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- JP2018112496A5 JP2018112496A5 JP2017003710A JP2017003710A JP2018112496A5 JP 2018112496 A5 JP2018112496 A5 JP 2018112496A5 JP 2017003710 A JP2017003710 A JP 2017003710A JP 2017003710 A JP2017003710 A JP 2017003710A JP 2018112496 A5 JP2018112496 A5 JP 2018112496A5
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- pancreatic
- eps8
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- exosome
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- 206010033616 Pancreatic disease Diseases 0.000 claims description 23
- 210000001808 Exosomes Anatomy 0.000 claims description 20
- 208000008443 Pancreatic Carcinoma Diseases 0.000 claims description 20
- 239000003153 chemical reaction reagent Substances 0.000 claims description 20
- 201000002528 pancreatic cancer Diseases 0.000 claims description 20
- 102000011302 Epidermal growth factor receptor kinase substrate 8 Human genes 0.000 claims 2
- 108050001599 Epidermal growth factor receptor kinase substrate 8 Proteins 0.000 claims 2
- 102100010933 EPS8 Human genes 0.000 description 17
- 108060002569 EPS8 Proteins 0.000 description 17
- 238000005259 measurement Methods 0.000 description 5
- 102000004965 antibodies Human genes 0.000 description 4
- 108090001123 antibodies Proteins 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- NALWOULWGHTVDA-UWVGGRQHSA-N Asp-Tyr Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NALWOULWGHTVDA-UWVGGRQHSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 239000012327 Ruthenium complex Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000038129 antigens Human genes 0.000 description 2
- 108091007172 antigens Proteins 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 108091005941 blue fluorescent protein Proteins 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- VNYDHJARLHNEGA-RYUDHWBXSA-N (2S)-1-[(2S)-2-azaniumyl-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carboxylate Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 VNYDHJARLHNEGA-RYUDHWBXSA-N 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N (2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-amino-3-carboxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]hexanoyl]amino]-3-carboxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-carbox Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- RNIPJYFZGXJSDD-UHFFFAOYSA-N 2,4,5-triphenyl-1H-imidazole Chemical compound C1=CC=CC=C1C1=NC(C=2C=CC=CC=2)=C(C=2C=CC=CC=2)N1 RNIPJYFZGXJSDD-UHFFFAOYSA-N 0.000 description 1
- 101710028178 ANPEP Proteins 0.000 description 1
- 102100016549 ANPEP Human genes 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- FRYULLIZUDQONW-IMJSIDKUSA-N Asp-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O FRYULLIZUDQONW-IMJSIDKUSA-N 0.000 description 1
- 210000004369 Blood Anatomy 0.000 description 1
- 102100016530 CD37 Human genes 0.000 description 1
- 101700044364 CD37 Proteins 0.000 description 1
- 102100019284 CD53 Human genes 0.000 description 1
- 101700029492 CD53 Proteins 0.000 description 1
- 102100005827 CD63 Human genes 0.000 description 1
- 101700052224 CD63 Proteins 0.000 description 1
- 102100019450 CD81 Human genes 0.000 description 1
- 101700062651 CD81 Proteins 0.000 description 1
- 102100008204 CD82 Human genes 0.000 description 1
- 101700087069 CD82 Proteins 0.000 description 1
- 102100016531 CD9 Human genes 0.000 description 1
- 101700017162 CD9 Proteins 0.000 description 1
