JP6060425B2 - Diagnosis of bladder cancer - Google Patents
Diagnosis of bladder cancer Download PDFInfo
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- JP6060425B2 JP6060425B2 JP2015020750A JP2015020750A JP6060425B2 JP 6060425 B2 JP6060425 B2 JP 6060425B2 JP 2015020750 A JP2015020750 A JP 2015020750A JP 2015020750 A JP2015020750 A JP 2015020750A JP 6060425 B2 JP6060425 B2 JP 6060425B2
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Description
本発明は、膀胱癌診断のための腫瘍マーカーおよび膀胱癌の診断用試薬に関する。 The present invention relates to a tumor marker for diagnosing bladder cancer and a reagent for diagnosing bladder cancer.
膀胱癌の初発症状は無症候性血尿で認められることが多く、診断法として主に尿細胞診、膀胱鏡検査が行われている。尿細胞診は進行性癌や低分化癌の検出率は良好であるが、表在性癌や高分化癌での有効性は満足のいくのもではない。一方、膀胱鏡検査は進行癌のみではなく、表在性癌や高分化癌の検出にも優れ確定診断に重要である反面、侵襲度が高く、身体負担を伴う検査である。また、表在性癌は高率に再発をきたし、治療後も定期的な膀胱鏡検査を含む経過観察が必要となる。さらに、進行性膀胱癌では特異的マーカーは存在せず、従来通り画像診断により経過観察が行われているのが現状である。近年、膀胱癌に対する尿中腫瘍マーカーとしてＢＴＡ (ｂｌａｄｄｅｒ ｔｕｍｏｒ ａｎｔｉｇｅｎ) やＮＭＰ２２ (ｎｕｃｌｅａｒ ｍａｔｒｉｘ ｐｒｏｔｅｉｎ ２２) が用いられるようになったが、表在性癌の検出に用いられるのみであり、また、尿細胞診の診断率を超える有効性は認められておらず、膀胱癌に対する種々の新規腫瘍マーカーの開発が望まれている。 The initial symptoms of bladder cancer are often observed in asymptomatic hematuria, and urine cytology and cystoscopy are mainly performed as diagnostic methods. Urine cytodiagnosis has a good detection rate for advanced cancer and poorly differentiated cancer, but the effectiveness in superficial cancer and well differentiated cancer is not satisfactory. On the other hand, cystoscopy is not only advanced cancer but also excellent for detection of superficial cancer and well-differentiated cancer, and is important for definitive diagnosis. However, it is highly invasive and involves physical burden. Superficial cancers relapse at a high rate, and follow-up including regular cystoscopy is required after treatment. Furthermore, there is no specific marker in advanced bladder cancer, and the current situation is that follow-up is performed by image diagnosis as usual. In recent years, BTA (bladder tumor antigen) or NMP22 (nuclear matrix protein 22) has been used as a urinary tumor marker for bladder cancer, but it is only used for detection of superficial cancer, and urine cells Effectiveness exceeding the diagnostic rate of diagnosis is not recognized, and development of various new tumor markers for bladder cancer is desired.
本発明は、プロテオーム解析技術を用いて尿、血清と組織と多角的にアプローチして、膀胱癌に対する新規腫瘍マーカーを探索することにより、有効な膀胱癌の診断方法を提供することを目的とする。 An object of the present invention is to provide an effective method for diagnosing bladder cancer by probing urine, serum, and tissue from various angles using proteome analysis technology and searching for a novel tumor marker for bladder cancer. .
本発明者らは、膀胱癌に対する新規腫瘍マーカーの探索において、一般的なプロテオーム解析に用いられている二次元電気泳動（ｔｗｏ−ｄｉｍｅｎｓｉｏｎａｌ ｇｅｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ, ２−ＤＥ）法ではなく、一次元目の等電点電気泳動（ｉｓｏ ｅｌｅｃｔｒｉｃ ｆｏｃｕｓｉｎｇ, ＩＥＦ）にアガロースゲルを用いる二次元電気泳動法、すなわち、アガロース二次元電気泳動（アガロース２−ＤＥ）法を用いた。この手法を用いることにより、従来困難であった微量にしか存在しない蛋白質や高分子量タンパク質の解析が可能となり、その結果、Ｇｅｎｅ Ｂａｎｋ データベースに登録された蛋白質の中から新たに膀胱癌のマーカーとなるポリペプチドを見出すことができた。本発明は、かかる研究成果に基づきなされたものである。 In the search for a novel tumor marker for bladder cancer, the present inventors do not use the two-dimensional electrophoresis (2-DE) method used for general proteome analysis, but the first dimension etc. A two-dimensional electrophoresis method using an agarose gel for isoelectric focusing (IEF), that is, an agarose two-dimensional electrophoresis (agarose 2-DE) method was used. By using this method, it is possible to analyze proteins and high molecular weight proteins that were present only in trace amounts, which has been difficult in the past, and as a result, it becomes a new bladder cancer marker from proteins registered in the Gene Bank database. The polypeptide could be found. The present invention has been made based on such research results.
即ち、本発明は以下の通りである。
（１）被験者から得た生体試料中の膀胱癌細胞の検出において、下記（ａ）、または（ｂ）のポリペプチドを腫瘍マーカーとして使用することを特徴とする、該ポリペプチドの使用方法：
（ａ）配列番号３に記載のアミノ酸配列を有するポリペプチド、
（ｂ）配列番号３に記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ（ａ）と同等の抗体産生能を有するかまたは（ａ）に対する特異的抗体と反応しうるポリペプチド。
（２）腫瘍マーカーとしての使用が、被検者から得た生体試料中に前記ポリペプチドを検出することである、上記（１）記載の方法。
（３）前記ポリペプチドの検出が、前記ポリペプチドに対する特異的抗体を用いた検出である、上記（２）記載の方法。
（４）腫瘍マーカーとしての使用が、被検者から得た生体試料中に、前記ポリペプチドに対する特異的抗体を検出することである、上記（１）記載の方法。
（５）前記抗体の検出が、前記ポリペプチドを用いた検出である、上記（４）記載の方法。
（６）腫瘍マーカーとしての使用が、被検者から得た生体試料中に、前記ポリペプチドをコードするヌクレオチド配列を有するポリヌクレオチドまたはその部分配列を有するヌクレオチド断片を検出することである、上記（１）記載の方法。
（７）前記検出が、配列番号１０に記載のヌクレオチド配列に基づいて作製したプローブおよび／またはプライマーにより行う、上記（６）記載の方法。
（８）生体試料が、尿、血清または組織である、上記（１）〜（７）のいずれか一つに記載の方法。
（９）配列番号３に記載のアミノ酸配列を有するポリペプチドに対する特異的抗体を含有する、膀胱癌の診断用試薬。
（１０）下記（ａ）、または（ｂ）を含む、膀胱癌マーカーポリペプチドに対する特異的抗体製造用組成物：
（ａ）配列番号３に記載のアミノ酸配列を有するポリペプチド；
（ｂ）配列番号３に記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ配列番号３に記載のアミノ酸配列からなるポリペプチドに対する抗体の産生を誘導するポリペプチド。
（１１）下記（ａ）、または（ｂ）を含む、膀胱癌の診断用試薬：
（ａ）配列番号３に記載のアミノ酸配列を有するポリペプチド；
（ｂ）配列番号３に記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ配列番号３に記載のアミノ酸配列からなるポリペプチドに対する特異的抗体と反応しうるポリペプチド。
（１２）配列番号３に記載のアミノ酸配列を有するポリペプチドに対する特異的抗体を検出するための、上記（１１）記載の診断用試薬。
（１３）下記(ｉ)、(ｉｉ)、(ｉｉｉ) のいずれかを含む、膀胱癌診断用核酸プローブ：
(ｉ)配列番号１０に記載のヌクレオチド配列と相補的なヌクレオチド配列を有するポリヌクレオチド；
(ｉｉ)配列番号１０に記載のヌクレオチド配列と相補的なヌクレオチド配列の部分配列を含むヌクレオチド断片；
(ｉｉｉ) 配列番号１０に記載のヌクレオチド配列を有するポリヌクレオチドと、ストリンジェントな条件下でハイブリダイズする、ポリヌクレオチドまたはヌクレオチド断片。
（１４）被検者から得た生体試料と、上記（１３）記載の膀胱癌診断用核酸プローブとを接触させることを含む、上記（６）記載の方法。
（１５）被検者から得た生体試料に含まれるポリヌクレオチドをポリメラーゼ連鎖反応（ＰＣＲ）で増幅することを含む、上記（６）記載の方法。
（１６）配列番号１０に記載のヌクレオチド配列の部分配列からなるヌクレオチド断片、及び配列番号１０に記載のヌクレオチド配列と相補的なヌクレオチド配列の部分配列からなるヌクレオチド断片を含む、膀胱癌診断用プライマー。
（１７）上記（１３）に記載の膀胱癌診断用核酸プローブを含む、膀胱癌診断用キット。
（１８）上記（１６）に記載の膀胱癌診断用プライマーを含む、膀胱癌診断用キット。
That is, the present invention is as follows.
(1) In the detection of bladder cancer cells in a biological sample obtained from a subject, the following (a) or (b) polypeptide is used as a tumor marker, and the method for using the polypeptide:
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3,
(B) has an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 3 and has an antibody-producing ability equivalent to (a) or A polypeptide capable of reacting with a specific antibody against (a).
(2) The method according to (1) above, wherein the use as a tumor marker is to detect the polypeptide in a biological sample obtained from a subject.
(3) The method according to (2) above, wherein the detection of the polypeptide is detection using a specific antibody against the polypeptide.
(4) The method according to (1) above, wherein the use as a tumor marker is to detect a specific antibody against the polypeptide in a biological sample obtained from a subject.
(5) The method according to (4) above, wherein the detection of the antibody is detection using the polypeptide.
(6) The use as a tumor marker is to detect a polynucleotide having a nucleotide sequence encoding the polypeptide or a nucleotide fragment having a partial sequence thereof in a biological sample obtained from a subject. 1) The method described.
(7) The method according to (6) above, wherein the detection is performed with a probe and / or primer prepared based on the nucleotide sequence set forth in SEQ ID NO: 10.
(8) The method according to any one of (1) to (7) above, wherein the biological sample is urine, serum or tissue.
(9) A diagnostic reagent for bladder cancer, comprising a specific antibody against the polypeptide having the amino acid sequence set forth in SEQ ID NO: 3.
