JP5785088B2 - ヒト胚性幹細胞の膵内分泌系への分化 - Google Patents
ヒト胚性幹細胞の膵内分泌系への分化 Download PDFInfo
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Description
本発明は、多能性幹細胞の分化を促進する方法を提供する。具体的には本発明は、膵内分泌系に特徴的なマーカーを発現している細胞でMAFAの発現を増加させるための方法を提供する。
幹細胞は、単一の細胞レベルにて自己複製し、分化して後代細胞を生成する、それら両方の能力で定義される未分化細胞であり、後代細胞には、自己複製前駆細胞、非再生前駆細胞、及び最終分化細胞が含まれる。幹細胞はまた、インビトロで複数の胚葉(内胚葉、中胚葉及び外胚葉)から様々な細胞系の機能的細胞に分化する能力によって、また移植後に複数の胚葉の組織を生じ、胚盤胞への注入後、全部ではないとしても殆どの組織を提供する能力によっても、特徴付けられる。
多能性幹細胞の特徴付け
多能性幹細胞は、ステージ特異的胚抗原(SSEA)3及び4の1以上、並びにTra−1−60及びTra−1−81と呼ばれる抗体によって検出可能なマーカーを発現し得る(Thomsonら、Science 282:1145,1998)。インビトロで多能性幹細胞を分化させると、SSEA−4、Tra−1−60、及びTra−1−81の発現が消失し(存在する場合)、SSEA−1の発現が増大する。未分化の多能性幹細胞は通常アルカリホスファターゼ活性を有し、これは、細胞を4%パラホルムアルデヒドで固定した後、製造業者(Vector Laboratories(Burlingame Calif.))によって述べられるようにVectorRedを基質として現像することによって検出することができる。未分化多能性幹細胞はまた、RT−PCRで検出されるように、一般にOct−4及びTERTも発現する。
使用が可能な多能性幹細胞の種類としては、妊娠期間中の任意の時期(必ずしもではないが、通常は妊娠約10〜12週よりも前)に採取した前胚性組織(例えば胚盤胞等)、胚性組織、胎児組織などの、妊娠後に形成される組織に由来する多能性細胞の株化細胞系が含まれる。非限定的な例は、例えばヒト胚幹細胞株H1、H7、及びH9(WiCell)などのヒト胚幹細胞又はヒト胚生殖細胞の確立株である。それらの細胞の最初の樹立又は安定化中に本開示の組成物を使用することも想定され、その場合、源となる細胞は、源となる組織から直接採取した一次多能性細胞である。フィーダー細胞の不在下で既に培養された多能性幹細胞集団から採取した細胞も好適である。例えば、BG01v(BresaGen,Athens,GA)などの変異ヒト胚性幹細胞株も好適である。
一実施形態では、多能性幹細胞は、一般にフィーダー細胞の層上で培養され、このフィーダー細胞は、多能性幹細胞を様々な方法で支持する。あるいは、多能性幹細胞を、フィーダー細胞を基本的に含まないにも関わらず、細胞を実質的に分化させることなく多能性幹細胞の増殖を支持するような培養システム中で培養する。フィーダー細胞不含培養における多能性幹細胞の分化を伴わない増殖は、あらかじめ他の細胞種を培養することにより条件づけした培地を使用して支持される。あるいはフィーダー細胞不含培養における多能性幹細胞の分化を伴わない増殖は、合成培地を使用して支持される。
一実施形態では、本発明は、多能性幹細胞から膵臓ホルモン産生細胞を作製するための方法を提供し、かかる方法は、
a.多能性幹細胞を培養する工程と、
b.多能性幹細胞を、胚体内胚葉系に特徴的なマーカーを発現している細胞に分化させる工程と、
c.胚体内胚葉系に特徴的なマーカーを発現している細胞を、膵臓内胚葉系に特徴的なマーカーを発現している細胞に分化させる工程と、
d.膵臓内胚葉系に特徴的なマーカーを発現している細胞を、膵内分泌系に特徴的なマーカーを発現している細胞に分化させる工程と、を含む。
多能性幹細胞は、当該技術分野のいかなる方法、又は本発明で提案されるいかなる方法によって胚体内胚葉系に特徴的なマーカーを発現する細胞に分化させてもよい。
