WO2012039430A1 - Method for isolating cancer stem cells - Google Patents

Method for isolating cancer stem cells Download PDF

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WO2012039430A1
WO2012039430A1 PCT/JP2011/071504 JP2011071504W WO2012039430A1 WO 2012039430 A1 WO2012039430 A1 WO 2012039430A1 JP 2011071504 W JP2011071504 W JP 2011071504W WO 2012039430 A1 WO2012039430 A1 WO 2012039430A1
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cancer stem
stem cells
antibody
sugar chain
cancer
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PCT/JP2011/071504
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French (fr)
Japanese (ja)
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三善英知
森脇健太
奥戸久美子
黒田俊一
武石俊作
澤木弘道
成松久
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株式会社Ｇｐバイオサイエンス
国立大学法人大阪大学
国立大学法人名古屋大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells

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  • the present invention relates to a method for isolating cancer stem cells, and more particularly, to a method for isolating cancer stem cells using sugar chains, and a method for selecting high-purity cancer stem cells. Moreover, it is related with the isolation method of the cancer stem cell regarding the process in the molecular determination for isolating a cancer stem cell.
  • Cancer stem cells are involved in cancer development, malignant transformation, resistance to treatment, and the like, and can be said to be the largest topic of current cancer research. It has been known that cells isolated by surface molecules such as CD133 have a remarkable tumor forming ability even with a very small number of cancer cells.
  • CD133 itself does not have a function of transforming cancer cells as stem cells and is currently considered to be one of surface molecules.
  • CD133 as a marker for cancer stem cells progresses and concerns. Stem Cells Dev. 2009, 1127-34, review. How powerful is CD133 as a cancer stem cell marker in brain tumors CancerTreat Rev. 2009: 403-8, review. The utilities and limitations of glycosylated human CD133 epitopes in defining cancer stem cells. J Mol Med 86, 1025-32, 2008 review. Strategy for Glycoproteomics: Identification of Glyco-Alteration Using Multiple Glycan Profiling Tools. J. Proteome 2009, 8, 1358-67
  • the present invention relates to a method for isolating cancer stem cells, a characteristic study of cancer stem cells, and a method for distinguishing cancer severity and recurrence.
  • the invention described in claim 1 A method for isolating cancer stem cells, which comprises selecting cancer stem cells based on differences in sugar chain structures on the cells.
  • the invention according to claim 2 After selecting the cancer cells, characterize the sugar chain structure of the selected cancer cells and correlate the cell characteristics with the characteristics of the sugar chain structure to make a molecular determination for isolating cancer stem cells.
  • the invention described in claim 3 After selecting cancer cells, a cell group having at least one of the characteristics of high tumorigenicity, high metastasis ability, and high drug resistance is selected, and the sugar chain structure of the selected cell group is selected.
  • the invention according to claim 4 4.
  • the selection of a cell group having any one or more of the high tumorigenic potential, high metastatic potential, and high drug resistance is performed by a cultured cell experiment and / or an animal experiment. It is the isolation method of the cancer stem cell of description.
  • the invention according to claim 5 The method for isolating cancer stem cells according to claim 3 or 4, wherein the characterization of the sugar chain structure of the selected cell group is performed by a sugar chain structure evaluation means.
  • the sugar chain structure evaluation means is based on any one of a lectin array, MS, HPLC, Western blotting, and electrophoretic electrophoresis, or a combination of a plurality of them. It is the isolation method of the cancer stem cell of description.
  • the invention described in claim 7 The cancer cell is selected using a CD marker, any one of sugar chains, or a combination of plural kinds thereof.
  • CD marker or any one of sugar chains, or a combination of a plurality of them is a CD marker alone, and one type of CD marker or a plurality of types of CD.
  • the cancer cells are selected by a combination of markers, the sugar chain alone is used, and when the cancer cells are selected by a combination of one type of sugar chain or a plurality of types of sugar chains, one type or a plurality of types of CDs are used. Any of the cases where the cancer cells are selected by a combination of a marker and one or more kinds of sugar chains is included.
  • the invention described in claim 8 The selection of the cancer cells is performed using any one of an anti-CD marker antibody, a lectin, and an anti-sugar chain antibody, or a combination of a plurality of these. It is the isolation method of the cancer stem cell as described in any one of Claim 6.
  • the invention according to claim 9 The selection of the cancer cells is performed using an anti-CD marker antibody, a lectin, an anti-glycan antibody, a column using a combination of a plurality of these, or affinity beads.
  • an anti-CD marker antibody or any one of lectins and anti-sugar chain antibodies, or a combination of plural types thereof refers to an anti-CD marker antibody alone.
  • the cancer cells can be selected by using a single lectin or a combination of multiple types of lectins.
  • cancer cells When performing the selection of the cancer cells by using only one type of anti-sugar chain antibody or a combination of a plurality of types of anti-sugar chain antibodies, When selecting the cancer cells by combining with one or more lectins, one or more anti-CD marker antibodies and one or When the cancer cells are selected by a combination with a plurality of types of anti-sugar chain antibodies, and when the cancer cells are selected by a combination of one type or a plurality of types of lectins and one or more types of anti-sugar chain antibodies. These include any of cases where the cancer cells are selected by a combination of one or more kinds of anti-CD marker antibodies, one or more kinds of lectins and one or more kinds of anti-sugar chain antibodies.
  • the invention according to claim 10 provides: 10. The isolation of cancer stem cells according to claim 8 or 9, wherein the lectin and the anti-sugar chain antibody are a lectin that recognizes the characterized sugar chain structure and an anti-sugar chain antibody. Is the method.
  • the invention according to claim 11 11 The cancer stem cell unit according to claim 10, wherein the anti-sugar chain antibody is any one of a natural antibody, a recombinant antibody, a part of the antibody, an artificial antibody including an aptamer, a nucleotide, and an artificial structure. It is a separation method.
  • the invention according to claim 12 The cancer stem cell unit according to claim 7, wherein CD133 or CD13 or a combination thereof is used as a CD marker, and ⁇ 2,6 sialic acid, ⁇ 2,3 sialic acid or a combination thereof is used as a sugar chain. It is a separation method.
  • the invention according to claim 13 The method for isolating cancer stem cells according to claim 12, wherein the isolation of cancer stem cells is isolation of cancer stem cells in liver cancer stem cells.
  • Anti-CD133 antibody or anti-CD13 antibody or a combination thereof is used as an anti-CD marker antibody, ⁇ 2,6-sialic acid recognition lectin or ⁇ 2,3-sialic acid recognition lectin or a combination thereof is used as a lectin, and anti- ⁇ 2 is used as an anti-sugar chain antibody.
  • the invention according to claim 15 is: The method for isolating cancer stem cells according to claim 14, wherein the isolation of cancer stem cells is isolation of cancer stem cells in liver cancer stem cells.
  • the invention according to claim 16 The method for isolating cancer stem cells according to claim 14 or 15, wherein the ⁇ 2,6-sialic acid-recognizing lectin is one of SNA, SSA, and TJA-I.
  • the ⁇ 2,6-sialic acid recognition lectin is not limited to any one of SNA, SSA, and TJA-I.
  • the ⁇ 2,3-sialic acid recognition lectin is not limited to MAL-I.
  • the invention according to claim 18 A cancer stem cell identification method comprising identifying cancer stem cells using an identification method based on gene expression levels.
  • the invention according to claim 19 19.
  • the invention according to claim 20 provides The method for identifying cancer stem cells according to claim 18 or 19, wherein the gene is a glycosyltransferase gene.
  • the invention according to claim 21 21 21.
  • the invention according to claim 22 A method for selecting cancer stem cells, which comprises using cells presenting both a CD marker and a sugar chain as a target for delivery of an object to cancer stem cells.
  • the invention described in claim 23 A cancer stem cell selection method characterized by presenting both an anti-CD antibody and a sugar chain recognition molecule as a target molecule to be presented on the surface of a DDS (drug delivery system) molecule.
  • the invention according to claim 24 provides 23.
  • the invention according to claim 25 provides The method for selecting cancer stem cells according to claim 23, wherein the anti-CD antibody is an anti-CD133 antibody.
  • the invention according to claim 26 provides The method for selecting cancer stem cells according to claim 23, wherein the sugar chain recognition molecule is a sialic acid recognition molecule.
  • the invention according to claim 27 provides 27.
  • the present invention is not limited to the above lectins such as SNA, and all other sialic acid recognizing lectins can be employed.
  • the invention according to claim 28 provides 24.
  • the anti-CD antibody includes a full-body antibody, a single chain antibody, a Fab fragment, a partial antibody (ie, a part of an antibody), an artificial antibody including a chimeric antibody, an aptamer, and a mimic molecule.
  • Item 24 The method for selecting cancer stem cells according to Item 23.
  • mimic molecule examples include mimic RNA.
  • the invention according to claim 30 provides 24.
  • the above-mentioned artificially created one includes, for example, an artificially produced one by a method such as “phage display”.
  • the invention of claim 31 is 26.
  • the sialic acid recognition molecule comprises a full-body antibody, a single chain antibody, a Fab fragment, a partial antibody (ie, a part of an antibody), an artificial antibody including a chimeric antibody, an aptamer, and a mimic molecule.
  • Item 27 The method for selecting cancer stem cells according to Item 26.
  • mimic molecule examples include mimic RNA.
  • the invention of claim 33 is 27.
  • the above-mentioned artificially created one includes, for example, an artificially produced one by a method such as “phage display”.
  • lectins include not only wild type ⁇ lectins, but also recombinant and artificially modified lectins.
  • the invention according to claim 34 provides 27.