(10) A composition for producing a specific antibody against a bladder cancer marker polypeptide, comprising the following (a) or (b):
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3;
(B) against a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in SEQ ID NO: 3 and consisting of the amino acid sequence described in SEQ ID NO: 3 A polypeptide that induces the production of an antibody.
(11) A diagnostic reagent for bladder cancer comprising the following (a) or (b):
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3;
(B) against a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in SEQ ID NO: 3 and consisting of the amino acid sequence described in SEQ ID NO: 3 A polypeptide capable of reacting with a specific antibody.
(12) The diagnostic reagent according to (11) above, for detecting a specific antibody against the polypeptide having the amino acid sequence described in SEQ ID NO: 3.
(13) Nucleic acid probe for bladder cancer diagnosis comprising any of the following (i), (ii), and (iii):
(i) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 10;
(ii) a nucleotide fragment comprising a partial sequence of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 10;
(iii) A polynucleotide or nucleotide fragment that hybridizes with the polynucleotide having the nucleotide sequence of SEQ ID NO: 10 under stringent conditions.
(14) The method according to (6) above, comprising contacting a biological sample obtained from a subject with the nucleic acid probe for bladder cancer diagnosis according to (13) above.
(15) The method according to (6) above, comprising amplifying a polynucleotide contained in a biological sample obtained from a subject by polymerase chain reaction (PCR).
(16) A bladder cancer diagnosis primer comprising a nucleotide fragment comprising a partial sequence of the nucleotide sequence set forth in SEQ ID NO: 10 and a nucleotide fragment comprising a partial sequence of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 10.
(17) A bladder cancer diagnosis kit comprising the bladder cancer diagnosis nucleic acid probe according to (13).
(18) A bladder cancer diagnosis kit comprising the bladder cancer diagnosis primer according to (16) .
本発明により、膀胱癌に対する７種類のポリペプチドからなる新規腫瘍マーカーが提供されるので、これらの腫瘍マーカーを用いることにより膀胱癌の診断が容易となる。また、これらのポリペプチドおよびその特異的抗体を用いた治療薬を提供すると共に、膀胱癌発症のメカニズムの解明に利用して新たな治療薬の開発を行うこともできる。さらに、本発明は治療薬の有効性の判定や、再発時における早期診断および予後予見の判断にも有用である。 According to the present invention, a novel tumor marker composed of seven kinds of polypeptides for bladder cancer is provided. Therefore, bladder cancer can be easily diagnosed by using these tumor markers. In addition to providing therapeutic agents using these polypeptides and their specific antibodies, new therapeutic agents can be developed by using them to elucidate the mechanism of bladder cancer development. Furthermore, the present invention is also useful for determining the effectiveness of therapeutic agents, early diagnosis at the time of recurrence, and judgment of prognosis.
腫瘍マーカーとして使用するポリペプチド
本発明は、膀胱癌に対する新規腫瘍マーカーとして、（ａ） 配列番号１〜７のいずれかに記載のアミノ酸配列を有するポリペプチド、（ｂ） 配列番号１〜７のいずれかに記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ（ａ） と同等の抗体産生能を有するかまたは（ａ） に対する特異的抗体と反応しうるポリペプチド、または、（ｃ） 上記（ａ） または（ｂ） のポリペプチドのアミノ酸配列の部分配列を有し、かつ（ａ） と同等の抗体産生能を有するかまたは（ａ） に対する特異的抗体と反応しうるポリペプチドを使用することを特徴とする。
Polypeptide used as a tumor marker The present invention provides a novel tumor marker for bladder cancer, (a) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 7, (b) any of SEQ ID NOs: 1 to 7 Or an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, added and / or inserted, and have an antibody-producing ability equivalent to (a) or specific for (a) A polypeptide capable of reacting with a target antibody, or (c) having a partial sequence of the amino acid sequence of the polypeptide of (a) or (b) above and having an antibody-producing ability equivalent to (a) or ( It is characterized in that a polypeptide capable of reacting with a specific antibody against a) is used.
これらのポリペプチドが膀胱癌の腫瘍マーカーであることは以下のようにして本発明者らにより確認された。
（１）膀胱移行上皮癌１０例において膀胱癌組織および膀胱正常組織を採取し、これよりタンパク質抽出液を調製した。
（２）アガロース二次元電気泳動（アガロース２−ＤＥ) 法により、これまで解析されていない高分子領域 (９７．４ｋＤａ以上) のタンパク質について、膀胱癌組織と膀胱正常組織における発現を二次元電気泳動パターンにおいて比較した。最も違いの見られた１例における癌組織と正常組織の高分子領域に認められたタンパク質スポット (図１参照) をすべて切り出し、質量分析により同定した。同定されたタンパク質を比較し、癌部にのみ同定されたタンパク質を文献により詳細に検討した結果、後出の表１に示す７種類のタンパク質を膀胱癌の新規腫瘍マーカー候補とした。
（３）上記で腫瘍マーカー候補とした７種類のタンパク質について、膀胱癌組織１０例と膀胱正常組織１０例における発現量を比較検討した。具体的には、各タンパク質に対する抗体を用意し、ウエスタンブロッティング法による確認実験を行い、これらの７種類のタンパク質について膀胱癌組織における発現の増加が確認された (図３Ａ）。
It was confirmed by the present inventors that these polypeptides are tumor markers for bladder cancer as follows.
(1) Bladder cancer tissue and normal bladder tissue were collected from 10 cases of bladder transitional cell carcinoma, and a protein extract was prepared therefrom.
(2) Two-dimensional electrophoresis of protein in the high molecular region (97.4 kDa or more) that has not been analyzed by two-dimensional electrophoresis using agarose two-dimensional electrophoresis (agarose 2-DE) method. The patterns were compared. All protein spots (see FIG. 1) found in the polymer region of cancer tissue and normal tissue in one case where the most difference was found were cut out and identified by mass spectrometry. As a result of comparing the identified proteins and examining in detail the proteins identified only in the cancer part, the following seven proteins shown in Table 1 were used as novel tumor marker candidates for bladder cancer.
(3) The expression levels in 10 cases of bladder cancer tissue and 10 cases of normal bladder tissue were compared and examined for the 7 types of proteins used as tumor marker candidates. Specifically, antibodies against each protein were prepared, and confirmation experiments were conducted by Western blotting, and increased expression in bladder cancer tissues was confirmed for these seven types of proteins (FIG. 3A).
また、膀胱癌患者尿と健常者尿での尿中タンパク質存在量を比較すると、ｓｔｒｕｃｔｕｒａ ｍａｉｎｔｅｎａｃｅ ｏｆ ｃｈｒｏｍｏｓｏｍｅｓ ｐｒｏｔｅｉｎ ３ （ＳＭＣ3)が増加していることが判明した (図３Ｃ）。
さらに、現在確立されている６種類の膀胱癌細胞株（ＲＴ４, ５６３７, Ｔ２４, ＥＪ, ＭＴＢ２, ＴＣＣＳＵＰ）においてマーカー候補タンパク質の発現を検討した結果、上記７種類のタンパク質の発現が確認された。
（４）上記ウエスタンブロッティング法により癌組織において有意に発現量の増加が確認されたタンパク質について、免疫化学染色により発現の確認を行った（図４）。７種類のタンパク質全てが癌組織で高発現していることを確認した。
Moreover, when the amount of protein in the urine was compared between the urine of the bladder cancer patient and the urine of the healthy subject, it was found that the structure maintenance of chromosomes protein 3 (SMC3) was increased (FIG. 3C).
Furthermore, as a result of examining the expression of marker candidate proteins in 6 types of currently established bladder cancer cell lines (RT4, 5637, T24, EJ, MTB2, and TCCSUP), the expression of the above 7 types of proteins was confirmed.
(4) The expression of the protein whose expression level was significantly increased in the cancer tissue by the Western blotting method was confirmed by immunochemical staining (FIG. 4). It was confirmed that all seven proteins were highly expressed in cancer tissues.
以上のように、表１に示す７種類のタンパク質、即ち、Ｎｕｅｒｏｂｌａｓｔ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ−ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ（ＡＨＮＡＫ）、Ｐｌｅｃｔｉｎ １、Ｅｐｉｐｌａｋｉｎ、Ｅｕｋａｒｙｏｔｉｃ ｔｒａｎｓｌａｔｉｏｎ ｉｎｉｔｉａｔｉｏｎ ｆａｃｔｏｒ ３ ｓｕｂｕｎｉｔ １０（ＥＩＦ３）、Ｖｉｇｉｌｉｎ、Ｓｔｒｕｃｔｕｒａｌ ｍａｉｎｔｅｎａｎｃｅ ｏｆ ｃｈｒｏｍｏｓｏｍｅｓ ｐｒｏｔｅｉｎ ３（ＳＭＣ３）、Ｒａｓ ＧＴＰａｓｅ−ａｃｔｉｖａｔｉｎｇ−ｌｉｋｅ ｐｒｏｔｅｉｎ（ＩＱＧＡＰ１）が癌組織中において発現が増加しているので、これらのタンパク質は膀胱癌に対する腫瘍マーカーとなりうる。これらのタンパク質は、タンパク質データベースであるＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ Ｐｒｏｔにそれぞれ、アクセッシション番号Ｑ０９６６６、Ｑ１５１４９、Ｐ５８１０７、Ｑ１４１５２、Ｑ００３４１、Ｑ９ＵＱＥ７、Ｐ４６９４０として登録されており、アミノ酸配列は、それぞれ配列番号１〜７に示す通りである。これらのタンパク質と膀胱癌との関連はこれまで全く知られておらず、本発明者らによって初めて見出されたものである。
腫瘍マーカーとしての使用
配列番号１〜７のいずれかに示すアミノ酸配列を有するポリペプチド、配列番号１〜７のいずれかに示すアミノ酸配列と実質的に同じアミノ酸配列を有するポリペプチド、またはこれらのポリペプチドの部分配列を有するポリペプチドは、腫瘍マーカーとして膀胱癌の診断に用いることができる。本願明細書において、腫瘍マーカーとしての使用とは以下のような態様を含む。
（１）生体試料中での上記ポリペプチドの発現を検出または発現量を測定する。
（２）生体試料中における上記ポリペプチドに対する特異的抗体の存在を検出または測定する。
（３）上記ポリペプチドをコードするポリヌクレオチド配列を有するポリペプチドまたはその部分配列を有するヌクレオチド断片の存在を検出または測定する。
As described above, the seven types of proteins shown in Table 1, namely, Neuroblast differentiation-associated protein (AHNAK),
Use as a tumor marker Polypeptide having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 7, polypeptide having substantially the same amino acid sequence as the amino acid sequence shown in any one of SEQ ID NOs: 1 to 7, or these polypeptides A polypeptide having a peptide partial sequence can be used for diagnosis of bladder cancer as a tumor marker. In the present specification, the use as a tumor marker includes the following aspects.
