JP2010136729A - ヒトパピローマウイルス関連疾患の評価 - Google Patents
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
Abstract
【解決手段】HPV感染患者においてHPV誘導性の前癌、を検出する方法であって、患者から集められた試料の初期遺伝子および後期遺伝子の発現mRNAレベルを測定して特定の遺伝子の発現レベルの比を計算することを含み、その比がHPV誘導性の前癌、を示す、方法。
【選択図】なし
Description
HPVに基づいた疾患の予後的および診断的評価の正確性および信頼性を改良するために他のアッセイと組み合わせることができるアッセイを提供するのが本発明の別の目的である。
現在HPVに感染してはいるが、検出可能なHPVに基づいた疾患がない患者を、疾患進行の危険性のある患者および疾患進行の危険度のない患者へ階級分けするための方法を提供するのが本発明の別の目的である。
HPVに基づいた疾患処置の有効性をモニタリングするための方法を提供するのも本発明の別の目的である。
HPVに基づいた疾患の段階を評価するため、アッセイデータのコンピューターに基づいた操作、分析およびデータ取り扱いを提供するのも本発明の別の目的である。
実施例1:核酸分析の一般法
核酸のアッセイはDigeneによりWO93/10263に記載されているDigeneハイブリッド捕捉HIV試験によりHIV RNAを検出する一般原理および方法に従った。簡単には、溶解後、50μlのプローブ混合物(DNAビオチニル化プローブを含んでいる)を各々のウェルに加えた。プレートをシールし、65℃で2時間ハイブリダイゼーションを起こすためにインキュベートした。ハイブリダイゼーション後、試料をストレプトアビジン被覆マイクロプレートへ移し、25μlの抗ハイブリッド抗体を各々のウェルに加えた。プレートは室温で1時間、1100RPMでかき混ぜた。ウェルは65℃の洗浄緩衝液で6回洗浄し、続いて蒸留水で1回洗浄した。100μlの化学発光基質を各々のウェルへ加え、プレートを室温で30分間インキュベートした。プレートはDML2000ルミノメーターで読み取った。データは信号対雑音として表された。検量線を用い、各々の試料から発生した化学発光信号を細胞当たりのmRNAコピーへ変換された。前記のアッセイは全溶解細胞または他の細胞成分から分離された核酸の両方で実行できる。
実施例2:HPVの定量
この実施例は開示された方法で使用するためのHPV E6/E7発現の測定を例示している。HPV mRNA(E6/E7およびmRNAを含む)を検出および定量する方法が開発された。この実施例ではCaSki細胞での発現を測定しているが、本方法は他の細胞株および臨床試料にも一般的に応用可能である。CaSki細胞は組み込まれた高危険度のHPV−16ゲノムを含んでいる(約600コピー/細胞)。CaSki細胞は、10%FBSおよび10mMピルビン酸ナトリウムを含んでいるRMPI1640培地でサブコンフルエントで維持された。この実施例のため、CaSki細胞をコンフルエントまで増殖させ、0.1%トリプシン−0.5mM EDTAでの処理により培養皿から除去した。トリパンブルーを用いて、生きている細胞を顕微鏡で計数した。細胞はウェル当たり10、102、103、104および105細胞の最終濃度でポリスチレン、組織培養処理、96ウェルプレートへ10μl容量で播種した。各々の希釈が実施され、3重に播種された。
実施例3:保存採取培地を用いるHPV mRNAの定量
CaSki細胞は0.25%トリプシン−EDTAと37℃で5分間インキュベートすることによりトリプシン処理された。細胞はSorvall RT6000遠心分離機中、800rpmで3分間遠心分離して懸濁液からペレット化した。細胞ペレットは500μlの1X PBSに再懸濁し、トリパンブルー溶液を顕微鏡で計数した。細胞を1X PBS中、50および500細胞/μlに希釈した。ゼロ点(10μlの1X PBS)を含む各々の細胞濃度の10μlを3mlのPreservCyt試薬へ加えた。細胞ペレットの可視化を助けるために、各々のタブに100μlの試料変換緩衝液を加えた。全ての試料はよく混合し、Sorvall RT6000遠心分離機中、3800rpmで15分間遠心分離して分離させた。上清液は捨て、遠心管は実験台上で2から5分キムタオル上に逆さにすることにより水を排出させた。全てのペレットは50μlの溶解試薬(50単位のプロテイナーゼK)で再懸濁し、混合物はストレプトアビジンで被覆されたプレートへ移された。プレートをプレートシーラーで覆い、15分ごとにかき混ぜながら、37℃で30分間インキュベートした(ヒートブロック)。
実施例4:エピソーム性および組み込まれたHPV16DNAを含んでいる細胞株中のE6/E7 RNAおよびL1 RNA発現の比較
試験された細胞株
以下の細胞株が上に概説された方法に従って試験された。
SiHa:ヒト癌細胞株(Friedl(1970))
W12:非腫瘍発生性ヒト子宮頚部ケラチノサイト細胞株(Stanley(1989))
HPV感染状態
HaCaT細胞株はWhiteらの方法(White(1998))によりHPV16で感染させ、エピソーム(非組込み、全配列、スプライスされていない)HPV感染を起こさせた。40細胞毎にHPV16の約1コピーが存在していた。これらの細胞は初期段階感染またはCIN I(子宮頚部上皮内異常増殖)の代表と考えられた。W12細胞は約100コピーのエピソームHPV16DNAを含んでおり、前癌性、不死化細胞またはCIN IIまたはCIN IIIを代表している。SiHa細胞はゲノム内へ組込まれたHPV16の1−2コピーを含んでいる。これらの細胞は癌を代表すると考えられた。
方法
