JP6707248B2 - Gastric cancer cell detection method and gastric cancer cell detection kit - Google Patents
Gastric cancer cell detection method and gastric cancer cell detection kit Download PDFInfo
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Description
本発明は、胃癌細胞の検出方法、及び胃癌細胞検出キットに関する。 The present invention relates to a method for detecting gastric cancer cells and a gastric cancer cell detection kit.
癌/精巣抗原は、癌細胞と精巣以外では発現が認められないタンパク質の総称である。
癌/精巣抗原の一つであるKitakyushu lung cancer antigen−1(以下、KK−LC−1ともいう。)は、肺癌よりT細胞に認識される腫瘍抗原として同定されている(例えば、非特許文献1参照。)。従って、KK−LC−1は、免疫応答を介した肺癌の治療、診断及び予防の標的分子としては有用と考えられる。
Cancer/testis antigen is a generic term for proteins that are not expressed in cancer cells and testis.
Kitakyushu lung cancer antigen-1 (hereinafter also referred to as KK-LC-1), which is one of cancer/testis antigens, has been identified as a tumor antigen recognized by T cells from lung cancer (for example, non-patent document). See 1.). Therefore, KK-LC-1 is considered to be useful as a target molecule for the treatment, diagnosis and prevention of lung cancer mediated by immune response.
しかしながら、一般的に癌/精巣抗原の発現率は4割以下であり、癌/精巣抗原を用いて癌細胞を高精度に検出する方法については未だ改良の余地がある。 However, the expression rate of cancer/testis antigen is generally 40% or less, and there is still room for improvement in the method of detecting cancer cells with high accuracy using cancer/testis antigen.
本発明は、上記事情に鑑みてなされたものであって、生体試料中の胃癌細胞の検出を高精度に検出できる胃癌細胞の検出方法、及び胃癌細胞検出キットを提供する。 The present invention has been made in view of the above circumstances, and provides a gastric cancer cell detection method and a gastric cancer cell detection kit capable of detecting gastric cancer cells in a biological sample with high accuracy.
本発明者らは、上記課題を解決すべく鋭意研究した結果、KK−LC−1の発現を検出する工程と、ヘリコバクター・ピロリ感染を検出する工程と、を兼ね備えることで、生体試料中の胃癌細胞の検出を高精度に行うことができることを見出し、本発明を完成させた。
すなわち、本発明は、下記の特徴を有する胃癌細胞の検出方法、及び胃癌細胞検出キットを提供する。
As a result of intensive studies to solve the above-mentioned problems, the present inventors have combined the step of detecting the expression of KK-LC-1 and the step of detecting Helicobacter pylori infection, thereby producing gastric cancer in a biological sample. They have found that cells can be detected with high accuracy, and have completed the present invention.
That is, the present invention provides a method for detecting gastric cancer cells and a gastric cancer cell detection kit having the following characteristics.
(1)生体試料中の胃癌細胞の検出方法であって、KK−LC−1の発現を検出する工程と、ヘリコバクター・ピロリ感染を検出する工程を有することを特徴とする胃癌細胞の検出方法。
(2)前記KK−LC−1の発現を検出する工程は、配列番号1で表されるプライマー及び配列番号2で表されるプライマーを用いてRT−PCRを行う工程を有する前記(1)に記載の胃癌細胞の検出方法。
(3)KK−LC−1検出キットとヘリコバクター・ピロリ感染検出キットを備えたことを特徴とする胃癌細胞検出キット。
(4)前記KK−LC−1検出キットは、配列番号1で表されるプライマー及び配列番号2で表されるプライマーを含む前記(3)に記載の胃癌細胞検出キット。
(1) A method for detecting gastric cancer cells in a biological sample, comprising a step of detecting KK-LC-1 expression and a step of detecting Helicobacter pylori infection.
(2) The step of detecting the expression of KK-LC-1 includes the step of performing RT-PCR using the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2, The method for detecting a gastric cancer cell described.
(3) A gastric cancer cell detection kit comprising a KK-LC-1 detection kit and a Helicobacter pylori infection detection kit.
(4) The KK-LC-1 detection kit according to (3) above, which comprises the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2.
本発明によれば、生体試料中の胃癌細胞の検出を高精度に行うことができる。 According to the present invention, gastric cancer cells in a biological sample can be detected with high accuracy.
<胃癌細胞の検出方法>
本発明の胃癌細胞の検出方法は、生体試料中の胃癌細胞の検出方法であって、KK−LC−1の発現を検出する工程と、ヘリコバクター・ピロリ感染を検出する工程を有する。
<Method for detecting gastric cancer cells>
The method for detecting gastric cancer cells of the present invention is a method for detecting gastric cancer cells in a biological sample, which comprises a step of detecting the expression of KK-LC-1 and a step of detecting Helicobacter pylori infection.