  • the invention of claim 35 is The cancer treatment method is characterized in that a cancer treatment target is a cell presenting both a CD marker and a sugar chain.
  • the invention according to claim 36 provides 36.
  • the invention of claim 37 is A method for treating cancer characterized in that an anticancer agent, RNAi, or a molecule that inhibits gene expression is placed on a molecule used in a DDS (drug delivery system) and delivered to a cancer stem cell group. .
  • the molecule to be delivered to the cancer stem cell group on the anticancer drug, RNAi, or molecule that inhibits gene expression is any molecule that can be used as a DDS (drug delivery system) technology. Can also be used. For example, a liposome can be mentioned.
  • the invention according to claim 38 provides 38.
  • the invention according to claim 39 provides 38.
  • the cancer according to claim 37, wherein the RNAi or the inhibition target of the molecule that inhibits gene expression is ST6GALNAC1, GCNT3, MGAT5, or a combination of two or more of these. This is a treatment method.
  • the invention according to claim 40 provides 38.
  • the invention of claim 41 is 38.
  • a method capable of determining a cell trait at the gene expression level as well as the conventional FACS technique in the future is developed, a method for identifying CSC in ST6GALNAC1, GCNT3, or MGAT5 And can be applied.
  • the isolation method of the cancer stem cell regarding the process in the molecular determination for isolating a cancer stem cell can be provided.
  • the present invention makes it possible to provide a method for isolating high-purity cancer stem cells. That is, according to the present invention, it becomes possible to make a dramatic progress in the development of means for killing cancer stem cells while at the same time advancing research on the characteristics of cancer stem cells. This is a very big breakthrough that brings the realization of a major theme that has never been possible to cure cancer.
  • the cell group itself isolated by the same CD133 is also a heterogeneous population, and further fractionation concentrates a cell population having high tumorigenic potential in a certain fraction. I understand that By utilizing this research result, we have invented a method for isolating higher-purity cancer stem cells.
  • cancer cells are selected using a CD marker or the like, and from among the selected cells, a cell culture experiment or an animal experiment is performed.
  • a cell group having one or more of the above, that is, a cell group having a high malignancy is selected.
  • the selected group is characterized with respect to the glycan structure of the group by glycan structure evaluation means such as lectin array, MS (mass spectrometry), HPLC (high performance liquid chromatography), western blotting, and electrophoretic electrophoresis. Do.
  • glycan structure evaluation means such as lectin array, MS (mass spectrometry), HPLC (high performance liquid chromatography), western blotting, and electrophoretic electrophoresis.
  • CD133 and CD13 double positive cells are highly biologically malignant and have a trait as a cancer stem cell (CD13 is a therapeutic target in human liver cancer stem cells. J. Clin. Invest. 2010 in press).
  • CD133 positive / SSA positive cells had significantly higher tumorigenicity than the CD133 positive / SSA negative cells (Table 1 shown in FIG. 2 and FIG. 3).
  • SSA was used as the 2,6-sialic acid recognition lectin
  • MAL-I Mesackia amrensis Lectin I
  • Cancer Stem Cell cDNA Synthesis A cancer stem cell fraction sample prepared by the method according to claim 21 of the present application is washed with PBS (Dulbecco PBS (-) manufactured by Nissui Pharmaceutical Co., Ltd.) and then a commercially available RNA extraction kit ( The extract was used as cancer stem cell total RNA using Qiagen RNeasy plus mini kit).
  • PBS Dulbecco PBS (-) manufactured by Nissui Pharmaceutical Co., Ltd.
  • RNA extraction kit The extract was used as cancer stem cell total RNA using Qiagen RNeasy plus mini kit).
  • Cancer stem cell cDNA was prepared using 1 ⁇ g of cancer stem cell total RNA as a starting material and using a commercially available cDNA synthesis kit (Quantitect RT kit manufactured by Qiagen).
  • glycosyltransferase gene in the cancer stem cell fraction sample was a combination of ST6GALNAC1, GCNT3, and MGAT5.
  • Quantitative PCR analysis of glycosyltransferase gene expression level The amount of glycosyltransferase gene expression was quantified in cancer stem cell cDNA using a quantitative PCR method. Regarding the quantification method, the method using a quantitative PCR array described in Non-Patent Document 4 was followed.
  • the cancer stem cell cDNA was used as a template and qPCRQuickGoldStar Mastermix Plus from Eurogentec was used as a PCR enzyme.
  • ST6GALNAC1 gene transcript-specific detection set reagent F primer; 5'-ggtgcccactggaggata-3 '(SEQ ID NO: 1), R primer; 5'-gaagccataagcactcacctg-3' (SEQ ID NO: 2), TaqMan probe: ctggcc (sequence) Number x 3).
  • GCNT3 gene transcript-specific detection set reagent F primer; 5'-atccctaagcaggagagaagc-3 '(SEQ ID NO: 4), R primer: 5'-gtggagaccgagcacagg-3 (SEQ ID NO: 5), TaqMan probe: ctcctcca (SEQ ID NO: ⁇ 6).
  • MGAT5 gene transcript-specific detection set reagent F primer; 5'-ggggatgctacagagaatcaa-3 '(SEQ ID NO: 7), R primer: 5'-gggtggccacatcacttg-3 (SEQ ID NO: 8), TaqMan probe: cttctgcc (SEQ ID NO: ⁇ 9).
  • the numerical value of the measurement result was calculated as the copy number of the target gene transcript in the measurement data by applying it to a calibration curve prepared with a separately measured standard sample.

Abstract

Proposed is a method for isolating cancer stem cells with higher purity by simple combination of markers. A method for isolating cancer stem cells is characterized by screening cancer stem cells by the difference in carbohydrate structure on the cells.

Description

がん幹細胞の単離方法Methods for isolating cancer stem cells
　この発明は、がん幹細胞の単離方法に関し、特に、糖鎖を利用したがん幹細胞の単離方法であって、高純度ながん幹細胞の選別方法に関する。また、がん幹細胞を単離するための分子決定におけるプロセスに関するがん幹細胞の単離方法に関する。 The present invention relates to a method for isolating cancer stem cells, and more particularly, to a method for isolating cancer stem cells using sugar chains, and a method for selecting high-purity cancer stem cells. Moreover, it is related with the isolation method of the cancer stem cell regarding the process in the molecular determination for isolating a cancer stem cell.
　がん幹細胞は、がんの発生、悪性形質転換、治療に対する抵抗性などに関与し、現在のがん研究最大のトピックスと言える。これまでＣＤ１３３などの表面分子によって単離された細胞は、非常に少数のがん細胞でも著しい腫瘍形成能をもつことが知られている。 Cancer stem cells are involved in cancer development, malignant transformation, resistance to treatment, and the like, and can be said to be the largest topic of current cancer research. It has been known that cells isolated by surface molecules such as CD133 have a remarkable tumor forming ability even with a very small number of cancer cells.
　しかし、近年、ＣＤ１３３以外にもがん幹細胞を単離できる様々な表面分子の報告があり、各がん細胞によって幹細胞に特徴的な表面分子は異なる可能性がある。またＣＤ１３３そのものに、がん細胞を幹細胞として形質転換させる機能はなく、現在のところ、あくまで表面分子の１つと考えられている。 However, in recent years, there have been reports of various surface molecules that can isolate cancer stem cells other than CD133, and the surface molecules characteristic of stem cells may differ depending on each cancer cell. In addition, CD133 itself does not have a function of transforming cancer cells as stem cells and is currently considered to be one of surface molecules.
　本発明はがん幹細胞の単離方法、がん幹細胞の特性研究、がんの重篤度や再発性の鑑別方法に関するものである。 The present invention relates to a method for isolating cancer stem cells, a characteristic study of cancer stem cells, and a method for distinguishing cancer severity and recurrence.
　がん幹細胞の単離において、単独のCD markerによる現状のがん幹細胞の単離方法では、その純度に改善すべき余地がある。また、がん種によって、そのマーカーが変わることも、実際の臨床応用への困難さになっている。 In the isolation of cancer stem cells, there is room for improvement in the purity of current methods for isolation of cancer stem cells using a single CD marker. In addition, the change of the marker depending on the type of cancer also makes it difficult for actual clinical application.
　簡単なマーカーの組み合わせにより、より高純度のがん幹細胞の単離方法が求められており、更に簡便に誰にでもできる方法の提案が要請されている。 There is a demand for a method of isolating higher-purity cancer stem cells by combining simple markers, and a proposal for a method that can be easily performed by anyone is required.
　請求項１記載の発明は、
　細胞上の糖鎖構造の相違によってがん幹細胞を選別することを特徴とするがん幹細胞の単離方法である。 The invention described in claim 1
A method for isolating cancer stem cells, which comprises selecting cancer stem cells based on differences in sugar chain structures on the cells.
　請求項２記載の発明は、
　癌細胞の選別を行った後、当該選別した癌細胞について糖鎖構造に関する特徴付けを行い、細胞特性と糖鎖構造上の特徴を関連付けることにより、がん幹細胞を単離する為の分子決定を行うことを特徴とする請求項１記載のがん幹細胞の単離方法である。 The invention according to claim 2
After selecting the cancer cells, characterize the sugar chain structure of the selected cancer cells and correlate the cell characteristics with the characteristics of the sugar chain structure to make a molecular determination for isolating cancer stem cells. The method for isolating cancer stem cells according to claim 1, wherein the method is performed.
