JPWO2006022114A1 - Brain tumor detection method, brain tumor detection substance used therefor, and pharmaceutical composition - Google Patents
Brain tumor detection method, brain tumor detection substance used therefor, and pharmaceutical composition Download PDFInfo
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- JPWO2006022114A1 JPWO2006022114A1 JP2006531442A JP2006531442A JPWO2006022114A1 JP WO2006022114 A1 JPWO2006022114 A1 JP WO2006022114A1 JP 2006531442 A JP2006531442 A JP 2006531442A JP 2006531442 A JP2006531442 A JP 2006531442A JP WO2006022114 A1 JPWO2006022114 A1 JP WO2006022114A1
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Abstract
確実に、且つ早期に脳腫瘍を検出可能な脳腫瘍の検出方法及びそれに用いる脳腫瘍の検出物質を提供する。さらに、脳腫瘍を治療するための医薬組成物を提供する。脳腫瘍に特異的に発現する糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆの検出に基づいて、脳腫瘍を検出することにより、脳腫瘍の早期発見及び治療に有益である。さらに、Ｎ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを使用して、確実に、且つ早期に脳腫瘍を検出することができる。また、脳腫瘍の検出物質は、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに特異的に結合するレクチンを有するため、糖鎖Ａ２Ｇ２Ｆに基づいて脳腫瘍を早期且つ簡易に検出することができる。さらに、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを有効成分として含む医薬組成物は、脳腫瘍細胞に対してアポトーシスを誘発するので、脳腫瘍細胞により引き起こされる疾患の予防および治療に有効である。A method for detecting a brain tumor capable of reliably and early detecting a brain tumor and a brain tumor detection material used therefor are provided. Further provided are pharmaceutical compositions for treating brain tumors. Detection of a brain tumor based on detection of an N-linked sugar chain A2G2F of a glycoprotein specifically expressed in the brain tumor is beneficial for early detection and treatment of the brain tumor. Furthermore, a brain tumor can be reliably and early detected using a lectin having binding specificity for the N-linked sugar chain A2G2F. Moreover, since the brain tumor detection substance has a lectin that specifically binds to the N-linked sugar chain A2G2F of the glycoprotein, the brain tumor can be detected early and easily based on the sugar chain A2G2F. Furthermore, since the pharmaceutical composition containing a lectin having binding specificity for the N-linked sugar chain A2G2F of the glycoprotein as an active ingredient induces apoptosis of brain tumor cells, the prevention of diseases caused by brain tumor cells and It is effective for treatment.
Description
本発明は、脳腫瘍の検出方法及びそれに用いる脳腫瘍の検出物質、並びに医薬組成物に関する。 The present invention relates to a brain tumor detection method, a brain tumor detection substance used therefor, and a pharmaceutical composition.
近年、多くの蛋白質に糖鎖が結合していることが知られるようになり、蛋白質に結合した糖鎖が細胞同士の認識や細胞接着，又はウイルスや微生物の宿主への感染等に重要な役割を果たしている他、蛋白質輸送のためのシグナルや蛋白質立体構造形成の際の補助的因子としても作用していることなどが明らかになりつつある。 In recent years, it has become known that sugar chains are bound to many proteins, and sugar chains bound to proteins play an important role in cell-to-cell recognition, cell adhesion, and infection of viruses and microorganisms to hosts. In addition to the above, it is becoming clear that it is also acting as a signal for protein transport and an auxiliary factor in the formation of protein three-dimensional structure.
また、複合糖質（糖蛋白質、糖脂質、プロテオグリカン）の糖鎖は、蛋白質、核酸に次ぐ第三の生命鎖として、その役割が注目されている。特に、糖蛋白質糖鎖がその糖蛋白質の機能発現に不可欠であることが報告されている。例えば、末梢神経系ミエリンにあるＰ０という糖蛋白質は細胞間接着因子として機能しており、このＰ０の糖鎖結合部位に改変を加えるとその細胞間接着活性が失われることが知られている。このことは糖鎖に接着活性があったためではなく、蛋白質の構造を細胞膜上に支えるようにして存在していた糖鎖がなくなったことにより、Ｐ０が接着に必要なコンフォメーションを維持できなくなったと考えられている。このように糖蛋白質糖鎖を有する分子は、神経系の発生においても、また他の様々な領域においても細胞と細胞間の認識は極めて重要な役割を果たしていることから、糖蛋白質糖鎖の重要性が認識されている。しかしながら、糖蛋白質糖鎖の精製には膨大な時間を要し、また細胞表面や組織中で発現している糖蛋白質糖鎖の系統的解析方法がないなどの理由から、糖蛋白質糖鎖の機能解明はあまり進んでいなかった。 In addition, the sugar chain of glycoconjugates (glycoproteins, glycolipids, proteoglycans) is attracting attention as a third life chain after proteins and nucleic acids. In particular, it has been reported that glycoprotein sugar chains are essential for the functional expression of the glycoprotein. For example, a glycoprotein called P0 in the peripheral nervous system myelin functions as an intercellular adhesion factor, and it is known that the intercellular adhesion activity is lost when a modification is made to the sugar chain binding site of P0. This is not because sugar chains had adhesive activity, but because the sugar chains that existed to support the protein structure on the cell membrane disappeared, P0 could not maintain the conformation necessary for adhesion. It is considered. In this way, molecules with glycoprotein sugar chains play an extremely important role in the development of the nervous system and the recognition between cells in various other regions. Sex is recognized. However, purification of glycoprotein sugar chains requires a huge amount of time, and there is no systematic analysis method for glycoprotein sugar chains expressed on cell surfaces and tissues. The elucidation has not progressed much.
そこで、本発明者らの１人である池中らはＨＰＬＣシステムを用いた自動化、簡易化による糖蛋白質糖鎖の系統的解析方法を開発した。そして、この方法により糖鎖の一種であるTriantennary trigalactosylated structure with one outer arm fucosylation （Ａ３Ｇ３Ｆ０）が肺癌患者血清中で増加していることを見出した（非特許文献１および２）。また、特許文献１には、糖蛋白質のＮ結合型糖鎖Ａ３Ｇ３Ｆ０に基づいて癌、特に肺癌を検出する癌検出方法及びそれに用いる癌検出物質が開示されている。さらに、非特許文献３には、神経細胞接着分子（Ｎ−ＣＡＭ）上に存在するポリシアル酸の発現が転移性の肺癌で高発現し、ポリシアル酸を制御するST8Sia II（ＳＴＸ）遺伝子の発現と腫瘍の悪性度との間に相関性があることが報告されている。
しかしながら、上記の特許文献および非特許文献には特に肺癌に特異的な糖蛋白質糖鎖しか開示されておらず、他臓器の癌における糖蛋白質糖鎖については一切開示されていなかった。特に脳における腫瘍は肺等の他臓器の癌と異なって、脳という特殊な臓器に存在するため、その発生・占拠部位によっては生きるのに重要な中枢が存在する脳の働きを障害し、様々な症状を引起こしていた。このような背景から、脳での病態による糖鎖の異常を解析し、特に脳腫瘍を確実に、且つ早期に検出する方法が望まれていた。 However, the above-mentioned patent documents and non-patent documents disclose only glycoprotein sugar chains specific to lung cancer, and none of the glycoprotein sugar chains in cancers of other organs. In particular, tumors in the brain, unlike cancers of other organs such as the lungs, exist in a special organ called the brain. Depending on the site of occurrence and occupation, the function of the brain, which has an important center for living, is impaired. Was causing serious symptoms. From such a background, there has been a demand for a method for analyzing a sugar chain abnormality due to a disease state in the brain and detecting a brain tumor reliably and at an early stage.
また、脳腫瘍の診断には、ＣＴスキャンや核磁気共鳴装置などの画像診断により行われているが、脳腫瘍に特異的な腫瘍マーカー等の役割を果たす検出物質の発見が期待されていた。 Diagnosis of a brain tumor is performed by image diagnosis such as a CT scan or a nuclear magnetic resonance apparatus, and the discovery of a detection substance that plays a role as a tumor marker specific to a brain tumor has been expected.