胚体内胚葉系に特徴的なマーカーを発現している細胞の形成は、以下の特定のプロトコルの前後に、マーカーの存在に関して試験することにより決定することができる。多能性幹細胞は、一般にかかるマーカーを発現しない。したがって、多能性細胞の分化は、細胞がそれらの発現を開始した際に検出される。
胚体内胚葉系に特徴的なマーカーを発現している細胞は、当該技術分野の任意の方法、又は本発明で提案する任意の方法により、膵臓内胚葉系に特徴的なマーカーを発現している細胞に分化され得る。
膵臓内胚葉系に特徴的なマーカーは、当業者に周知であり、膵臓内胚葉系の特徴を示す追加のマーカーが、継続して同定されている。これらのマーカーは、本発明に従って処理された細胞が分化して膵臓内胚葉系の特徴を示す性質を獲得したことを確認するために使用され得る。膵臓内胚葉系に特異的なマーカーとしては、例えば、HLXB9、PTF−1 α、PDX1、HNF6、HNF−1 βなどの転写因子の1つ以上のものの発現が挙げられる。
膵臓内胚葉系に特徴的なマーカーを発現している細胞は、当該技術分野の任意の方法、又は本発明で提案する任意の方法により、膵内分泌系に特徴的なマーカーを発現している細胞へと分化させることができる。
膵内分泌系に特徴的なマーカーは当業者に周知であり、膵内分泌系の特徴を示す追加のマーカーが継続して同定されている。これらのマーカーは、本発明に従って処理された細胞が分化して膵内分泌系の特徴を示す性質を獲得したことを確認するために使用され得る。膵内分泌系に特異的なマーカーとしては、例えばNGN3、NEUROD又はISL1などの転写因子の1種以上の発現が挙げられる。
一実施形態では、本発明は、膵内分泌系に特徴的なマーカーを発現している細胞を、十分な量のサイクリン依存性キナーゼ阻害剤を含む培地中で培養することで、MAFAの発現の増加を生じさせる工程を含む、膵内分泌系に特徴的なマーカーを発現している細胞でMAFAの発現を増加させるための方法を提供する。
ウシ胎児血清の非存在下での、ヒト胚性幹細胞の細胞株H1の膵内分泌細胞への分化
ヒト胚性幹細胞株H1の継代数52の細胞を、MATRIGEL(登録商標)をコートしたディッシュ(1:30希釈)上で培養し、細胞を膵内分泌系に特徴的なマーカーを発現している細胞へと分化させるために、以下の分化プロトコルを実施した。
a.2% BSA(カタログ#152401,MP Biomedical,Ohio)、100ng/mLのアクチビンA(R&D Systems,MN)、20ng/mLのWNT−3a(カタログ#1324−WN−002,R&D Systems,MN)、8ng/mLのbFGF(カタログ#100−18B,PeproTech,NJ)を加えたRPMI培地で1日処理した後に、2% BSA、100ng/mLのアクチビンA、8ng/mLのbFGFを加えたRPMI培地で更に2日間にわたって処理し(ステージ1)、次いで
b.DMEM/F12+2% BSA+50ng/mLのFGF7+0.25μMシクロパミン−KAAD(#239804,Calbiochem,CA)で2日間にわたって処理し(ステージ2)、次いで
c.DMEM/F12+1% B27(Invitrogen,CA)+50ng/mLのFGF7+0.25μMシクロパミン−KAAD+2μMレチノイン酸(RA)(Sigma,MO)+100ng/mLのノギン(R&D Systems,MN)で4日間にわたって処理し(ステージ3)、次いで
d.DMEM/F12+1% B27(Invitrogen,CA)+100ng/mLノギン+1μMのDAPT(γセクレターゼ阻害剤)(カタログ#565784,Calbiochem,CA)+1μMのALK5阻害剤II(カタログ#616452,Calbiochem,Ca)+100ng/mLのネトリン−4(R&D Systems,MN)で3日間にわたって処理し(ステージ4)、次いで
e.DMEM/F12+1% B27(Invitrogen,CA)+1μMのALK5阻害剤II(Calbiochem,Ca)で7日間にわたって処理した(ステージ5)。
実施例1に概説された分化プロトコルに従って処理された細胞に対する、EMDキナーゼ阻害剤ライブラリII由来の化合物の効果のスクリーニング