- 229940088598 Enzyme Drugs 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010020195 FLAG peptide Proteins 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 102100004115 ICAM1 Human genes 0.000 description 1
- TWVKGYNQQAUNRN-ACZMJKKPSA-N Ile-Ser Chemical compound CC[C@H](C)[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O TWVKGYNQQAUNRN-ACZMJKKPSA-N 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 102100005918 LAMP1 Human genes 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 108060001084 Luciferase family Proteins 0.000 description 1
- CIOWSLJGLSUOME-BQBZGAKWSA-N Lys-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC(O)=O CIOWSLJGLSUOME-BQBZGAKWSA-N 0.000 description 1
- 108010009254 Lysosomal-Associated Membrane Protein 1 Proteins 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 229940072417 Peroxidase Drugs 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108090000437 Peroxidases Proteins 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- -1 Rab5 Proteins 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N Rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 101710043164 Segment-4 Proteins 0.000 description 1
- QZOSVNLXLSNHQK-UHFFFAOYSA-N Tyrosyl-Aspartate Chemical compound OC(=O)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 QZOSVNLXLSNHQK-UHFFFAOYSA-N 0.000 description 1
- 101700038759 VP1 Proteins 0.000 description 1
- GIAZPLMMQOERPN-YUMQZZPRSA-N Val-Pro Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(O)=O GIAZPLMMQOERPN-YUMQZZPRSA-N 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 101700005460 hemA Proteins 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108060006184 phycobiliprotein family Proteins 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
Description
［３３］膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８測定試薬を含むことを特徴とする、膵臓癌患者と膵臓癌以外の膵臓疾患患者とを鑑別するための試薬。
［３４］前記ＥＰＳ８測定試薬が、免疫学的測定試薬である、［３３］に記載の試薬。［３５］前記免疫学的測定試薬が、ＥＰＳ８に結合する抗体又は該抗体断片を含む、［３４］に記載の試薬。
［３６］前記免疫学的測定試薬が、さらに、エクソソーム特異的抗原に結合する抗体又は該抗体断片を含む、［３５］に記載の試薬。
［３７］前記エクソソーム特異的抗原が、ＣＤ６３、ＣＤ９、ＣＤ８１、ＣＤ３７、ＣＤ５３、ＣＤ８２、ＣＤ１３、ＣＤ１１、ＣＤ８６、ＩＣＡＭ−１、Ｒａｂ５、Ａｎｎｅｘｉｎ Ｖ、又はＬＡＭＰ１である、［３６］に記載の試薬。
［３８］さらに、エクソソーム単離試薬を含む、［３３］〜［３７］のいずれか一つに記載の試薬。
［３９］前記膵臓癌以外の膵臓疾患が、膵炎である、［３３］〜［３８］のいずれか一つに記載の試薬。
［４０］前記検体が、血液である、［３３］〜［３９］のいずれか一つに記載の試薬。
[33] the pancreatic disorder in a specimen collected from a patient, exosomes characterized in that it comprises a EPS8 measuring reagent having the reagent for differentiating pancreatic diseases patients other than pancreatic cancer patients and pancreatic cancer.
[34] The reagent according to [33], wherein the EPS8 measurement reagent is an immunological measurement reagent. [35] The reagent according to [34], wherein the immunological measurement reagent contains an antibody that binds to EPS8 or an antibody fragment thereof.
[36] The reagent according to [35], wherein the immunological measurement reagent further comprises an antibody or an antibody fragment that binds to an exosome-specific antigen.
[37] The reagent according to [36], wherein the exosome-specific antigen is CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, or LAMP1.
[38] The reagent according to any one of [33] to [37], further comprising an exosome isolation reagent.
[39] The reagent according to any one of [33] to [38], wherein the pancreatic disease other than pancreatic cancer is pancreatitis.
[40] The reagent according to any one of [33] to [39], wherein the specimen is blood.