　請求項３記載の発明は、
　癌細胞の選別を行った後、高造腫瘍能、高転移能、高薬剤耐性能の中のいずれか一つ以上の特徴を有する細胞群を選別し、当該選別した細胞群について糖鎖構造に関する特徴付けを行い、細胞特性と糖鎖構造上の特徴を関連付けることにより、がん幹細胞を単離する為の分子決定を行うことを特徴とする請求項１記載のがん幹細胞の単離方法である。 The invention described in claim 3
After selecting cancer cells, a cell group having at least one of the characteristics of high tumorigenicity, high metastasis ability, and high drug resistance is selected, and the sugar chain structure of the selected cell group is selected. The method for isolating cancer stem cells according to claim 1, wherein molecular determination for isolating cancer stem cells is performed by characterizing and associating cell characteristics with features on the sugar chain structure. is there.
　請求項４記載の発明は、
　前記高造腫瘍能、高転移能、高薬剤耐性能の中のいずれか一つ以上の特徴を有する細胞群の選別は、培養細胞実験及び／又は動物実験により行うことを特徴とする請求項３記載のがん幹細胞の単離方法である。 The invention according to claim 4
4. The selection of a cell group having any one or more of the high tumorigenic potential, high metastatic potential, and high drug resistance is performed by a cultured cell experiment and / or an animal experiment. It is the isolation method of the cancer stem cell of description.
　請求項５記載の発明は、
　前記選別した細胞群についての糖鎖構造に関する特徴付けは糖鎖構造評価手段により行うことを特徴とする請求項３又は請求項４記載のがん幹細胞の単離方法である。 The invention according to claim 5
The method for isolating cancer stem cells according to claim 3 or 4, wherein the characterization of the sugar chain structure of the selected cell group is performed by a sugar chain structure evaluation means.
　請求項６記載の発明は、
　前記糖鎖構造評価手段は、レクチンアレイ、ＭＳ、ＨＰＬＣ、ウエスタンブロッティング、電機泳動の中のいずれかの手法、又は、これらの中の複数種の組み合わせによるものであることを特徴とする請求項５記載のがん幹細胞の単離方法である。 The invention described in claim 6
6. The sugar chain structure evaluation means is based on any one of a lectin array, MS, HPLC, Western blotting, and electrophoretic electrophoresis, or a combination of a plurality of them. It is the isolation method of the cancer stem cell of description.
　請求項７記載の発明は、
　前記癌細胞の選別は、ＣＤマーカー、又は、糖鎖の中のいずれか一種、又は、これらの中の複数種の組み合せを用いて行うことを特徴とする請求項２～請求項６のいずれか一項記載のがん幹細胞の単離方法である。 The invention described in claim 7
The cancer cell is selected using a CD marker, any one of sugar chains, or a combination of plural kinds thereof. A method for isolating cancer stem cells according to one item.
　なお、ここで「ＣＤマーカー、又は、糖鎖の中のいずれか一種、又は、これらの中の複数種の組み合せ」とは、ＣＤマーカー単独であって、一種類のＣＤマーカーあるいは複数種のＣＤマーカーの組み合わせによって前記癌細胞の選別を行う場合、糖鎖単独であって、一種類の糖鎖あるいは複数種の糖鎖の組み合わせによって前記癌細胞の選別を行う場合、一種類または複数種のＣＤマーカーと一種類または複数種の糖鎖との組み合わせによって前記癌細胞の選別を行う場合のいずれをも含むものである。 Here, “CD marker, or any one of sugar chains, or a combination of a plurality of them” is a CD marker alone, and one type of CD marker or a plurality of types of CD. When the cancer cells are selected by a combination of markers, the sugar chain alone is used, and when the cancer cells are selected by a combination of one type of sugar chain or a plurality of types of sugar chains, one type or a plurality of types of CDs are used. Any of the cases where the cancer cells are selected by a combination of a marker and one or more kinds of sugar chains is included.
　請求項８記載の発明は、
　前記癌細胞の選別は、抗ＣＤマーカー抗体、又は、レクチン、抗糖鎖抗体の中のいずれか一種、又は、これらの中の複数種の組み合せを用いて行うことを特徴とする請求項２～請求項６のいずれか一項記載のがん幹細胞の単離方法である。 The invention described in claim 8
The selection of the cancer cells is performed using any one of an anti-CD marker antibody, a lectin, and an anti-sugar chain antibody, or a combination of a plurality of these. It is the isolation method of the cancer stem cell as described in any one of Claim 6.
　請求項９記載の発明は、
　前記癌細胞の選別は、抗ＣＤマーカー抗体、又は、レクチン、抗糖鎖抗体の中のいずれか一種、又は、これらの中の複数種の組み合せを用いたカラム、アフィニティビーズを用いて行うことを特徴とする請求項２～請求項６のいずれか一項記載のがん幹細胞の単離方法である。 The invention according to claim 9
The selection of the cancer cells is performed using an anti-CD marker antibody, a lectin, an anti-glycan antibody, a column using a combination of a plurality of these, or affinity beads. The method for isolating cancer stem cells according to any one of claims 2 to 6, which is characterized by the following.
　なお、前記において、「抗ＣＤマーカー抗体、又は、レクチン、抗糖鎖抗体の中のいずれか一種、又は、これらの中の複数種の組み合せ」とは、抗ＣＤマーカー抗体単独であって、一種類の抗ＣＤマーカー抗体あるいは複数種の抗ＣＤマーカー抗体の組み合わせによって前記癌細胞の選別を行う場合、レクチン単独であって、一種類のレクチンあるいは複数種のレクチンの組み合わせによって前記癌細胞の選別を行う場合、抗糖鎖抗体単独であって、一種類の抗糖鎖抗体あるいは複数種の抗糖鎖抗体の組み合わせによって前記癌細胞の選別を行う場合、一種類または複数種の抗ＣＤマーカー抗体と一種類または複数種のレクチンとの組み合わせによって前記癌細胞の選別を行う場合、一種類または複数種の抗ＣＤマーカー抗体と一種類または複数種の抗糖鎖抗体との組み合わせによって前記癌細胞の選別を行う場合、一種類または複数種のレクチンと一種類または複数種の抗糖鎖抗体との組み合わせによって前記癌細胞の選別を行う場合、一種類または複数種の抗ＣＤマーカー抗体と一種類または複数種のレクチンと一種類または複数種の抗糖鎖抗体との組み合わせによって前記癌細胞の選別を行う場合のいずれをも含むものである。 In the above description, “an anti-CD marker antibody, or any one of lectins and anti-sugar chain antibodies, or a combination of plural types thereof” refers to an anti-CD marker antibody alone, When selecting the cancer cells by using a combination of different types of anti-CD marker antibodies or a plurality of types of anti-CD marker antibodies, the cancer cells can be selected by using a single lectin or a combination of multiple types of lectins. When performing the selection of the cancer cells by using only one type of anti-sugar chain antibody or a combination of a plurality of types of anti-sugar chain antibodies, When selecting the cancer cells by combining with one or more lectins, one or more anti-CD marker antibodies and one or When the cancer cells are selected by a combination with a plurality of types of anti-sugar chain antibodies, and when the cancer cells are selected by a combination of one type or a plurality of types of lectins and one or more types of anti-sugar chain antibodies. These include any of cases where the cancer cells are selected by a combination of one or more kinds of anti-CD marker antibodies, one or more kinds of lectins and one or more kinds of anti-sugar chain antibodies.
　請求項１０記載の発明は、
　前記レクチン及び、抗糖鎖抗体は、前記特徴付けが行われた糖鎖構造を認識するレクチン及び、抗糖鎖抗体であることを特徴とする請求項８又は９記載のがん幹細胞の単離方法である。 The invention according to claim 10 provides:
10. The isolation of cancer stem cells according to claim 8 or 9, wherein the lectin and the anti-sugar chain antibody are a lectin that recognizes the characterized sugar chain structure and an anti-sugar chain antibody. Is the method.
　請求項１１記載の発明は、
　前記抗糖鎖抗体が、天然の抗体、リコンビナント抗体、抗体の一部、アプタマーを含む人工抗体、ヌクレオチド、人工構造体のいずれかであることを特徴とする請求項１０記載のがん幹細胞の単離方法である。 The invention according to claim 11
11. The cancer stem cell unit according to claim 10, wherein the anti-sugar chain antibody is any one of a natural antibody, a recombinant antibody, a part of the antibody, an artificial antibody including an aptamer, a nucleotide, and an artificial structure. It is a separation method.
　請求項１２記載の発明は、
　ＣＤマーカーとしてＣＤ１３３又はＣＤ１３あるいはこれらの組み合わせを用い、糖鎖としてα２，６シアル酸、又は、α２，３シアル酸あるいはこれらの組み合せを用いることを特徴とする請求項７記載のがん幹細胞の単離方法である。 The invention according to claim 12
The cancer stem cell unit according to claim 7, wherein CD133 or CD13 or a combination thereof is used as a CD marker, and α2,6 sialic acid, α2,3 sialic acid or a combination thereof is used as a sugar chain. It is a separation method.
　請求項１３記載の発明は、
　がん幹細胞の単離が、肝臓癌幹細胞におけるがん幹細胞の単離であることを特徴とする請求項１２記載のがん幹細胞の単離方法である。 The invention according to claim 13
The method for isolating cancer stem cells according to claim 12, wherein the isolation of cancer stem cells is isolation of cancer stem cells in liver cancer stem cells.