継代数44のヒト胚性幹細胞株H1の細胞を、MATRIGEL(商標)をコートした24ウェルディッシュ(1:30希釈)上に播種し、実施例1に記載の方法に従ってステージ5まで分化させた。これに続き、細胞をEMD Calbiochem化合物ライブラリ(カタログ#539745,Calbiochem,San Diego,Ca)由来の化合物を最終濃度1μMで含有しているDMEM/F12+1% B27で、4日間にわたって処理した。ビヒクルを含有しているウェルを対照として含めた。プロトコルを通して、培地は毎日交換した。全てのサンプルは2つ組複製で処理した。この処理の最後に、PCR解析用にRNAを回収した。サンプルはリアルタイムPCRにより、インスリン、グルカゴン、MAFA、及びArx4の発現について解析した。結果は、リアルタイムPCRでの測定結果により、処理サンプル対未処理対照の、インスリン/グルカゴン比(図1、パネルa)、又はMAFAとARX4の比(図1、パネルb)として表わされる。各ウェル#について対応するPubChem化合物ID#は、表1に列挙する。
実施例1に概説された分化プロトコルに従って処理した細胞でのインスリン及びMAFA発現に対する、サイクリン依存性キナーゼ阻害剤処理の効果
実施例2でインスリン発現とグルカゴン発現の比、又はMAFAとARX4の比を増加させたいくつかの化合物は、サイクリン依存性キナーゼ阻害剤であった。このような化合物の1つはPubChem化合物ID# 5330797(5−アミノ−3−((4−(アミノスルホニル)フェニル)アミノ)−N−(2,6−ジフルオロフェニル)−1H−1,2,4−トリアゾール−1−カルボチオアミド)(カタログ# 217714;Calbiochem,San Diego,Ca)である。これらの観察結果を確証づけるために、MATRIGEL(登録商標)をコートした10cm2のディッシュ上で、継代数42のヒト胚性幹細胞株H1の細胞を培養し、実施例1に記載の方法に従って、ステージ5まで処理した。ステージ5以降は、1% B27と1μMのPubChem化合物ID# 5330797を含有するDMEM/F12で、細胞を6日間にわたって処理した。培地は1日おきに交換した。リアルタイムPCR用の細胞サンプルを化合物での処理前、化合物処理の2日目及び5日目に採取した。
サイクリン依存性キナーゼ阻害剤処理は、島様クラスタにおいてMAFA発現を上昇させた
継代数52のヒト胚性幹細胞株H1の細胞を、MATRIGEL(登録商標)をコートしたディッシュ(1:30希釈)上で培養し、実施例1に記載の方法に従って分化させた。膵内分泌系に特徴的なマーカーを発現している細胞を更に成熟させるために、追加のステージ(ステージ6)を加えた。この実施例では、ステージ6はDMEM/F12+1% B27(Invitrogen,CA)での7日間の処理から構成される。培地は毎日交換した。
本発明の方法により作製されたインスリン産生細胞の、FACS解析
継代数42のヒト胚性幹細胞株H1の細胞を、MATRIGEL(登録商標)をコートしたプレート上で培養し、以下のプロトコルを使用してインスリン産生細胞へと分化させた:
a.2% BSA(カタログ# 152401,MP Biomedical,Ohio)、100ng/mLのアクチビンA(R&D Systems,MN)、20ng/mLのWNT−3a(カタログ# 1324−WN−002,R&D Systems,MN)、8ng/mLのbFGF(カタログ# 100−18B,PeproTech,NJ)を加えたRPMI培地で1日処理した後に、2% BSA、100ng/mLのアクチビンA、8ng/mLのbFGFを加えたRPMI培地で更に2日間にわたって処理し(ステージ1)、次いで
b.DMEM/F12+2% BSA+50ng/mLのFGF7+0.25μMシクロパミン−KAAD(#239804,Calbiochem,CA)で2日間にわたって処理し(ステージ2)、次いで
c.DMEM/F12+1% B27(Invitrogen,CA)+50ng/mLのFGF7+0.25μMシクロパミン−KAAD+2μMレチノイン酸(RA)(Sigma,MO)+100ng/mLのノギン(R & D Systems,MN)で4日間にわたって処理し(ステージ3)、次いで