[KK−LC−1の発現を検出する工程]
KK−LC−1は、発明者らが肺癌から同定した抗原である。肺癌におけるKK−LC−1の発現頻度は、約3割〜4割であった(非特許文献1参照。)。
一方、実施例において後述するように、本発明者は、KK−LC−1が胃癌症例の約8割で発現していることを明らかにした。また、胃癌症例の腫瘍部位及び正常部位におけるKK−LC−1の発現パターンを分析したところ、KK−LC−1は、癌特異的に発現していることを明らかにした。
従って、KK−LC−1の発現を検出することにより、生体試料中の胃癌細胞を高精度に検出することができる。
[Step of detecting expression of KK-LC-1]
KK-LC-1 is an antigen that the inventors identified from lung cancer. The expression frequency of KK-LC-1 in lung cancer was about 30 to 40% (see Non-Patent Document 1).
On the other hand, as described later in Examples, the present inventor has revealed that KK-LC-1 is expressed in about 80% of gastric cancer cases. In addition, analysis of the expression pattern of KK-LC-1 in the tumor site and normal site of gastric cancer cases revealed that KK-LC-1 was expressed in a cancer-specific manner.
Therefore, gastric cancer cells in a biological sample can be detected with high accuracy by detecting the expression of KK-LC-1.
検出に用いる生体試料としては、特に限定されず、培養細胞であってもよいが、被験者から得られたものが好ましく、胃癌発症が疑われている発症被疑者、又は胃癌治療を受けている患者等の被験者の切除組織、胃癌細胞を含む血液、尿等から得られたものであることがより好ましい。 The biological sample used for detection is not particularly limited and may be cultured cells, but is preferably one obtained from a subject, a suspected person suspected of developing gastric cancer, or a patient undergoing gastric cancer treatment. More preferably, it is obtained from a resected tissue, blood containing gastric cancer cells, urine, etc. of the subject.
KK−LC−1の発現を検出する工程としては、例えば、生体試料中のKK−LC−1遺伝子を検出する工程が挙げられる。該工程は、KK−LC−1遺伝子のフラグメント、又はその相補配列からなるオリゴヌクレオチドを、該生体試料からの抽出物とインキュベートする工程を含むことが好ましく、該生体試料からの抽出物のゲノムDNA、mRNA、又は該mRNAに対するcDNAとハイブリダイズさせる工程を含むことがより好ましい。 Examples of the step of detecting the expression of KK-LC-1 include the step of detecting the KK-LC-1 gene in a biological sample. The step preferably includes a step of incubating an oligonucleotide consisting of a fragment of the KK-LC-1 gene or a complementary sequence thereof with an extract from the biological sample, and the genomic DNA of the extract from the biological sample. It is more preferable to include a step of hybridizing with the mRNA, or the cDNA for the mRNA.
本発明において、オリゴヌクレオチドは、ヌクレオチドが数個〜数十個または数百個結合したものを意味する。
本発明に用いられるオリゴヌクレオチドは、KK−LC−1遺伝子又はそのフラグメントを得るためのPCRプライマー又はハイブリダイゼーションプローブとして使用され得る。
本発明に用いられるオリゴヌクレオチドの長さとしては、7塩基以上が好ましく、15塩基以上がより好ましく、20塩基以上が更に好ましい。これらのオリゴヌクレオチドは、例えば、Applied Biosystems Incorporated(ABI,850 Lincoln Center Dr.,Foster City,CA 94404)392型シンセサイザー等によって合成される。
In the present invention, an oligonucleotide means an oligonucleotide in which several to several tens or several hundred nucleotides are bound.
The oligonucleotide used in the present invention can be used as a PCR primer or a hybridization probe for obtaining the KK-LC-1 gene or a fragment thereof.
The length of the oligonucleotide used in the present invention is preferably 7 bases or more, more preferably 15 bases or more, still more preferably 20 bases or more. These oligonucleotides are synthesized by, for example, Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) type 392 synthesizer.
KK−LC−1の発現を検出する工程としては、RT−PCRを行う工程を有することが好ましく、配列番号1(ATGAACTTCTATTTACTCCTAGCGAGC)で表されるプライマー及び配列番号2(TTAGGTGGATTTCCGGTGAGG)で表されるプライマーを用いてRT−PCR法を行う工程を有することがより好ましい。
実施例において後述するように、本発明者は、KK−LC−1の発現解析に用いられていた従来のリアルタイムPCR法では、ゲノムDNAを検出してしまう問題点を見出し、上記配列番号1及び2で表されるプライマーを用いたRT−PCR法により、KK−LC−1をより高精度に検出できることを見出した。
The step of detecting the expression of KK-LC-1 preferably has a step of performing RT-PCR, and comprises the primer represented by SEQ ID NO: 1 (ATGAACTTCTTATTACTCCTAGCGAGC) and the primer represented by SEQ ID NO: 2 (TTAGGTGGATTTTCCGGGTGAGG). It is more preferable to have a step of performing the RT-PCR method using the same.
As will be described later in Examples, the present inventor has found a problem that genomic DNA is detected in the conventional real-time PCR method used for the expression analysis of KK-LC-1, and the above-mentioned SEQ ID NO: 1 and It was found that KK-LC-1 can be detected with higher accuracy by the RT-PCR method using the primer represented by 2.