　請求項１４記載の発明は、
　抗ＣＤマーカー抗体として抗ＣＤ１３３抗体または抗ＣＤ１３抗体あるいはこれらの組み合わせを用い、レクチンとしてα２，６シアル酸認識レクチン又はα２，３シアル酸認識レクチンあるいはこれらの組み合わせを用い、抗糖鎖抗体として抗α２，６シアル酸抗体または抗α２，３シアル酸抗体あるいはこれらの組み合わせを用いることを特徴とする請求項８～請求項１１のいずれか一項記載のがん幹細胞の単離方法である。 The invention according to claim 14
Anti-CD133 antibody or anti-CD13 antibody or a combination thereof is used as an anti-CD marker antibody, α2,6-sialic acid recognition lectin or α2,3-sialic acid recognition lectin or a combination thereof is used as a lectin, and anti-α2 is used as an anti-sugar chain antibody. The method for isolating cancer stem cells according to any one of claims 8 to 11, wherein an antibody, 6, sialic acid antibody, anti-α2,3-sialic acid antibody, or a combination thereof is used.
　請求項１５記載の発明は、
　がん幹細胞の単離が、肝臓癌幹細胞におけるがん幹細胞の単離であることを特徴とする請求項１４記載のがん幹細胞の単離方法である。 The invention according to claim 15 is:
The method for isolating cancer stem cells according to claim 14, wherein the isolation of cancer stem cells is isolation of cancer stem cells in liver cancer stem cells.
　請求項１６記載の発明は、
　前記α２，６シアル酸認識レクチンが、ＳＮＡ、ＳＳＡ、ＴＪＡ－Ｉのいずれかであることを特徴とする請求項１４又は１５記載のがん幹細胞の単離方法である。 The invention according to claim 16
The method for isolating cancer stem cells according to claim 14 or 15, wherein the α2,6-sialic acid-recognizing lectin is one of SNA, SSA, and TJA-I.
　なお、請求項１４、１５記載の発明のように、全てのα２，６シアル酸認識レクチン、全ての抗α２，６シアル酸抗体を用いても、同様の効果を得ることができるものであり、本発明において、前記α２，６シアル酸認識レクチンが、ＳＮＡ、ＳＳＡ、ＴＪＡ－Ｉのいずれかに限定されるものではない。 In addition, as in the inventions of claims 14 and 15, even if all α2,6-sialic acid recognition lectins and all anti-α2,6-sialic acid antibodies are used, the same effect can be obtained. In the present invention, the α2,6-sialic acid recognition lectin is not limited to any one of SNA, SSA, and TJA-I.
　請求項１７記載の発明は、
　前記α２，３シアル酸認識レクチンが、ＭＡＬ－Ｉであることを特徴とする請求項１４又は１５記載のがん幹細胞の単離方法である。 The invention described in claim 17
The method for isolating cancer stem cells according to claim 14 or 15, wherein the α2,3-sialic acid-recognizing lectin is MAL-I.
　なお、請求項１４、１５記載の発明のように、全てのα２，３シアル酸認識レクチン、全ての抗α２，３シアル酸抗体を用いても、同様の効果を得ることができるものであり、本発明において、前記α２，３シアル酸認識レクチンが、ＭＡＬ－Ｉに限定されるものではない。 In addition, as in the inventions of claims 14 and 15, even if all α2,3-sialic acid-recognizing lectins and all anti-α2,3-sialic acid antibodies are used, the same effect can be obtained. In the present invention, the α2,3-sialic acid recognition lectin is not limited to MAL-I.
　請求項１８記載の発明は、
　遺伝子発現レベルによる同定方法を用いてがん幹細胞の同定を行うことを特徴とするがん幹細胞の同定方法である。 The invention according to claim 18
A cancer stem cell identification method comprising identifying cancer stem cells using an identification method based on gene expression levels.
　請求項１９記載の発明は、
　前記遺伝子の発現レベルをＰＣＲにより増幅させた際のコピー数によって定量することを特徴とする請求項１８記載のがん幹細胞の同定方法である。 The invention according to claim 19
19. The method for identifying cancer stem cells according to claim 18, wherein the expression level of the gene is quantified by the copy number when amplified by PCR.
　請求項２０記載の発明は、
　前記遺伝子を糖転移酵素遺伝子とすることを特徴とする請求項１８又は１９記載のがん幹細胞の同定方法である。 The invention according to claim 20 provides
The method for identifying cancer stem cells according to claim 18 or 19, wherein the gene is a glycosyltransferase gene.
　請求項２１記載の発明は、
　前記糖転移酵素遺伝子が、ＳＴ６ＧＡＬＮＡＣ１、ＧＣＮＴ３、ＭＧＡＴ５の中のいずれか一種、またはこれらの中の２種類以上の組み合わせであることを特徴とする請求項２０記載のがん幹細胞の同定方法である。 The invention according to claim 21
21. The method for identifying cancer stem cells according to claim 20, wherein the glycosyltransferase gene is any one of ST6GALNAC1, GCNT3, and MGAT5, or a combination of two or more thereof.
　請求項２２記載の発明は、
　がん幹細胞への目的物送達の対象として、ＣＤマーカー及び糖鎖の両方を提示している細胞を用いることを特徴とするがん幹細胞の選別方法である。 The invention according to claim 22
A method for selecting cancer stem cells, which comprises using cells presenting both a CD marker and a sugar chain as a target for delivery of an object to cancer stem cells.
　請求項２３記載の発明は、
　ＤＤＳ（薬物送達システム）分子の表面に提示するターゲット用分子として、抗ＣＤ抗体及び糖鎖認識分子の両方を提示していることを特徴とするがん幹細胞の選別方法である。 The invention described in claim 23
A cancer stem cell selection method characterized by presenting both an anti-CD antibody and a sugar chain recognition molecule as a target molecule to be presented on the surface of a DDS (drug delivery system) molecule.
　請求項２４記載の発明は、
　前記ＣＤマーカーがＣＤ１３３、前記糖鎖がシアル酸であることを特徴とする請求項２２記載のがん幹細胞の選別方法である。 The invention according to claim 24 provides
23. The method for selecting cancer stem cells according to claim 22, wherein the CD marker is CD133 and the sugar chain is sialic acid.
　請求項２５記載の発明は、
　前記抗ＣＤ抗体が、抗ＣＤ１３３抗体であることを特徴とする請求項２３記載のがん幹細胞の選別方法である。 The invention according to claim 25 provides
The method for selecting cancer stem cells according to claim 23, wherein the anti-CD antibody is an anti-CD133 antibody.
　請求項２６記載の発明は、
　前記糖鎖認識分子がシアル酸認識分子であることを特徴とする請求項２３記載のがん幹細胞の選別方法である。 The invention according to claim 26 provides
The method for selecting cancer stem cells according to claim 23, wherein the sugar chain recognition molecule is a sialic acid recognition molecule.
　請求項２７記載の発明は、
　前記シアル酸認識分子が、ＳＮＡ、ＳＳＡ、 ＴＪＡ－Ｉ、ＭＡＬ－Ｉ、ＡＣＧ、ＡＢＡ、ＡＣＡ、Ｊａｃａｌｉｎ、ＭＡＨのいずれかであることを特徴とする請求項２６記載のがん幹細胞の選別方法である。 The invention according to claim 27 provides
27. The method for sorting cancer stem cells according to claim 26, wherein the sialic acid recognition molecule is any one of SNA, SSA, TJA-I, MAL-I, ACG, ABA, ACA, Jacalin, and MAH. is there.
　なお、前記のＳＮＡなどのレクチンに限定されるものではなく、その他のシアル酸認識レクチンは全て採用可能である。 It should be noted that the present invention is not limited to the above lectins such as SNA, and all other sialic acid recognizing lectins can be employed.
　請求項２８記載の発明は、
　前記糖鎖認識分子がシアル酸認識抗体であることを特徴とする請求項２３記載のがん幹細胞の選別方法である。 The invention according to claim 28 provides
24. The method for selecting cancer stem cells according to claim 23, wherein the sugar chain recognition molecule is a sialic acid recognition antibody.
　請求項２９記載の発明は、
　前記抗ＣＤ抗体が、フルボディの抗体、１本鎖抗体、Ｆａｂフラグメント、部分抗体（すなわち、抗体の一部）、キメラ抗体を含む人工抗体及び、アプタマー、mimic分子を含むことを特徴とする請求項２３記載のがん幹細胞の選別方法である。 The invention according to claim 29 provides
The anti-CD antibody includes a full-body antibody, a single chain antibody, a Fab fragment, a partial antibody (ie, a part of an antibody), an artificial antibody including a chimeric antibody, an aptamer, and a mimic molecule. Item 24. The method for selecting cancer stem cells according to Item 23.
　前記のmimic分子としては、例えば、mimic RNA 等があげられる。 Examples of the mimic molecule include mimic RNA.
　請求項３０記載の発明は、
　前記抗ＣＤ抗体が、wild type の抗体及び、人工的に作られたものを含むことを特徴とする請求項２３記載のがん幹細胞の選別方法である。 The invention according to claim 30 provides
24. The method for selecting cancer stem cells according to claim 23, wherein the anti-CD antibody comprises a wild type antibody and an artificially produced antibody.
　前記の人工的に作られたものとしては、例えば、phage display 等の手法による人工的に作られたものが含まれる。 The above-mentioned artificially created one includes, for example, an artificially produced one by a method such as “phage display”.
　請求項３１記載の発明は、
　前記抗ＣＤ１３３抗体が、ペプチド及び、ヌクレオチドを対象とすることを特徴とする請求項２５記載のがん幹細胞の選別方法である。ＣＤ１３３を認識する以上、ペプチドやヌクレオチド、その他の分子も対象となるからである。 The invention of claim 31 is
26. The method for selecting cancer stem cells according to claim 25, wherein the anti-CD133 antibody targets peptides and nucleotides. This is because as long as it recognizes CD133, peptides, nucleotides, and other molecules are also targeted.
