラーゲン;ジグリセリン(EO)PO付加物;NOFより「Lipidure HM」及び「Lipidure PBM」などの名称で市販されている2−メタクリロイルオキシエチルホスホリルコリンのホモポリマー又はコポリマー;パンテノール;アラントイン;NOFより「Wilbride S 753」の名称で市販されているPEG/PPG/ポリブチレングリコール-8/5/3グリセリン;旭化成ケミカルズ株式会社より「AMINOCOAT」の名称で市販されているトリメチルグリシン;スウィートチェスナット(Castanea sativa)エキス、ヘーゼルナットタンパク質加水分解物、チューベローズ(Polianthes tuberosa)多糖類、アルガンツリー種子油(Argania spinosa kernel oil)、丸善製薬株式会社より「真珠エキス」(登録商標)の名称で市販されているコンキオリン含有真珠エキスなどの各種植物エキスが挙げられる。
[0073] 皮膚軟化剤としては、ポリメタクリル酸グリセリル、メチルグルセス−20(methyl gluceth-20)などが挙げられる。
[0074] 老化防止剤としては、例えば、アシルアミノ酸(具体的には、SEDERMA社より「Maxilip」、「Matrixyl 3000」、「Biopeptide CL」の名称で市販されているもの、SEPPIC社より「Sepilift」の名称で市販されているものなどが挙げられる。);エンドウ(Pisum sativum)エキス;ダイズタンパク質加水分解物;マンヌロン酸メチルシラノール;加水分解ペポカボチャ種子油粕;セネデスムスエキスなどが挙げられる。
[0075] 抗汚染剤としては、例えば、ワサビノキ種子エキス(Moringa pterygosperma seed extracts)(具体的には、LSN社より「Purisoft」の名称で市販されているものが挙げられる。);シアバターエキス(具体的には、SILAB社より「Detoxyl」の名称で市販されているもの、セイヨウキズタエキス(ivy extract)、フィチン酸、ヒマワリ種子エキスのブレンド(例えば、SEDERMA社より「OSMOPUR」の名称で市販されているもの)などが例示される。)などが挙げられる。
[0076] 角質溶解剤としては、例えば、α−ヒドロキシ酸(具体的には、グリコール酸、乳酸、クエン酸、リンゴ酸、マンデル酸及び酒石酸などが例示される)、β−ヒドロキシ酸(具体的には、サリチル酸などが例示される)、それらのエステル(具体的には、乳酸C12−13アルキル)及びこれらのヒドロキシ酸を含む植物エキス(具体的には、ロゼリソウ(Hibiscus sabdriffa)エキスなどが例示される)などが挙げられる。
[0078] 抗炎症剤としては、例えば、ビサボロール、アラントイン、トラネキサム酸、酸化亜鉛、硫黄酸化物及びその誘導体、コンドロイチン硫酸塩、グリチルリチン酸及びその誘導体(グリチルリチン酸塩など)などが挙げられる。
[0079] また、本発明の外用剤用組成物は、メラニン生成メカニズム(ステージI)に関与するメラニン細胞特異的糖タンパク質であるPmel17などの構造タンパク質の合成を阻害するために、ホワイトニング剤を少なくとも1種含んでいてもよい。ホワイトニング剤としては、BASF社より「Cytovector」(商標)の名称で市販されているフェルラ酸含有サイトベクター(水、グリコール、レシチン、フェルラ酸、ヒドロキシエチルセルロース)などが挙げられる。
[0082] さらに、必要に応じて、本発明による外用剤用組成物は、ビタミンC化合物などの抗酸化作用をも有するホワイトニング剤を含んでいてもよい。例えば、アスコルビン酸塩、脂肪酸又はソルビン酸のアスコルビルエステル、その他のアスコルビン酸誘導体などが挙げられる。具体的には、リン酸アスコルビル塩(リン酸アスコルビルマグネシウム、リン酸アスコルビルナトリウムなど)、アスコルビン酸のサッカリドエステル(アスコルビル−2−グルコシド、2−O−α−D−グルコピラノシル L−アスコルビン酸、6−O−β−D−ガラクトピラノシル L−アスコルビン酸など)が例示される。このタイプの活性成分は、DKSH社より「Ascorbyl glucoside」(商標)の名称で市販されている。
[0083] さらに、本発明の水中油型乳化組成物は、他のホワイントニング剤を含んでいてもよい。例えば、植物エキス(房咲水仙などのエキス)、アルブチン、コウジ酸、エラグ酸、システイン、4−チオレゾルシン、レゾルシノールもしくはルシノール又はそれらの誘導体、グリチルリチン酸及びヒドロキノン−β−グルコシドなどの色素沈着抑制剤を含んでいてもよい。
[0084] さらに、必要に応じて、本発明による外用剤用組成物は、有機及び/又は無機日焼け止め剤を含んでいてもよい。