配列番号1で表されるプライマー及び配列番号2で表されるプライマーを用いたKK−LC−1の検出方法としては、一例として、(a)生体試料からmRNAを抽出する工程と、(b)逆転写反応により、前記mRNAから該mRNAに相補するcDNAを合成する工程と、(c)配列番号1で表されるプライマー及び配列番号2で表されるプライマーを用いて、前記cDNAから、KK−LC−1のcDNAの配列を有する標的塩基配列を増幅する工程と、(d)前記標的塩基配列の増幅産物を検出する工程と、を有するものが挙げられる。
以下、各工程について説明する。
As a method for detecting KK-LC-1 using the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2, for example, (a) a step of extracting mRNA from a biological sample; and (b) Using the step of synthesizing a cDNA complementary to the mRNA from the mRNA by a reverse transcription reaction and (c) the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2, KK- One having a step of amplifying a target base sequence having the sequence of the LC-1 cDNA and (d) detecting an amplification product of the target base sequence is included.
Hereinafter, each step will be described.
まず、工程(a)において、生体試料からmRNAを抽出する。生体試料からのmRNA抽出は、トリゾルを用いる等、定法により行うことができる。 First, in step (a), mRNA is extracted from a biological sample. Extraction of mRNA from a biological sample can be performed by a standard method such as using Trisol.
次いで、工程(b)において、逆転写反応により、前記mRNAから該mRNAに相補するcDNAを合成する。
逆転写に用いられる逆転写酵素としては、従来公知のものが用いられ、例えば、Moloney Murine Leukemia Virus由来の逆転写酵素等が挙げられる。
逆転写に用いられるプライマーの長さは、通常用いられるプライマーの長さと同様、10〜40塩基が好ましく、20〜30塩基がより好ましい。
また、逆転写反応により合成されたcDNAは、鋳型となったmRNAとのハイブリッドを形成しているため、RNaseH等のRNaseを用いて、工程(c)の前に、予めmRNAを分解しておくことが好ましい。
Then, in step (b), a cDNA complementary to the mRNA is synthesized from the mRNA by a reverse transcription reaction.
As the reverse transcriptase used for reverse transcription, conventionally known ones can be used, and examples thereof include a reverse transcriptase derived from Moloney Murine Leukemia Virus.
The length of the primer used for reverse transcription is preferably 10 to 40 bases, and more preferably 20 to 30 bases, like the length of a primer usually used.
Further, since the cDNA synthesized by the reverse transcription reaction forms a hybrid with the mRNA serving as the template, RNase such as RNaseH is used to decompose the mRNA in advance before the step (c). Preferably.
次いで、工程(c)において、配列番号1で表されるプライマー及び配列番号2で表されるプライマーを用いて、前記cDNAから、KK−LC−1のcDNAの配列を有する標的塩基配列を増幅する。 Then, in step (c), a target base sequence having the sequence of KK-LC-1 cDNA is amplified from the cDNA using the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2. ..
DNAポリメラーゼとはプライマーがアニールした鋳型DNAと相補的な塩基配列を持つDNA鎖を合成する酵素の総称である。
本発明に用いられるDNAポリメラーゼとしては、特に限定されないが、Taq DNAポリメラーゼ、Tth DNAポリメラーゼ、Vent DNAポリメラーゼ等の熱安定性DNAポリメラーゼを用いることが好ましく、試験開始前の伸長を防ぐためにホットスタート機能を持つDNAポリメラーゼを使用することがより好ましい。
DNA polymerase is a general term for an enzyme that synthesizes a DNA chain having a base sequence complementary to a template DNA to which a primer is annealed.
The DNA polymerase used in the present invention is not particularly limited, but it is preferable to use a thermostable DNA polymerase such as Taq DNA polymerase, Tth DNA polymerase, Vent DNA polymerase and the like, which has a hot start function to prevent extension before the start of the test. It is more preferable to use a DNA polymerase having
配列番号1で表されるプライマー及び配列番号2で表されるプライマーは、KK−LC−1のmRNA及びゲノムDNAのいずれも認識するが、PCRによる増幅産物の大きさからその違いを見分けることができる。従って、上記配列番号1及び2で表されるプライマーを用いたRT−PCR法により、KK−LC−1の発現をより高精度に検出できる。 The primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2 recognize both KK-LC-1 mRNA and genomic DNA, but the difference can be distinguished from the size of the amplification product by PCR. it can. Therefore, the expression of KK-LC-1 can be detected with higher accuracy by the RT-PCR method using the primers represented by SEQ ID NOS: 1 and 2.
次いで、工程(d)において、前記標的塩基配列の増幅産物を検出する。
工程(d)における代表的な検出方法としては、標的塩基配列の増幅を電気泳動により評価する方法が挙げられる。この方法は、標的塩基配列の増幅産物及び核酸分子量マーカーに対して、電気泳動を行い、両者の泳動度を対比することにより、所定の分子量を有する標的塩基配列が増幅された否かを評価する方法である。
電気泳動による検出に用いる試薬としては、二本鎖DNAに結合して蛍光を発する臭化エチジウムやサイバーグリーンが好ましい。
Then, in step (d), an amplification product of the target base sequence is detected.