一方、脳腫瘍の治療は腫瘍全体を完全に摘除することが基本であるが、悪性の脳腫瘍においては境界が明確でないため、完全には摘出できないことが多く、放射線治療、化学療法や免疫療法を併用している。腫瘍細胞の病態細胞の増殖を阻害して、腫瘍細胞に起因する疾病を治療するものとして、例えば、マイトマイシンＣ、ブレオマイシンなどの抗生物質が知られているが、これらはいずれも所定の細胞に作用して、それを壊死すなわちネクローシスを起こさせて病態細胞を排除するものである。しかしながら、このネクローシスによる細胞死は、正常細胞または生体に対する副作用が強いために充分な効果が上げられておらず、正常細胞にも新たな疾患を惹起するという重大な欠陥がある。他方、生理的な条件下で細胞自らが積極的に惹起するようにプログラムされた細胞死すなわちアポトーシスは、周囲の細胞に影響を与えずに、細胞表面の湾曲、核クロマチンの凝縮、染色体ＤＮＡの断片化等の独特の形態異常を起して細胞が死ぬ現象であり、これを誘導しうることが可能であれば、悪性腫瘍等の疾患に対する治療に際して非常に有利である。 On the other hand, brain tumor treatment is based on the complete removal of the entire tumor, but in malignant brain tumors, the boundaries are not clear, so it is often impossible to remove the tumor completely, and combined with radiotherapy, chemotherapy, and immunotherapy. is doing. Antibiotics such as mitomycin C and bleomycin are known as agents that inhibit the growth of tumor cells and treat diseases caused by tumor cells. Thus, it causes necrosis, that is, necrosis, and eliminates pathological cells. However, this cell death due to necrosis is not sufficiently effective due to strong side effects on normal cells or living organisms, and there is a serious defect that normal cells also cause new diseases. On the other hand, cell death, or apoptosis, programmed to actively evoke cells under physiological conditions, without affecting the surrounding cells, cell surface curvature, nuclear chromatin condensation, chromosomal DNA It is a phenomenon in which cells die due to unique morphological abnormalities such as fragmentation, and if this can be induced, it is very advantageous in the treatment of diseases such as malignant tumors.
そこで、本発明は、確実に、且つ早期に脳腫瘍を検出可能な脳腫瘍の検出方法及びそれに用いる脳腫瘍の検出物質を提供することを目的とする。本発明は、また、脳腫瘍細胞に対してアポトーシスを誘導し、脳腫瘍の有効な予防および治療のための医薬組成物を提供することを目的とする。 Therefore, an object of the present invention is to provide a brain tumor detection method capable of reliably and early detecting a brain tumor and a brain tumor detection material used therefor. Another object of the present invention is to provide a pharmaceutical composition for inducing apoptosis of brain tumor cells and effectively preventing and treating brain tumors.
上記課題に鑑みて鋭意検討した結果、正常な脳組織のＮ結合型糖鎖の発現パターンは固体差なく保存されていることが報告されている（非特許文献１）ことから、正常組織と神経膠腫細胞とのＮ結合型糖鎖の発現パターンを比較したところ、糖蛋白質のＮ結合型糖鎖のうち特定なものが神経膠腫細胞で増加していることに着目し、その特定のＮ結合型糖鎖を同定した。そして、同定されたＮ結合型糖鎖Ａ２Ｇ２Ｆが正常組織には発現されず、神経膠腫細胞のみに発現されることを見出した。さらに、レンズマメ（ＬＣＡ）レクチンが、神経膠腫細胞に対してアポトーシスを誘導することを見出し、本発明に想到した。 As a result of intensive studies in view of the above problems, it has been reported that the expression pattern of N-linked sugar chains in normal brain tissue is preserved without any solid difference (Non-Patent Document 1). When the expression patterns of N-linked sugar chains with glioma cells were compared, it was noted that specific ones of N-linked sugar chains of glycoproteins increased in glioma cells. A conjugated sugar chain was identified. The inventors have found that the identified N-linked sugar chain A2G2F is not expressed in normal tissues but is expressed only in glioma cells. Furthermore, the inventors have found that lentil (LCA) lectin induces apoptosis of glioma cells and arrived at the present invention.
本発明の請求の範囲第１項記載の脳腫瘍の検出方法は、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆの検出に基づいて脳腫瘍を検出することを特徴とする。 The method for detecting a brain tumor according to claim 1 of the present invention is characterized in that a brain tumor is detected based on detection of an N-linked sugar chain A2G2F of a glycoprotein.
本発明の請求の範囲第２項記載の脳腫瘍の検出方法は、請求の範囲第１項において、前記Ｎ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを使用することを特徴とする。 The method for detecting a brain tumor according to claim 2 of the present invention is characterized in that, in claim 1, the lectin having binding specificity to the N-linked sugar chain A2G2F is used.
本発明の請求の範囲第３項記載の脳腫瘍の検出方法は、請求の範囲第２項において、前記レクチンが、レンズマメレクチンであることを特徴とする。 The brain tumor detection method according to claim 3 of the present invention is characterized in that, in claim 2, the lectin is a lentil lectin.
本発明の請求の範囲第４項記載の脳腫瘍の検出方法は、請求の範囲第１項において、前記脳腫瘍が、神経膠腫であることを特徴とする。 The method for detecting a brain tumor according to claim 4 of the present invention is characterized in that, in claim 1, the brain tumor is a glioma.
本発明の請求の範囲第５項記載の脳腫瘍の検出物質は、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを含有することを特徴とする。 The brain tumor detection substance according to claim 5 of the present invention is characterized by containing a lectin having binding specificity for the N-linked sugar chain A2G2F of glycoprotein.
本発明の請求の範囲第６項記載の脳腫瘍の検出物質は、請求の範囲第５項において、前記レクチンが、レンズマメレクチンであることを特徴とする。 The brain tumor detection substance according to claim 6 of the present invention is characterized in that, in claim 5, the lectin is a lentil lectin.
本発明の請求の範囲第７項記載の医薬組成物は、脳腫瘍細胞に対してアポトーシスを誘発する医薬組成物であって、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを有効成分として含むことを特徴とする。 The pharmaceutical composition according to claim 7 of the present invention is a pharmaceutical composition for inducing apoptosis of brain tumor cells, and has a binding specificity for N-linked sugar chain A2G2F of glycoprotein. As an active ingredient.
本発明の請求の範囲第８項記載の医薬組成物は、請求の範囲第７項において、前記レクチンが、レンズマメレクチンであることを特徴とする。 The pharmaceutical composition according to claim 8 of the present invention is characterized in that, in claim 7, the lectin is a lentil lectin.
本発明の請求の範囲第９項記載の医薬組成物は、請求の範囲第７項において、前記脳腫瘍細胞が、神経膠腫細胞であることを特徴とする。 The pharmaceutical composition according to claim 9 of the present invention is characterized in that, in claim 7, the brain tumor cell is a glioma cell.
本発明の請求の範囲第１項記載の脳腫瘍の検出方法によれば、脳腫瘍に特異的に発現する糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに基づいて、確実に、且つ早期に脳腫瘍を検出することができる。さらに、脳腫瘍の早期発見及び治療に有益である。 According to the method for detecting a brain tumor described in claim 1 of the present invention, the brain tumor can be reliably and early detected based on the N-linked sugar chain A2G2F of the glycoprotein specifically expressed in the brain tumor. Can do. Furthermore, it is useful for the early detection and treatment of brain tumors.
本発明の請求の範囲第２項記載の脳腫瘍の検出方法によれば、前記Ｎ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを使用して、確実に、且つ早期に脳腫瘍を検出することができる。さらに、脳腫瘍の早期発見及び治療に有益である。 According to the method for detecting a brain tumor according to claim 2 of the present invention, a brain tumor is reliably and early detected using the lectin having binding specificity to the N-linked sugar chain A2G2F. be able to. Furthermore, it is useful for the early detection and treatment of brain tumors.
本発明の請求の範囲第３項記載の脳腫瘍の検出方法によれば前記Ｎ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレンズマメレクチンを使用して神経膠腫を検出することができる。さらに、神経膠腫の早期発見及び治療に有益である。 According to the method for detecting a brain tumor according to claim 3 of the present invention, glioma can be detected using the lentil lectin having binding specificity to the N-linked sugar chain A2G2F. Furthermore, it is useful for the early detection and treatment of glioma.