酵素としては、例えば、アルカリホスファターゼ、ペルオキシダーゼ、ガラクトシダーゼ、グルクロニダーゼ、ルシフェラーゼ等が挙げられる。
蛍光物質としては、例えば、フルオレッセイン イソチオシアナート（ＦＩＴＣ）、ローダミンＢ−イソチオシアナート（ＲＩＴＣ）等が挙げられる。その他の蛍光物質としては、例えば、ｑｕａｎｔｕｍ ｄｏｔ（Ｓｃｉｅｎｃｅ, ２８１, ２０１６−２０１８, １９９８）、フィコエリスリン等のフィコビリ蛋白質、ＧＦＰ（Ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｔｅｉｎ）、ＲＦＰ（Ｒｅｄｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｔｅｉｎ）、ＹＦＰ （Ｙｅｌｌｏｗ ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｔｅｉｎ）、ＢＦＰ（Ｂｌｕｅ ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｔｅｉｎ）等の蛍光を発する蛋白質が挙げられる。
発光物質としては、例えば、アクリジニウム及びその誘導体、ルテニウム錯体化合物、ロフィン等が挙げられる。ルテニウム錯体化合物としては、電子供与体と共に電気化学的に発光する、Ｃｌｉｎ.Ｃｈｅｍ.３７,９,１５３４−１５３９, １９９１に示されたものが好ましい。
放射性同位元素としては、例えば、３Ｈ、１４Ｃ、３５Ｓ、３２Ｐ、１２５Ｉ、１３１Ｉ等が挙げられる。
タグ配列を含むポリペプチドとしては、ＦＬＡＧペプチド（ＦＬＡＧタグ、Ａｓｐ Ｔｙｒ Ｌｙｓ Ａｓｐ Ａｓｐ Ａｓｐ Ａｓｐ Ｌｙｓ）、ポリヒスチジン（Ｈｉｓタグ、Ｈｉｓ Ｈｉｓ Ｈｉｓ Ｈｉｓ Ｈｉｓ Ｈｉｓ）、ｍｙｃエピトープペプチド（ｍｙｃタグ、Ｇｌｕ Ｇｌｎ Ｌｙｓ Ｌｅｕ Ｉｌｅ Ｓｅｒ Ｇｌｕ Ｇｌｕ Ａｓｐ Ｌｅｕ）、ヘマグルチニンエピトープペプチド（ＨＡタグ、Ｔｙｒ Ｐｒｏ Ｔｙｒ Ａｓｐ Ｖａｌ Ｐｒｏ Ａｓｐ Ｔｙｒ Ａｌａ）等が挙げられる。
Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.
Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine B-isothiocyanate (RITC). Other fluorescent substances include, for example, quantum dot (Science, 281, 2016-2018, 1998), phycobiliproteins such as phycoerythrin, GFP (Green Fluorescent Protein ) , RFP (Redfluorescent Protein ) , and YFP (YFP). And proteins that emit fluorescence such as BFP (Blue Fluorescent Protein).
Examples of the light-emitting substance include acridinium and its derivatives, ruthenium complex compounds, lophine and the like. As the ruthenium complex compound, those described in Clin. Chem. 37, 9, 1534-1439, 1991, which emit electrochemically with an electron donor, are preferred.
Examples of the radioisotope include 3 H, 14 C, 35 S, 32 P, 125 I, 131 I and the like.
Examples of the polypeptide containing a tag sequence include a FLAG peptide (FLAG tag, Asp Tyr Lys Asp Asp Asp Lys), polyhistidine (His tag, His His His His His), and myc epitope peptide (myc tag, Glu GlnLy Ly). Ile Ser Glu Glu Asp Leu), hemagglutinin epitope peptide (HA tag, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala) and the like.
また、前記測定方法３の工程（２Ｂ）において、被検者より採取した検体より単離したエクソソームを用いて、検体中の、エクソソームが有するＥＰＳ８濃度を測定することもできる。具体的には、以下の工程を含む方法が挙げられる。
［測定方法４］
（１Ｃ）膵臓疾患患者より検体を採取する工程；
（２Ｃ）工程（１Ｃ）で採取した検体からエクソソームを単離する工程；
（３Ｃ）工程（２Ｃ）で単離したエクソソームが有するＥＰＳ８濃度を測定する工程；（４Ｃ）工程（３Ｃ）で測定したＥＰＳ８濃度から、該検体中の、エクソソームが有するＥＰＳ８濃度を決定する工程；
（５Ｃ）工程（４Ｃ）で決定したＥＰＳ８濃度が、膵臓癌以外の膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８濃度よりも高い場合には該被検者は膵臓癌に罹患している可能性が高いと判定し、膵臓癌以外の膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８濃度と同じか又は低い場合には該被検者は膵臓癌以外の膵臓疾患に罹患している可能性が高いと判定する工程。
In the step (2B) of the measurement method 3, the exosome concentration of the exosome in the sample can be measured using the exosome isolated from the sample collected from the subject. Specifically, a method including the following steps can be mentioned.