As a typical detection method in step (d), a method of evaluating amplification of the target base sequence by electrophoresis can be mentioned. In this method, an amplification product of a target base sequence and a nucleic acid molecular weight marker are electrophoresed, and their mobilities are compared to evaluate whether or not a target base sequence having a predetermined molecular weight has been amplified. Is the way.
As a reagent used for detection by electrophoresis, ethidium bromide or cyber green, which binds to double-stranded DNA and emits fluorescence, is preferable.
工程(d)におけるその他の検出方法としては、特に限定されるものではなく、蛍光色素等によるオリゴヌクレオチドの標識、高速液体クロマトグラフィー、マススペクトル、融解曲線分析、増殖曲線分析等が挙げられる。 Other detection methods in step (d) are not particularly limited, and include labeling of oligonucleotide with a fluorescent dye or the like, high performance liquid chromatography, mass spectrum, melting curve analysis, growth curve analysis and the like.
蛍光色素等によるオリゴヌクレオチドの標識としては、例えば、配列番号1で表されるプライマー及び配列番号2で表されるプライマーの少なくともいずれか一方を、標識物質により標識しておく方法が挙げられる。かかる方法により、標識物質を指標として標的塩基配列の増幅を検出することができる。このような標識物質としては、例えば、蛍光色素、エネルギー吸収性物質、ラジオアイソトープ、化学発光体、酵素、抗体等が挙げられる。かかる標識物質を用いて、オリゴヌクレオチドを標識する位置については、特に限定するものではないが、伸長反応を阻害しないような位置が好ましい。 Examples of labeling the oligonucleotide with a fluorescent dye include a method of labeling at least one of the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2 with a labeling substance. With such a method, amplification of the target base sequence can be detected using the labeling substance as an index. Examples of such labeling substances include fluorescent dyes, energy absorbing substances, radioisotopes, chemiluminescers, enzymes, antibodies and the like. The position of labeling the oligonucleotide with such a labeling substance is not particularly limited, but a position that does not inhibit the extension reaction is preferable.
また、KK−LC−1の発現を検出する工程としては、例えば、KK−LC−1タンパク質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(ELISA) 、免疫組織学的染色法等の免疫学的方法によりKK−LC−1タンパク質の発現を検出する工程も挙げられる。 In addition, as the step of detecting the expression of KK-LC-1, for example, a specific antibody against the KK-LC-1 protein is prepared, and a Western blotting method using the specific antibody, a solid phase enzyme immunoassay method ( And a step of detecting the expression of KK-LC-1 protein by an immunological method such as ELISA) or an immunohistological staining method.
ウエスタンブロット法は、例えば、KK−LC−1タンパク質を含有する生体試料をSDS−PAGEにより分離し、ゲル中の分離されたタンパク質をPVDF膜等のメンブレンに転写し、KK−LC−1タンパク質を認識しうる抗体 (一次抗体) と反応させて免疫複合体を形成させ、該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。形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、生体試料中に存在するKK−LC−1タンパク質の量を測定できる。 The Western blotting method, for example, separates a biological sample containing the KK-LC-1 protein by SDS-PAGE, transfers the separated protein in the gel to a membrane such as a PVDF membrane, and detects the KK-LC-1 protein. In this method, an immune complex is formed by reacting with a recognizable antibody (primary antibody), and the immune complex is detected using a labeled secondary antibody. The amount of KK-LC-1 protein present in the biological sample can be measured by measuring the amount of labeled secondary antibody bound to the formed immune complex.
免疫組織学的染色法は、例えば、スライドガラス上に固定された切除組織切片や細胞などを、抗KK−LC−1抗体と反応させて免疫複合体を形成させ、これと結合した標識二次抗体により検出するものである。 The immunohistological staining method is, for example, a method in which a resected tissue section or cell fixed on a slide glass is reacted with an anti-KK-LC-1 antibody to form an immune complex, and a labeled secondary antibody bound to this is formed. It is detected by an antibody.
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、KK−LC−1タンパク質を構成するアミノ酸配列の一部に基づいて作製した合成ペプチドをウサギ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ことができる。
モノクローナル抗体の場合、KK−LC−1タンパク質を構成するアミノ酸配列の一部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得ることができる。
また、形成した免疫複合体の検出は、二次抗体を用いる方法に代えて、一次抗体を予め同位体などで標識する方法を用いたものであってもよい。
The specific antibody 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 rabbit is immunized with a synthetic peptide prepared based on a part of the amino acid sequence constituting the KK-LC-1 protein, and an antibody produced from the serum of this animal is used in a conventional method. Can be obtained by
In the case of a monoclonal antibody, a non-human animal such as a mouse is immunized with a synthetic peptide prepared based on a part of the amino acid sequence constituting the KK-LC-1 protein, and antibody-producing cells contained in the spleen are collected and It can be obtained from hybridoma cells obtained by fusing with tumor cells.