本発明の請求の範囲第４項記載の脳腫瘍の検出方法によれば、前記Ｎ結合型糖鎖Ａ２Ｇ２Ｆに基づいて神経膠腫を、確実に、且つ早期に検出することができる。 According to the method for detecting a brain tumor according to claim 4 of the present invention, glioma can be reliably and early detected based on the N-linked sugar chain A2G2F.
本発明の請求の範囲第５項記載の脳腫瘍の検出物質によれば、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを含有するため、糖鎖Ａ２Ｇ２Ｆに基づいて脳腫瘍を検出できる。また、本発明の検出物質を用いることにより脳腫瘍をより早期且つ簡易に検出することができる。 According to the substance for detecting brain tumor according to claim 5 of the present invention, since the lectin having binding specificity to the N-linked sugar chain A2G2F of glycoprotein is contained, the brain tumor is detected based on the sugar chain A2G2F. It can be detected. Moreover, a brain tumor can be detected earlier and more easily by using the detection substance of the present invention.
本発明の請求の範囲第６項記載の脳腫瘍の検出物質によれば、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレンズマメレクチンを含有するため、糖鎖Ａ２Ｇ２Ｆに基づいて脳腫瘍を検出できる。また、本発明の検出物質を用いることにより脳腫瘍をより早期且つ簡易に検出することができる。 According to the brain tumor detection substance according to claim 6 of the present invention, since it contains lentil lectin having binding specificity to the N-linked sugar chain A2G2F of the glycoprotein, the brain tumor is based on the sugar chain A2G2F. Can be detected. Moreover, a brain tumor can be detected earlier and more easily by using the detection substance of the present invention.
本発明の請求の範囲第７項記載の医薬組成物によれば、アポトーシス誘導に基づく細胞増殖阻害活性を有するため、周囲の正常細胞に影響を与えずに、脳腫瘍細胞の増殖のみを選択的に阻害しうるので、脳腫瘍細胞により引き起こされる疾患の予防および治療に有効である。 According to the pharmaceutical composition of claim 7 of the present invention, since it has a cell growth inhibitory activity based on the induction of apoptosis, only the growth of brain tumor cells can be selectively performed without affecting surrounding normal cells. Since it can inhibit, it is effective in the prevention and treatment of diseases caused by brain tumor cells.
本発明の請求の範囲第８項記載の医薬組成物によれば、脳腫瘍細胞に対して選択的にアポトーシスを誘発することができる。 According to the pharmaceutical composition of claim 8 of the present invention, apoptosis can be selectively induced to brain tumor cells.
本発明の請求の範囲第９項記載の医薬組成物によれば、アポトーシスの誘導により、神経膠腫細胞を選択的に排除することができる。 According to the pharmaceutical composition of claim 9 of the present invention, glioma cells can be selectively eliminated by induction of apoptosis.
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明でいうＡ２Ｇ２Ｆとは、糖蛋白糖鎖（Ｇａｌβ１，４−ＧｌｃＮＡｃβ１，２−Ｍａｎα１，３−）（Ｇａｌβ１，４−ＧｌｃＮＡｃβ１，２−Ｍａｎα１，６−）Ｍａｎβ１，４−ＧｌｃＮＡｃβ１，４−（Ｆｕｃα１，６−）ＧｌｃＮＡｃであり、Ａ２Ｇ２Ｆという各記号の意味は次の通りである。Ａｎ：Ｍ３糖鎖構造に結合するＧｌｃＮＡｃのアンテナの数、Ｇｎ：非還元末端に付けられたガラクトース残基の数、Ｆ：還元末端のＧｌｕＮＡｃ残基に結合したフコースを有するもの。 A2G2F in the present invention is a glycoprotein sugar chain (Galβ1,4-GlcNAcβ1,2-Manα1,3-) (Galβ1,4-GlcNAcβ1,2-Manα1,6-) Manβ1,4-GlcNAcβ1,4- (Fucα1 , 6-) GlcNAc, and the meaning of each symbol A2G2F is as follows. An: number of GlcNAc antennas that bind to the M3 sugar chain structure, Gn: number of galactose residues attached to the non-reducing end, and F: fucose attached to the GluNAc residue at the reducing end.
また、本発明の検出方法により検出可能な脳腫瘍とは、脳や脳の周辺組織などの頭蓋骨の内部にある組織に発生する腫瘍のことであり、脳組織自体から発生する「原発性脳腫瘍」と、他の臓器の癌が脳に転移してくる「転移性脳腫瘍」が挙げられる。原発性脳腫瘍として、原発性脳腫瘍の約３割を占める神経膠腫、また神経膠腫のうちで最も悪性度の高い神経膠芽腫などが挙げられる。 The brain tumor that can be detected by the detection method of the present invention is a tumor that occurs in a tissue inside the skull, such as the brain and surrounding tissues of the brain, and a “primary brain tumor” that occurs from the brain tissue itself. Examples include “metastatic brain tumors” in which cancers of other organs metastasize to the brain. Examples of primary brain tumors include glioma, which accounts for about 30% of primary brain tumors, and glioblastoma with the highest malignancy among gliomas.
本発明の脳腫瘍の検出方法は、摘出された脳組織のＮ結合型糖鎖を、ＨＰＬＣを用いた糖蛋白質糖鎖システムにより解析し、脳腫瘍に特有の糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆの検出に基づいて脳腫瘍を検出する方法である。また、前記Ｎ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを使用して脳腫瘍を検出する方法である。 In the method for detecting a brain tumor of the present invention, an N-linked sugar chain of an extracted brain tissue is analyzed by a glycoprotein sugar chain system using HPLC, and an N-linked sugar chain A2G2F of a glycoprotein peculiar to a brain tumor is detected. Is a method of detecting a brain tumor based on the above. Further, the present invention is a method for detecting a brain tumor using a lectin having binding specificity for the N-linked sugar chain A2G2F.
本発明の脳腫瘍の検出方法に用いるレクチンとは、植物・動物・微生物に存在するタンパク質または糖タンパク質のうち、糖に対する特異的結合活性をもった物質である。本発明の脳腫瘍の検出方法に用いるレクチンとしては、例えばレンズマメ（ＬＣＡ：Lens culinaris Agglutinin）レクチンが好ましい。このレンズマメレクチンは、ポリペプチド鎖のアスパラギンに結合した糖鎖（Ａｓｎ型（Ｎ型）糖鎖）ＧｌｃＮＡｃ残基のＣ−６位に結合したα−Ｆｕｃに特異的に結合することが知られている。なお、神経膠芽腫細胞及び神経膠芽腫組織において、０．１％以上存在する糖鎖構造の中でＡ２Ｇ２Ｆのみが、ＬＣＡレクチンが特異的に結合するフコシル化されたα１−６ＧｌｃＮＡｃ構造を有する。 The lectin used in the brain tumor detection method of the present invention is a substance having a specific binding activity to sugar among proteins or glycoproteins present in plants, animals and microorganisms. As the lectin used in the brain tumor detection method of the present invention, for example, a lentil (LCA) lectin is preferable. This lentil lectin is known to specifically bind to α-Fuc bound to the C-6 position of the sugar chain (Asn type (N type) sugar chain) GlcNAc residue bound to the asparagine of the polypeptide chain. Yes. In glioblastoma cells and glioblastoma tissues, only A2G2F has a fucosylated α1-6GlcNAc structure to which LCA lectin specifically binds among the sugar chain structures present in 0.1% or more. .
本発明の脳腫瘍の検出方法により、高精度で神経膠腫を検出でき、さらにＡ２Ｇ２Ｆに対して結合特異性を有するレクチン（ＬＣＡレクチン）を利用することにより神経膠腫や神経膠芽腫などの脳腫瘍をより早期且つ簡易に検出できる。また、Ａ２Ｇ２Ｆを特異的に認識する抗体を利用することにより神経膠腫や神経膠芽腫などの脳腫瘍をより早期且つ簡易に検出してもよい。 By the method for detecting a brain tumor of the present invention, a glioma can be detected with high accuracy, and further, a brain tumor such as glioma or glioblastoma by using a lectin (LCA lectin) having binding specificity to A2G2F. Can be detected earlier and more easily. In addition, brain tumors such as glioma and glioblastoma may be detected earlier and easily by using an antibody that specifically recognizes A2G2F.