(1) The expression of the polypeptide in the biological sample is detected or the expression level is measured.
(2) Detect or measure the presence of a specific antibody against the polypeptide in the biological sample.
(3) Detect or measure the presence of a polypeptide having a polynucleotide sequence encoding the polypeptide or a nucleotide fragment having a partial sequence thereof.
生体試料中での上記ポリペプチドの発現を検出または発現量を測定して膀胱癌の診断を行うには、例えば、このポリペプチドに対する特異的抗体を作製して、特異的抗体を使用したウエスタンブロット法、固相酵素免疫検定法（ＥＬＩＺＡ） 、免疫組織学的染色法などの免疫学的方法によりポリペプチドの発現の有無または発現の程度を測定する。 In order to diagnose bladder cancer by detecting the expression of the above polypeptide in a biological sample or measuring the expression level, for example, a specific antibody against this polypeptide is prepared, and a Western blot using the specific antibody is used. The presence or degree of expression of the polypeptide is measured by an immunological method such as a method, a solid phase enzyme immunoassay (ELIZA), or an immunohistological staining method.
ウエスタンブロット法は、例えばポリペプチドを含有する生体試料液をアクリルアミドゲル電気泳動によりポリペプチドを分離し、これをメンブランに転写し、ポリペプチドを認識しうる抗体 (一次抗体) と反応させ、生成する免疫複合体を標識した二次抗体を用いて検出する方法である。生成した免疫複合体と結合した標識二次抗体の標識量を測定することにより、存在するポリペプチドの量を測定できる。 Western blotting is performed by separating a polypeptide from a biological sample solution containing the polypeptide, for example, by acrylamide gel electrophoresis, transferring the polypeptide to a membrane, and reacting with an antibody that can recognize the polypeptide (primary antibody). In this method, the immune complex is detected using a labeled secondary antibody. By measuring the amount of labeled secondary antibody bound to the produced immune complex, the amount of polypeptide present can be measured.
免疫組織学的染色法は、スライドガラス上に固定された組織切片や細胞などを、抗体と反応させて免疫複合体を生成させ、これと結合した標識二次抗体により検出するものであり、組織や細胞における対象ポリペプチドの発現部位を解析するのに有効である。 In the immunohistological staining method, tissue sections or cells fixed on a slide glass are reacted with an antibody to produce an immune complex, which is detected by a labeled secondary antibody bound thereto. It is effective for analyzing the expression site of the target polypeptide in cells and cells.
使用する特異的抗体は、ポリクローナル抗体でもモノクローナル抗体でもよく、特異的抗体は既知の抗体作製方法によって得ることができる。ポリクローナル抗体の場合、配列番号１〜７のいずれかのアミノ酸配列またはその一部のアミノ酸配列に基づいて作製した合成ペプチドをマウスなどの非ヒト動物に免疫し、この動物の血清などから産生された抗体を常法により得る。モノクローナル抗体の作製も常法により行うことができる。例えば、配列番号１〜７のいずれかのアミノ酸配列またはその一部のアミノ酸配列に基づいて作製した合成ペプチドをマウスなどの非ヒト動物に免疫し、脾臓などに含まれる抗体産生細胞を採取し、骨髄腫細胞と融合させて得たハイブリドーマ細胞より製造することができる。 The specific antibody to be used may be a polyclonal antibody or a monoclonal antibody, and the specific antibody can be obtained by a known antibody production method. In the case of a polyclonal antibody, a non-human animal such as a mouse was immunized with a synthetic peptide prepared based on any one of the amino acid sequences of SEQ ID NOS: 1 to 7 or a part of the amino acid sequence, and the antibody was produced from the serum of this animal. Antibodies are obtained by conventional methods. Monoclonal antibodies can also be prepared by conventional methods. For example, a non-human animal such as a mouse is immunized with a synthetic peptide prepared based on any one of the amino acid sequences of SEQ ID NOs: 1 to 7 or a part thereof, and antibody-producing cells contained in the spleen are collected, It can be produced from hybridoma cells obtained by fusing with myeloma cells.
抗体の作製に使用するポリペプチドとしては、配列番号１〜７のいずれかに示すアミノ酸配列を有するポリペプチドの他、１または複数のアミノ酸が欠失、置換、付加および／または挿入されたアミノ酸配列を有するポリペプチドであって、配列番号１〜７のいずれかに示すアミノ酸配列からなるポリペプチドに対する抗体を産生しうるポリペプチド、また、これらのポリペプチドのアミノ酸配列の一部を有するポリペプチド断片で同様の抗体産生能をもつものであってもよい。このようなポリペプチド断片は、膀胱癌マーカーポリペプチドに対する抗体産生を誘導することができれば、その長さは特に制限されないが、通常、少なくとも１５アミノ酸残基、好ましくは、少なくとも２０アミノ酸残基、特に好ましくは、少なくとも２５アミノ酸残基を含むものである。 As a polypeptide used for the production of an antibody, in addition to the polypeptide having the amino acid sequence shown in any of SEQ ID NOs: 1 to 7, an amino acid sequence in which one or more amino acids are deleted, substituted, added and / or inserted A polypeptide capable of producing an antibody against the polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 1 to 7, and a polypeptide fragment having a part of the amino acid sequence of these polypeptides It may have the same antibody-producing ability. The length of such a polypeptide fragment is not particularly limited as long as it can induce antibody production against a bladder cancer marker polypeptide, but is usually at least 15 amino acid residues, preferably at least 20 amino acid residues, particularly Preferably, it contains at least 25 amino acid residues.
使用するポリペプチドまたはポリペプチド断片は、配列番号１〜７のアミノ酸配列の情報に基づいて化学的に合成しても、また配列番号８〜１４に示す遺伝子の配列情報に基づいて既知の遺伝子工学的手段によって製造してもよい。 The polypeptide or polypeptide fragment to be used can be chemically synthesized based on the amino acid sequence information of SEQ ID NOs: 1 to 7 or known genetic engineering based on the sequence information of the genes shown in SEQ ID NOs: 8 to 14. May be manufactured by conventional means.
上記ポリペプチドまたはポリペプチド断片は、抗体誘導に好ましい成分と組み合わせて抗体製造用組成物とすることができる。抗体産生に際しては、産生能を高めるために、例えば完全フロイントアジュバントや不完全フロイントアジュバントなどのアジュバントを使用してもよい。 The polypeptide or polypeptide fragment can be combined with a component preferable for antibody induction to form a composition for producing an antibody. In the production of antibodies, an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant may be used in order to increase the production ability.
また、腫瘍マーカーとしての使用とは、配列番号１〜７のいずれかに示すアミノ酸配列を有するポリペプチド、または、その一部のアミノ酸配列を有するペプチドを使用して、生体試料中にそれらに対する特異的抗体を検出することも含む。 In addition, the use as a tumor marker means that a polypeptide having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 7 or a peptide having a part of the amino acid sequence is used and specific for them in a biological sample. Detecting specific antibodies.
膀胱癌マーカーポリペプチドに対する特異的抗体を検出するためには、以下の（ａ）、（ｂ）または（ｃ）などが使用できる。
（ａ）配列番号１〜７のいずれかに記載のアミノ酸配列を有するポリペプチド；
（ｂ）配列番号１〜７のいずれかに記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ配列番号１〜７のいずれかに記載のアミノ酸配列からなるポリペプチドに対する特異的抗体と反応しうるポリペプチド；
（ｃ）上記（ａ）または（ｂ）のポリペプチドのアミノ酸配列の部分配列を有し、かつ配列番号１〜７のいずれかに記載のアミノ酸配列からなるポリペプチドに対する特異的抗体と反応しうるポリペプチド断片。
In order to detect a specific antibody against a bladder cancer marker polypeptide, the following (a), (b) or (c) can be used.
(A) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 7;
(B) any one of SEQ ID NOS: 1 to 7 having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NOS: 1 to 7 A polypeptide capable of reacting with a specific antibody against the polypeptide comprising the amino acid sequence described in 1.
(C) It can react with a specific antibody against a polypeptide having a partial sequence of the amino acid sequence of the polypeptide of (a) or (b) and comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7 Polypeptide fragment.
使用するポリペプチドまたはポリペプチド断片は、配列番号１〜７のアミノ酸配列の情報に基づいて化学的に合成しても、また配列番号８〜１４に示す遺伝子の配列情報に基づいて既知の遺伝子工学的手段によって製造してもよい。 The polypeptide or polypeptide fragment to be used can be chemically synthesized based on the amino acid sequence information of SEQ ID NOs: 1 to 7 or known genetic engineering based on the sequence information of the genes shown in SEQ ID NOs: 8 to 14. May be manufactured by conventional means.
また、腫瘍マーカーとしての使用には、上記ポリペプチドをコードするヌクレオチド配列を有するポリヌクレオチド、またはその部分配列を有するヌクレオチド断片を検出する方法も挙げられる。このような検出には、配列番号8 〜１４に記載のヌクレオチド配列に基づいて作成したプローブやプライマー、またはそれらを組み合わせて用いることができる。 The use as a tumor marker also includes a method for detecting a polynucleotide having a nucleotide sequence encoding the above polypeptide or a nucleotide fragment having a partial sequence thereof. For such detection, probes and primers prepared based on the nucleotide sequences described in SEQ ID NOs: 8 to 14, or combinations thereof can be used.
使用するプローブとしては、下記（ｉ）、（ｉｉ）、（ｉｉｉ）などが挙げられる：
（ｉ）配列番号８〜１４のいずれかに記載のヌクレオチド配列と相補的なヌクレオチド配列を有するポリヌクレオチド；
（ｉｉ）配列番号８〜１４のいずれかに記載のヌクレオチド配列と相補的なヌクレオチド配列の部分配列を含むヌクレオチド断片；
（ｉｉｉ）配列番号８〜１４のいずれかに記載のヌクレオチド配列を有するポリヌクレオチドと、ストリンジェントな条件下でハイブリダイズする、ポリヌクレオチドまたはヌクレオチド断片。
Examples of the probe used include the following (i), (ii), (iii) and the like:
(I) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of any one of SEQ ID NOs: 8 to 14;
(Ii) a nucleotide fragment comprising a partial sequence of a nucleotide sequence complementary to the nucleotide sequence of any one of SEQ ID NOs: 8 to 14;
(Iii) A polynucleotide or nucleotide fragment that hybridizes with the polynucleotide having the nucleotide sequence of any one of SEQ ID NOs: 8 to 14 under stringent conditions.