　請求項３２記載の発明は、
　前記シアル酸認識分子がフルボディの抗体、１本鎖抗体、Ｆａｂフラグメント、部分抗体（すなわち、抗体の一部）、キメラ抗体を含む人工抗体及び、アプタマー、mimic分子を含むことを特徴とする請求項２６記載のがん幹細胞の選別方法である。 The invention according to claim 32 provides
The sialic acid recognition molecule comprises a full-body antibody, a single chain antibody, a Fab fragment, a partial antibody (ie, a part of an antibody), an artificial antibody including a chimeric antibody, an aptamer, and a mimic molecule. Item 27. The method for selecting cancer stem cells according to Item 26.
　前記のmimic分子としては、例えば、mimic RNA 等があげられる。 Examples of the mimic molecule include mimic RNA.
　請求項３３記載の発明は、
　前記シアル酸認識分子が、wild type の抗体及び、人工的に作られたものを含むことを特徴とする請求項２６記載のがん幹細胞の選別方法である。 The invention of claim 33 is
27. The method for selecting cancer stem cells according to claim 26, wherein the sialic acid recognition molecule includes a wild type antibody and an artificially produced antibody.
　前記の人工的に作られたものとしては、例えば、phage display 等の手法による人工的に作られたものが含まれる。 The above-mentioned artificially created one includes, for example, an artificially produced one by a method such as “phage display”.
　なお、レクチンにおいてはwild type のレクチンだけでなく、リコンビナント化したものや、人工的に手を加えたものも含む。 In addition, lectins include not only wild type レ lectins, but also recombinant and artificially modified lectins.
　請求項３４記載の発明は、
　前記シアル酸認識分子が、ペプチド及び、ヌクレオチドを対象とすることを特徴とする請求項２６記載のがん幹細胞の選別方法である。シアル酸を認識する以上、ペプチドやヌクレオチド、その他の分子も対象となるからである。 The invention according to claim 34 provides
27. The method for selecting cancer stem cells according to claim 26, wherein the sialic acid recognition molecule targets peptides and nucleotides. This is because as long as sialic acid is recognized, peptides, nucleotides, and other molecules are also targeted.
　請求項３５記載の発明は、
　がん治療の対象を、ＣＤマーカー及び糖鎖の両方を提示している細胞とすることを特徴とするがんの治療方法である。 The invention of claim 35 is
The cancer treatment method is characterized in that a cancer treatment target is a cell presenting both a CD marker and a sugar chain.
　請求項３６記載の発明は、
　前記ＣＤマーカーＣＤ１３３で、前記糖鎖がシアル酸であることを特徴とする請求項３５記載のがんの治療方法である。 The invention according to claim 36 provides
36. The cancer treatment method according to claim 35, wherein the sugar chain is sialic acid in the CD marker CD133.
　請求項３７記載の発明は、
　ＤＤＳ（薬物送達システム）で使用される分子に、抗がん剤、RNAi、あるいは、遺伝子発現を阻害する分子をのせてがん幹細胞群に送達することを特徴とするがんの治療方法である。 The invention of claim 37 is
A method for treating cancer characterized in that an anticancer agent, RNAi, or a molecule that inhibits gene expression is placed on a molecule used in a DDS (drug delivery system) and delivered to a cancer stem cell group. .
　ここで、抗がん剤、RNAi、あるいは、遺伝子発現を阻害する分子をのせてがん幹細胞群に送達させる分子は、ＤＤＳ（薬物送達システム）技術として使用される分子であればいずれであっても使用可能である。例えば、リポソームを挙げることができる。 Here, the molecule to be delivered to the cancer stem cell group on the anticancer drug, RNAi, or molecule that inhibits gene expression is any molecule that can be used as a DDS (drug delivery system) technology. Can also be used. For example, a liposome can be mentioned.
　請求項３８記載の発明は、
　前記RNAiが、siRNA または、shRNAであるることを特徴とする請求項３７記載のがんの治療方法である。 The invention according to claim 38 provides
38. The cancer treatment method according to claim 37, wherein the RNAi is siRNA or shRNA.
　請求項３９記載の発明は、
　前記RNAi、あるいは、前記遺伝子発現を阻害する分子の阻害対象が、ST6GALNAC1、GCNT3 、MGAT5のいずれか、またはこれらの中の２種類以上の組み合わせであることを特徴とする請求項３７記載のがんの治療方法である。 The invention according to claim 39 provides
38. The cancer according to claim 37, wherein the RNAi or the inhibition target of the molecule that inhibits gene expression is ST6GALNAC1, GCNT3, MGAT5, or a combination of two or more of these. This is a treatment method.
　請求項４０記載の発明は、
　治療対象を、ST6GALNAC1、GCNT3、又は、MGAT5 の発現にかかわる転写因子を抑制する薬剤もしくは自然物質とすることを特徴とする請求項３７記載のがんの治療方法である。 The invention according to claim 40 provides
38. The method of treating cancer according to claim 37, wherein the treatment target is a drug or natural substance that suppresses a transcription factor involved in the expression of ST6GALNAC1, GCNT3, or MGAT5.
　請求項４１記載の発明は、
　ST6GALNAC1、GCNT3、又は、MGAT5 の働きを阻害することを特徴とする請求項３７記載のがんの治療方法である。 The invention of claim 41 is
38. The cancer treatment method according to claim 37, wherein the function of ST6GALNAC1, GCNT3, or MGAT5 is inhibited.
　本発明によれば、将来的に従来のFACSの技術だけでなく、遺伝子発現レベルで細胞の形質を決定できる方法が開発されれば、ST6GALNAC1、GCNT3、またはMGAT5 を対象にCSC を同定する方法へと応用できる。 According to the present invention, if a method capable of determining a cell trait at the gene expression level as well as the conventional FACS technique in the future is developed, a method for identifying CSC in ST6GALNAC1, GCNT3, or MGAT5 And can be applied.
　この発明によれば、高純度ながん幹細胞の選別方法を提供することができる。また、がん幹細胞を単離するための分子決定におけるプロセスに関するがん幹細胞の単離方法を提供することができる。 According to this invention, it is possible to provide a method for selecting cancer stem cells with high purity. Moreover, the isolation method of the cancer stem cell regarding the process in the molecular determination for isolating a cancer stem cell can be provided.
　本発明により、高純度のがん幹細胞の単離方法を提供することが可能になる。すなわち、本発明によれば、がん幹細胞の特性研究を進歩させると同時に、がん幹細胞を死滅させる手段の開発を飛躍的に進歩させることが可能になる。これは癌根治というこれまで成しえなかった大きなテーマを現実のものにする、非常に大きなブレークスルーとなるものである。 The present invention makes it possible to provide a method for isolating high-purity cancer stem cells. That is, according to the present invention, it becomes possible to make a dramatic progress in the development of means for killing cancer stem cells while at the same time advancing research on the characteristics of cancer stem cells. This is a very big breakthrough that brings the realization of a major theme that has never been possible to cure cancer.
ＣＤ１３３陽性／シアル酸認識レクチン陰性と、ＣＤ１３３陽性／シアル酸認識レクチン陽性の癌細胞群に対するレクチンマイクロアレイによる解析結果を示す図。The figure which shows the analysis result by a lectin microarray with respect to a cancer cell group of CD133 positive / sialic acid recognition lectin negative and CD133 positive / sialic acid recognition lectin positive. 癌細胞接種後のマウスの写真であって、ＣＤ１３３陽性、レクチンＸ陽性がん細胞の腫瘍形成能に関する実験結果を表す図。It is a photograph of the mouse | mouth after cancer cell inoculation, Comprising: The figure showing the experimental result regarding the tumor formation ability of CD133 positive and a lectin X positive cancer cell. 図２に示す実験における腫瘤がみられた数を表す表。The table | surface showing the number by which the mass in the experiment shown in FIG. 2 was seen.
（糖鎖構造の相違によるがん幹細胞の選別、単離方法）
　本願の発明者等の研究により、同じＣＤ１３３によって単離された細胞群自体もヘテロな集団であり、更に分画することで、ある種の画分に造腫瘍形成能が高い細胞集団が集中していることがわかってきた。この研究成果を利用することで、より高い純度のがん幹細胞の単離方法を発明したものである。 (Selection and isolation of cancer stem cells based on differences in sugar chain structure)
According to the study by the inventors of the present application, the cell group itself isolated by the same CD133 is also a heterogeneous population, and further fractionation concentrates a cell population having high tumorigenic potential in a certain fraction. I understand that By utilizing this research result, we have invented a method for isolating higher-purity cancer stem cells.
　本発明が提案するがん幹細胞の単離方法においては、ＣＤマーカーなどによって癌細胞選別を行い、選別した細胞の中から培養細胞実験や動物実験などにより、造腫瘍能、薬剤耐性能などの中のいずれか一つ以上が高い細胞群、すなわち、悪性度の高い細胞群の選別を行う。 In the method of isolating cancer stem cells proposed by the present invention, cancer cells are selected using a CD marker or the like, and from among the selected cells, a cell culture experiment or an animal experiment is performed. A cell group having one or more of the above, that is, a cell group having a high malignancy is selected.
　そして、その選別したグループについて、レクチンアレイ、ＭＳ（質量分析法）、ＨＰＬＣ（高速液体クロマトグラフィー）、ウエスタンブロッティング、電機泳動等の糖鎖構造評価手段により、そのグループの糖鎖構造に関する特徴付けを行う。特徴的な糖鎖構造を利用して更にグループ分けすることで、前述した、造腫瘍能、薬剤耐性能などが高いグループを高度に絞り込むと同時に、糖鎖構造上の特徴を特定するのである。 The selected group is characterized with respect to the glycan structure of the group by glycan structure evaluation means such as lectin array, MS (mass spectrometry), HPLC (high performance liquid chromatography), western blotting, and electrophoretic electrophoresis. Do. By further grouping using the characteristic sugar chain structure, the above-mentioned group having high tumorigenic ability and drug resistance is highly narrowed down and at the same time, the characteristics on the sugar chain structure are specified.