また、本発明の脳腫瘍の検出物質としては、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに特異的に結合するレクチンを含有するものであれば特に制限されるものではない。また、Ｎ結合型糖鎖Ａ２Ｇ２Ｆに特異的に結合するレクチンとしては、ＬＣＡレクチン等を挙げることができる。さらに、本発明の脳腫瘍の検出物質が、Ｎ結合型糖鎖Ａ２Ｇ２Ｆを特異的に認識する抗体からなるものであってもよい。Ａ２Ｇ２Ｆを認識する抗体としては、モノクローナル抗体やポリクローナル抗体等を例示することができる。これら抗体は、慣用のプロトコールを用いて、実験動物にＡ２Ｇ２Ｆの糖鎖を投与することにより産生することが可能である。 The substance for detecting a brain tumor of the present invention is not particularly limited as long as it contains a lectin that specifically binds to the N-linked sugar chain A2G2F of glycoprotein. Examples of the lectin that specifically binds to the N-linked sugar chain A2G2F include LCA lectin. Furthermore, the substance for detecting a brain tumor of the present invention may comprise an antibody that specifically recognizes an N-linked sugar chain A2G2F. Examples of antibodies that recognize A2G2F include monoclonal antibodies and polyclonal antibodies. These antibodies can be produced by administering A2G2F sugar chains to experimental animals using conventional protocols.
また、Ａ２Ｇ２Ｆを特異的に結合するレクチンやＡ２Ｇ２Ｆを特異的に認識する抗体を化学蛍光試薬や放射性同位体等で標識することができる。化学蛍光試薬として例えばフルオロセイン，ロダミン，ルミノールなど、放射性同位体として例えば３Ｈ，１４Ｃ，３５Ｓ，３３Ｐ，３２Ｐ，１２５Ｉなどが挙げられる。この標識により、Ａ２Ｇ２Ｆが例えば脳腫瘍マーカーとして、従来のＣＴスキャン，核磁気共鳴装置などの画像診断による診断方法を併用することにより、高い精度で、早期且つ確実に脳腫瘍を検出または診断することが可能である。 In addition, a lectin that specifically binds A2G2F or an antibody that specifically recognizes A2G2F can be labeled with a chemical fluorescent reagent, a radioisotope, or the like. Examples of the chemical fluorescent reagent include fluorescein, rhodamine and luminol, and examples of the radioisotope include 3H, 14C, 35S, 33P, 32P and 125I. With this label, A2G2F can detect or diagnose brain tumors early and reliably with high accuracy by using diagnostic methods based on image diagnosis such as conventional CT scans and nuclear magnetic resonance devices as brain tumor markers, for example. It is.
本発明の医薬組成物は、脳腫瘍細胞に対してアポトーシスを誘発する医薬組成物であって、糖蛋白質のＮ結合型糖鎖Ａ２Ｇ２Ｆに対して結合特異性を有するレクチンを有効成分として含む。本発明の有効成分として用いるレクチンとは、植物・動物・微生物に存在するタンパク質または糖タンパク質のうち、糖に対する特異的結合活性をもった物質である。また、本発明の有効成分として用いるレクチンとしては、レンズマメ（ＬＣＡ：Lens culinaris Agglutinin）レクチンが好ましい。 The pharmaceutical composition of the present invention is a pharmaceutical composition that induces apoptosis of brain tumor cells, and contains, as an active ingredient, a lectin having binding specificity for the N-linked sugar chain A2G2F of glycoprotein. The lectin used as an active ingredient of the present invention is a substance having a specific binding activity to sugar among proteins or glycoproteins present in plants, animals and microorganisms. Moreover, as a lectin used as an active ingredient of this invention, a lentil (LCA: Lens culinaris Agglutinin) lectin is preferable.
本発明の医薬組成物は、その使用目的に応じ、医薬剤や製剤成分と複合してアポトーシスを誘導するための医薬組成物として用いてもよい。併用される医薬剤としては、抗癌剤、抗ウイルス剤、抗自己免疫疾患剤を挙げることができる。このように、本発明の医薬組成物に、抗癌剤などの医薬剤を包合させることにより薬剤を病巣局所に集約させ、脳腫瘍細胞により引き起こされる疾患の予防および治療に有効である。 The pharmaceutical composition of the present invention may be used as a pharmaceutical composition for inducing apoptosis in combination with a pharmaceutical agent or a pharmaceutical ingredient depending on the purpose of use. Examples of the pharmaceutical agent used in combination include an anticancer agent, an antiviral agent, and an antiautoimmune disease agent. Thus, the pharmaceutical composition of the present invention is effective for the prevention and treatment of diseases caused by brain tumor cells by encapsulating a pharmaceutical agent such as an anticancer agent in the pharmaceutical composition, thereby concentrating the drug locally.
また、本発明の医薬組成物は、医薬製剤としてこの分野で慣用されている各種の投与形態で使用される。例えば、粉末、錠剤、丸剤、液剤、懸濁剤、乳剤、顆粒剤、カプセル剤、注射剤（液剤、懸濁剤等）等の形で製剤化することができ、所望に応じ溶解補助剤、緩衝剤、等張化剤、安定剤、保存剤、無痛化剤など注射液に慣用されている添加剤や、充填剤、増量剤、結合剤、付湿剤、崩壊剤、表面活性剤、あるいは賦形剤等を併用してもよい。さらに、本発明の医薬組成物は、経口的又は非経口的に投与される。その投与量は、治療すべき脳腫瘍の程度により増減され、特に限定されるものではない。 In addition, the pharmaceutical composition of the present invention is used in various dosage forms conventionally used in this field as a pharmaceutical preparation. For example, it can be formulated in the form of powder, tablets, pills, solutions, suspensions, emulsions, granules, capsules, injections (solutions, suspensions, etc.), and solubilizing agents as desired Additives commonly used in injection solutions such as buffers, isotonic agents, stabilizers, preservatives, soothing agents, fillers, extenders, binders, moisturizers, disintegrants, surface active agents, Or an excipient | filler etc. may be used together. Furthermore, the pharmaceutical composition of the present invention is administered orally or parenterally. The dosage is increased or decreased depending on the degree of brain tumor to be treated and is not particularly limited.
なお、本発明は、上記実施形態に限定されるものではない。上記実施形態は、例示であり、本発明の特許請求の範囲に記載された技術的思想と実質的に同一な構成を有し、同様な作用効果を奏するものは、いかなるものであっても本発明の技術的範囲に包含される。 The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.
（神経膠芽腫組織、及び神経膠腫細胞株におけるＮ結合型糖蛋白質糖鎖の発現解析）
３例の正常脳組織、１５例の神経膠芽腫組織、及び３種の神経膠腫細胞株を用いて糖蛋白質糖鎖分析を行った。コントロールとしての３例の正常脳組織（男性:女性＝２:１、平均年齢５５．２）は、てんかん性の手術を受けた患者からインフォームドコンセントを得て組織を収集した。また、１５例の神経膠芽腫組織は、新潟大学病院で神経膠芽腫を有する患者（表１参照）からインフォームドコンセントを得て組織を収集した。(Expression analysis of N-linked glycoprotein sugar chain in glioblastoma tissue and glioma cell line)
Glycoprotein sugar chain analysis was performed using 3 normal brain tissues, 15 glioblastoma tissues, and 3 glioma cell lines. Three normal brain tissues (male: female = 2: 1, mean age 55.2) as controls were collected with informed consent from patients undergoing epileptic surgery. In addition, 15 glioblastoma tissues were collected by obtaining informed consent from patients with glioblastoma (see Table 1) at Niigata University Hospital.
