JP2017530370A - Susd2タンパク質のマーカーとしての使用 - Google Patents
Susd2タンパク質のマーカーとしての使用 Download PDFInfo
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- JP2017530370A JP2017530370A JP2017526737A JP2017526737A JP2017530370A JP 2017530370 A JP2017530370 A JP 2017530370A JP 2017526737 A JP2017526737 A JP 2017526737A JP 2017526737 A JP2017526737 A JP 2017526737A JP 2017530370 A JP2017530370 A JP 2017530370A
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Abstract
Description
1)抗SUSD2モノクローナル抗体を抗体とした免疫蛍光抗体法。
2)抗SUSD2モノクローナル抗体を抗体としたフローサイトメトリー法。
3)抗SUSD2モノクローナル抗体を抗体とした磁気ビーズ分離。
定する。
クローナル抗体(該抗体はR&D Systemsから購入し、カタログ番号がAF2419)であり、EGFP検出用の抗体は鶏由来のポリクローナル抗体(該抗体はAbcamから購入し、カタログ番号がAB13970である)であり、NGN3検出用の抗体はヒツジ由来のポリクローナル抗体である(該抗体はR&DSystemから購入し、カタログ番号がAF3444である)である。
ansretinoic acid、NOGGIN及びSANT−1を1ml:10μl:2μmol:250ng:0.25μmolの配合比率で混合して、細胞培養液VIIを得る方法により調製される。
たヤギ由来の抗マウス抗原サブタイプ2bポリクローナル抗体(カタログ番号115−605−207)、AlexaFluor 488で標識したロバ由来の抗兎ポリクローナル抗体(カタログ番号711−545−152)を使用して二重染色される。
Paired−End DNA Sample Prep Kitを使用してcDNAライブラリーを構築した。次にIllumina Hiseq2000を使用してシークエンシングした。Tophatソフトウェアを使用してシークエンシング結果(raw reads)とヒト参照ゲノム(the human reference genome、NCBI Build 37、hg19)を比較し、マッピング配列(Mapping reads)とNCBIの参照データベース(RefSeq Genes hg19)を順方向又は逆方向マッチングしてトランスクリプトームを再構築した。すべての遺伝子の発現存在度を計算して、RPKMで標準化した後、下記標準に応じてNGN3−EGFP+とNGN3−EGFP−細胞間での遺伝子発現差異の有意性を判断する:1)発現倍数変化(fold change)が2より大きく又は0.5未満である。2)P値が0.05未満である。Rソフトウェアのヒートマップパッケージ(heatmap package)を使用して、RPKMに基づき、取得したデータに対して、log10でヒートマップ分析を行う。識別的遺伝子発現(Differential Gene Expression、DGE)によってGO分析を行う。
の標識とすることができることが説明される。次に、SUSD2遺伝子を発現させるタンパク質も同様であるか否かを同定する。
来のポリクローナル抗体(該抗体はR&D Systemsから購入し、カタログ番号がAF3444である)とマウス由来のモノクローナル抗体(該抗体はDSHBから購入し、カタログ番号がF25A1B3である)であり、NEUROD1検出用の抗体はヤギ由来のポリクローナル抗体(該抗体はR&D Systemsから購入し、カタログ番号がAF2746である)であり、PDX1検出の抗体はヤギ由来のポリクローナル抗体(該抗体はR&D Systemsから購入し、カタログ番号がAF2419である)であり、CHROMOGRANINA検出用の抗体は兎由来のポリクローナル抗体(該抗体は中杉金橋から購入し、カタログ番号がZA−0507である)である。
Fluor 488で標識したロバ由来の抗ヤギポリクローナル抗体(カタログ番号705−545−147)であり、Alexa Fluor 488で標識したヤギ由来の抗マウス抗原サブタイプ2bポリクローナル抗体(カタログ番号115−545−207)、Alexa Fluor 647で標識したヤギ由来の抗マウス抗原サブタイプ2bポリクローナル抗体(カタログ番号115−605−207)、Alexa Fluor
488で標識したロバ由来の抗兎ポリクローナル抗体(カタログ番号711−545−152)、Alexa Fluor 488で標識したロバ由来の抗鶏ポリクローナル抗体(カタログ番号703−545−155)である。
echnologiesから購入し、カタログ番号が4367659である)を行い、増幅プライマーは表1に示され、内部参照プライマーはGAPDHであり、具体的な操作は取扱い書を参照する。
ないことを表明する。
ソーティングして得られたSUSD2陽性細胞、SUSD2陰性細胞及びソーティングしていない膵臓内胚葉細胞を、免疫不全マウス(6−8週間)の腎嚢胞に移植する。移植19週間後、移植物を取って凍結切片に対する免疫組織化学染色を行う。