これらのプローブを用いて、被検者から得た検体、例えば、血液、尿、組織から調製した生体試料において、膀胱癌マーカーポリペプチドをコードするポリヌクレオチドを検出することができる。 Using these probes, a polynucleotide encoding a bladder cancer marker polypeptide can be detected in a sample obtained from a subject, for example, a biological sample prepared from blood, urine, or tissue.
また、生体試料に含まれるポリヌクレオチドについて、特定のプライマーを用いたポリメラーゼ連鎖反応（ＰＣＲ）での増幅の有無をみることによっても、膀胱癌マーカーポリペプチドをコードするポリヌクレオチドの存在を検出しうる。 The presence of a polynucleotide encoding a bladder cancer marker polypeptide can also be detected by examining the presence or absence of amplification in a polymerase chain reaction (PCR) using a specific primer for a polynucleotide contained in a biological sample. .
プライマーについては、例えば、配列番号８〜１４のいずれかに記載のヌクレオチド配列の部分配列からなるヌクレオチド断片をフォワードプライマーとし、配列番号８〜１４のいずれかに記載のヌクレオチド配列と相補的なヌクレオチド配列の部分配列からなるヌクレオチド断片をリバースプライマーとして使用できる。
膀胱癌の診断
配列番号１〜７のいずれかのアミノ酸配列からなるポリペプチドなどを、腫瘍マーカーとして膀胱癌の診断に用いるには、腫瘍が疑われる被検者の組織、尿、血清などの生体試料を採取し、上記のようにして生体試料中のこれらのポリペプチド、特異的抗体、ポリペプチドをコードするポリヌクレオチドの検出または測定を行うことにより、膀胱癌の判定を行う。検出または測定は、各ポリペプチドやポリヌクレオチドをそれぞれ単独で、あるいはそれらを組み合わせて行うことができる。
For the primer, for example, a nucleotide fragment consisting of a partial sequence of the nucleotide sequence described in any of SEQ ID NOs: 8 to 14 is used as a forward primer, and a nucleotide sequence complementary to the nucleotide sequence described in any of SEQ ID NOs: 8 to 14 A nucleotide fragment consisting of the partial sequence can be used as a reverse primer.
Diagnosis of bladder cancer In order to use a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 7 as a tumor marker for diagnosis of bladder cancer, a living body such as tissue, urine, serum, etc. of a subject suspected of having a tumor A sample is collected, and bladder cancer is determined by detecting or measuring these polypeptides, specific antibodies, and polynucleotides encoding the polypeptides in the biological sample as described above. Detection or measurement can be performed individually or in combination for each polypeptide or polynucleotide.
抗体を用いた膀胱癌の判定では、本発明において確認された７種類のタンパク質の抗体を１種またはそれらを組み合わせることで、実際の臨床検体、即ち、組織、血清や尿検体でのタンパク質の存在量の増加をELIZA 法や免疫染色法を用いて検出し、正常組織、健常者の尿や血清と比較して、上記ポリペプチドの発現が増加している場合に膀胱癌の可能性があると判定する。 In the determination of bladder cancer using an antibody, the presence of the protein in an actual clinical specimen, that is, a tissue, serum, or urine specimen is obtained by combining one or a combination of seven kinds of antibodies of the protein confirmed in the present invention. When the increase in the amount of the above polypeptide is detected compared to normal tissue or urine or serum of normal subjects, the increase in the amount is detected using ELIZA method or immunostaining method. judge.
実施例２および３では、本発明において確認された７種類のポリペプチドの抗体を用い、膀胱全摘除術症例１０例の病理標本を用いて、膀胱癌組織に反応することを確認している。実際の臨床検体を用い７種類とも癌組織と反応性を認め、診断での有用性を確認している。さらにＳＭＣ３に関しては尿検体にて存在量の増加の確認をしており、診断での有用性を確認している。
膀胱癌診断試薬
本発明の膀胱癌診断試薬は、配列番号１〜７のいずれかのアミノ酸配列からなるポリペプチドに対する特異的抗体を含有するものであり、この抗体は検出方法に応じてこの分野で既知の標識物質や方法により標識されていてもよい。また、本発明の膀胱癌診断用試薬は、配列番号１〜７のいずれかのアミノ酸配列もしくは実質的に同一のアミノ酸配列からなるポリペプチド、またはその部分配列を有するポリペプチド断片を含有するものであってもよい。
In Examples 2 and 3, it was confirmed that the antibodies of seven types of polypeptides confirmed in the present invention were reacted with bladder cancer tissues using pathological specimens of 10 cases of total cystectomy. Using clinical specimens, all seven types have been found to be reactive with cancer tissues and confirmed their usefulness in diagnosis. Furthermore, regarding SMC3, the increase in the abundance is confirmed in the urine specimen, and its usefulness in diagnosis is confirmed.
Bladder cancer diagnostic reagent The bladder cancer diagnostic reagent of the present invention contains a specific antibody against a polypeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7, and this antibody is used in this field depending on the detection method. It may be labeled with a known labeling substance or method. The bladder cancer diagnostic reagent of the present invention contains a polypeptide consisting of any one of the amino acid sequences of SEQ ID NOS: 1 to 7, or a polypeptide fragment having a partial sequence thereof. There may be.
診断用試薬は、必要に応じて免疫反応用ウェル、染色剤、検出用の酵素標識抗体、洗浄液、抗体希釈液、検体希釈液、酵素基質、酵素基質液希釈液などと組み合わせてキットとしてもよい。 Diagnostic reagents may be combined with immune reaction wells, staining agents, enzyme-labeled antibodies for detection, washing solutions, antibody dilutions, specimen dilutions, enzyme substrates, enzyme substrate solution dilutions, and the like as necessary. .
また、本発明の腫瘍マーカーは、膀胱癌の判定に用いる以外に、膀胱癌治療薬の有効性の判定、再発時における早期診断および予後予見の判断にも利用できる。
膀胱癌治療薬
本発明において確認された７種類のタンパク質はいずれも膀胱癌病理組織で確認されているため、７種類のタンパク質の抗体を１種または複数組み合わせることにより、膀胱癌特異的に癌の縮小や消失を得られる可能性がある。即ち、分子標的薬としての役割を担う可能性がある。さらに、７種類のタンパク質と既存の化学療法剤や放射線療法との組み合わせを行うことで、相乗的に治療効果を得られる可能性がある。
The tumor marker of the present invention can be used not only for determination of bladder cancer, but also for determination of the effectiveness of therapeutic agents for bladder cancer, early diagnosis at the time of recurrence, and determination of prognosis.
Bladder cancer therapeutic agent Since all the seven types of proteins identified in the present invention have been confirmed in bladder cancer pathological tissue, by combining one or more antibodies of the seven types of proteins, the cancer of the bladder cancer is specifically identified. There is a possibility to obtain reduction or disappearance. That is, it may play a role as a molecular target drug. Furthermore, there is a possibility that a therapeutic effect can be obtained synergistically by combining seven types of proteins with existing chemotherapeutic agents and radiation therapy.
膀胱癌のプロテオーム解析
（腫瘍マーカーとして使用できるポリペプチドの同定）
（１）使用する膀胱癌組織および正常組織
膀胱移行上皮癌10例を膀胱全摘出術施行時に切除し、同時に肉眼的に正常と考えられる膀胱粘膜を膀胱正常組織として採取した。また、同定したタンパク質の確認実験で使用する膀胱正常組織として、限局性前立腺癌の診断で、前立腺全摘出術を施行した際に切除した膀胱組織を採取した。
Proteomic analysis of bladder cancer (identification of polypeptides that can be used as tumor markers)
(1) Bladder cancer tissue and normal tissue to be used Ten cases of bladder transitional cell carcinoma were excised at the time of total cystectomy, and at the same time, the bladder mucosa considered to be normal macroscopically was collected as normal bladder tissue. In addition, as normal bladder tissue used in the confirmation experiment of the identified protein, bladder tissue excised when radical prostatectomy was performed in the diagnosis of localized prostate cancer was collected.
組織の採取後、生理的食塩水でよく洗浄し、血液成分を取り除いた後、速やかに液体窒素で凍結させ、使用するまで −８０℃で保存した。
（２）組織試料の調製
テフロン（登録商標）ガラスホモジナイザーを用いて凍結した組織約２０ｍｇを、質量の３０倍量タンパク抽出液で処理してタンパク質を抽出した。ホモジナイズ後、直径０．３５−０．５０ｍｍのガラスビーズ（Ａｓ Ｏｎｅ Ｃｏｒｐｏｒａｔｉｏｎ, Ｏｓａｋａ, Ｊａｐａｎ）の存在下ですばやく攪拌した。このホモジェネートは１１２，０００ｘｇ、２０分間の超遠心により脂質、膜成分を分離し、上清をタンパク質抽出液として使用した。
（３）アガロース二次元電気泳動
一次元目の等電点電気泳動（ＩＥＦ）のゲルは、長さ１８０ｍｍ、直径３．４ｍｍのガラス管を用いて作成し、二次元目のＳＤＳ−ＰＡＧＥは１９５×１２０×１．５ｍｍの大きさのものを作成した。アガロース二次元電気泳動（アガロース ２−ＤＥ）の特性を活かすために、２種類の濃度のＳＤＳ−ＰＡＧＥ ｇｅｌを目的に応じて作成した。すなわち、低分子量（ｌｏｗ ｍｏｌｅｃｕｌａｒ ｍａｓｓ，ＬＭＭ ）タンパク質の分離を目的とした12％均一ゲルと高分子量（ｈｉｇｈ ｍｏｌｅｃｕｌａｒ ｍａｓｓ，ＨＭＭ）タンパク質の分離を目的とした6-10％濃度勾配ゲルである。二次元目のＳＤＳ−ＰＡＧＥはＬａｅｍｍｌｉの方法によって行った。
After the tissue was collected, it was thoroughly washed with physiological saline to remove blood components, immediately frozen with liquid nitrogen, and stored at −80 ° C. until use.
(2) Preparation of Tissue Sample Protein was extracted by treating about 20 mg of tissue frozen with a Teflon (registered trademark) glass homogenizer with a protein extract of 30 times the mass. After homogenization, the mixture was rapidly stirred in the presence of glass beads having a diameter of 0.35-0.50 mm (As One Corporation, Osaka, Japan). In this homogenate, lipids and membrane components were separated by ultracentrifugation at 112,000 × g for 20 minutes, and the supernatant was used as a protein extract.