まず、検体の前処理として、脳組織を冷ＰＢＳで灌流し、その重量を測定した後、９倍量の−２０℃のアセトンを加えた。なお、ＰＢＳは、ＮａＣｌ ８ｇ、ＫＣｌ ０．２ｇ、Ｎａ２ＨＰＯ４ １．４４ｇ、ＫＨ２ＰＯ４ ０．２４ｇを1リットルの脱イオン水に溶かして、ｐＨ７．４として調製したものを使用した。脳組織を、９倍量の冷アセトン中でポリトロン型ホモジナイザーを用いてホモジナイズし、−２０℃で６０分間放置した後、４℃、８０００Ｇで２０分間遠心した。この上清を除去後、再度同様の操作を行った後、沈殿物を凍結乾燥し、検体を得た。乾燥重量２ｍｇの検体をGlycoPrepTM１０００（Oxford Glycosystems，Oxford，UK）を用いて検体をヒドラジン分解して蛋白質から糖鎖を切り離した。次に、切り離された糖鎖をGlycoTagTM（Takara，Tokyo，Japan）を用いてピリジルアミノ化による蛍光標識を行った後、ノイラミニダーゼ（neuraminidase）（Arthrobacter ureafaciens由来，Nacalai Tesque，Kyoto，Japan）で糖鎖を処理して、その側鎖にあるシアル酸を切断した。電荷を有するシアル酸が切断された結果、検体の脳組織内に存在する中性の糖鎖を得ることができた。First, as pretreatment of the specimen, brain tissue was perfused with cold PBS, and its weight was measured. Then, 9 times the amount of acetone at −20 ° C. was added. PBS prepared by dissolving NaCl 8 g, KCl 0.2 g, Na 2 HPO 4 1.44 g, KH 2 PO 4 0.24 g in 1 liter of deionized water to a pH of 7.4 was used. The brain tissue was homogenized in 9 times amount of cold acetone using a polytron homogenizer, left at −20 ° C. for 60 minutes, and then centrifuged at 4 ° C. and 8000 G for 20 minutes. After removing the supernatant, the same operation was performed again, and then the precipitate was lyophilized to obtain a specimen. A sample having a dry weight of 2 mg was subjected to hydrazine decomposition using GlycoPrep ™ 1000 (Oxford Glycosystems, Oxford, UK) to separate sugar chains from the protein. Next, after the separated sugar chain is fluorescently labeled by pyridylamination using GlycoTag ™ (Takara, Tokyo, Japan), the sugar chain is neuraminidase (derived from Arthrobacter ureafaciens, Nacalai Tesque, Kyoto, Japan). To cleave the sialic acid in the side chain. As a result of the cleavage of the charged sialic acid, neutral sugar chains present in the brain tissue of the specimen could be obtained.
検体中の様々な大きさの糖鎖を、その大きさ別に分けるために、蛍光標識された上記中性の糖鎖群を含む検体を順相ＨＰＬＣ（Asahipak NH2P-50 size-fractionation column (4.6×50 mm; Shodex, Tokyo, Japan)）にかけ、糖鎖の量を蛍光検出器で検出して測定した。順相ＨＰＬＣの測定条件を詳しく述べると、流速は ０．６ ｍｌ／ｍｉｎ、カラムの温度は３０℃、溶媒Ａを２０％アセトニトリル、０．３％酢酸を含み、トリエチルアミンでｐＨ７．０に調整した水として、溶媒Ｂは９３％アセトニトリル、０．３％酢酸を含み、トリエチルアミンでｐＨ７．０に調整した水として、最初溶媒Ａ：溶媒Ｂの比を９７：３でスタートさせ、溶媒Ｂを、検体を投入して１分後に１９％、３５分後に５７％まで直線的に増加させた。ここから溶出してくる蛍光標識糖鎖を励起波長３１０ｎｍ、検出波長３８０ｎｍで検出し、標準糖鎖（PA-sugar chain Ｍ２Ａ,Ｍ３Ｂ,Ｍ４Ｂ,Ｍ５Ａ,Ｍ６Ｂ,Ｍ７Ａ,Ｍ８Ａ,Ｍ９Ａ(Takara,Tokyo,Japan)）の溶出時間にしたがってＭ２〜Ｍ１０の画分を分取した。Ｍ１０溶出時間はＭ９Ａ溶出時間＋１．５分として設定した。 In order to classify sugar chains of various sizes according to their size, samples containing the above-mentioned neutral sugar groups labeled with fluorescence were analyzed by normal phase HPLC (Asahipak NH2P-50 size-fractionation column (4.6 × 50 mm; Shodex, Tokyo, Japan)), and the amount of sugar chain was detected by a fluorescence detector and measured. The measurement conditions of normal phase HPLC are described in detail. The flow rate is 0.6 ml / min, the column temperature is 30 ° C., the solvent A contains 20% acetonitrile and 0.3% acetic acid, and the pH is adjusted to 7.0 with triethylamine. As water, solvent B contains 93% acetonitrile and 0.3% acetic acid, and water adjusted to pH 7.0 with triethylamine was first started at a solvent A: solvent B ratio of 97: 3. Was increased linearly to 19% after 1 minute and 57% after 35 minutes. The fluorescently labeled sugar chains eluted from this are detected at an excitation wavelength of 310 nm and a detection wavelength of 380 nm, and standard sugar chains (PA-sugar chains M2A, M3B, M4B, M5A, M6B, M7A, M8A, M9A (Takara, Tokyo, The fractions M2 to M10 were collected according to the elution time of Japan)). The M10 elution time was set as M9A elution time +1.5 minutes.
順相ＨＰＬＣで大きさ別に分離された糖類を、今度はその糖鎖分子の構造上の特性によって分類するために逆相ＨＰＬＣ（Cosmosil 5C18-P reversed-phase column (4.6×150 mm; Nacali Tesque)）にかけた。なお、ＨＰＬＣシステムとしてPU-2089 plus HPLC pump（Nihon Ｂunkou, Tokyo, Japan)、 FP-2055 plus fluorescence detector (Nihon Bunkou)、AS-2055 plus autosampler (Nihon Bunkou)を用いた。コントロールシステム及びピークの溶出時間、相対含有量の解析にはJasco Bowin(Nihon Bunkou)を使用した。逆相ＨＰＬＣの測定条件を詳しく述べると、流速は１．５ｍｌ／ｍｉｎ 、カラムの温度は３０℃、溶媒Ｃを０．１Ｍの酢酸アンモニウム緩衝液（ｐＨ４．０）、溶媒Ｄは溶媒Ｃに０．５％の１ブタノールを加えたものとして、最初溶媒Ｃ：溶媒Ｄの比を９４：６でスタートさせ、溶媒Ｄを、検体を投入して４５分後に８０%まで、直線的に増加させた。ここから溶出してくる蛍光標識糖鎖を励起波長３２０ｎｍ、検出波長４００ｎｍで検出し、その溶出パターンを次の手順で比較検討し、検体中の糖鎖の構造と含有量を決定した。 In order to classify the saccharides separated by size by normal phase HPLC, this time according to the structural characteristics of the glycan molecule, Cosmosil 5C18-P reversed-phase column (4.6 × 150 mm; Nacali Tesque) ) As the HPLC system, PU-2089 plus HPLC pump (Nihon Bunkou, Tokyo, Japan), FP-2055 plus fluorescence detector (Nihon Bunkou), and AS-2055 plus autosampler (Nihon Bunkou) were used. Jasco Bowin (Nihon Bunkou) was used for the control system and the analysis of peak elution time and relative content. The measurement conditions of the reverse phase HPLC are described in detail. The flow rate is 1.5 ml / min, the column temperature is 30 ° C., the solvent C is 0.1 M ammonium acetate buffer (pH 4.0), and the solvent D is 0 in the solvent C. Assuming 5% 1-butanol was added, the solvent C: solvent D ratio was initially started at 94: 6, and solvent D was increased linearly to 80% 45 minutes after loading the sample. . The fluorescently labeled sugar chains eluted from this were detected at an excitation wavelength of 320 nm and a detection wavelength of 400 nm, and the elution patterns were compared and examined by the following procedure to determine the structure and content of sugar chains in the specimen.