d.DMEM/F12+1% B27(Invitrogen,CA)+100ng/mLノギン+1μMのDAPT(γセクレターゼ阻害剤)(カタログ# 565784,Calbiochem,CA)+1μMのALK5阻害剤II(カタログ# 616452,Calbiochem,Ca)+100ng/mLのネトリン−4(R&D Systems,MN)で3日間にわたって処理し(ステージ4)、次いで
e.DMEM/F12+1% B27(Invitrogen,CA)+1μMのALK5阻害剤II(Calbiochem,Ca)で7日間にわたって処理し(ステージ5)、次いで
f.DMEM/F12+1% B27で7日間にわたって処理し(ステージ6)、次いで
g.アキュターゼで5分間にわたって処理した後に、接着しているあらゆる残りの細胞を掻き取りにより取り外した。次いで細胞懸濁液を40μmのセルストレイナーで濾過した。ストレイナー上に残った細胞を基本培地ですすぐことで取り外し、超低接着性の培養プレート上で、DMEM−高グルコース(カタログ# 11995−073,Invitrogen,Ca)+1% B27+20ng/mLのアクチビンA(AA)、1μmのCDK阻害剤III(カタログ# 217714,Calbiochem,Ca)中に懸濁した状態で、5日間にわたって培養した(ステージ7)。
CDK阻害剤誘導型のMAFA発現の動態
継代数42のヒト胚性幹細胞株H1の細胞を、MATRIGEL(登録商標)をコートしたプレート上で培養し、以下のプロトコルを使用してインスリン産生細胞へと分化させた:
a.2% BSA(カタログ# 152401,MP Biomedical,Ohio)、100ng/mLのアクチビンA(R&D Systems,MN)、20ng/mLのWNT−3a(カタログ# 1324−WN−002,R&D Systems,MN)、8ng/mLのbFGF(カタログ# 100−18B,PeproTech,NJ)を加えたRPMI培地で1日処理した後に、2% BSA、100ng/mLのアクチビンA、8ng/mLのbFGFを加えたRPMI培地で更に2日間にわたって処理し(ステージ1)、次いで
b.DMEM/F12+2% BSA+50ng/mLのFGF7+0.25μMシクロパミン−KAAD(#239804,Calbiochem,CA)で2日間にわたって処理し(ステージ2)、次いで
c.DMEM/F12+1% B27(Invitrogen,CA)+50ng/mLのFGF7+0.25μMシクロパミン−KAAD+2μMレチノイン酸(RA)(Sigma,MO)+100ng/mLのノギン(R & D Systems,MN)で4日間にわたって処理し(ステージ3)、次いで
d.DMEM/F12+1% B27(Invitrogen,CA)+100ng/mLノギン+1μMのDAPT(γセクレターゼ阻害剤)(カタログ# 565784,Calbiochem,CA)+1μMのALK5阻害剤II(カタログ# 616452,Calbiochem,Ca)+100ng/mLのネトリン−4(R&D Systems,MN)で3日間にわたって処理し(ステージ4)、次いで
e.DMEM/F12+1% B27(Invitrogen,CA)+1μMのALK5阻害剤II(Calbiochem,Ca)で7日間にわたって処理し(ステージ5)、次いで
f.DMEM/F12+1% B27で7日間にわたって処理し(ステージ6)、次いで
g.アキュターゼで5分間にわたって処理した後に、接着しているあらゆる残りの細胞を掻き取りにより取り外した。次いで細胞懸濁液を40μmのセルストレイナーで濾過した。ストレイナー上に残った細胞を基本培地ですすぐことで取り外し、超低接着性の培養プレート上で、DMEM−高グルコース(カタログ# 11995−073,Invitrogen,Ca)+1% B27+20ng/mLのアクチビンA(AA)、2μmのCDK阻害剤III(カタログ# 217714,Calbiochem,Ca)中に懸濁した状態で、1〜8日間にわたって培養した(ステージ7)。
実施例1に概説された分化プロトコルに従って処理された細胞に対する、BIOMOL(商標)キナーゼ阻害剤ライブラリ由来の化合物の効果のスクリーニング