[0085] 有機日焼け止め剤の例としては、ジベンゾイルメタン誘導体(例えば、ブチルメトキシジベンゾイルメタン(HOFFMANN LA ROCHEからParsol 1789という商品名で市販されている製品)など);桂皮酸誘導体(例えばエチルヘキシルメトキシ桂皮酸エステル(HOFFMANN LA ROCHEからParsol MCXという商品名で市販されている製品)、サリチル酸塩、パラアミノ安息香酸など);β−β’−ジフェニルアクリル酸誘導体;ベンゾフェノン誘導体;ベンジリデンショウノウ誘導体(テレフタリリデン−ジショウノウスルホン酸;フェニルベンズイミダゾール誘導体;トリアジン誘導体;フェニルベンゾトリアゾール誘導体;アントラニル酸誘導体などが挙げられ、いずれもコーティング又はカプセル化されていてもよい。
[0089] 本発明のスクリーニング法により選択されるモータリン及び/又はHsp60の発現レベルを低下させる化合物は、しみ、ケロイド、又は色素沈着過度など、過剰なメラニンによって引き起こされる疾患又は症状の治療に使用する医薬組成物に使用することができる。
[0092] 本発明は培養皮膚細胞のメラニン生成又は色素沈着を操作する方法も提供し、これは、幹細胞をin vitroで皮膚細胞に分化させる工程と、培養皮膚細胞中のモータリン及び/又はHsp60の量を調節する工程とを含む。
[0094] 以降で本発明を実施例に基づいて詳細に説明するが、本発明はそれに限定されないことに留意されたい。
細胞培養、処理及びトランスフェクション
ヒトメラノーマG361は、日本の東北大学生物医学的研究用細胞資源センターから購入し、加湿したインキュベータ(36℃、5%CO2)内で10%のウシ胎児血清を添加したDMEM-Dulbecco's Modified Eagle's Medium (Invitrogen)中で培養した。明るい肌及び暗い肌のドナー由来のヒト初代メラノサイトは、ScienCell Research Laboratoriesから購入し、推奨されたメラノサイト増殖培地で培養した。細胞は、推奨されたプロトコルに従い、Fugene (Roche)を使用して、モータリン又はHSP60タンパク質をコードする発現プラスミドで形質転換した。通常、70〜80%コンフルエントの6cmの培養皿に、3〜5μgのプラスミドDNAを使用した。細胞はプラスミドで48時間インキュベートし、その後に細胞を回収して、下記のようなプロトコルによって発現を示す遺伝子を調べた。shRNAトランスフェクションについては、Oligofectamine(商標) Transfection Reagent (Life technologies(商標))を製造業者の指示に従って使用した。トランスフェクトした細胞を、ピューロマイシン(1μg/ml)を添加した培地で24〜48時間インキュベートし、次に下記のようなアッセイのために処理した。
細胞は、ジアシルグリセロール(OAG、20μg/ml)、3−イソブチル−1−メチルキサンチン(IBMX−100μM)(Merck Millipore)、PD98059(20μg/ml)(Life Technologies)及び過酸化水素(150〜300μM)などの色素誘導物質で48時間処理した。
細胞はジアシルグリセロール(OAG、20μg/ml)で24時間処理し、その後に下記のような通常の培地又はビタミンC(0.1〜0.3mM)で24〜48時間回収した。
細胞はTXCで以下のように処理した。
−モータリンshRNAベクターの構築
モータリンを標的とするshRNAを発現するプラスミドを、既報(Miyagishi他(2004)J Gene Med, 6: 715-723及びYoo他、(2010)J. Gene Medicine 12, 586-595)にしたがって、U6プロモータベクターで生成した。モータリンについて使用した3つの標的サイトは#007-GCAACAAGCTGAAAGAAGA(配列番号:1)、#008-GCCAGAAGGACAACATATG(配列番号:2)及び#009-GAATGAGGCTAGACCTTTA(配列番号:3)であった。通常、モータリン標的shRNAを生成するために、センスオリゴヌクレオチド5' -gatcccGCAACAAGCTGAAAGAAGAttcaagagaTCTTCTTTCAGCTTGTTGCttttttggaaa-3'(配列番号:4)及びそれとコグネートのアンチセンスオリゴヌクレオチド5'-agcttttccaaaaaaGCAACAAGCTGAAAGAAGAttcaagagaTCTTCTTTCAGCTTGTTGCgg-3'(配列番号:5)をアニールすることによって、ヒトモータリンを標的とするshRNAを発現するDNA断片を生成した。19−ヌクレオチドのモータリン標的配列は大文字で表示され、9−ヌクレオチドヘアピン及び指向性クローニングに必要な配列は小文字で示されている。