RNA分析は実施例1に従ってまたは以下の方法で行われた。特異的HPV16遺伝子配列を含んでいる一本鎖、ビオチニル化DNAプローブが調製された。HaCaTおよびSiHa細胞株については、細胞はコンフルエントまで増殖させ、細胞を採取し、全RNAはRNeasyキット(Qiagen Inc.,Santa Clarita,CA)を用いて精製された。W12では、全細胞が分析に使用された。完全HPVゲノムを含んでいるRNA標品は完全HPV16ゲノムを含んでいるプラスミドからの(+)センスRNAのT7RNAポリメラーゼによる転写により調製された。RNAは次に50μl当たり103、104、105、106、および107コピーに希釈された。細胞RNAの一部を50μlに希釈し、50μlのプローブ混合物(ビオチニル化、一本鎖DNAプローブを含んでいる)を加えて65℃で2時間RNA試料へハイブリダイズさせた。ハイブリダイゼーション反応液はストレプトアビジン被覆マイクロプレートへ移し、25μlの検出試薬1を各々のウェルに加えた。(検出試薬1はアルカリ性ホスファターゼ−抗RNA:DNAモノクローナル抗体結合体を含んでいる。)1時間の振盪しながらのインキュベーションの間に、RNA:DNAハイブリッドはストレプトアビジン被覆プレート上へ捕捉され、同時に抗ハイブリッド抗体結合体と反応する。いくつかの洗浄工程後、化学発光基質(Tropix CDP−starおよびEmrald)をウェルへ加え、室温で30分間インキュベーション後にマイクロプレートルミノメーターで光出力を測定した。
定量
HPV mRNAの定量は以下のように実施された。RNA標品からの結果は検量線の作製に使用された。回帰方程式はコピーの対数に対する信号対雑音比から1を引いたものの[(S/N)−1]対数から計算された。回帰方程式は続いて細胞RNA試料中のmRNAコピー数を計算するために使用された。
比の結果
各々の細胞型に対するHPV16 E6、E7、E2、E4、L1およびL2の比が計算された。結果は表2に示されている。これらの結果はエピソームにおける初期感染では(E6+E7)/L1の比は約0.7であり、前癌性不死化細胞株においては該比は約4であり、および癌性細胞株においては該比は無限大に到達することを示している。
本明細書に引用された出版物およびそれらが引用されている資料は特に本明細書において援用される。
文献
Claims (1)
- HPV感染患者においてHPV誘導性の前癌を検出する方法であって、以下の工程:
該患者から集められた試料のHPV発現mRNAレベルを測定し、ここでmRNAはE6 mRNAおよびE7 mRNAからなる第1のmRNA、およびL1 mRNAおよびE2 mRNAからなる第2のmRNAを含む、工程;
L1またはE2に対するE6+E7の比が決定されるように、E6+E7の発現レベルおよびL1またはE2の発現レベルを決定し、ここで4よりも大きな比がHPV誘導性の前癌を示している、工程
を含む上記方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US6942697P | 1997-12-12 | 1997-12-12 | |
US7048698P | 1998-01-05 | 1998-01-05 | |
US8216798P | 1998-04-17 | 1998-04-17 |
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JP2000524461A Division JP2001526045A (ja) | 1997-12-12 | 1998-12-11 | ヒトパピローマウイルス関連疾患の評価 |
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JP2010136729A true JP2010136729A (ja) | 2010-06-24 |
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JP2000539170A Pending JP2002508190A (ja) | 1997-12-12 | 1998-12-11 | 万能収集媒質 |
JP2000524461A Withdrawn JP2001526045A (ja) | 1997-12-12 | 1998-12-11 | ヒトパピローマウイルス関連疾患の評価 |
JP2010053090A Pending JP2010136729A (ja) | 1997-12-12 | 2010-03-10 | ヒトパピローマウイルス関連疾患の評価 |
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JP2000539170A Pending JP2002508190A (ja) | 1997-12-12 | 1998-12-11 | 万能収集媒質 |
JP2000524461A Withdrawn JP2001526045A (ja) | 1997-12-12 | 1998-12-11 | ヒトパピローマウイルス関連疾患の評価 |
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US (5) | US6969585B2 (ja) |
EP (2) | EP1038022B1 (ja) |
JP (3) | JP2002508190A (ja) |
AT (1) | ATE299946T1 (ja) |
AU (2) | AU748022B2 (ja) |
BR (2) | BR9814271A (ja) |
CA (2) | CA2313641A1 (ja) |
DE (1) | DE69830927T2 (ja) |
DK (1) | DK1038022T3 (ja) |
ES (1) | ES2246546T3 (ja) |
NO (2) | NO330906B1 (ja) |
PT (1) | PT1038022E (ja) |
WO (2) | WO1999029890A2 (ja) |
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