0.5% fetal bovine serum(FBS、Hyclone))を使用して37℃で30分間消化し、5分間おきにピペットで軽くピペッティングして細胞を分散させる。消化した単細胞をPRMI 1640 with 0.5%FBSに移し替え、0.5%のBSAと2mMのEDTAを含有するPBSで二回洗浄する。残った組織塊を収集して、上記ステップにより再消化する。得られた細胞懸濁液を40μmのセルストレーナー(BD Biosciences)で濾過し、得られた単細胞懸濁液を0.5%のBSAと2mMのEDTAを含有するPBSに保存し、氷に置いて後続分析に用いる。
る)を使用して、上記単細胞懸濁液に対して磁気ビーズ分離を行い、SUSD2陽性細胞とSUSD2陰性細胞を得る。
Claims (10)
- 被測定細胞集団における膵臓内分泌前駆細胞又は新生膵臓内分泌細胞のソーティング又は補助ソーティングの方法であって、
被測定細胞集団における細胞でSUSD2遺伝子を発現させるか否かを検出し、いずれかの細胞がSUSD2遺伝子を発現させると、該当細胞を膵臓内分泌前駆細胞又は新生内分泌細胞とし、又はこれらの候補とし、いずれかの細胞がSUSD2遺伝子を発現させないと、該当細胞を膵臓内分泌前駆細胞又は新生内分泌細胞としなく、又はこれらの候補としないステップを含み、
前記SUSD2遺伝子のヌクレオチド配列は、配列表における配列2である、ソーティング又は補助ソーティングの方法。 - 前記被測定細胞集団における細胞でSUSD2遺伝子を発現させるか否かは、被測定細胞集団における細胞に、SUSD2遺伝子を発現させるタンパク質を含有するか否かを検出することにより行われ、前記被測定細胞集団における細胞にSUSD2遺伝子を発現させるタンパク質を含有するか否かを検出する方法は、
1)抗SUSD2モノクローナル抗体を抗体とした免疫蛍光抗体法、或いは
2)抗SUSD2モノクローナル抗体を抗体としたフローサイトメトリー法、或いは
3)抗SUSD2モノクローナル抗体を抗体とした磁気ビーズ分離である、ことを特徴とする請求項1に記載の方法。 - 前記被測定細胞が膵臓内胚葉細胞である、ことを特徴とする請求項1又は2に記載の方法。
- 前記膵臓内胚葉細胞がヒト由来膵臓内胚葉細胞である、ことを特徴とする請求項1〜3のいずれか1項に記載の方法。
- マーカーとしてのSUSD2タンパク質、そのコード遺伝子、又は前記SUSD2タンパク質の前駆体タンパク質をコードするmRNAの膵臓内分泌前駆細胞及び/又は新生膵臓内分泌細胞の同定、スクリーニング又はソーティングでの使用であって、
前記SUSD2タンパク質のアミノ酸配列は、配列表における配列1に示され、
前記SUSD2タンパク質のコード遺伝子のヌクレオチド配列は、配列表における配列2に示される、使用。 - 前記SUSD2タンパク質に特異的に結合できる抗体の、膵臓内分泌前駆細胞及び/又は新生膵臓内分泌細胞を同定、スクリーニング又はソーティングする試薬の調製での使用。
- 前記抗体は蛍光標識を有し、前記抗体はモノクローナル抗体である、ことを特徴とする請求項6に記載の使用。
- 前記使用は、膵臓内胚葉細胞から膵臓内分泌前駆細胞及び/又は新生膵臓内分泌細胞をスクリーニング又はソーティングすることであり、
前記スクリーニング又はソーティングする方法は、免疫磁気ビーズ分離法及びフローサイトメトリー法であり、
前記同定方法は、免疫蛍光抗体法及びフローサイトメトリー分析法である、ことを特徴とする請求項6又は7に記載の使用。 - 前記SUSD2タンパク質の前駆体タンパク質をコードできるmRNAに特異的に結合できるプライマー対、プローブ又はこれらの相補鎖の膵臓内分泌前駆細胞及び/又は新生
内分泌細胞の同定用試薬の調製での使用。 - 前記プライマー対は、配列表における配列3に示される一本鎖DNA分子と、配列表における配列4に示される一本鎖DNA分子からなる、ことを特徴とする請求項9に記載の使用。
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CN201410386506.0A CN104215768B (zh) | 2014-08-07 | 2014-08-07 | Susd2蛋白作为标记物的用途 |
CN201410386506.0 | 2014-08-07 | ||
PCT/CN2015/085990 WO2016019842A1 (zh) | 2014-08-07 | 2015-08-04 | Susd2蛋白作为标记物的用途 |
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CN106706915A (zh) * | 2016-12-30 | 2017-05-24 | 山东大学齐鲁医院 | Susd2在制备高级别浆液性卵巢癌诊断和/或预后判断的试剂盒中的应用 |
US20230072362A1 (en) * | 2021-09-07 | 2023-03-09 | Wisconsin Alumni Research Foundation | Cardiac fibroblast derived extracellular matrix |
US20230251262A1 (en) * | 2022-02-09 | 2023-08-10 | Universitaesklinikum Hamburg-Eppendorf | Enrichment, detection and characterization of circulating tumor cells with susd2 and enpp1 |
CN116536251B (zh) * | 2023-07-06 | 2023-09-19 | 北京北启生物医药有限公司 | 一种无饲养层的化学诱导多能干细胞单克隆建株方法 |
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