[0001]
　The present invention relates to a method of destruction exosomes, it relates to a method of separating destruction kits and normal cell-derived exosomes of exosomes.
Background technique
[0002]

　exosomes is a type of extracellular granules are secreted from the cell. Specifically, exosomes are vesicles with a diameter of about 30 nm ~ 100 nm emitted from the cells having a vesicle structure comprising a lipid bilayer membrane. The exosomes, cells secreting the exosomes (hereinafter also referred to as secretory cells.) By being incorporated into other different cells and, it is known to transmit information between the secreting cells and the other cells and that (non-Patent documents 1 and 2).
[0003]

　film structure exosomes are generally known to be stable as compared to membrane structures of a cell. For example, Non-Patent Document 3, a temperature of -20 ° C., even when the freeze-thaw process 4 times exosomes without adding a stabilizing agent such as sugars,, inclusions stably in said exosomes experimental results indicate that it has been maintained is described. On the other hand, cells referred to as animal cells generally, unless a stabilizing agent as described above, it is known that not withstand the freeze-thaw process. Therefore, exosomes, as compared to animal cells, are presumed to have a stable film structure.
[0004]
　Further, in the exosomes, a cell, the formation process of the film structure are different. In addition, when compared with the film structure of exosomes, a membrane structure of cells, both, or surface area of ​​the membrane, the type of surface proteins present on the surface of the membrane are different. Even in light of these circumstances, the film structure of the exosomes are considered to be stable structure compared to membrane structures of a cell. Incidentally, exosomes is not a living cell, which is not subject to cell death program by apoptosis that can be applied to general cellular.
[0005]

　Recently, the that there is a correlation between the exosomes and various diseases, various reports have been made. In particular, we focus on exosomes cancer cells release, the impact of the exosomes exerts against a particular disease that cancer have been reported various studies. For example, if the factors that induce angiogenesis cancer cells are contained in exosomes to release incorporated into healthy vascular endothelial cells, induced angiogenesis near cancer tissues by factors that induce the angiogenesis It is has been reported about the potential to advance the cancer metastasis or wet (non-Patent documents 4-6).
[0006]
　Further, there is in recent years, the correlation between the exosomes and various diseases, also becoming clear that the in exosomes include information associated with a disease. For example, in Non-Patent Document 3, the exosomes present in the cancer patient blood, a cancer biomarker substance (PSA, HER2, protein EGFR such as micro such miR-21, miR-200a RNA, Apbb1ip, messenger RNA, etc. ASPN, DNA of KRAS like, and various metabolites, etc.), it has been reported that has been encapsulated.
[0007]
　In particular, micro-RNA that is contained in exosomes as the substance becomes a cancer biomarker, is promising as a test indicator of cancer. Micro RNA organisms, already, miRBase are database to (URL: //www.mirbase.org/: http). Among them, micro-RNA of human, 2588 or in 2015 are known. The expression profile of the micro-RNA of human described above, be different depending on the type of organ and is also known to be different between normal cells and cancer cells. In view of these circumstances, in recent years, by the blood test for analyzing a micro-RNA that is contained in exosomes in the blood, it has been intensively studied to develop a technique for diagnosing the site or progression of cancer (Non-patent Document 7).
[0008]