Further, the detection of the formed immune complex may be performed by using a method of labeling the primary antibody with an isotope or the like in advance, instead of the method using the secondary antibody.
[ヘリコバクター・ピロリ感染を検出する工程]
ヘリコバクター・ピロリは、胃癌発生のclass1(確実に発癌誘導する)発癌物質としてWHO/IARCにより認定されている。また、本邦における胃癌患者の80%以上は、ヘリコバクター・ピロリに感染していることが明らかとなっている(Helicobacter pylori infection and the development of gastric cancer. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M, Taniyama K, Sasaki N, SchlemperRJ. N Engl J Med. 2001 Sep 13;345(11):784-9.参照)。
実施例において後述するように、本発明者は、KK−LC−1を発現している胃癌症例で抗ヘリコバクター・ピロリIgGが高値であることを見出した。従って、KK−LC−1の発現を検出する工程と、ヘリコバクター・ピロリ感染を検出する工程と、を兼ね備えることで、生体試料中の胃癌細胞を高精度に行うことができる。
[Process to detect Helicobacter pylori infection]
Helicobacter pylori is certified by WHO/IARC as a class 1 (certainly inducing carcinogenic) carcinogen of gastric carcinogenesis. Further, it has been revealed that 80% or more of gastric cancer patients in Japan are infected with Helicobacter pylori (Helicobacter pylori infection and the development of gastric cancer. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M, Taniyama K, Sasaki N, Schlemper RJ. N Engl J Med. 2001
As described later in Examples, the present inventor has found that anti-Helicobacter pylori IgG has high levels in gastric cancer cases expressing KK-LC-1. Therefore, by combining the step of detecting the expression of KK-LC-1 and the step of detecting the Helicobacter pylori infection, the gastric cancer cells in the biological sample can be performed with high accuracy.
検出に用いる生体試料としては、[KK−LC−1の発現を検出する工程]で挙げたものに加えて、唾液、糞便等が挙げられる。 Examples of the biological sample used for detection include saliva, feces, and the like in addition to those described in [Step of detecting expression of KK-LC-1].
ヘリコバクター・ピロリ感染を検出する工程としては、ヘリコバクター・ピロリの発現を検出する工程、又はその感染によって起こりうる炎症反応もしくは免疫応答を検出する工程が挙げられる。 The step of detecting Helicobacter pylori infection includes a step of detecting the expression of Helicobacter pylori, or a step of detecting an inflammatory reaction or an immune response that may be caused by the infection.
ヘリコバクター・ピロリの発現を検出する工程としては、KK−LC−1の発現を検出する工程と同様に、ヘリコバクター・ピロリ由来の遺伝子を検出する工程、又は、ヘリコバクター・ピロリ由来のタンパク質を検出する工程が挙げられる。 As a step of detecting the expression of Helicobacter pylori, a step of detecting a gene derived from Helicobacter pylori or a step of detecting a protein derived from Helicobacter pylori, as in the step of detecting the expression of KK-LC-1. Is mentioned.
ヘリコバクター・ピロリ由来のタンパク質としては、ウレアーゼA、ウレアーゼB等のウレアーゼ;FlaA、FlaB、FliD、FliK、FlgE、FlgM等の鞭毛タンパク質;空胞化毒素(VacA);毒素随伴タンパク質(CagA);好中球活性化タンパク質(NapA)等が挙げられる。 Examples of Helicobacter pylori-derived proteins include urease such as urease A and urease B; flagellar proteins such as FlaA, FlaB, FliD, FliK, FlgE, and FlgM; vacuolating toxin (VacA); toxin-associated protein (CagA); Examples include sphere activating protein (NapA).
ヘリコバクター・ピロリ由来の遺伝子としては、上述したヘリコバクター・ピロリ由来のタンパク質をコードする遺伝子が挙げられる。
これらのヘリコバクター・ピロリ由来のタンパク質、遺伝子の検出方法としては、KK−LC−1を検出する工程で述べた方法と同様のものが挙げれらる。
Examples of Helicobacter pylori-derived genes include the genes encoding the above-mentioned Helicobacter pylori-derived proteins.
Examples of the method for detecting these Helicobacter pylori-derived proteins and genes include the same methods as those described in the step of detecting KK-LC-1.
ヘリコバクター・ピロリ感染によって起こりうる炎症反応もしくは免疫応答を検出する工程としては、抗ヘリコバクター・ピロリIgG抗体を検出する工程、ヘリコバクター・ピロリ感染により、胃粘膜上皮細胞から放出されるIL−8を検出する工程、IL−8が産生誘導するTNF−α、IL−1、IL−6等を検出する工程等が挙げられる。 As a step of detecting an inflammatory reaction or an immune response which may be caused by Helicobacter pylori infection, a step of detecting an anti-Helicobacter pylori IgG antibody and a detection of IL-8 released from gastric mucosal epithelial cells by the infection with Helicobacter pylori And the step of detecting IL-8 production-induced TNF-α, IL-1, IL-6 and the like.