　このプロセスにより造腫瘍能や薬剤耐性（この時点では幾つかのグループの特徴が特定されるので、転移性や分化度等の特徴付けも行われている）等の特性の異なるグループに分けられると同時に、その糖鎖構造上の特徴が付加されるため、あるグループの細胞を選別する為の抗体やレクチンのセットが決定されることになる。 When this process is divided into groups with different characteristics such as tumorigenicity and drug resistance (characteristics of several groups are specified at this point, metastaticity and differentiation degree are also characterized) At the same time, since the features of the sugar chain structure are added, a set of antibodies and lectins for selecting a certain group of cells is determined.
　こうして決定された抗体やレクチンを使用して、カラムやアフィニティビーズを構築することで、簡便に誰にでも高純度のがん幹細胞を単離することが可能となるのである。 By constructing columns and affinity beads using the antibodies and lectins thus determined, it becomes possible for anyone to easily isolate high-purity cancer stem cells.
　ＣＤ１３３とＣＤ１３のダブル陽性細胞は、極めて生物学的悪性度の高い、がん幹細胞としての形質をもつ(CD13 is a therapeutic target in human liver cancer stem cells. J. Clin. Invest. 2010 in press)。 CD133 and CD13 double positive cells are highly biologically malignant and have a trait as a cancer stem cell (CD13 is a therapeutic target in human liver cancer stem cells. J. Clin. Invest. 2010 in press).
　この細胞と親株を、レクチンアレイを用いて網羅的に糖鎖構造を解析したところ、α２，６シアル酸認識レクチンあるいはα２，３シアル酸認識レクチンとの親和性が高いことがわかった（図１）。 When the sugar chain structure of this cell and the parent strain was comprehensively analyzed using a lectin array, it was found that the affinity to α2,6-sialic acid recognition lectin or α2,3-sialic acid recognition lectin was high (FIG. 1). ).
　次に、ＣＤ１３３とＳＳＡ(Sambucus sieboldiana)を用いて、細胞株からがん幹細胞の単離することに成功した。高純度のがん幹細胞群であることの確認は、マウスにより造腫瘍能を観察することによって行った（図２）。 Next, using CD133 and SSA (Sambucus sieboldiana), we succeeded in isolating cancer stem cells from cell lines. Confirmation of the high-purity cancer stem cell group was performed by observing tumorigenicity with mice (FIG. 2).
　その結果ＣＤ１３３陽性／ＳＳＡ陰性の細胞に比べて、ＣＤ１３３陽性／ＳＳＡ陽性の細胞の方が、著しく造腫瘍能が高いことが分かった（図２及び、図３に示す表１）。 As a result, it was found that the CD133 positive / SSA positive cells had significantly higher tumorigenicity than the CD133 positive / SSA negative cells (Table 1 shown in FIG. 2 and FIG. 3).
　尚、本実施例においては、２，６シアル酸認識レクチンとしてＳＳＡを、α２，３シアル酸認識レクチンとしてＭＡＬ－Ｉ(Maackia amrensis Lectin Ｉ)を用いた。 In this example, SSA was used as the 2,6-sialic acid recognition lectin, and MAL-I (Maackia amrensis Lectin I) was used as the α2,3-sialic acid recognition lectin.
（がん幹細胞の遺伝子発現プロファイル）
　がん幹細胞ｃＤＮＡ合成
　本願の特許請求の範囲の請求項２１記載の方法で調製したがん幹細胞画分試料を、ＰＢＳ（日水製薬社製ダルベッコＰＢＳ（－））で洗浄後に市販ＲＮＡ抽出キット（キアゲン社製 RNeasy plus miniキット）を用いて抽出物をがん幹細胞トータルＲＮＡとした。 (Gene expression profile of cancer stem cells)
Cancer Stem Cell cDNA Synthesis A cancer stem cell fraction sample prepared by the method according to claim 21 of the present application is washed with PBS (Dulbecco PBS (-) manufactured by Nissui Pharmaceutical Co., Ltd.) and then a commercially available RNA extraction kit ( The extract was used as cancer stem cell total RNA using Qiagen RNeasy plus mini kit).
　がん幹細胞トータルＲＮＡ１μｇを出発材料として、市販ｃＤＮＡ合成キット（キアゲン社製　QuantitectRTキット）を用いて調製したｃＤＮＡをがん幹細胞ｃＤＮＡとした。 Cancer stem cell cDNA was prepared using 1 μg of cancer stem cell total RNA as a starting material and using a commercially available cDNA synthesis kit (Quantitect RT kit manufactured by Qiagen).
　なお、前記のがん幹細胞画分試料における糖転移酵素遺伝子は、ＳＴ６ＧＡＬＮＡＣ１と、ＧＣＮＴ３と、ＭＧＡＴ５とを組み合わせたものを使用した。 The glycosyltransferase gene in the cancer stem cell fraction sample was a combination of ST6GALNAC1, GCNT3, and MGAT5.
　糖転移酵素遺伝子発現レベルの定量ＰＣＲ解析
　定量ＰＣＲ法を用いてがん幹細胞ｃＤＮＡについて糖転移酵素遺伝子の発現量を定量した。定量方法については非特許文献４に記載した定量ＰＣＲアレイを用いる方法に従った。 Quantitative PCR analysis of glycosyltransferase gene expression level The amount of glycosyltransferase gene expression was quantified in cancer stem cell cDNA using a quantitative PCR method. Regarding the quantification method, the method using a quantitative PCR array described in Non-Patent Document 4 was followed.
　がん幹細胞ｃＤＮＡを鋳型として用いて、Eurogentec社製のqPCRQuickGoldStar Mastermix PlusをＰＣＲ酵素として用いた。 The cancer stem cell cDNA was used as a template and qPCRQuickGoldStar Mastermix Plus from Eurogentec was used as a PCR enzyme.
　なお、後述のように、ＳＳＡ陽性がん幹細胞で顕著に発現が亢進していた遺伝子は３種あり、定量ＰＣアレイに搭載されているこれらのＰＣＲプライマー及びプローブは以下の通りである。 As described later, there are three types of genes whose expression was remarkably enhanced in SSA positive cancer stem cells, and these PCR primers and probes mounted on the quantitative PC array are as follows.
　ＳＴ６ＧＡＬＮＡＣ１遺伝子転写産物特異的検出セット試薬（Ｆプライマー；5’-ggtgcccactggaggata-３’（配列番号×１）、Ｒプライマー；5’-gaagccataagcactcacctg-３’（配列番号×２）、TaqManプローブ；ctggcc（配列番号×３）。 ST6GALNAC1 gene transcript-specific detection set reagent (F primer; 5'-ggtgcccactggaggata-3 '(SEQ ID NO: 1), R primer; 5'-gaagccataagcactcacctg-3' (SEQ ID NO: 2), TaqMan probe: ctggcc (sequence) Number x 3).
　ＧＣＮＴ３遺伝子転写産物特異的検出セット試薬（Ｆプライマー；5’-atccctaagcaggagagaagc-３’（配列番号×４）、Ｒプライマー；5’-gtggagaccgagcacagg-３（配列番号×５）、TaqManプローブ；ctcctcca（配列番号×６）。 GCNT3 gene transcript-specific detection set reagent (F primer; 5'-atccctaagcaggagagaagc-3 '(SEQ ID NO: 4), R primer: 5'-gtggagaccgagcacagg-3 (SEQ ID NO: 5), TaqMan probe: ctcctcca (SEQ ID NO: × 6).
　ＭＧＡＴ５遺伝子転写産物特異的検出セット試薬（Ｆプライマー；5’-ggggatgctacagagaatcaa-３’（配列番号×７）、Ｒプライマー；5’-gggtggccacatcacttg-３（配列番号×８）、TaqManプローブ；cttctgcc（配列番号×９）。 MGAT5 gene transcript-specific detection set reagent (F primer; 5'-ggggatgctacagagaatcaa-3 '(SEQ ID NO: 7), R primer: 5'-gggtggccacatcacttg-3 (SEQ ID NO: 8), TaqMan probe: cttctgcc (SEQ ID NO: × 9).
　がん幹細胞ｃＤＮＡ試料溶液にDEPC Treated Water（和光純薬製）を加えて２ｍＬとし、さらにＰＣＲ酵素（Eurogentec社製）２ｍＬを加えたものをＰＣＲ反応液とした。 DEPC 幹 Treated Water (Wako Pure Chemical Industries) was added to the cancer stem cell cDNA sample solution to make 2 mL, and PCR enzyme (Eurogentec) 2 mL was used as the PCR reaction solution.
　遺伝子転写産物特異的検出セット試薬が配置してある定量ＰＣＲアレイ用測定プレートの各ウェルにＰＣＲ反応液７．５Ｌずつを文注した後に混和し、シール材で密封した後に遠心分離機を用いて１５００ｒｐｍで反応ウェル内の反応液をスピンダウンしてからＰＣＲ反応測定装置（Roche社製　LighCycler480）にセットして定量ＰＣＲアレイ反応サイクルプログラムによって測定した。定量ＰＣＲアレイ反応サイクルプログラムは、５０℃／２分、９５℃／３分の後に、９５℃／１５秒、６０℃／１分２０秒を１サイクルとして５５回繰返した。検出は測定装置付属のソフトウェアで各資料中の標的遺伝子転写産物に由来する蛍光強度の変化を測定した後に、２次微分極大法によって数値化を施した。 After 7.5 L of PCR reaction solution is poured into each well of the measurement plate for quantitative PCR array where the gene transcript-specific detection set reagent is placed, mix, seal with a sealant, and then use a centrifuge. The reaction solution in the reaction well was spun down at 1500 rpm and then set in a PCR reaction measuring device (LighCycler 480 manufactured by Roche) and measured by a quantitative PCR array reaction cycle program. The quantitative PCR array reaction cycle program was repeated 55 times, with 50 ° C./2 minutes and 95 ° C./3 minutes followed by 95 ° C./15 seconds and 60 ° C./1 minute 20 seconds. For the detection, the change in fluorescence intensity derived from the target gene transcript in each sample was measured with software attached to the measurement device, and then numericalization was performed by the second derivative maximum method.