(3) Agarose two-dimensional electrophoresis A first-dimensional isoelectric focusing (IEF) gel was prepared using a glass tube having a length of 180 mm and a diameter of 3.4 mm, and the second-dimensional SDS-PAGE was 195. The thing of the magnitude | size of * 120 * 1.5mm was created. In order to take advantage of the characteristics of agarose two-dimensional electrophoresis (agarose 2-DE), two concentrations of SDS-PAGE gel were prepared according to the purpose. That is, a 12% homogeneous gel for the purpose of separating low molecular mass (LMM) protein and a 6-10% concentration gradient gel for the purpose of separating high molecular mass (HMM) protein. Second-dimensional SDS-PAGE was performed by the Laemmli method.
尿では１０ｍｌの尿を脱塩・濃縮して得られた２００μｌ中にタンパク質８００μｇ相当を含有するタンパク質抽出液をＩＥＦゲルのアルカリ側に入れ、１Ｍ尿素、０．２５Ｍチオ尿素溶液をその上から注入した。組織では、１００μＬ、タンパク質１．０ｍｇ相当のタンパク質抽出液をＩＥＦゲルのアルカリ側に入れ、４Ｍ尿素、１Ｍチオ尿素溶液をその上から注入した。 In the case of urine, a protein extract containing 800 μg of protein in 200 μl obtained by desalting and concentrating 10 ml of urine is placed on the alkali side of the IEF gel, and 1 M urea and 0.25 M thiourea solution are injected from above. did. In the tissue, 100 μL of protein extract equivalent to 1.0 mg of protein was placed on the alkali side of the IEF gel, and 4M urea and 1M thiourea solution were injected from above.
一次元目ＩＥＦは４℃下で７００Ｖ、２０時間の泳動で行った。一次元目の泳動後、長さ３００ｍｍ、直径５ｍｍのガラス管にゲルを移し、１０％ＴＣＡ 、５％スルホサリチル酸溶液で１時間還流してタンパク質を固定し、１時間蒸留水で洗浄した。固定したＩＥＦゲルは二次元目のＳＤＳ−ＰＡＧＥゲルの上端に設置し１％ａｇａｒで隙間を埋めた。ＳＤＳ泳動バッファー（２％ＳＤＳ，１０％ｇｌｙｃｅｒｏｌ，５％２−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ，０．０２％ｂｒｏｍｏｐｈｅｎｏｌ ｂｌｕｅ，０．０５Ｍ Ｔｒｉｓ−ＨＣｌ ｐＨ６．５）をＩＥＦゲル上に注入し、４０ｍＡで１時間、その後８０ｍＡで二次元目の泳動を行った。泳動が終了したゲルはＣＢＢ Ｒ−３５０（ＰｈａｓｔＧｅｌ Ｂｌｕｅ Ｒ，ＧＥ Ｈｅａｌｔｈｃａｒｅ，Ｌｉｔｔｌｅ Ｃｈａｌｆｏｎｔ，ＵＫ）で染色した。
（４）質量分析解析
２−ＤＥゲルから切り出したタンパクスポットを、５０％ｖ／ｖアセトニトリルで脱色した後に１００％アセトニトリルで脱水、乾燥させた。０．５ｎｇ／μＬトリプシン（Ｒｏｃｈｅ Ｄｉａｇｎｏｓｔｉｃｓ，Ｍａｎｎｈｅｉｍ，Ｇｅｒｍａｎｙ）、５０ｍＭ ＮＨ４ＨＣＯ３ ｂｕｆｆｅｒ（ｐＨ８．０）で３７℃２４時間タンパク質のゲル内消化を行った。この消化産物ペプチドを５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ９．０）と７０％アセトニトリル溶液で溶出させた。ゲル内消化後のペプチド混合物を液体クロマトグラフィー質量分析計［液体クロマトグラフィー：Ｎａｎｏｓｐａｃｅ ＳＩ−２，（Ｓｈｉｓｅｉｄｏ Ｆｉｎｅ Ｃｈｅｍｉｃａｌｓ，Ｔｏｋｙｏ，Ｊａｐａｎ）質量分析計：ＬＣＱ−ＤＥＣＡ（Ｔｈｅｒｍｏ Ｆｉｎｎｉｇａｎ，Ｓａｎ Ｊｏｓｅ，ＣＡ，ＵＳＡ）］により分析した。
（５）タンパク質の同定
上記に記した質量分析計によって得られた各トリプシン消化ペプチドのＭＳスペクトルとタンデムＭＳスペクトル（ＭＳ／ＭＳスペクトル）を質量分析計に装備されているタンパク質同定用プログラム（ＳＥＱＵＥＳＴ Ｓｅａｒｃｈ Ｖｅｒ２．０， ｈｔｔｐ:／／ｆｉｅｌｄｓ.ｓｃｒｉｐｐｓ.ｅｄｕ／ｓｅｑｕｅｓｔ／ｉｎｄｅｘ.ｈｔｍｌ）を用いて、Ｓｅｑｕｅｎｃｅ Ｔａｇ法によりデータベース検索を行った。このプログラムは、測定で得たＭＳスペクトル、ＭＳ／ＭＳスペクトルと、データベース上の各タンパク質を理論的に酵素消化して得られるＭＳスペクトル、ＭＳ／ＭＳスペクトルを比較する。その結果として一致度合いの高いタンパク質を選び出し、可能性に応じて一致度合いの評点（Ｓｃｏｒｅ）を付ける。本研究では、タンパク質のＳｃｏｒｅが７０以上のものを同定されたタンパク質とした。また、Ｓｃｏｒｅが７０未満の場合は、信憑性の高いＭＳ／ＭＳスペクトルが２つ以上観測されていることを確認し、同定されていると判断した。この基準は一般的な質量分析計の同定基準以上のものであり、十分に信頼できるものである。
（６）結果
（６−１）高分子量タンパク質領域のアガロース二次元電気泳動ゲル地図とタンパク質の同定
本発明者らは、アガロース ２−ＤＥ法を用いることで、今まで解析されていない高分子領域（９７．４ｋＤａ以上）に焦点をあてた。すなわち、６−１０％グラディエントゲルを用いて１０例の膀胱癌組織と同一人物から得た膀胱正常粘膜における高分子量タンパク質の発現量を比較した。得られた二次元電気泳動パターンは、癌と正常部ではそのパターンが大きく異なった。そのため、最も違いが認められた１症例 （ＴＣＣ，ｐＴ２，Ｇｒａｄｅ ｉｉ）の癌部と正常粘膜における高分子領域（９７．４ｋＤａ以上）に認められたタンパク質スポットをすべて切り出し、同定した（図１）。癌部からは７３スポット切り出し、５６スポット、５３種類のタンパク質を同定し、正常粘膜からは４８スポット切り出し、３４スポット、３３種類のタンパク質を同定した。同定されたタンパク質を比較し、癌部でのみ同定されたタンパク質を文献的に詳細に検討した結果、表１に示す７種類のタンパク質を新規腫瘍マーカー候補とした。
The first dimension IEF was performed by electrophoresis at 700 V for 20 hours at 4 ° C. After the first-dimensional migration, the gel was transferred to a glass tube having a length of 300 mm and a diameter of 5 mm, and the protein was fixed by refluxing with 10% TCA and 5% sulfosalicylic acid solution for 1 hour, followed by washing with distilled water for 1 hour. The fixed IEF gel was placed on the upper end of the second-dimensional SDS-PAGE gel, and the gap was filled with 1% agar. SDS running buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.02% bromophenol blue, 0.05M Tris-HCl pH 6.5) was injected onto the IEF gel, 1 hour at 40 mA, then 80 mA. The second dimension of electrophoresis was performed. The gel after electrophoresis was stained with CBB R-350 (PastGel Blue R, GE Healthcare, Little Chalfont, UK).
(4) Mass spectrometry analysis The protein spot cut out from the 2-DE gel was decolorized with 50% v / v acetonitrile and then dehydrated and dried with 100% acetonitrile. In-gel digestion of proteins was performed at 37 ° C. for 24 hours with 0.5 ng / μL trypsin (Roche Diagnostics, Mannheim, Germany), 50 mM NH 4 HCO 3 buffer (pH 8.0). The digested product peptide was eluted with 50 mM Tris-HCl (pH 9.0) and 70% acetonitrile solution. The peptide mixture after in-gel digestion was analyzed using a liquid chromatography mass spectrometer [liquid chromatography: Nanospace SI-2, (Shiseido Fine Chemicals, Tokyo, Japan) mass spectrometer: LCQ-DECA (Thermo Finnigan, San Jose, CA, USA). )].
(5) Identification of protein A protein identification program (SEQUEST Search) equipped with a mass spectrometer for the MS spectrum and tandem MS spectrum (MS / MS spectrum) of each tryptic peptide obtained by the mass spectrometer described above. Ver2.0, http://fields.scripts.edu/sequence/index.html), and database search was performed by the Sequence Tag method. This program compares the MS spectrum and MS / MS spectrum obtained by measurement with the MS spectrum and MS / MS spectrum obtained by theoretically enzymatically digesting each protein on the database. As a result, a protein having a high degree of matching is selected, and a score (Score) of the degree of matching is given according to the possibility. In this study, proteins with a score of 70 or more were identified as proteins. Moreover, when Score was less than 70, it was determined that two or more highly reliable MS / MS spectra were observed and determined to be identified. This criterion is more than a general mass spectrometer identification criterion and is sufficiently reliable.
(6) Results (6-1) Agarose two-dimensional electrophoresis gel map of high molecular weight protein region and protein identification The present inventors have used the agarose 2-DE method to analyze a high molecular region that has not been analyzed so far. Focused on (97.4 kDa and above). That is, the expression level of high molecular weight protein in normal bladder mucosa obtained from the same person as 10 cases of bladder cancer tissue was compared using 6-10% gradient gel. The obtained two-dimensional electrophoresis pattern was greatly different between cancer and normal part. Therefore, all the protein spots observed in the high molecular area (97.4 kDa or more) in the cancerous part and normal mucosa of one case (TCC, pT2, Grade ii) in which the difference was most recognized were cut out and identified (FIG. 1). . 73 spots were extracted from the cancer area, 56 spots and 53 types of proteins were identified, and 48 spots were extracted from the normal mucosa, 34 spots and 33 types of proteins were identified. As a result of comparing the identified proteins and examining in detail the proteins identified only in the cancerous part, seven types of proteins shown in Table 1 were used as novel tumor marker candidates.