また、ヘリコバクター・ピロリ感染を検出する工程としては、尿素試験法を用いて生ずる同位元素で標識されたCO2を測定する工程も挙げられる。
具体的には、安定性同位元素又は放射性同位元素で標識した尿素を経口投与する。ヘリコバクター・ピロリのもつウレアーゼにより同位元素で標識した尿素が加水分解され、アンモニアと同位元素で標識されたCO2が発生し、呼気から放出される。この呼気を採取し、同位元素で標識されたCO2と自然界に存在するCO2との比率により、ヘリコバクター・ピロリの存在を測定することができる。
Further, the step of detecting Helicobacter pylori infection also includes the step of measuring CO 2 labeled with an isotope produced using a urea test method.
Specifically, urea labeled with a stable isotope or a radioactive isotope is orally administered. The urease possessed by Helicobacter pylori hydrolyzes the isotope-labeled urea to generate ammonia and isotope-labeled CO 2, which are released from the breath. The breath was collected, by the ratio of the CO 2 present in the labeled CO 2 and nature isotopically can measure the presence of Helicobacter pylori.
<胃癌細胞検出キット>
本発明の胃癌細胞検出キットは、KK−LC−1検出キットとヘリコバクター・ピロリ感染検出キットを備えたものであり、具体的には、上述した<胃癌細胞検出方法>で、KK−LC−1及びヘリコバクター・ピロリの検出に用いられる試薬を含むものが挙げられる。
一例として、抗KK−LC−1抗体と抗ヘリコバクター・ピロリIgGに対する抗体とを備えたELISAキット;抗KK−LC−1抗体とヘリコバクター・ピロリ由来のタンパク質に対する抗体(例えば、抗CagA抗体)とを備えたELISAキット等が挙げられる。
<Gastric cancer cell detection kit>
The gastric cancer cell detection kit of the present invention comprises a KK-LC-1 detection kit and a Helicobacter pylori infection detection kit. Specifically, in the above-mentioned <gastric cancer cell detection method>, KK-LC-1 And those containing reagents used for the detection of Helicobacter pylori.
As an example, an ELISA kit comprising an anti-KK-LC-1 antibody and an antibody against anti-Helicobacter pylori IgG; an anti-KK-LC-1 antibody and an antibody against a protein derived from Helicobacter pylori (for example, an anti-CagA antibody). An equipped ELISA kit and the like can be mentioned.
また、KK−LC−1遺伝子とヘリコバクター・ピロリ由来の遺伝子を検出するためのプライマーセットを含むものも挙げられる。一例として、本発明の胃癌細胞検出キットは、逆転写酵素と、逆転写用プライマーと、RNaseと、KK−LC−1遺伝子用プライマーと、ヘリコバクター・ピロリ由来の遺伝子用プライマーと、を有する。
増幅産物の大きさから、ゲノムDNAの検出を排除できる観点から、胃癌細胞検出キット中のKK−LC−1検出キットは、配列番号1で表されるプライマー及び配列番号2で表されるプライマーを含むことが好ましい。
このように、本発明の胃癌細胞検出方法に必要な試薬等をキット化することにより、生体試料中の胃癌細胞をより簡便にかつ高精度に検出をすることができる。
Moreover, the thing containing the primer set for detecting the KK-LC-1 gene and the gene derived from Helicobacter pylori is also mentioned. As an example, the gastric cancer cell detection kit of the present invention has a reverse transcriptase, a reverse transcription primer, RNase, a KK-LC-1 gene primer, and a Helicobacter pylori-derived gene primer.
From the viewpoint of eliminating the detection of genomic DNA from the size of the amplification product, the KK-LC-1 detection kit in the gastric cancer cell detection kit comprises the primer represented by SEQ ID NO: 1 and the primer represented by SEQ ID NO: 2. It is preferable to include.
In this way, by preparing the reagents and the like required for the method for detecting gastric cancer cells of the present invention as a kit, gastric cancer cells in a biological sample can be detected more easily and highly accurately.
次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
[胃癌における癌/精巣抗原の発現についての検討]
胃癌と診断され、胃の切除手術を受けた患者のうち、切除組織から癌部位の採取可能であった55例について、癌/精巣抗原の発現を検討した。
胃癌切除手術直後、新鮮胃癌組織より腫瘍部位及び正常部位を採取し、RNAlater(Ambion社)に一晩浸漬した。浸漬した各部位をRNAlaterより取り出し、5mm角の大きさに細切りし、−80℃に保存した。
保存した部位をアルミホイルに包み、液体窒素に浸漬し完全に凍結させた後にハンマーで破砕した。破砕した組織からEZ1 RNA Tissue Mini Kit(Qiagen社)を用いてRNAを抽出した。詳細には、破砕した組織をRLT液に添加し、23Gニードル付1mLシリンジで完全に溶解させ、組織を溶解したRLT液よりRNAを抽出した。オリゴp(dN)6ランダムプライマー及びSuperscriptII
reverse(Invitrogen社)を用いて、抽出したRNAからcDNAを合成した。
[Study on expression of cancer/testis antigen in gastric cancer]
Of the patients who were diagnosed with gastric cancer and underwent gastrectomy, the expression of cancer/testis antigen was examined in 55 cases in which the cancerous site could be collected from the resected tissue.