HSP60を標的とするshRNAを発現するベクターを上述したように生成した。使用した2つの標的サイトは-5' GTGAGATGAGGAGCCAGTACC 3'(配列番号:6)及び5' TTCAAGCATTAAGGCTCGGGC 3'(配列番号:7)であった。
Hsp60及びモータリンを過剰発現させるために、レトロウィルス発現システムを使用した。既報(Wadhwa他、2006 International J Cancer, 118: 2973-2980)にしたがって、完全長のHSP60又はモータリンタンパク質をコードするcDNAをベクターのBamHIサイト内にクローニングした。レトロウィルスを産生するために、Plat−E、すなわちエコトロピック・マウス白血病ウィルス(MuLV)パッケージング細胞株に、pVPack-GP(gal及びpolを発現する)及びpVPack VSVG(envを発現する)ベクター(カリフォルニア州Stratagene)と、pCXneoレトロウィルスベクター又はpCX4neo/モータリンをFuGENE6(Boehringer Mannheim)を使用してトランスフェクトした。48時間後に培養上清を採取し、0.45μmのフィルタで濾過して、感染用のウィルス株として使用した。MCF7細胞(6ウェルディッシュで2×105/ウェル)を8μg/mlのポリブレンで1時間、37℃で処理し、その後に200μlの濾過したウィルスストックを1時間、細胞に感染させた。プレートは15分おきに傾けて、ウィルス粒子を細胞全体に広げた。1時間後、2mlのDMEMを培養物に加えて、ウィルスストックを希釈し、次にプレートを37℃でさらに48時間インキュベートした。安定した発現細胞株が得られるまで、G418(1mg/ml)を含む培地内で細胞を選別した。
細胞(約10000個/ウェル)を96ウェルのNunc-Immuno maxisorpプレートで培養した。約70%コンフルエントで、上述のように細胞にshRNAプラスミド(100ng/ウェル)をトランスフェクトした。トランスフェクションの24時間後、ピューロマイシン添加培地内で細胞を選別し、次に下記のようにチロシナーゼELISAのために処理した。細胞をPBSで2回洗浄し、RIPA溶解緩衝液(Santa Cruz Biotehnology)中に溶解させた。溶解物中のタンパク質濃度を測定し(Pierce BCA Protein Assayキット、Thermo Scientific)、コーティング緩衝液(0.1Mの重炭酸ナトリウム、0.02%のアジ化ナトリウム、pH9.6)中でサンプルを1μg/μlの濃度まで希釈した。コーティング緩衝液で100μlの最終ボリュームとして5μgのタンパク質サンプルを各ウェルに加えた。プレートを10分間優しく振盪し、次にしっかり密封して室温で2時間放置した。溶液を廃棄し、プレートを洗浄緩衝液(PBS0.5%のTween 20)で5分間洗浄した。ブロッキング緩衝液(200μl)を各ウェルに加え、プレートを室温で2時間インキュベートし、その後に洗浄緩衝液で2回(毎回3分間)洗浄した。抗チロシナーゼ抗体(50μl、ブロッキング緩衝液中に5μg/ml)を各ウェルに加え、プレートを密封して1〜2時間室温に維持し、その後に洗浄緩衝液で洗浄した(3×10分間)。プレートを2次抗体(アルカリホスファターゼ、APヤギ抗ラビットIgG)で1時間インキュベートし、その後に洗浄緩衝液で3〜5回洗浄した。100μlのAP(−ニトロフェニルホスフェート、Disodium Salt、PNPP)用基質を各ウェルに加えた。プレートを再び密封して30分間放置し、その後にチロシナーゼ発現の定量的評価を表す405nmの吸光度を測定した。
細胞を96ウェルプレートで培養し、プレートの表面に24時間付着させ、その後に上述のとおり図の説明文で示したようにトランスフェクション及び処理をした。細胞をPBSで洗浄した。各々に水酸化カリウム(KOH−0.85N、100μl)を加えた。プレートを暗所で一晩優しく振盪した。色素の吸光度を405nmで測定した。標準的な精製メラニン(シグマ)を使用して、メラニン含有量を計算した。
細胞を、12ウェル培養ディッシュに配置したカバーガラス上で培養し、処理した。上述の処理の最後に、カバーガラスを冷リン酸緩衝食塩水(PBS)で洗浄し、細胞を予め冷却したメタノール:アセトン(1:1v/v)混合物で5〜10分間固定した。固定した細胞をPBSで洗浄し、PBS中の0.2%のトリトンX−100で10分間透過性処理して、PBS中び2%ウシ血清アルブミン(BSA)で20分間ブロッキングした。細胞を、抗モータリン、抗HSP60、抗チロシナーゼ、及び抗メラノソーム抗体で染色した。Alexa-488又はAlexa-594結合抗体(Molecular probes)で2次染色することにより、免疫染色を視覚化した。PBS中の0.2%トリトンX−100(PBST)で3回から4回洗浄した後、細胞にFluoromount(Difco)を重ねた。落射蛍光のカールツァイス顕微鏡で細胞を検査した。