[Measurement method 4]
(1C) collecting a sample from a patient with pancreatic disease;
(2C) a step of isolating exosomes from the sample collected in step (1C);
(3C) a step of measuring the EPS8 concentration of the exosome isolated in the step (2C); (4C) a step of determining the EPS8 concentration of the exosome in the sample from the EPS8 concentration measured in the step (3C);
(5C) If the EPS8 concentration determined in step (4C) is higher than the EPS8 concentration of exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer, the subject suffers from pancreatic cancer. Is determined to be likely to be present, and if the EPS8 concentration of the exosomes in a sample collected from a patient with pancreatic disease other than pancreatic cancer is equal to or lower than the EPS8 concentration of the exosome, the subject suffers from pancreatic disease other than pancreatic cancer. A step of determining that it is highly probable that
＜工程（５Ｃ）＞
工程（５Ｃ）は、工程（４Ｃ）で決定された、該膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８濃度と、膵臓癌以外の膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８濃度とを対比する工程であり、膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８濃度が、膵臓癌以外の膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８濃度よりも高い場合には該膵臓疾患患者は膵臓癌に罹患している可能性が高いと判定され、膵臓癌以外の膵臓疾患患者より採取した検体中の、エクソソームが有するＥＰＳ８濃度と同じか又は低い場合には、該膵臓疾患患者は膵臓癌以外の膵臓疾患に罹患している可能性が高いと判定される。
<Step (5C)>
In the step (5C), the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient and the exosome in the sample collected from the patient with pancreatic disease other than pancreatic cancer, determined in the step (4C). A step of comparing the EPS8 concentration with the EPS8 concentration, wherein the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient is higher than the EPS8 concentration of the exosome in the sample collected from the pancreatic disease patient other than pancreatic cancer. It is determined that the patient with pancreatic disease is likely to be suffering from pancreatic cancer, and in a sample collected from a patient with pancreatic disease other than pancreatic cancer, when the exosome has the same or lower EPS8 concentration, It is determined that the pancreatic disease patient is highly likely to be suffering from a pancreatic disease other than pancreatic cancer.
５．膵臓疾患患者において、膵臓癌と膵臓癌以外の膵臓疾患とを鑑別するための試薬
本発明の、膵臓疾患患者において、膵臓癌と膵臓癌以外の膵臓疾患とを鑑別するための試薬は、エクソソームが有するＥＰＳ８測定試薬を含有することを特徴とする試薬であり、本発明の、膵臓疾患患者において、膵臓癌と膵臓癌以外の膵臓疾患とを鑑別する方法に用いることができる。
5. In a patient with pancreatic disease, a reagent for distinguishing between pancreatic cancer and a pancreatic disease other than pancreatic cancer of the present invention, a reagent for distinguishing between pancreatic cancer and a pancreatic disease other than pancreatic cancer in a patient with pancreatic disease is an exosome. a reagent which is characterized by containing a EPS8 measurement reagent having, of the present invention, the pancreatic disorder patients, can be used in a method for differentiating pancreatic diseases other than pancreatic cancer and pancreatic cancer.
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| EP2176665B1 (en) * | 2007-08-16 | 2016-03-02 | The Royal Institution for the Advancement of Learning / McGill University | Tumor cell-derived microvesicles |
| US20100255514A1 (en) * | 2007-08-16 | 2010-10-07 | The Royal Institution For The Advancement Of Learning/Mcgill University | Tumor cell-derived microvesicles |
| ES2703363T3 (en) * | 2008-02-01 | 2019-03-08 | Massachusetts Gen Hospital | Use of microvesicles in the diagnosis and prognosis of brain tumors |
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| CN103992382B (en) * | 2014-05-14 | 2015-03-11 | 南方医科大学 | Oligopeptide formed by combining targeted EPS8 (Epidermal Growth Factor Receptor pathway substrate No.8) with EGFR (Epidermal Growth Factor Receptor) and application of oligopeptide |
| JP5852759B1 (en) * | 2015-04-01 | 2016-02-03 | 株式会社キュービクス | Detection of pancreatic cancer by gene expression analysis |
| JP2016211925A (en) * | 2015-05-01 | 2016-12-15 | 地方独立行政法人東京都健康長寿医療センター | Method of evaluating tolerance of cancer to docetaxel or paclitaxel, method of evaluating malignant alteration of cancer, and kits used for the same in cancer |
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2017
- 2017-01-12 JP JP2017003710A patent/JP6873460B2/en active Active
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