Immediately after gastric cancer resection surgery, a tumor site and a normal site were collected from fresh gastric cancer tissue and immersed in RNAlater (Ambion) overnight. Each dipped site was taken out from RNAlater, cut into 5 mm square pieces, and stored at -80°C.
The preserved part was wrapped in aluminum foil, immersed in liquid nitrogen to be completely frozen, and then crushed with a hammer. RNA was extracted from the crushed tissue using EZ1 RNA Tissue Mini Kit (Qiagen). Specifically, the crushed tissue was added to the RLT solution and completely dissolved by a 1 mL syringe with a 23G needle, and RNA was extracted from the RLT solution in which the tissue was dissolved. Oligo p(dN)6 random primer and Superscript II
cDNA was synthesized from the extracted RNA using reverse (Invitrogen).
β−actinの発現量、並びに、他の癌/精巣抗原として、Melanoma antigen−encoding gene(MAGE)A1、Melanoma antigen−encoding gene(MAGE)A3、及びNew York Esophageal squamous cell carcinoma−1(NY−ESO−1)の発現量をTaqman Gene Expression Assays(Hs99999903_m1、Hs00607097_m1、Hs200366532_m1、Hs00265824_m1)を用いて測定した。
5μLのcDNA、10μLのFastStart Universal Probe
Master(Roche社)、1μLのTaqman Gene Expression Assaysを含む20μLの反応液を作製し、HT−7900(Life Technologies)にて反応回数45サイクルのリアルタイムPCRを実施した。
MAGE−A1、MAGE−A3、及びNY−ESO−1に関しては、Ct値が45以下を結果として採用した。β―actinに関しては、Ct値が28以下を結果として採用した。
β―actinのCt値と各癌/精巣抗原のCt値の差をΔCtとし、ΔCtの有無を各癌/精巣抗原の発現の有無とした。結果を図1に示す。
The expression level of β-actin and other cancer/testis antigens include Melanoma antigen-encoding gene (MAGE) A1, Melanoma antigen-encoding gene (MAGE) A3, and New York Esophagecomacq. The expression level of -1) was measured using Taqman Gene Expression Assays (Hs999999993_m1, Hs00607097_m1, Hs200363632_m1, Hs00265824_m1).
5 μL of cDNA, 10 μL of FastStart Universal Probe
Master (Roche), 20 μL of a reaction solution containing 1 μL of Taqman Gene Expression Assays was prepared, and real-time PCR was performed with HT-7900 (Life Technologies) at a reaction frequency of 45 cycles.
For MAGE-A1, MAGE-A3, and NY-ESO-1, a Ct value of 45 or less was adopted as a result. Regarding β-actin, a Ct value of 28 or less was adopted as a result.
The difference between the Ct value of β-actin and the Ct value of each cancer/testis antigen was defined as ΔCt, and the presence or absence of ΔCt was defined as the presence or absence of expression of each cancer/testis antigen. The results are shown in Figure 1.
KK−LC−1に関しては、RT−PCRで発現を確認した。2μLのcDNA、0.2μLのrTaq(TAKARA社)、125μMdNTP、及び500nMのKK−LC−1特異的プライマー(配列番号1(ATGAACTTCTATTTACTCCTAGCGAGC)及び配列番号2(TTAGGTGGATTTCCGGTGAGG))を含む反応液を調製した。40サイクルでの増幅の有無を確認した。 Regarding KK-LC-1, the expression was confirmed by RT-PCR. A reaction solution containing 2 μL of cDNA, 0.2 μL of rTaq (TAKARA), 125 μM dNTP, and 500 nM of a KK-LC-1 specific primer (SEQ ID NO: 1 (ATGAACTTCATTTTACTCCTAGCGAGC) and SEQ ID NO: 2 (TTAGGTGGATTTCCGGGTGAGG)) was prepared. The presence or absence of amplification in 40 cycles was confirmed.
図1に示すように、胃癌症例の胃癌部位において、55例中41例(75%)でKK−LC−1の発現が認められた。それに対して、MAGE−A1、MAGE−A3、及びNY−ESO−1の発現は、それぞれ20例(36%)、27例(49%)、及び11例(20%)であった。
この結果から、KK−LC−1は、他の癌/精巣抗原よりも胃癌において高頻度に発現していることが明らかとなった。
As shown in FIG. 1, in the gastric cancer site of gastric cancer cases, KK-LC-1 expression was observed in 41 out of 55 cases (75%). In contrast, the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 was 20 cases (36%), 27 cases (49%), and 11 cases (20%), respectively.
From this result, it was revealed that KK-LC-1 was more frequently expressed in gastric cancer than other cancer/testis antigens.