　測定結果の数値は別途測定した標準試料をもって作成した検量線にあてはめることで、測定資料中の標的遺伝子転写産物のコピー数として算出した。 The numerical value of the measurement result was calculated as the copy number of the target gene transcript in the measurement data by applying it to a calibration curve prepared with a separately measured standard sample.
　ＳＳＡ陽性がん幹細胞における糖転位酵素遺伝子の発現レベル
　ＳＳＡ陽性がん幹細胞画分試料について測定した結果、３種類の糖転位酵素遺伝子（ＳＴ６ＧＡＬＮＡＣ、ＧＣＮＴ３及び、ＭＧＡＴ５）で発現レベルの顕著な更新が認められた。発現レベルをトータルＲＮＡ１．８８ナノグラム中に含まれる転写産物のコピー数として表したのが表１である。
Figure JPOXMLDOC01-appb-T000001
 Expression level of glycosyltransferase gene in SSA-positive cancer stem cells As a result of measuring the SSA-positive cancer stem cell fraction sample, a marked update of the expression level was recognized in three types of glycosyltransferase genes (ST6GALNAC, GCNT3 and MGAT5) It was. Table 1 shows the expression level as the copy number of the transcript contained in 1.88 nanograms of total RNA.
Figure JPOXMLDOC01-appb-T000001
　これらの結果からＳＳＡ陽性がん幹細胞において、これらの遺伝子産物がＳＳＡレクチンに結合し得る糖鎖の生合成を担っていることが示された。 These results indicate that these gene products are responsible for the biosynthesis of sugar chains that can bind to SSA lectins in SSA positive cancer stem cells.
　以上、添付図面を参照して本発明の好ましい実施形態を説明したが、本発明はかかる実施形態に限定されるものではなく、特許請求の範囲の記載から把握される技術的範囲において種々の形態に変更可能である。 The preferred embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to such embodiments, and various forms are possible within the technical scope grasped from the description of the claims. Can be changed.

Claims (41)

  1. 　細胞上の糖鎖構造の相違によってがん幹細胞を選別することを特徴とするがん幹細胞の単離方法。 A method for isolating cancer stem cells, which comprises selecting cancer stem cells based on the difference in sugar chain structure on the cells.
  2. 　癌細胞の選別を行った後、当該選別した癌細胞について糖鎖構造に関する特徴付けを行い、細胞特性と糖鎖構造上の特徴を関連付けることにより、がん幹細胞を単離する為の分子決定を行うことを特徴とする請求項１記載のがん幹細胞の単離方法。 After selecting the cancer cells, characterize the sugar chain structure of the selected cancer cells and correlate the cell characteristics with the characteristics of the sugar chain structure to make a molecular determination for isolating cancer stem cells. The method for isolating cancer stem cells according to claim 1, wherein the method is performed.
  3. 　癌細胞の選別を行った後、高造腫瘍能、高転移能、高薬剤耐性能の中のいずれか一つ以上の特徴を有する細胞群を選別し、当該選別した細胞群について糖鎖構造に関する特徴付けを行い、細胞特性と糖鎖構造上の特徴を関連付けることにより、がん幹細胞を単離する為の分子決定を行うことを特徴とする請求項１記載のがん幹細胞の単離方法。 After selecting cancer cells, a cell group having at least one of the characteristics of high tumorigenicity, high metastasis ability, and high drug resistance is selected, and the sugar chain structure of the selected cell group is selected. The method for isolating cancer stem cells according to claim 1, wherein molecular determination for isolating cancer stem cells is performed by characterizing and associating cell characteristics with features on the sugar chain structure.
  4. 　前記高造腫瘍能、高転移能、高薬剤耐性能の中のいずれか一つ以上の特徴を有する細胞群の選別は、培養細胞実験及び／又は動物実験により行うことを特徴とする請求項３記載のがん幹細胞の単離方法。 4. The selection of a cell group having any one or more of the high tumorigenic potential, high metastatic potential, and high drug resistance is performed by a cultured cell experiment and / or an animal experiment. The method for isolating a cancer stem cell as described.
  5. 　前記選別した細胞群についての糖鎖構造に関する特徴付けは糖鎖構造評価手段により行うことを特徴とする請求項３又は請求項４記載のがん幹細胞の単離方法。 The method for isolating cancer stem cells according to claim 3 or 4, wherein the characterization of the sugar chain structure of the selected cell group is performed by a sugar chain structure evaluation means.
  6. 　前記糖鎖構造評価手段は、レクチンアレイ、ＭＳ、ＨＰＬＣ、ウエスタンブロッティング、電気泳動の中のいずれかの手法、又は、これらの中の複数種の組み合わせによるものであることを特徴とする請求項５記載のがん幹細胞の単離方法。 6. The sugar chain structure evaluation means is based on any one of a lectin array, MS, HPLC, Western blotting, electrophoresis, or a combination of plural kinds thereof. The method for isolating a cancer stem cell as described.
  7. 　前記癌細胞の選別は、ＣＤマーカー、又は、糖鎖の中のいずれか一種、又は、これらの中の複数種の組み合わせを用いて行うことを特徴とする請求項２～請求項６のいずれか一項記載のがん幹細胞の単離方法。 The cancer cell is selected using any one of a CD marker, a sugar chain, or a combination of a plurality of these among cancer markers. The method for isolating cancer stem cells according to one item.
  8. 　前記癌細胞の選別は、抗ＣＤマーカー抗体、又は、レクチン、抗糖鎖抗体の中のいずれか一種、又は、これらの中の複数種の組み合わせを用いて行うことを特徴とする請求項２～請求項６のいずれか一項記載のがん幹細胞の単離方法。 The selection of the cancer cells is performed using any one of an anti-CD marker antibody, a lectin, and an anti-sugar chain antibody, or a combination of a plurality of these. The method for isolating cancer stem cells according to claim 6.
  9. 　前記癌細胞の選別は、抗ＣＤマーカー抗体、又は、レクチン、抗糖鎖抗体の中のいずれか一種、又は、これらの中の複数種の組み合わせを用いたカラム、アフィニティビーズを用いて行うことを特徴とする請求項２～請求項６のいずれか一項記載のがん幹細胞の単離方法。 The selection of the cancer cells is performed using an anti-CD marker antibody, a lectin, an anti-sugar chain antibody, a column using a combination of a plurality of types, or affinity beads. The method for isolating cancer stem cells according to any one of claims 2 to 6, characterized in that:
  10. 　前記レクチン及び、抗糖鎖抗体は、前記特徴付けが行われた糖鎖構造を認識するレクチン及び、抗糖鎖抗体であることを特徴とする請求項８又は９記載のがん幹細胞の単離方法。 10. The isolation of cancer stem cells according to claim 8 or 9, wherein the lectin and the anti-sugar chain antibody are a lectin that recognizes the characterized sugar chain structure and an anti-sugar chain antibody. Method.
  11. 　前記抗糖鎖抗体が、天然の抗体、リコンビナント抗体、抗体の一部、アプタマーを含む人工抗体、ヌクレオチド、人工構造体のいずれかであることを特徴とする請求項１０記載のがん幹細胞の単離方法 11. The cancer stem cell unit according to claim 10, wherein the anti-sugar chain antibody is any one of a natural antibody, a recombinant antibody, a part of the antibody, an artificial antibody including an aptamer, a nucleotide, and an artificial structure. Separation method
  12. 　ＣＤマーカーとしてＣＤ１３３又はＣＤ１３あるいはこれらの組み合わせを用い、糖鎖としてα２，６シアル酸、又は、α２，３シアル酸あるいはこれらの組み合せを用いることを特徴とする請求項７記載のがん幹細胞の単離方法。 The cancer stem cell unit according to claim 7, wherein CD133 or CD13 or a combination thereof is used as a CD marker, and α2,6 sialic acid, α2,3 sialic acid or a combination thereof is used as a sugar chain. Separation method.
  13. 　がん幹細胞の単離が、肝臓癌幹細胞におけるがん幹細胞の単離であることを特徴とする請求項１２記載のがん幹細胞の単離方法。 The method for isolating cancer stem cells according to claim 12, wherein the isolation of cancer stem cells is isolation of cancer stem cells in liver cancer stem cells.
  14. 　抗ＣＤマーカー抗体として抗ＣＤ１３３抗体または抗ＣＤ１３抗体あるいはこれらの組み合わせを用い、レクチンとしてα２，６シアル酸認識レクチン又はα２，３シアル酸認識レクチンあるいはこれらの組み合わせを用い、抗糖鎖抗体として抗α２，６シアル酸抗体または抗α２，３シアル酸抗体あるいはこれらの組み合わせを用いることを特徴とする請求項８～請求項１１のいずれか一項記載のがん幹細胞の単離方法。 Anti-CD133 antibody or anti-CD13 antibody or a combination thereof is used as an anti-CD marker antibody, α2,6-sialic acid recognition lectin or α2,3-sialic acid recognition lectin or a combination thereof is used as a lectin, and anti-α2 is used as an anti-sugar chain antibody. The method for isolating cancer stem cells according to any one of claims 8 to 11, characterized by using a 1,6 sialic acid antibody, an anti-α2,3 sialic acid antibody, or a combination thereof.