逆相ＨＰＬＣに検体を投入する前に、予め、グルコースオリゴマー（３〜２２個のブドウ糖が結合した標準糖鎖）を投入し、その溶出時間を測定しておき、検体の溶出時間の内的標準とする。検体のピークが内部標準のどれくらいの位置に匹敵するかを計算し、グルコースユニット（ＧＵ）として表現する。逆相ＨＰＬＣで認められるピーク毎にそのピークに含有する糖鎖を採取し、もう一度これを順相ＨＰＬＣに投入する。ＧＵと同様な方法で、今度はマンノースユニット（ＭＵ）を計算し、逆相ＨＰＬＣで認められる各々のピークについて、ＧＵ及びＭＵの２つの指標を用いて、横軸にＧＵ、縦軸にＭＵをとって２次元のグラフ上にプロットする。２次元でその糖鎖の特性を判別するこの方法を、予め構造が分かっている糖鎖（Takara，Seikagaku Corporation (Tokyo, Japan), Oxford GlycoSystemsから購入）について行った後、脳組織由来の糖鎖について行い、その２次元マップ上で点が重複するか否かで、構造を同定した。図１は標準糖鎖の２次元マップであり、横軸はグルコースユニット値を示し、縦軸はマンノースユニット値を示す。また、図１のグラフ上にプロットされた番号の糖鎖構造式を図２に示す。すなわち、番号１はＡ０Ｇ０Ｆ、２はＡ２Ｇ１Ｆ０（３）ＦＢ、３はＡ２Ｇ１Ｆ０（６）ＦＢ、４はＡ２Ｇ２，５はＡ２Ｇ２Ｆ、６はＡ３Ｇ３、７はＢＡ２、８はＭ２Ｂ、９はＭ３Ｂ、１０はＭ４Ｂ、１１はＭ５Ａ、１２はＭ６Ｂ、１３はＭ７Ａ、１４はＭ７Ｂ、１５はＭ８Ａ、１６はＭ９Ａを示している。 Before the sample is put into the reverse phase HPLC, a glucose oligomer (standard sugar chain to which 3 to 22 glucoses are bound) is put in advance and its elution time is measured, and the internal standard of the sample elution time is measured. And Calculate how much the peak of the sample is comparable to the internal standard and express it as a glucose unit (GU). For each peak observed in reverse phase HPLC, the sugar chain contained in that peak is collected and once again put into normal phase HPLC. In the same way as GU, this time calculate mannose unit (MU), and for each peak recognized by reversed-phase HPLC, GU on the horizontal axis and MU on the vertical axis using two indicators, GU and MU. Plot it on a two-dimensional graph. This method of distinguishing the characteristics of sugar chains in two dimensions is performed on sugar chains of known structure (purchased from Takara, Seikagaku Corporation (Tokyo, Japan), Oxford GlycoSystems), and then sugar chains derived from brain tissue. The structure was identified by whether or not the points overlap on the two-dimensional map. FIG. 1 is a two-dimensional map of standard sugar chains, where the horizontal axis indicates glucose unit values and the vertical axis indicates mannose unit values. FIG. 2 shows the sugar chain structural formulas of the numbers plotted on the graph of FIG. That is, number 1 is A0G0F, 2 is A2G1F0 (3) FB, 3 is A2G1F0 (6) FB, 4 is A2G2, 5 is A2G2F, 6 is A3G3, 7 is BA2, 8 is M2B, 9 is M3B, 10 is M4B , 11 is M5A, 12 is M6B, 13 is M7A, 14 is M7B, 15 is M8A, and 16 is M9A.
図３は、ヒト正常脳組織（Ａ）および神経膠芽腫組織（Ｂ）のＮ結合型糖鎖の逆相ＨＰＬＣ分析における全フラクション（Ｍ２〜Ｍ１１）の溶出パターンを示す。ピーク５は、神経膠芽腫組織（Ｂ）のＭ６及びＭ７画分のみに確認され、ヒト正常脳組織（Ａ）では確認されなかった。このピーク５は、図１に示す２次元マップにより糖鎖Ａ２Ｇ２Ｆであると同定された。このことより、糖鎖Ａ２Ｇ２Ｆは正常脳組織に発現されず、神経膠腫細胞にのみ発現される、神経膠腫に特異的な糖鎖であることがわかった。 FIG. 3 shows the elution patterns of all fractions (M2 to M11) in reversed-phase HPLC analysis of N-linked sugar chains of human normal brain tissue (A) and glioblastoma tissue (B). Peak 5 was confirmed only in the M6 and M7 fractions of glioblastoma tissue (B) and not in human normal brain tissue (A). This peak 5 was identified as sugar chain A2G2F by the two-dimensional map shown in FIG. From this, it was found that sugar chain A2G2F is a glycoma-specific sugar chain that is not expressed in normal brain tissue but is expressed only in glioma cells.
また、正常脳組織（Ｎｏｒｍａｌ Ｂｒａｉｎ）、神経膠芽腫組織（ＧＢＭ）、及び神経膠腫細胞株（Ｃｅｌｌ Ｌｉｎｅ）の分離、同定された糖蛋白質のＮ結合型糖鎖の含有率（％）を表２示す。 In addition, separation of normal brain tissue (Normal Brain), glioblastoma tissue (GBM), and glioma cell line (Cell Line), and the content (%) of N-linked sugar chain of the identified glycoprotein Table 2 shows.
正常脳組織においては、Ｎ結合型糖鎖全体のうち平均４６．４％が同定された。また、分岐の数を考慮した場合、２分岐構造を持つ糖鎖が６．２７％と最も多く認められたものの、３分岐構造及び４分岐構造を持つ高分岐型糖鎖は殆ど認められなかった。オリゴマンノース系列ではＭ５Ａが最も多く認められ、次いでＭ６Ｂ，Ｍ９Ａが多く認められた。さらに、Ａ２Ｇ２においては２．７８％認められた。 In normal brain tissue, an average of 46.4% of all N-linked sugar chains was identified. In addition, when the number of branches was taken into consideration, sugar chains having a 2-branched structure were most frequently observed at 6.27%, but hardly branched sugar chains having 3-branched structures and 4-branched structures were observed. . In the oligomannose series, M5A was observed most frequently, followed by M6B and M9A. Furthermore, 2.78% was observed in A2G2.
Ａ２Ｇ２Ｆは正常脳組織には認められなかったが、神経膠芽腫細胞株及び神経膠芽腫組織で認められた(ｐ＜０．０１)。また、標準検体（Ａ２Ｇ２Ｆ）とのco-injectionにより単一のピークとなることを確認した。Ａ２Ｇ２Ｆは神経膠芽腫組織において２．９±１．９３％含まれており、神経膠腫細胞株において５．６０±１．１４％含まれていた。２例の神経膠芽腫組織についてイオン交換ＨＰＬＣを行い、Ａ２Ｇ２Ｆのシアル酸化率を調べたところ、６２．２０％と５６．１９％であった。Ａ２Ｇ２は神経膠芽腫組織で１２．６６±８．３３％認められ、正常脳組織での２．７８±０．６０％に比べ有意に高かった。Ａ３Ｇ３は神経膠芽腫組織で１．００±０．７９％、神経膠腫細胞株で３．５１±２．１３％認められた。神経膠芽腫組織には肺癌や肝細胞癌で認められている高分岐型糖鎖は認められなかった。 A2G2F was not found in normal brain tissue, but was found in glioblastoma cell lines and glioblastoma tissue (p <0.01). Moreover, it confirmed that it became a single peak by co-injection with a standard sample (A2G2F). A2G2F was contained in 2.9 ± 1.93% in glioblastoma tissues and 5.60 ± 1.14% in glioma cell lines. Ion exchange HPLC was performed on two glioblastoma tissues, and the sialylation rate of A2G2F was determined to be 62.20% and 56.19%. A2G2 was found to be 12.66 ± 8.33% in glioblastoma tissue and was significantly higher than 2.78 ± 0.60% in normal brain tissue. A3G3 was found to be 1.00 ± 0.79% in glioblastoma tissue and 3.51 ± 2.13% in glioma cell lines. The hyperbranched glycan observed in lung cancer and hepatocellular carcinoma was not found in the glioblastoma tissue.
（レクチン染色）
ＨＰＬＣの結果神経膠芽腫に含有され、正常脳では検出されなかったＡ２Ｇ２Ｆに対して、レクチン染色を行った。細胞においてＬＣＡレクチンが特異的に結合するｆｕｃｏｓｙｌａｔｅｄ α１−６ＧｌｃＮＡｃ構造は、０．１％以上存在する糖鎖構造の中でＡ２Ｇ２Ｆのみであったことから、コア（core）のα1,6-fucosylation構造に特異的に結合するＬＣＡ(レンズマメレクチン；Seikagaku Corporation,Tokyo,Japan)を使用した。(Lectin staining)
As a result of HPLC, lectin staining was performed on A2G2F that was contained in glioblastoma and was not detected in normal brain. Since the fucosylated α1-6GlcNAc structure to which LCA lectin specifically binds in cells was only A2G2F among the sugar chain structures present in 0.1% or more, it was changed to the core α1,6-fucosylation structure. LCA (Lens bean lectin; Seikagaku Corporation, Tokyo, Japan) that specifically binds was used.