（ａ）：図１に示す２−ＤＥゲル（Ａ）におけるタンパク質スポットから抽出された各タンパク質に付した番号
（ｂ）：ＣＳ ａｎａｌｙｚｅｒ ２．０により算出した２−ＤＥゲルにおけるタンパク質の位置から得られた実験的分子量（ＭＭ）
（ｃ）：アミノ酸配列から算出した理論的分子量（ＭＭ）およびｐＩ
（ｄ）：ＳＥＱＵＥＳＴ ｓｃｏｒｅ、候補のＳＥＱＵＥＳＴ検索での最も高いスコア
（ｅ）：ｓｅｑｕｅｎｃｅ ｃｏｖｅｒａｇｅ、候補タンパク質のアミノ酸配列のＭＳ／ＭＳ解析により確認されたペプチドの一致率
（ｆ）：ｎｕｍｂｅｒ ｏｆ ｐｅｐｔｉｄｅｓ ｍａｔｃｈｅｄ ｂｙ ＭＳ／ＭＳ ｓｅｑｕｅｎｃｅ、ＭＳ／ＭＳデータを与えるＳＥＱＵＥＳＴ検索でのタンパク質配列に用いた断片数
（６−２）癌と正常組織におけるタンパク質発現の変化
癌と正常部で同定されたタンパク質をＨｕｍａｎ ｐｒｏｔｅｉｎ ｒｅｆｅｒｅｎｃｅ Ｄａｔａｂａｓｅ（ｈｔｔｐ：／／ｗｗｗ.ｈｐｒｄ．ｏｒｇ／）によりｓｕｂｃｅｌｌｕｌａｒ ｌｏｃａｔｉｏｎとＢｉｏｌｏｇｙ ｐｒｏｃｅｓｓの２つ項目で分類した。その結果を図２に示す。高分子領域では癌で核内タンパクが増え、機能分類としても転写・翻訳に関連した働きをもつタンパク質が大きく増加することが確認された。
(A): Number assigned to each protein extracted from the protein spot in the 2-DE gel (A) shown in FIG. 1 (b): Obtained from the protein position in the 2-DE gel calculated by CS analyzer 2.0 Experimental molecular weight (MM)
(C): Theoretical molecular weight (MM) and pI calculated from the amino acid sequence
(D): SEQUEST score, highest score in candidate SEQUEST search (e): sequence coverage, peptide match rate confirmed by MS / MS analysis of amino acid sequence of candidate protein (f): number of peptides matched by MS / MS sequence, number of fragments used in protein sequence in SEQUEST search that gives MS / MS data (6-2) Changes in protein expression in cancer and normal tissue Protein identified in cancer and normal part Human protein reference Database (Http://www.hprd.org/), and classified into two items, subcellular location and Biology process. The result is shown in FIG. In the high molecular area, it has been confirmed that proteins in the nucleus increase due to cancer, and that proteins with functions related to transcription / translation also greatly increase in functional classification.
腫瘍マーカー候補タンパク質のウエスタンブロッティング法による確認
膀胱癌組織で発現が増加を示した上記７種類のタンパク質についてウエスタンブロッティング法による確認実験を行った。ウエスタンブロッティング法で使用するＡＨＮＡＫ、ＳＭＣ３、ｐｌｅｃｔｉｎ−１、ＩＱＧＡＰ１に対する特異的抗体は市販の抗体を使用した。即ち、ＡＨＮＡＫ抗体はＡＢＮＯＶＡ（Ｔｈａｉｐｅｉ，Ｔｈａｉｗａｎ）から、抗ＳＭＣ３抗体はＣＥＬＬ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ（Ｄａｎｖｅｒｓ，ＵＳＡ）、抗ｐｌｅｃｔｉｎ−１抗体はＢｅｎｄｅｒ ＭｅｄＳｙｓｔｅｍｓ（Ｖｉｅｎｎａ，Ａｕｓｔｒｉａ）、抗ＩＱＧＡＰ１抗体はＢＤ Ｂｉｏｓｃｉｅｎｃｅ（Ｆｒａｎｋｌｉｎ Ｌａｋｅｓ，ＵＳＡ）より購入し, それぞれ１：１０００の希釈倍率で使用した。また、抗体が市販されていないＥｐｉｐｌａｋｉｎ、ＥＩＦ−３、Ｖｉｇｉｌｉｎについては、理論的なアミノ酸配列をもとに作成した合成ペプチドからマウスポリクローン性抗体を作成し、それぞれ１：１０００の希釈倍率で使用した。キャリブレーションにはウサギ抗ヒトアクチン抗体（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ，Ｄａｎｖｅｒｓ，ＭＡ，ＵＳＡ）を１：１０００の希釈倍率で使用した。
Confirmation of Tumor Marker Candidate Proteins by Western Blotting Method Confirmation experiments by Western blotting method were conducted on the above seven proteins whose expression was increased in bladder cancer tissues. Commercial antibodies were used as specific antibodies for AHNAK, SMC3, lectin-1, and IQGAP1 used in the Western blotting method. That is, the AHNAK antibody is from ABNOVA (Thaipei, Thailand), the anti-SMC3 antibody is CELL Signaling Technology (Danvers, USA), the anti-plectin-1 antibody is Bender MedSystem, and the Bender MedSystem (AustrAGB). USA) and each used at a dilution factor of 1: 1000. For Epiplatin, EIF-3, and Vigilin, for which antibodies are not commercially available, mouse polyclonal antibodies are prepared from synthetic peptides prepared based on the theoretical amino acid sequence and used at a dilution ratio of 1: 1000, respectively. did. For calibration, a rabbit anti-human actin antibody (Cell Signaling Technology, Danvers, MA, USA) was used at a dilution ratio of 1: 1000.
タンパク質抽出液を１０−２０％ＳＤＳ−ＰＡＧＥゲル（ＤＲＣ，Ｔｏｋｙｏ,Ｊａｐａｎ）で展開し、ＰＶＤＦ膜に転写した。転写の終了したＰＶＤＦ膜はＢｌｏｃｋ Ａｃｅ(大日本製薬、大阪）でブロッキングを行い、２％ｎｏｒｍａｌ ｓｗｉｎｅ ｓｅｒｕｍ （Ｄａｋｏ Ｃｙｔｏｍａｔｉｏｎ，Ｄｅｎｍａｒｋ）／ＴＢＳを抗体希釈バッファーとして使用してウエスタンブロットを行った。ＨＲＰ標識二次抗体は１：１０００で希釈して使用し、検出にはＩｍｍｏｂｉｌｏｎ Ｗｅｓｔｅｒｎ ｄｅｔｅｃｔｉｏｎ ｒｅｇｅｎｔｓ（Ｍｉｌｌｉｐｏｒｅ，Ｂｉｌｌｅｒｉｃａ，ＵＳＡ）による化学発光を用いた。 The protein extract was developed on a 10-20% SDS-PAGE gel (DRC, Tokyo, Japan) and transferred to a PVDF membrane. After the transfer, the PVDF membrane was blocked with Block Ace (Dainippon Pharmaceutical Co., Osaka), and Western blotted using 2% normal spine serum (Dako Cytomation, Denmark) / TBS as an antibody dilution buffer. HRP-labeled secondary antibody was used at a dilution of 1: 1000, and chemiluminescence by Immobilon Western detection reagents (Millipore, Billerica, USA) was used for detection.
(結果)
前立腺癌患者の手術時に切除した膀胱正常粘膜１０例と膀胱癌組織１０例における発現量を比較検討したところ、腫瘍マーカー候補とした７種類のタンパク質はすべて膀胱癌組織で発現の増加を確認した（図３Ａ）。膀胱癌患者尿（尿細胞診 ＣｌａｓｓＶ）と健常者尿での尿中タンパク存在量を比較したところ、ＳＭＣ３が癌患者尿で存在量が増加していることを確認した（図３Ｂ）。また、現在確立されている６種類の膀胱癌細胞株、すなわちＲＴ４，５６３７，Ｔ２４，ＥＪ，ＭＴＢ２，ＴＣＣＳＵＰにおけるマーカー候補タンパク質の発現比較を行い、発現を確認した。ＡＨＮＡＫにおいては、ＲＴ４，５６３７，ＥＪと比較しＴ２４，ＭＴＢ２，ＴＣＣＳＵＰで発現量が強く増加しているのが確認された（図３Ｃ ）。
(result)
When the expression levels of 10 normal bladder mucosa excised at the time of surgery for prostate cancer patients and 10 bladder cancer tissues were compared, all seven proteins as tumor marker candidates were confirmed to increase in expression in the bladder cancer tissues ( FIG. 3A). When urinary protein abundance was compared between urine of bladder cancer patients (urinary cytology class V) and normal urine, it was confirmed that the abundance of SMC3 was increased in urine of cancer patients (FIG. 3B). In addition, the expression of marker candidate proteins was compared in 6 types of currently established bladder cancer cell lines, namely RT4, 5637, T24, EJ, MTB2, and TCCSUP, to confirm the expression. In AHNAK, it was confirmed that the expression level was strongly increased in T24, MTB2, and TCCSUP compared to RT4, 5637 and EJ (FIG. 3C).