細胞を6ウェルプレートで増殖させ、処理した。上述したトランスフェクション又は薬剤処理の後、細胞をRIPA溶解緩衝液で溶解させた。細胞溶解物(30μg)をSDS−PAGE上で分離し、その後に半乾燥トランスファブロッタを使用してニトロセルロース膜(Millipore)上に転写した。イムノアッセイは抗モータリン、抗HSP70及び抗チロシナーゼ抗体で行った。形成された免疫複合体は、ホースラディッシュ・ペルオキシダーゼ結合抗ラビット/マウス免疫グロブリンGで視覚化した。LAS 3000 mini(富士フイルム)でバンドを検出した。
メラニン生成に関与する遺伝子をスクリーニングするアッセイの確立
図1は、コントロール及び明るい肌のドナー由来のOAG処理した初代メラノサイト(A)及びヒトメラノーマ細胞G361(B)におけるメラノソーム誘導の免疫組織化学的分析の結果を示す。細胞を検出するために核染色法としてPI染色法を使用した。明るい肌のドナー由来の初代メラノサイト及びヒトメラノーマ細胞G361の両方で、OAGによりメラノソーム産生が誘導された。
図4は、OAGでメラニン生成を誘導した際のメラノソーム及びHsp60の免疫染色の結果を示す。OAGによるメラニン生成の誘導の結果、Hsp60が上方制御された。
図11は、様々な濃度でTXC処理した後の、OAG処理又はOAG未処理のG361細胞内のメラニン含有量を示す。TXC処理は、内因性のメラニン及び細胞内でOAGに誘導されたメラニンの減少を引き起こした。
Claims (13)
- メラニン生成又は色素沈着を調節するための候補化合物をスクリーニングする方法であって、
被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とを、in vitroで接触させる工程と、
前記被験化合物から、モータリン及び/又はHsp60の発現レベルを変化させる化合物を選択する工程と
を含む方法。 - 脱色素、美白、又はメラニン生成の抑制のための候補化合物をスクリーニングする方法であって、
被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とを接触させる工程と、
前記被験化合物から、モータリン及び/又はHsp60の発現レベルを減少させる化合物を選択する工程と
を含む方法。 - メラニン生成又は色素沈着を誘導するための候補化合物をスクリーニングする方法であって、
被験化合物と、モータリン及び/又はHsp60を発現可能な細胞とを接触させる工程と、
前記被験化合物から、モータリン及び/又はHsp60の発現レベルを増加させる化合物を選択する工程と
を含む方法。 - 前記被験化合物と細胞とを接触させる工程の前に、メラニン生成及び/又は色素沈着を誘導する工程をさらに含む、請求項1〜3のいずれか1項に記載の方法。
- モータリン及び/又はHsp60の発現レベルは、ELISAによって決定される、請求項1〜4のいずれか1項に記載の方法。
- モータリン及び/又はHsp60を発現可能な前記細胞は、ヒトメラノーマ細胞又はメラノサイトを含む、請求項1〜5のいずれか1項に記載の方法。
- 色素沈着又はメラニン生成を誘導するための医薬組成物であって、(i)Hsp60遺伝子及び/又はモータリン遺伝子を含む発現ベクター、又は(ii)Hsp60タンパク質及び/又はモータリンタンパク質を含む、医薬組成物。
- 前記組成物は、皮膚の障害を治療するために使用される、請求項7に記載の医薬組成物。
- 前記皮膚の障害は、癌、色素沈着過度、発疹及びケロイドからなる群から選択される、請求項8に記載の医薬組成物。
- モータリン、HSP60、及びミトコンドリア中の他のタンパク質に基づいてミトコンドリア機能を操作する工程を含む、色素沈着又はメラニン生成に介入する方法。
- モータリン、HSP60、及びミトコンドリア中の他のタンパク質に関与する細胞の酸化ストレス反応を操作する工程を含む、色素沈着又はメラニン生成に介入する方法。
- 皮膚の美白のために、請求項1〜6のいずれか1項に記載の方法により得られる、モータリン及びHsp60から選択される少なくとも1つの遺伝子の発現抑制剤の化粧品への使用。
- 培養皮膚細胞のメラニン生成又は色素沈着を操作する方法であって、
幹細胞を皮膚細胞にin vitroで分化させる工程と、
前記培養皮膚細胞中のモータリン及び/又はHsp60の量を調節する工程と
を含む方法。
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