継代数51のヒト胚性幹細胞株H1の細胞を、MATRIGEL(登録商標)をコートした24ウェルディッシュ(1:30希釈)上に播種し、実施例1に記載の方法に従って、ステージ5まで分化させた。これに続き、細胞をDMEM/F12+1% B27中で1日間生育させ、次いでBIOMOL(商標)化合物ライブラリ(カタログ# 2832,BIOMOL,Plymouth Meeting,Pa)由来の化合物を最終濃度4μMで含有しているDMEM/F12+1% B27中で、6日間にわたって処理した。ビヒクルを含有しているウェルを対照として含めた。処理プロトコルを通し、ビヒクル又は化合物を含有している培地は1日おきに交換した。全てのサンプルは2つ組複製で処理した。この処理の最後に、PCR解析用にRNAを回収した。サンプルはリアルタイムPCRにより、インスリン、グルカゴン、MAFA、及びARX4の発現について解析した。結果は、リアルタイムPCRでの測定結果により、処理サンプル対未処理対照の、インスリン/グルカゴン比(表2)、又はMAFAとArx4の比(表2)として表わされる。各英数字ウェル#に対する、対応するカタログ#、CAS#、及び化合物名又はID数を表3に列挙する。
実施例1に概説された分化プロトコルに従って処理した細胞でのインスリン及びMAFA発現に対する、サイクリン依存性キナーゼ阻害剤の効果
継代数51のヒト胚性幹細胞株H1の細胞を、MATRIGEL(商標)をコートした24ウェルディッシュ(1:30希釈)上に播種し、実施例1に記載の方法に従ってステージ5まで分化させた。これに続き、細胞をDMEM/F12+1 %B27中で8日間にわたって生育させ、次いでサイクリン依存性キナーゼ阻害剤を最終濃度0.6125、1.25、又は5.0μMで含有しているDMEM/F12+1 %B27中で、4日間にわたって処理した。6種の阻害剤を試験した:PubChem ID# 5330812(EMD cat# 217714)、PubChem ID# 4566(EMD cat#217713)、PubChem ID# 5330797(EMD cat# 219476)、PubChem ID# 73292(EMD cat#341251)、PubChem ID #4592(EMD cat#495620)、及びPubChem ID# 160355(EMD cat #557360)。ビヒクルを含有しているウェルを対照として含めた。処理プロトコルを通し、ビヒクル又は化合物を含有している培地は1日おきに交換した。全てのサンプルは2つ組複製で処理した。この処理の最後に、PCR解析用にRNAを回収した。サンプルはリアルタイムPCRにより、インスリン、グルカゴン、MAFA、及びARX4の発現について解析した。結果は、リアルタイムPCRにより測定されたものとして、ビヒクル処理対照に対する倍数変化として記載される。
ウシ胎児血清を欠き、25mMグルコースを含有しているDMEM(DMEM−HG)での、ヒト胚性幹細胞の細胞株H1の膵内分泌細胞への分化
ヒト胚性幹細胞株H1の細胞を、MATRIGEL(登録商標)をコートしたディッシュ(1:30希釈)上で培養し、以下のプロトコルを用いて、膵内分泌系に特徴的なマーカーを発現している細胞へと分化させた:
a.2% BSA(カタログ# 152401,MP Biomedical,Ohio)、100ng/mLのアクチビンA(R&D Systems,MN)、20ng/mLのWNT−3a(カタログ# 1324−WN−002,R&D Systems,MN)、8ng/mLのbFGF(カタログ# 100−18B,PeproTech,NJ)を加えたRPMI培地で1日処理した後に、2% BSA、100ng/mLのアクチビンA、8ng/mLのbFGFを加えたRPMI培地で更に2日間にわたって処理し(ステージ1)、次いで
b.2% BSA+50ng/mLのFGF7+0.25μMシクロパミン−KAAD(#239804,Calbiochem,CA)を加えたRPMI培地で2日間にわたって処理し(ステージ2)、次いで
c.DMEM−HG+1% B27(Invitrogen,CA)+50ng/mLのFGF7+0.25μMシクロパミン−KAAD+2μMレチノイン酸(RA)(Sigma,MO)+100ng/mLのノギン(R & D Systems,MN)で6日間にわたって処理し(ステージ3)、次いで
d.DMEM−HG+1% B27(Invitrogen,CA)+100ng/mLノギン+1μMのALK5阻害剤II(カタログ# 616452,Calbiochem,Ca)で3日間にわたって処理し(ステージ4)、次いで