免疫組織化学染色による確認
ウエスタンブロッティング法で癌組織において有意に発現量の増加が確認されたタンパク質について免疫組織化学染色による検討を行った。ヒト膀胱正常組織、同病変部組織アレイ（Ｆｏｌｉｏ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｃｏｌｕｍｂｕｓ，ＯＨ，ＵＳＡ）を恒温室にて５５℃、２０分間加温した。標本を脱パラフィン処理し、０．０１Ｍクエン酸緩衝液（ｐＨ６．０ ）に切片を浸し、５分間３回マイクロウエーブ処理して抗原性を賦活化した。その後、３％過酸化水素水にて10分間放置し内因性ペルオキシダーゼを阻止し、ＰＢＳ（Ｔｗｅｅｎ２０含有) にて洗浄した。次いで、正常ヤギ血清（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ Ｉｎｃ．，Ｕ.Ｓ.Ａ 、１：２０）を滴下し、室温で１５分間放置後、抗ＡＨＮＡＫ抗体（ＡＢＮＯＶＡ，Ｔｈａｉｐｅｉ，Ｔｈａｉｗａｎ，１：１０００）、抗ＳＭＣ３抗体（ＣＥＬＬ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ，Ｄａｎｖｅｒｓ，ＵＳＡ，１：４００ ）、抗Ｐｌｅｃｔｉｎ−１抗体（Ｂｅｎｄｅｒ ＭｅｄＳｙｓｔｅｍｓ，Ｖｉｅｎｎａ，Ａｕｓｔｒｉａ，１：２００）、抗ＩＱＧＡＰ１抗体（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ，Ｆｒａｎｋｌｉｎ Ｌａｋｅｓ，ＵＳＡ）、抗Ｅｐｉｐｌａｋｉｎ抗体、抗ＥＩＦ−３抗体、抗Ｖｉｇｉｌｉｎ抗体を切片上に滴下し、４℃にて一晩(１６時間) 反応させた。ＰＢＳ（Ｔｗｅｅｎ２０含有）にて洗浄後、Ｄａｋｏ ＣｈｅｍＭａｔｅ ＥＮＶＩＳＩＯＮキット／ＨＲＰ（ＤＡＢ）ポリマー試薬（Ｄａｋｏ Ｃｙｔｏｍａｔｉｏｎ，Ｄｅｎｍａｒｋ）を切片上に滴下し、室温で３０分間反応させた。
ＰＢＳで洗浄後、Ｓｔａｂｌｅ ＤＡＢ（ファルマ，東京）溶液を切片上に滴下し、３分間発色を行い、マイヤーヘマトキシリン溶液（Ｗａｋｏ，大阪）にて核染色し、脱水、透徹、封入をした。
（結果）
組織マイクロアレイ（Ｆｏｌｉｏ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｃｏｌｕｍｂｕｓ，ＯＨ，ＵＳＡ）を用いて、免疫組織化学染色による確認実験を行った。ｖｉｇｉｌｉｎ、ｅｐｉｐｌａｋｉｎ、ＥＩＦ−３、ＩＱＧＡＰ１、ＳＭＣ３、ＡＨＮＡＫおよびｐｌｅｃｔｉｎ−１の７種類のタンパク質について検討した。その結果、図４において７種類全てのタンパク質が膀胱癌組織において発現していることが確認できた。
Confirmation by immunohistochemical staining Proteins whose expression level was significantly increased in cancer tissues by Western blotting were examined by immunohistochemical staining. A normal tissue of a human bladder and a tissue array of the affected part (Folio Biosciences, Columbia, OH, USA) were heated at 55 ° C. for 20 minutes in a thermostatic chamber. The specimen was deparaffinized, the section was immersed in 0.01 M citrate buffer (pH 6.0), and subjected to microwave treatment for 3 minutes for 5 minutes to activate antigenicity. Thereafter, the cells were left in 3% hydrogen peroxide solution for 10 minutes to block endogenous peroxidase and washed with PBS (containing Tween 20). Subsequently, normal goat serum (Vector Laboratories Inc., USA, 1:20) was added dropwise and allowed to stand at room temperature for 15 minutes, followed by anti-AHNAK antibody (ABNOVA, Thaipei, Thaiwan, 1: 1000), anti-SMC3 antibody (CELL Signaling Technology, Danvers, USA, 1: 400), anti-Plectin-1 antibody (Bender MedSystems, Vienna, Austria, 1: 200), anti-IQGAP1 antibody (BD Bioscience, FrankinP, anti-LinklinLampinLampinL) An EIF-3 antibody and an anti-Vigilin antibody were dropped onto the section and reacted at 4 ° C. overnight (16 hours). After washing with PBS (containing Tween 20), Dako Chemmate ENVISION kit / HRP (DAB) polymer reagent (Dako Cytomation, Denmark) was dropped onto the section and reacted at room temperature for 30 minutes.
After washing with PBS, a Stable DAB (Pharmacia, Tokyo) solution was dropped on the section, color was developed for 3 minutes, and nuclear staining was performed with Mayer's hematoxylin solution (Wako, Osaka), followed by dehydration, penetration, and encapsulation.
(result)
A confirmation experiment by immunohistochemical staining was performed using a tissue microarray (Folio Biosciences, Columbias, OH, USA). Seven proteins, vigilin, epiplatin, EIF-3, IQGAP1, SMC3, AHNAK and plectin-1, were examined. As a result, it was confirmed in FIG. 4 that all seven proteins were expressed in bladder cancer tissue.
ＳＭＣ３を腫瘍マーカーとして用いた膀胱癌診断
（１）ＳＭＣ３抗体による膀胱正常組織と膀胱癌部の免疫組織化学染色
図５に示す結果から明らかなように、正常組織における移行上皮細胞の核はほとんどＳＭＣ３に対し染色されていないが、癌細胞の核はＳＭＣ３に対し濃染されており、癌細胞においてＳＭＣ３の発現が有意に上昇していることを確認した。
（２）尿細胞診における利用
尿細胞診は、尿中に存在する細胞片等を遠心分離して集め、腫瘍細胞をＰａｐａｎｉｃｏｌａｕ染色により判定する腫瘍診断法の一つである。通常、ＣｌａｓｓＶ、ＣｌａｓｓＩＶは陽性、ＣｌａｓｓＩ、ＣｌａｓｓＩＩは陰性と判断される。ただし尿細胞診のみでは膀胱癌と診断できない。
Diagnosis of bladder cancer using SMC3 as a tumor marker (1) Immunohistochemical staining of bladder normal tissue and bladder cancer site with SMC3 antibody As is clear from the results shown in FIG. 5, the nucleus of transitional epithelial cells in normal tissue is almost SMC3 However, it was confirmed that the nuclei of cancer cells were intensely stained for SMC3 and that the expression of SMC3 was significantly increased in the cancer cells.
(2) Use in urine cytology
Urine cytodiagnosis is one of the tumor diagnostic methods in which cell fragments and the like present in urine are collected by centrifugation, and tumor cells are determined by Papanicolau staining. Usually, Class V and Class IV are judged positive, and Class I and Class II are judged negative. However, bladder cancer cannot be diagnosed by urine cytology alone.
本実施例では、通常の尿細胞診（ｐａｐａｎｉｃｏｌａｕ染色）においてＣｌａｓｓＶが得られ、膀胱癌として診断された患者尿を用い、ＳＭＣ３による尿細胞診を行う。抗ＳＭＣ３抗体を用いた免疫組織化学染色の結果、全体の約７０％以上の異型細胞の核がＳＭＣ３に対し濃染された（図６、矢印は濃染した核を示す）。ＣｌａｓｓＩＩ症例においても異型細胞の核がＳＭＣ３に対し濃染していた（ｄａｔａ ｎｏｔ ｓｈｏｗｎ）。従って、尿細胞診において、ＳＭＣ３は膀胱癌診断のための有用なマーカーとなりうる。
（３）尿中のＳＭＣ３の定量
１．Ｐａｐａｎｉｃｏｌａｕ染色における尿細胞診でｃｌａｓｓＶである１０例、２．膀胱癌患者であるが、尿細胞診においては ｃｌａｓｓＩＩであった１０例、３．健常者尿（非膀胱癌患者尿）１０例の計３０例で、尿中ＳＭＣ３の存在量をウエスターンブロット法にて比較検討した。ｃｌａｓｓＶの症例においては、９例で尿中ＳＭＣ３の存在量の増加が認められた。また、尿細胞診ではｃｌａｓｓＩＩで正常と判断された症例については、４例において、ＳＭＣ３の存在量が健常者尿に比較して増加していた（図７）。このことより膀胱癌診断において尿中ＳＭＣ３を定量することは、尿細胞診の診断率を超える可能性が示唆された。
In this example, Class V is obtained in normal urine cytodiagnosis (papanicolau staining), and urine cytology by SMC3 is performed using patient urine diagnosed as bladder cancer. As a result of immunohistochemical staining using an anti-SMC3 antibody, about 70% or more of the nuclei of atypical cells were darkly stained for SMC3 (FIG. 6, arrows indicate darkly stained nuclei). Also in Class II cases, the nuclei of atypical cells were intensely stained for SMC3 (data not shown). Therefore, SMC3 can be a useful marker for bladder cancer diagnosis in urine cytology.