以下の手順により、神経膠芽腫組織（ｎ＝１０）及び３種類の神経膠腫細胞株、コントロールとして正常脳組織 (ｎ＝３)において、ＬＣＡ（レンズマメレクチン）を用いた染色を行い顕微鏡観察した。まず、それぞれの試料の凍結切片を用い、蛍光試薬ＦＩＴＣで標識されたＬＣＡ−ＦＩＴＣ（Seikagaku Corporation,Tokyo,Japan; 500 μg/ml: diluted 1:50）を加え２０分間反応後、蛍光顕微鏡にて観察した。なお、糖鎖Ａ２Ｇ２Ｆに対し、非免疫マウスイムノグロブリンをネガティブコントロールとして用いた。 According to the following procedure, staining with LCA (lens bean lectin) was performed on glioblastoma tissue (n = 10), three types of glioma cell lines, and normal brain tissue (n = 3) as a control, and observed with a microscope. did. First, using frozen sections of each sample, LCA-FITC (Seikagaku Corporation, Tokyo, Japan; 500 μg / ml: diluted 1:50) labeled with a fluorescent reagent FITC was added, reacted for 20 minutes, and then fluorescence microscope Observed. In addition, non-immune mouse immunoglobulin was used as a negative control for sugar chain A2G2F.
結果を図４に示す。図４−Ａは神経膠腫細胞株の免疫組織化学染色を示し、図４−Ｂは神経膠芽腫組織の免疫組織化学染色を示す。神経膠腫細胞株ではＬＣＡにより非常に強く染色され（図４−Ａ）、神経膠芽腫組織においてもＬＣＡにより強く染色された（図４−Ｂ）。正常脳組織においては実質には明らかな染色を認めなかった（図示せず）。この結果より、Ａ２Ｇ２Ｆと特異的に結合するＬＣＡレクチンが神経膠芽腫組織及び神経膠腫細胞株と特異的に結合するのが明らかとなった。したがって、脳腫瘍のみに発現するＡ２Ｇ２Ｆに対して結合特異性を有する蛍光試薬で標識されたレクチンを用いて脳腫瘍を検出することが可能である。 The results are shown in FIG. FIG. 4-A shows immunohistochemical staining of a glioma cell line, and FIG. 4-B shows immunohistochemical staining of glioblastoma tissue. Glioma cell lines stained very strongly with LCA (FIG. 4-A), and glioblastoma tissues also stained strongly with LCA (FIG. 4-B). In normal brain tissue, no clear staining was observed in the parenchyma (not shown). From this result, it was revealed that LCA lectin that specifically binds to A2G2F specifically binds to glioblastoma tissue and glioma cell line. Therefore, it is possible to detect a brain tumor using a lectin labeled with a fluorescent reagent having binding specificity for A2G2F expressed only in the brain tumor.
（細胞増殖及びアポトーシス測定）
Ｕ２５１，Ｔ９８Ｇ (Riken Cell Bank,Tsukuba,Japan)、及び神経膠芽腫患者組織から培養されたＯＮ−１２神経膠腫細胞株を１０％のＦＣＳ，５０ｍＭの２−メルカプトエタノール，１０ｍＭのヘペス（Ｈｅｐｅｓ）(ｐＨ ７．４)，２ｍＭのグルタミン，１００Ｕ／ｍｌのペニシリン, １００μg／ｍｌのストレプトマイシンを含むＭＥＭ培地 (Invitrogen, Tokyo,Japan)中で培養したものを試料として使用した。(Cell proliferation and apoptosis measurement)
U251, T98G (Riken Cell Bank, Tsukuba, Japan), and ON-12 glioma cell lines cultured from glioblastoma patient tissue were treated with 10% FCS, 50 mM 2-mercaptoethanol, 10 mM Hepes. ) (PH 7.4), cultured in MEM medium (Invitrogen, Tokyo, Japan) containing 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin was used as a sample.
（１）Ｕ２５１，Ｔ９８Ｇ，ＯＮ−１２の各細胞を、９６wellプレートに５×１０３ずつ細胞を播種し、２４時間後に各濃度のＬＣＡレクチンを加え、さらに２４時間培養を継続した。その後、テトラカラーワン（TetraColor ONE）試薬 (Seikagaku Corporation，Tokyo, Japan)を１０μｌ加え、４時間後に４２０ｎｍの吸光度を測定後、各細胞の細胞増殖能を検討した。(1) Each cell of U251, T98G, ON-12 was seeded at 5 × 10 3 cells in a 96-well plate, 24 hours later, each concentration of LCA lectin was added, and the culture was further continued for 24 hours. Thereafter, 10 μl of TetraColor ONE reagent (Seikagaku Corporation, Tokyo, Japan) was added, and after 4 hours, the absorbance at 420 nm was measured, and the cell growth ability of each cell was examined.
（２）Ｕ２５１，Ｔ９８Ｇ，ＯＮ−１２の各細胞１×１０4を培養スライド(BD Falcon,Bedford,MA)で培養し、２５μg／ｍｌのＬＣＡレクチンを加え２４時間培養後、Hoechst 33342 (bisbenzimide H33342 Fluorochrome : Calbiochem-Novabiochem. Co., CA) を１０μl／ｍｌ加え、核クロマチンを蛍光顕微鏡にて観察した。(2) 1 × 10 4 cells of U251, T98G, ON-12 were cultured on culture slides (BD Falcon, Bedford, Mass.), 25 μg / ml LCA lectin was added and cultured for 24 hours, and then Hoechst 33342 (bisbenzimide H33342 Fluorochrome: Calbiochem-Novabiochem. Co., CA) was added at 10 μl / ml, and nuclear chromatin was observed with a fluorescence microscope.
（３）Ｕ２５１，Ｔ９８Ｇ，ＯＮ−１２の各細胞１×１０6を２５μg／ｍｌのＬＣＡレクチンを加え２４時間培養後、５０μg／ｍｌのヨウ化プロピジウム(Sigma, Tokyo, Japan)を用いて核染色を行い、フローサイトメトリー法にてhypodiploid DNAを測定した。(3) 1 × 10 6 cells of U251, T98G and ON-12 were added with 25 μg / ml LCA lectin, cultured for 24 hours, and then stained with 50 μg / ml propidium iodide (Sigma, Tokyo, Japan). The hypodiploid DNA was measured by flow cytometry.
（４）ＭＥＢＳＴＡＩＮアポトーシスキット（Medical & Biological Laboratories CO., Nagoya, Japan）を用いて、ビオチン化ｄＵＴＰでＤＮＡ末端をニックエンドラベルし、さらにＦＩＴＣ標識アビジンで染色することにより、ＤＮＡの断片化を指標に、アポトーシスによる細胞死を細胞個々に検出した。具体的には、各細胞に２５μg／ｍｌのＬＣＡレクチンを加え２４時間培養後、細胞をトリプシン処理により回収し、ＰＢＳ／０．２％ＢＳＡ（Bovine serum albumin）で数回洗浄行い、４％ＰＦＡ(paraformaldehyde)にて３０分間固定を行った後、ＰＢＳ／０．２％ＢＳＡで洗浄後、７０％エタノール１ｍｌを加えて、−２０℃で３０分間さらに固定を行った。次いで、ＰＢＳ／０．２％ＢＳＡにて洗浄後、細胞沈渣に対してＴｄＴ（ターミナル・デオキシヌクレオチジル・トランスフェラーゼ）反応液を３０μl加え撹拌し、３７℃で１時間反応させた。次いで、ＰＢＳ／０．２％ＢＳＡにて洗浄後、細胞沈渣を５００μlのＰＢＳ／０．２％ＢＳＡに懸濁させフローサイトメトリー法にて測定した。 (4) DNA fragmentation is indicated by nick-end labeling of DNA ends with biotinylated dUTP and staining with FITC-labeled avidin using MEBSTAIN apoptosis kit (Medical & Biological Laboratories CO., Nagoya, Japan) In addition, cell death due to apoptosis was detected for each cell. Specifically, 25 μg / ml LCA lectin was added to each cell and cultured for 24 hours. The cells were collected by trypsin treatment, washed several times with PBS / 0.2% BSA (Bovine serum albumin), and 4% PFA. After fixing with (paraformaldehyde) for 30 minutes, after washing with PBS / 0.2% BSA, 1 ml of 70% ethanol was added and further fixed at −20 ° C. for 30 minutes. Next, after washing with PBS / 0.2% BSA, 30 μl of TdT (terminal deoxynucleotidyl transferase) reaction solution was added to the cell sediment and stirred, followed by reaction at 37 ° C. for 1 hour. Next, after washing with PBS / 0.2% BSA, the cell sediment was suspended in 500 μl of PBS / 0.2% BSA and measured by flow cytometry.