  15. 　がん幹細胞の単離が、肝臓癌幹細胞におけるがん幹細胞の単離であることを特徴とする請求項１４記載のがん幹細胞の単離方法。 The method for isolating cancer stem cells according to claim 14, wherein the isolation of cancer stem cells is isolation of cancer stem cells in liver cancer stem cells.
  16. 　前記α２，６シアル酸認識レクチンが、ＳＮＡ、ＳＳＡ、ＴＪＡ－Ｉのいずれかであることを特徴とする請求項１４又は１５記載のがん幹細胞の単離方法。 The method for isolating cancer stem cells according to claim 14 or 15, wherein the α2,6-sialic acid recognizing lectin is one of SNA, SSA, and TJA-I.
  17. 　前記α２，３シアル酸認識レクチンが、ＭＡＬ－Ｉであることを特徴とする請求項１４又は１５記載のがん幹細胞の単離方法。 The method for isolating cancer stem cells according to claim 14 or 15, wherein the α2,3-sialic acid-recognizing lectin is MAL-I.
  18. 　遺伝子発現レベルによる同定方法を用いてがん幹細胞の同定を行うことを特徴とするがん幹細胞の同定方法。 A method for identifying cancer stem cells, comprising identifying cancer stem cells using an identification method based on gene expression levels.
  19. 　前記遺伝子の発現レベルをＰＣＲにより増幅させた際のコピー数によって定量することを特徴とする請求項１８記載のがん幹細胞の同定方法。 19. The method for identifying cancer stem cells according to claim 18, wherein the expression level of the gene is quantified based on the copy number when amplified by PCR.
  20. 　前記遺伝子を糖転移酵素遺伝子とすることを特徴とする請求項１８又は１９記載のがん幹細胞の同定方法。 20. The method for identifying a cancer stem cell according to claim 18 or 19, wherein the gene is a glycosyltransferase gene.
  21. 　前記糖転移酵素遺伝子が、ＳＴ６ＧＡＬＮＡＣ１、ＧＣＮＴ３、ＭＧＡＴ５の中のいずれか一種、またはこれらの中の２種類以上の組み合わせであることを特徴とする請求項２０記載のがん幹細胞の同定方法。 21. The method for identifying a cancer stem cell according to claim 20, wherein the glycosyltransferase gene is any one of ST6GALNAC1, GCNT3, and MGAT5, or a combination of two or more thereof.
  22. 　がん幹細胞への目的物送達の対象として、ＣＤマーカー及び糖鎖の両方を提示している細胞を用いることを特徴とするがん幹細胞の選別方法。 A method for selecting cancer stem cells, which uses cells presenting both a CD marker and a sugar chain as a target for delivery of an object to cancer stem cells.
  23. 　ＤＤＳ分子の表面に提示するターゲット用分子として、抗ＣＤ抗体及び糖鎖認識分子の両方を提示していることを特徴とするがん幹細胞の選別方法。 A screening method for cancer stem cells, wherein both an anti-CD antibody and a sugar chain recognition molecule are presented as target molecules to be presented on the surface of a DDS molecule.
  24. 　前記ＣＤマーカーがＣＤ１３３、前記糖鎖がシアル酸であることを特徴とする請求項２２記載のがん幹細胞の選別方法。 The method for selecting cancer stem cells according to claim 22, wherein the CD marker is CD133 and the sugar chain is sialic acid.
  25. 　前記抗ＣＤ抗体が、抗ＣＤ１３３抗体であることを特徴とする請求項２３記載のがん幹細胞の選別方法。 The method for selecting cancer stem cells according to claim 23, wherein the anti-CD antibody is an anti-CD133 antibody.
  26. 　前記糖鎖認識分子がシアル酸認識分子であることを特徴とする請求項２３記載のがん幹細胞の選別方法。 24. The method for selecting cancer stem cells according to claim 23, wherein the sugar chain recognition molecule is a sialic acid recognition molecule.
  27. 　前記シアル酸認識分子が、ＳＮＡ、ＳＳＡ、 ＴＪＡ－Ｉ、ＭＡＬ－Ｉ、ＡＣＧ、ＡＢＡ、ＡＣＡ、Ｊａｃａｌｉｎ、ＭＡＨのいずれかであることを特徴とする請求項２６記載のがん幹細胞の選別方法。 27. The method for selecting cancer stem cells according to claim 26, wherein the sialic acid recognition molecule is any one of SNA, SSA, TJA-I, MAL-I, ACG, ABA, ACA, Jacalin, and MAH.
  28. 　前記糖鎖認識分子がシアル酸認識抗体であることを特徴とする請求項２３記載のがん幹細胞の選別方法。 24. The method for selecting cancer stem cells according to claim 23, wherein the sugar chain recognition molecule is a sialic acid recognition antibody.
  29. 　前記抗ＣＤ抗体が、フルボディの抗体、１本鎖抗体、Ｆａｂフラグメント、部分抗体、キメラ抗体を含む人工抗体及び、アプタマー、mimic分子を含むことを特徴とする請求項２３記載のがん幹細胞の選別方法。 24. The cancer stem cell according to claim 23, wherein the anti-CD antibody comprises a full-body antibody, a single chain antibody, a Fab fragment, a partial antibody, an artificial antibody including a chimeric antibody, an aptamer, and a mimic molecule. Sorting method.
  30. 　前記抗ＣＤ抗体が、wild type の抗体及び、人工的に作られたものを含むことを特徴とする請求項２３記載のがん幹細胞の選別方法。 24. The method for selecting cancer stem cells according to claim 23, wherein the anti-CD antibody includes a wild type antibody and an artificially produced antibody.
  31. 　前記抗ＣＤ１３３抗体が、ペプチド及び、ヌクレオチドを対象とすることを特徴とする請求項２５記載のがん幹細胞の選別方法。 26. The method for selecting cancer stem cells according to claim 25, wherein the anti-CD133 antibody targets peptides and nucleotides.
  32. 　前記シアル酸認識分子がフルボディの抗体、１本鎖抗体、Ｆａｂフラグメント、部分抗体、キメラ抗体を含む人工抗体及び、アプタマー、mimic分子を含むことを特徴とする請求項２６記載のがん幹細胞の選別方法。 27. The cancer stem cell according to claim 26, wherein the sialic acid recognition molecule comprises a full-body antibody, a single chain antibody, a Fab fragment, a partial antibody, an artificial antibody including a chimeric antibody, an aptamer, and a mimic molecule. Sorting method.
  33. 　前記シアル酸認識分子が、wild type の抗体及び、人工的に作られたものを含むことを特徴とする請求項２６記載のがん幹細胞の選別方法。 27. The method for selecting cancer stem cells according to claim 26, wherein the sialic acid recognition molecule includes a wild type antibody and an artificially produced one.
  34. 　前記シアル酸認識分子が、ペプチド及び、ヌクレオチドを対象とすることを特徴とする請求項２６記載のがん幹細胞の選別方法。 27. The method for selecting cancer stem cells according to claim 26, wherein the sialic acid recognition molecule targets peptides and nucleotides.
  35. 　がん治療の対象を、ＣＤマーカー及び糖鎖の両方を提示している細胞とすることを特徴とするがんの治療方法。 A cancer treatment method, characterized in that a cancer treatment target is a cell presenting both a CD marker and a sugar chain.
  36. 　前記ＣＤマーカーＣＤ１３３で、前記糖鎖がシアル酸であることを特徴とする請求項３５記載のがんの治療方法。 36. The method for treating cancer according to claim 35, wherein in the CD marker CD133, the sugar chain is sialic acid.
  37. 　ＤＤＳで使用される分子に、抗がん剤、RNAi、あるいは、遺伝子発現を阻害する分子をのせてがん幹細胞群に送達することを特徴とするがんの治療方法。 A method for treating cancer, which comprises delivering an anticancer agent, RNAi, or a molecule that inhibits gene expression to a molecule used in DDS to a cancer stem cell group.
  38. 　前記RNAiが、siRNA または、shRNAであるることを特徴とする請求項３７記載のがんの治療方法。 The method for treating cancer according to claim 37, wherein the RNAi is siRNA or shRNA.
  39. 　前記RNAi、あるいは、前記遺伝子発現を阻害する分子の阻害対象が、ST6GALNAC1、GCNT3 、MGAT5のいずれか、またはこれらの中の２種類以上の組み合わせであることを特徴とする請求項３７記載のがんの治療方法。
    38. The cancer according to claim 37, wherein the RNAi or the inhibition target of the molecule that inhibits gene expression is ST6GALNAC1, GCNT3, MGAT5, or a combination of two or more of these. Treatment methods.
  40. 　治療対象を、ST6GALNAC1、GCNT3、又は、MGAT5 の発現にかかわる転写因子を抑制する薬剤もしくは自然物質とすることを特徴とする請求項３７記載のがんの治療方法。 38. The method for treating cancer according to claim 37, wherein the treatment target is a drug or a natural substance that suppresses a transcription factor involved in the expression of ST6GALNAC1, GCNT3, or MGAT5.
  41. 　ST6GALNAC1、GCNT3、又は、MGAT5 の働きを阻害することを特徴とする請求項３７記載のがんの治療方法。 38. The method for treating cancer according to claim 37, wherein the function of ST6GALNAC1, GCNT3, or MGAT5 is inhibited.
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