(3) Determination of SMC3 in
本明細書で使用する配列番号１〜１４は、以下に説明する配列を表す。配列番号１〜７ のアミノ酸配列を有するポリペプチドは、これまで膀胱癌との関連は知られていなかった。
（配列番号：１）
ＡＨＮＡＫは細胞間橋に局在する高分子量タンパク質として、扁平上皮より分離され、その後の研究で上皮細胞では細胞間橋に発現するが、上皮細胞以外では核内に発現することが確認されている。神経芽細胞腫や肺小細胞癌で発現が抑制されるという報告がある。ＡＨＮＡＫの機能は細胞間接着、腫瘍形成、細胞増殖などに関連すると報告されているが、現段階ではほとんど分かっていない。
Ｓｗｉｓｓ−Ｐｒｏｔ．ａｃｃｅｓｓｉｏｎ Ｎｏ：Ｑ０９６６６，Ｇｅｎｅ Ｎａｍｅ：ＡＨＮＡＫ
（配列番号：２）
Ｐｌｅｃｔｉｎは、元来グリオーマ細胞から抽出された分子量約５００ｋＤａのプラキンファミリーに属する細胞内タンパク質である。ヘミデスモゾームに存在しケラチンと結合し細胞を安定化させる働きがあり、自己免疫性水疱症の発症に関連があるとの報告が多い。Ｐｌｅｃｔｉｎには４つのスプライシングフォームが存在し、今回同定されたｐｌｅｃｔｉｎ−１はその一つである。
Ｓｗｉｓｓ−Ｐｒｏｔ．ａｃｃｅｓｓｉｏｎ Ｎｏ：Ｑ１５１４９，Ｇｅｎｅ Ｎａｍｅ：ＰＬＥＣ１
（配列番号：３）
Ｅｐｉｐｌａｋｉｎはデスモゾーム等の構成成分であるプラキンファミリーの一種であり表皮下水疱症の自己抗原として同定されたタンパク質である。癌との関連を示した報告例はない。
Ｓｗｉｓｓ−Ｐｒｏｔ．ａｃｃｅｓｓｉｏｎ Ｎｏ：Ｐ５８１０７，Ｇｅｎｅ Ｎａｍｅ：ＥＰＰＫ１
（配列番号：４）
Ｅｕｋａｒｙｏｔｉｃ ｔｒａｎｓｌａｔｉｏｎ ｉｎｉｔｉａｔｉｏｎ ｆａｃｔｏｒ ３はリボゾームにおける翻訳開始に関与するとタンパクであり、多くのサブユニットをもつ。Ｅｕｋａｒｙｏｔｉｃ ｔｒａｎｓｌａｔｉｏｎ ｉｎｉｔｉａｔｉｏｎ ｆａｃｔｏｒ ３ ｓｕｂｕｎｉｔ １０と異なるサブユニットに関してノックダウンした膀胱癌以外の培養細胞株でその増殖が抑制されるとの報告がある。
Ｓｗｉｓｓ−Ｐｒｏｔ．ａｃｃｅｓｓｉｏｎ Ｎｏ：Ｑ１４１５２，Ｇｅｎｅ Ｎａｍｅ： ＥＩＦ３Ａ
（配列番号：５）
ＶｉｇｉｌｉｎはＨＤＬ結合タンパクの一種であり、細胞内のコレステロール量の調節に働くタンパク質である。近年、細胞質だけでなく核内にも存在し、核内のｔＲＮＡが細胞質へ移動する機序に関与すると報告されている。
Ｓｗｉｓｓ−Ｐｒｏｔ．ａｃｃｅｓｓｉｏｎ Ｎｏ：Ｑ００３４１，Ｇｅｎｅ Ｎａｍｅ：ＨＤＬＢＰ
（配列番号：６）
Ｓｔｒｕｃｔｕｒａｌ ｍａｉｎｔｅｎａｎｃｅ ｏｆ ｃｈｒｏｍｏｓｏｍｅｓ ｐｒｏｔｅｉｎ ３（ＳＭＣ３）はｃｈｒｏｍｓｏｍｅのコヒーシンに存在し、ＤＮＡ修復に関与するタンパク質であるが、細胞質に存在するものは基底膜に豊富で分泌性のタンパク質である。
Ｓｗｉｓｓ−Ｐｒｏｔ．ａｃｃｅｓｓｉｏｎ Ｎｏ：Ｑ９ＵＱＥ７，Ｇｅｎｅ Ｎａｍｅ：ＳＭＣ３
（配列番号：７）
Ｒａｓ ＧＴＰａｓｅ−ａｃｔｉｖａｔｉｎｇ−ｌｉｋｅ ｐｒｏｔｅｉｎ（ＩＱＧＡＰ１）は細胞骨格を構成する分子や細胞接着に関与する分子間で相互に作用するタンパクであり、近年では胃癌の２つの培養細胞株で発現が上昇するとの報告がある。
Ｓｗｉｓｓ−Ｐｒｏｔ．ａｃｃｅｓｓｉｏｎ Ｎｏ：Ｐ４６９４０，Ｇｅｎｅ Ｎａｍｅ：ＩＱＧＡＰ１
（配列番号：８〜１４）それぞれ、配列番号１〜７の膀胱癌マーカーポリペプチドをコードする遺伝子の配列を示す。
SEQ ID NOs: 1 to 14 used in the present specification represent the sequences described below. So far, the polypeptide having the amino acid sequence of SEQ ID NOs: 1 to 7 has not been known to be associated with bladder cancer.
(SEQ ID NO: 1)
AHNAK is isolated from the squamous epithelium as a high molecular weight protein localized in the intercellular bridge, and in subsequent studies, it is expressed in the intercellular bridge in epithelial cells, but it is confirmed that it is expressed in the nucleus other than epithelial cells. . There are reports that expression is suppressed in neuroblastoma and small cell lung cancer. Although the function of AHNAK has been reported to be related to cell-cell adhesion, tumor formation, cell proliferation, etc., little is known at this stage.
Swiss-Prot. accession No: Q09666, Gene Name: AHNAK
(SEQ ID NO: 2)
Plectin is an intracellular protein belonging to the Plakin family with a molecular weight of about 500 kDa originally extracted from glioma cells. There are many reports that it exists in hemidesmosome and has a function to bind to keratin and stabilize cells, and is related to the onset of autoimmune blistering. There are four splicing forms in Plectin, and plectin-1 identified this time is one of them.
Swiss-Prot. accession No: Q15149, Gene Name: PLEC1
(SEQ ID NO: 3)
Epiplatin is a protein of the Plakin family that is a constituent component of desmosome and the like, and is a protein identified as an autoantigen of subepidermal blistering. There are no reports showing an association with cancer.
Swiss-Prot. accession No: P58107, Gene Name: EPPK1
(SEQ ID NO: 4)
Eukaryotic
Swiss-Prot. accession No: Q14152, Gene Name: EIF3A
(SEQ ID NO: 5)
Vigilin is a kind of HDL-binding protein and is a protein that functions to regulate intracellular cholesterol levels. In recent years, it has been reported that it exists not only in the cytoplasm but also in the nucleus, and is involved in the mechanism by which tRNA in the nucleus moves to the cytoplasm.
Swiss-Prot. accession No: Q00341, Gene Name: HDLBP
(SEQ ID NO: 6)
Structural maintenance of chromosomes protein 3 (SMC3) is a protein involved in DNA repair, and is present in the cytoplasm, but is abundant and secreted in the basement membrane.
Swiss-Prot. accession No: Q9UQE7, Gene Name: SMC3
(SEQ ID NO: 7)
Ras GTPase-activating-like protein (IQGAP1) is a protein that interacts between molecules that make up the cytoskeleton and molecules that are involved in cell adhesion, and has recently been reported to increase expression in two cultured cell lines of gastric cancer There is.
Swiss-Prot. accession No: P46940, Gene Name: IQGAP1
(SEQ ID NOs: 8-14) show the sequences of the genes encoding the bladder cancer marker polypeptides of SEQ ID NOs: 1-7, respectively.
Claims (18)
（ａ）配列番号３に記載のアミノ酸配列を有するポリペプチド、
（ｂ）配列番号３に記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ（ａ）と同等の抗体産生能を有するかまたは（ａ）に対する特異的抗体と反応しうるポリペプチド。 In detecting bladder cancer cells in a biological sample obtained from a subject, the following (a) or (b) polypeptide is used as a tumor marker, and the method of using the polypeptide:
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3,
(B) has an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 3 and has an antibody-producing ability equivalent to (a) or A polypeptide capable of reacting with a specific antibody against (a).
（ａ）配列番号３に記載のアミノ酸配列を有するポリペプチド；
（ｂ）配列番号３に記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ配列番号３に記載のアミノ酸配列からなるポリペプチドに対する抗体の産生を誘導するポリペプチド。 A composition for producing a specific antibody against a bladder cancer marker polypeptide, comprising the following (a) or (b):
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3;
(B) against a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in SEQ ID NO: 3 and consisting of the amino acid sequence described in SEQ ID NO: 3 A polypeptide that induces the production of an antibody.
（ａ）配列番号３に記載のアミノ酸配列を有するポリペプチド；
（ｂ）配列番号３に記載のアミノ酸配列において１もしくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を有し、かつ配列番号３に記載のアミノ酸配列からなるポリペプチドに対する特異的抗体と反応しうるポリペプチド。 A diagnostic reagent for bladder cancer, comprising the following (a) or (b):
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3;
(B) against a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in SEQ ID NO: 3 and consisting of the amino acid sequence described in SEQ ID NO: 3 A polypeptide capable of reacting with a specific antibody.
(ｉ)配列番号１０に記載のヌクレオチド配列と相補的なヌクレオチド配列を有するポリヌクレオチド；
(ｉｉ)配列番号１０に記載のヌクレオチド配列と相補的なヌクレオチド配列の部分配列を含むヌクレオチド断片；
(ｉｉｉ) 配列番号１０に記載のヌクレオチド配列を有するポリヌクレオチドと、ストリンジェントな条件下でハイブリダイズする、ポリヌクレオチドまたはヌクレオチド断片。 Nucleic acid probe for diagnosing bladder cancer, comprising any of the following (i), (ii), (iii):
(i) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 10;
(ii) a nucleotide fragment comprising a partial sequence of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 10;
(iii) A polynucleotide or nucleotide fragment that hybridizes with the polynucleotide having the nucleotide sequence of SEQ ID NO: 10 under stringent conditions.
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| JP2013172643A Pending JP2014036656A (en) | 2008-03-14 | 2013-08-22 | Diagnosis of bladder cancer |
| JP2015020750A Expired - Fee Related JP6060425B2 (en) | 2008-03-14 | 2015-02-04 | Diagnosis of bladder cancer |
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| JP2010502905A Active JP5445969B2 (en) | 2008-03-14 | 2009-03-13 | Diagnosis of bladder cancer |
| JP2013172643A Pending JP2014036656A (en) | 2008-03-14 | 2013-08-22 | Diagnosis of bladder cancer |
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| CN102639555B (en) | 2009-09-24 | 2015-11-25 | 加利福尼亚大学董事会 | Bladder cancer specific ligand peptide |
| KR101247610B1 (en) * | 2009-12-11 | 2013-03-26 | 서울대학교산학협력단 | Dimeric core-shell nanoparticles labeled with Raman active molecule localized at interparticle junction, use thereof and method for preparing thereof |
| CN105785044B (en) * | 2016-04-12 | 2018-01-19 | 河北特温特生物科技发展有限公司 | Carcinoma of urinary bladder detection kit, the application of detection method and human complement factor H GAP-associated protein GAPs wherein |
| JP6782949B2 (en) * | 2017-03-31 | 2020-11-11 | 株式会社Ｈｉｒｏｔｓｕバイオサイエンス | Predicting therapeutic effects and / or recurrence monitoring of cancer patients |
| JP7100323B2 (en) * | 2017-07-11 | 2022-07-13 | 株式会社Ｈｉｒｏｔｓｕバイオサイエンス | Cancer detection method using tissue specimens |
| EP4354143A1 (en) * | 2021-06-10 | 2024-04-17 | The Kitasato Institute | Kit for diagnosis of cancer and use thereof |
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| US7919467B2 (en) * | 2000-12-04 | 2011-04-05 | Immunotope, Inc. | Cytotoxic T-lymphocyte-inducing immunogens for prevention, treatment, and diagnosis of cancer |
| US7998695B2 (en) * | 2005-02-10 | 2011-08-16 | Oncotherapy Science, Inc. | Method of diagnosing bladder cancer |
| EP1938104A2 (en) * | 2005-10-17 | 2008-07-02 | Institute for Systems Biology | Tissue-and serum-derived glycoproteins and methods of their use |
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| JP5445969B2 (en) | 2014-03-19 |
| WO2009113674A1 (en) | 2009-09-17 |
| JP2015092192A (en) | 2015-05-14 |
| JPWO2009113674A1 (en) | 2011-07-21 |
| JP2014036656A (en) | 2014-02-27 |
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