（５）細胞内のカスパーゼ−３をＦＩＴＣ結合ラビット抗活性カスパーゼ−３モノクローナル抗体（BD PharMingen, Tokyo, Japan）を用いて検討した。具体的には、２５μg／ｍｌのＬＣＡレクチンを、Ｕ２５１，Ｔ９８Ｇ，ＯＮ−１２の各細胞に加え２４時間以上培養して細胞沈渣を回収した後、ＦＩＴＣ結合ラビット抗活性化カスパーゼ−３モノクローナル抗体（BD PharMingen, Tokyo, Japan）を２０μl加え撹拌し、４℃で１時間反応させた。次いで、ＰＢＳ／０．２％ＢＳＡにて洗浄後、細胞沈渣を５００μlのＰＢＳ／０．２％ＢＳＡに懸濁させフローサイトメトリー法にて測定した。 (5) Intracellular caspase-3 was examined using a FITC-conjugated rabbit anti-active caspase-3 monoclonal antibody (BD PharMingen, Tokyo, Japan). Specifically, 25 μg / ml of LCA lectin was added to each cell of U251, T98G, ON-12 and cultured for 24 hours or longer to recover the cell sediment, and then FITC-conjugated rabbit anti-activated caspase-3 monoclonal antibody ( (BD PharMingen, Tokyo, Japan) was added in an amount of 20 μl, stirred, and reacted at 4 ° C. for 1 hour. Next, after washing with PBS / 0.2% BSA, the cell sediment was suspended in 500 μl of PBS / 0.2% BSA and measured by flow cytometry.
上述した（１）〜（５）の細胞増殖又はアポトーシス測定の結果を図５〜９に示す。図５は、Ｕ２５１，Ｔ９８Ｇ，ＯＮ−１２細胞にそれぞれＬＣＡレクチンを添加した時のＵ２５１，Ｔ９８Ｇ，ＯＮ−１２細胞の平均の生存率の結果を示すグラフである。図５からわかるように、Ｕ２５１，Ｔ９８Ｇ，ＯＮ−１２細胞はＬＣＡレクチン添加により濃度依存性に細胞増殖能の抑制が認められた。 The results of the above-described cell proliferation or apoptosis measurements (1) to (5) are shown in FIGS. FIG. 5 is a graph showing the results of the average survival rate of U251, T98G and ON-12 cells when LCA lectin was added to U251, T98G and ON-12 cells, respectively. As can be seen from FIG. 5, in the cells of U251, T98G, ON-12, suppression of cell proliferation ability was recognized in a concentration-dependent manner by adding LCA lectin.
図６は、Ｔ９８Ｇ細胞にＬＣＡレクチンを添加してないもの（Ａ）と、ＬＣＡレクチンを添加したもの(Ｂ)を培養後、Hoechst 33342の蛍光色素を使用してアポトーシス細胞のクロマチンの凝集を蛍光顕微鏡下で観察した結果を示す顕微鏡写真である。図６−ＢのＬＣＡレクチンを添加した細胞では、クロマチンの凝集部分に色素が集まり（矢印で示した箇所）、強い青色蛍光染色が認められた。この結果は、クロマチン凝集がアポトーシスに伴うもっとも特徴的な形態変化の一つであることから、ＬＣＡレクチンによってアポトーシスが誘導されることを示した。 FIG. 6 shows that the chromatin aggregates of apoptotic cells were fluorescent using the Hoechst 33342 fluorescent dye after culturing the T98G cells without LCA lectin (A) and those with LCA lectin added (B). It is a microscope picture which shows the result observed under the microscope. In the cells to which the LCA lectin of FIG. 6-B was added, dyes gathered at the chromatin aggregated portions (locations indicated by arrows), and strong blue fluorescent staining was observed. This result indicated that apoptosis was induced by LCA lectin since chromatin aggregation was one of the most characteristic morphological changes associated with apoptosis.
図７は、フローサイトメトリー法によるhypodiploid DNAの検出結果を示すグラフである。図７−Ａは、Ｔ９８Ｇ細胞にＬＣＡレクチンを添加してないコントロール核の典型的なＤＮＡヒストグラムであり、図７−Ｂは、Ｔ９８Ｇ細胞にＬＣＡレクチンを添加して培養した細胞のＤＮＡヒストグラムである。図７に示したように核染色によりhypodiploid DNAの検討を行ったところＴ９８Ｇ細胞では、無処置では０．２９％であったが、ＬＣＡレクチンを加えると３１．９９％であり、ＬＣＡレクチン添加によるhypodiploid DNAの増加が認められた。 FIG. 7 is a graph showing the detection results of hypodiploid DNA by flow cytometry. Fig. 7-A is a typical DNA histogram of a control nucleus without addition of LCA lectin to T98G cells, and Fig. 7-B is a DNA histogram of cells cultured with addition of LCA lectin to T98G cells. . As shown in FIG. 7, when hypodiploid DNA was examined by nuclear staining, it was 0.29% without treatment in T98G cells, but it was 31.99% when LCA lectin was added. An increase in hypodiploid DNA was observed.
図８は、ＭＥＢＳＴＡＩＮアポトーシスキットを用いたフローサイトメトリー法によるアポトーシス細胞の検出結果を示すグラフである。ＭＥＢＳＴＡＩＮアポトーシスキットを用いた染色によりヌクレオソームにおけるＤＮＡの断片化の観察では、Ｔ９８Ｇ細胞のＬＣＡレクチンを添加してないもの(図８−Ａ)では２．０８％であったが、ＬＣＡレクチンを添加したもの(図８−Ｂ)では１６．４５％であり、ＬＣＡレクチンを添加することによりＤＮＡの断片化の増加が認められた。この結果より、神経膠腫細胞はＬＣＡレクチンでアポトーシスが誘導されることがわかった。 FIG. 8 is a graph showing the detection results of apoptotic cells by flow cytometry using MEBSTAIN apoptosis kit. In the observation of DNA fragmentation in the nucleosome by staining with the MEBSTAIN apoptosis kit, it was 2.08% in the case where the LCA lectin of T98G cells was not added (FIG. 8-A), but the LCA lectin was added. In the sample (FIG. 8B), it was 16.45%, and an increase in DNA fragmentation was observed by adding LCA lectin. From this result, it was found that apoptosis of glioma cells was induced by LCA lectin.
図９は、フローサイトメトリー法による活性化カスパーゼ−３の検出結果を示すグラフである。図９−Ａ，Ｂ，Ｃは、それぞれＴ９８Ｇ，Ｕ２５１，ＯＮ１２の各細胞におけるカスパーゼ−３陽性細胞の検出結果を示す。また、図中の実線は、ＬＣＡレクチンを添加したものを示し、破線はＬＣＡレクチンを添加していないものを示す。Ｔ９８Ｇ，Ｕ２５１，ＯＮ１２の各細胞は、それぞれＬＣＡレクチンを添加した場合、３６．７８％，２３．０３％，２０．８６％であり、ＬＣＡレクチンを添加していないものと比較し、活性化カスパーゼ−３の増加が認められた。これらの結果からわかるように、神経膠腫細胞にＬＣＡレクチンはアポト-シスを誘導し、それはカスパーゼ経路依存性であることが明らかとなった。 FIG. 9 is a graph showing the detection result of activated caspase-3 by flow cytometry. 9-A, B, and C show the detection results of caspase-3 positive cells in T98G, U251, and ON12 cells, respectively. Moreover, the solid line in a figure shows what added LCA lectin, and a broken line shows what did not add LCA lectin. The cells of T98G, U251, and ON12 were 36.78%, 23.03%, and 20.86%, respectively, when LCA lectin was added, and activated caspases compared to those without LCA lectin. An increase of -3 was observed. As can be seen from these results, LCA lectin induced apoptosis in glioma cells, and it was revealed that it was caspase pathway-dependent.
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