血清採取可能であった25症例について、Anti−IgG ヘリコバクター・ピロリ
ELISA Kit Quantiative(Phoenix pharmaceutics)を用いて血清中のヘリコバクター・ピロリに対するIgGを測定した。各癌/精巣抗原について、発現の有無で群分けし、群間における抗体価について比較した。結果を図2〜3に示す。
With respect to 25 cases in which serum could be collected, IgG for Helicobacter pylori in serum was measured using Anti-IgG Helicobacter pylori ELISA Kit Quantative (Phoenix pharmaceuticals). Each cancer/testis antigen was divided into groups according to the presence or absence of expression, and antibody titers were compared between the groups. The results are shown in FIGS.
図2に示すように、KK−LC−1陽性患者及び陰性患者の血清中の抗ヘリコバクター・ピロリ IgG価は、それぞれ67.5±7.6U及び15.8±7.5Uであり、KK−LC−1陽性の胃癌患者血清で高い値を示した(p=0.0046)。
以上の結果から、KK−LC−1は胃癌において高頻度に発現しており、その発現はヘリコバクター・ピロリ感染に対するIgG産生と相関することが示された。
一方、図2に示すように他の癌/精巣抗原の発現と、ヘリコバクター・ピロリ感染に対するIgG産生との相関は見られなかった。
As shown in FIG. 2, the anti-Helicobacter pylori IgG titers in sera of KK-LC-1 positive patients and negative patients were 67.5±7.6 U and 15.8±7.5 U, respectively, and KK- The serum level of LC-1 positive gastric cancer patients showed a high value (p=0.0046).
From the above results, it was shown that KK-LC-1 was frequently expressed in gastric cancer, and its expression was correlated with IgG production against Helicobacter pylori infection.
On the other hand, as shown in FIG. 2, no correlation was observed between the expression of other cancer/testis antigens and IgG production against Helicobacter pylori infection.
[RT−PCRとリアルタイムPCRの比較]
RT−PCR及びリアルタイムPCRをそれぞれ用いて、胃癌29症例の腫瘍部におけるKK−LC−1の発現の検出精度を比較した。
〔リアルタイムPCR〕
β―actinの発現量、並びに、KK−LC−1の発現量をTaqman Gene
Expression Assays(Hs02386421_g1)を用いて測定した。
5μLのcDNA、10μLのFastStart Universal Probe
Master(Roche社)、1μLのTaqman Gene Expression Assaysを含む20μLの反応液を作製し、HT−7900(Life Technologies)にて反応回数45サイクルのリアルタイムPCRを実施した。
KK−LC−1に関しては、Ct値が45以下を結果として採用した。β―actinに関しては、Ct値が28以下を結果として採用した。
β―actinのCt値と各癌/精巣抗原のCt値の差をΔCtとし、ΔCtの有無を各癌/精巣抗原の発現の有無とした。結果を図4に示す。図4中、ΔCtが−25以上をKK−LC−1の発現ありと判断した。
[Comparison of RT-PCR and real-time PCR]
RT-PCR and real-time PCR were used to compare the detection accuracy of KK-LC-1 expression in the tumor part of 29 cases of gastric cancer.
[Real-time PCR]
The expression level of β-actin and the expression level of KK-LC-1 were measured using Taqman Gene.
It was measured using Expression Assays (Hs02386421_g1).
5 μL of cDNA, 10 μL of FastStart Universal Probe
Master (Roche), 20 μL of a reaction solution containing 1 μL of Taqman Gene Expression Assays was prepared, and real-time PCR was performed with HT-7900 (Life Technologies) at a reaction frequency of 45 cycles.
Regarding KK-LC-1, a Ct value of 45 or less was adopted as a result. Regarding β-actin, a Ct value of 28 or less was adopted as a result.
The difference between the Ct value of β-actin and the Ct value of each cancer/testis antigen was defined as ΔCt, and the presence or absence of ΔCt was defined as the presence or absence of expression of each cancer/testis antigen. The results are shown in Fig. 4. In FIG. 4, it was determined that KK-LC-1 was expressed when ΔCt was −25 or more.
〔RT−PCR〕
[胃癌における癌/精巣抗原の発現についての検討]で述べた実験条件に従って342bpの増幅産物の有無でKK−LC−1の発現の有無を判断した。結果を図4に示すように、リアルタイムPCRによる KK−LC−1の発現頻度は、RT−PCRにおける結果と比較すると高値であった。このプローブを用いたリアルタイムPCRでは、ゲノムDNAのコンタミネーションを検出してしまうおそれがあり、上記配列番号1及び配列番号2のプライマーを用いたRT−PCR法による方が正確にKK−LC−1の発現頻度を測定できることが示唆された。
[RT-PCR]
The presence or absence of KK-LC-1 expression was determined by the presence or absence of the 342 bp amplification product according to the experimental conditions described in [Study on expression of cancer/testis antigen in gastric cancer]. As shown in the results in FIG. 4, the expression frequency of KK-LC-1 by real-time PCR was higher than that in RT-PCR. Real-time PCR using this probe may detect contamination of genomic DNA, and thus RT-PCR using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 described above is more accurate than KK-LC-1. It was suggested that the frequency of expression of the can be measured.
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