e.DMEM−HG+1% B27(Invitrogen,CA)+1μMのALK5阻害剤II(Calbiochem,Ca)で7日間にわたって処理した(ステージ5)。
実施例9に概説された分化プロトコルに従って処理された細胞に対する、EMDキナーゼ阻害剤ライブラリI由来の化合物の効果のスクリーニング
継代数45のヒト胚性幹細胞株H1の細胞を、MATRIGEL(登録商標)をコートした24ウェルディッシュ(1:30希釈)上に播種し、実施例9に記載の方法に従ってステージ5まで分化させた。これに続き細胞を供給し、ステージ5の1、3及び5日目に、DMEM−HG、1% B27(Invitrogen,CA)と、1μMのALK5阻害剤II(Calbiochem,Ca)と、DMSO(カタログ# 539744,Calbiochem,San Diego,Ca)に溶解し、2.5μMの最終濃度で処理したEMD Calbiochem化合物ライブラリ1由来の化合物とを含む培地で処理した。ビヒクルを含有しているウェルを対照として含めた。培地はステージ5では1日おきに交換したことを除き、プロトコルを通して毎日交換した。全てのサンプルは2つ組複製で処理した。
実施例9に概説された分化プロトコルに従って処理された細胞に対する、EMDキナーゼ阻害剤ライブラリII由来の化合物の効果のスクリーニング
継代数46のヒト胚性幹細胞株H1の細胞を、MATRIGEL(登録商標)をコートした24ウェルディッシュ(1:30希釈)上に播種し、実施例9に記載の方法に従ってステージ5まで分化させた。これに続き細胞を供給し、ステージ5の1、3及び5日目に、DMEM−HGと、1% B27(Invitrogen,CA)と、1μMのALK5阻害剤II(Calbiochem,Ca)(ステージ5)と、DMSO(表1及び6,Calbiochem,San Diego,Ca)に溶解させ最終濃度2.5μMに処理したEMD Calbiochem化合物ライブラリII由来の化合物と、を含む培地で処理した。ビヒクルを含有しているウェルを対照として含めた。培地はステージ5では1日おきに交換したことを除き、プロトコルを通して毎日交換した。全てのサンプルは2つ組複製で処理した。
膵内分泌系に特徴的なマーカーを発現している細胞でMAFA発現を促進する低分子阻害剤での、膵内分泌系に特徴的なマーカーを発現している細胞の細胞周期の進行の阻害
細胞周期の進行に由来して生じる細胞増殖は、細胞外増殖因子で細胞を刺激することにより活性化及び維持され得る。増殖因子は増殖因子受容体の細胞外ドメインへと結合し、受容体の細胞内ドメインの立体構造スイッチを誘導する。このシフトは受容体二量体化及び受容体の細胞内ドメイン上に位置するチロシンキナーゼの活性化を開始し、下流の複数のセリン/スレオニンキナーゼのリン酸化及び活性化を導き、最終的には細胞周期の進行と細胞増殖をもたらす。
Claims (1)
- 膵内分泌系に特徴的なマーカーを発現している細胞内でインスリン及びMAFAの発現を増加させるための方法であって、
(a)ヒト多能性幹細胞を連続的に分化させて膵内分泌系に特徴的なマーカーを発現している細胞を得る工程、と
(b)膵内分泌系に特徴的なマーカーを発現している細胞を、5-アミノ−3−((4-(アミノスルホニル)フェニル)アミノ)−N−(2,6−ジフルオロフェニル)−1H−1,2,4−トリアゾール−1−カルボチオアミド、6-シクロヘキシルメトキシ−2−(4’−スルファモイルアニリノ)プリン、13-オキソ−12,13−ジヒドロピリド[1,2−a:3,4−b’]ジインドール−5−イウム クロリド及び2−ブロモー12,13−ジヒドロ−5H−インドール[2,3−a]ピロロ[3,4−c]カルバゾール−5,7(6H)−ジオンからなる群より選ばれる追加量のサイクリン依存性キナーゼ阻害剤を含む培地中で培養することで、追加されたサイクリン依存性キナーゼ阻害剤を含む培地中で培養されない、膵内分泌系に特徴的なマーカーを発現している細胞に比較してインスリン及びMAFAの発現の増加を生じさせる工程
とを含んでなる、方法。
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