　as a technique that focuses on the separation and recovery of exosomes (collecting) and removing method, for example, are as follows.
　Human body fluids (blood, saliva, urine, etc.) as a method for recovering exosomes from or cell culture, ultracentrifugation method described in Non-Patent Document 8 and the like, most commonly at 2015 it is a technique that is used. For example, from cell culture supernatants or body fluids when performing the recovery of exosomes, after removal of the large contaminants such as cell debris in such as a filter, 4 ° C., the desired by performing 70 minutes ultracentrifugation under the conditions of 100,000G it is possible to recover the exosomes. Further, Non-Patent Document 8, by utilizing the coprecipitant such as PEG, a method of recovering by precipitation of exosomes in cell culture supernatants or body fluids are also described [8].
[0009]
　Further, Patent Document 1, a technique for adsorbing and removing the exosomes is disclosed utilizing an antibody that specifically binds to exosomes. Specifically, Patent Document 1, by using an antibody that specifically binds to the membrane 4 times transmembrane protein of the CD 9, such as CD63 and CD81 present on the vesicle surface of exosomes, adsorbing and removing the exosomes how to have been described.
[0010]
　Patent Document 2, in order to adsorb and remove exosomes cancer cell-derived cancer cells secreted a method that uses an antibody to specifically adsorb the exosomes derived from the cancer cells have been described.
[0011]
　Non-Patent Document 9 and 10, by chemically treating the cell membranes of exosomes, disruption removing the exosomes itself is disclosed. Specifically, Non-Patent Document 9, a method of using a surfactant have been described, in Non-Patent Document 10, a method of using phenol, guanidine, and surfactants are described.
CITATION
Patent Literature
[0012]
Patent Document 1: WO 2013/099925 Pamphlet
Patent Document 2: WO 2007/103572 pamphlet
Patent Document 3: JP-T 2009-526862 Patent Publication
Non-Patent Document
[0013]
Non-Patent Document 1: Salido-Guadarrama I, et al, Onco Targets Ther 2014 Jul 21; 7:... 1327-38
Non-Patent Document 2: Valadi H et al, Nat Cell Biol 2007, 9: 654-659.
Non- Patent Document 3: Int J. Biol Sci 2013, 9; 10:... 1021-31
non-Patent Document 4: cell Technology, Volume 32 No. 1, pp16-22
non-Patent Document 5: Jikken Medicine Volume 29 No. 1, p410 -414
non-patent Document 6:. Ueda, et al, Proc Natl, Acad Sci USA, 106, 10746-10751, 2009....
non-Patent Document 7: Genomics Proteomics Bioinformatics 13 (2015 ) 17-24
non-Patent Document 8 : produced by Exxon Toso one woods parsing ma center have Ritzッsu nn (Yodosha)
non-Patent Document 9: Anal Biochem, 2012 Dec 15 ; 431 (2):. 96-8.
non-Patent Document 10: Mol Immunol, 2012 Apr; 50 (4)..: 278-86.
non-Patent Document 11: Journal of Extracellular Vesicles 2015, 4: 27522
non-Patent Document 12: Gynecologic Oncology 2011, 122: 437-446
non-Patent Document 13: Biochimica et Biophysica Acta, 1778 (2008), 357-375
non-Patent Document 14: Nucleic Acids Symposium Series, 2000 , 44 49-50
non-Patent Document 15: Prog Nucl . eic Acid Res Mol Biol 2005; 80: 349-74
Non-Patent Document 16: Biochemistry, 1979, 18 ( 23), pp 5143-5149
Summary of the Invention
Problems that the Invention is to Solve
[0014]
　In research field regarding exosomes, 2015 now, as well as capture the biomarkers of cancer being contained in said exosomes efficiently, which can be reproduced with high quantitative detection of the biomarkers with high sensitivity development of the system has been demanded. Further, in order to realize such a system, techniques and to efficiently destroy the exosomes derived cancer cells by a simple operation, without destroying the exosomes from normal cells, selectively exosomes derived from a cancer cell there is a quantitative extraction it is necessary to establish such a technology for (separation) technology and to destroy, the biomarkers are contained in exosomes in. Further, if it is possible to realize the system described above, it is believed possible future, also be applied to such a system for early detection and treatment of cancer.
　However, the background section at said various prior art techniques are all, did not satisfy the required level necessary to implement the system described above. Specifically, the present inventors have found that for the background terms in various prior art described technology, it has been found that there is the following problem.
[0015]
　First, the method described in Non-Patent Document 9 and 10 chemically treating the cell membranes of exosomes is a method of destroying both the exosomes derived exosomes and normal cells from cancer cells.
　Further, the method described in Non-Patent Document 9 and 10, assuming that the application of future the method the disease treatment of cancer metastasis and the like, non-organic compounds utilized in the process of cell membrane in vivo specifically there is a high probability that result in action. In particular, materials that phenol is because it has a strong protein denaturing effect compared with other compounds, in the case where the method described in Non-Patent Document 10 to apply the disease treatment of cancer metastasis or the like, phenol the exosomes present in the living body is considered necessary to eliminate the influence on different components. Therefore, the methods described in Non-Patent Documents 9 and 10 apply to the diseases in the future cancer metastasis or the like is considered to be virtually impossible. In view of these circumstances, in recent years tend to build destruction removal method exosomes to use a substance having a non-specific expression hard biosafety action are required in vivo.
[0016]
　As a method of extracting a biomarker that is contained in exosomes, method for extracting biomarkers such as a micro RNA has become known from exosomes cancer from cells were collected by antibodies immunization. For example, Non-Patent Document 11, after collecting the exosomes by immunosorbent technique, a technique for extracting a biomarker such as a micro RNA from the exosomes are described. However, the technique described above is that there is room for improvement in terms of selectively extract biomarkers from cancer cells. Specifically, in the method for extracting the biomarkers described above, as an antibody for use in collecting exosomes, also a characteristic protein present in exosomes derived from cancer cells in the case of using those recognized as antigens since according characteristic protein is present also common to some extent exosomes from normal cells in that they collected to determine the exosomes derived from cancer cells and exosomes of normal cell-derived, sufficient selectivity there is a can not be obtained. Further, Non-Patent Document 12, characteristic protein present in exosomes derived from cancer cells, by being lost from decomposed with exosomes surface by proteases in vivo, it is difficult to collect the exosomes it has been reported that the case also made.
[0017]
　Similarly, the method described in Patent Documents 1 and 2, although there is a technique of exosomes adsorbed and removed by using a secure antibody to a living body, as long as exosomes amount that can be adsorbed to the antibody because there, there is a disadvantage that it is not possible to adsorb and remove a particular amount or more of exosomes. This disadvantage is particularly that they tend to more actualized in the case of adsorbing and removing exosomes using column or the like filled densely antibodies have been identified. The reasons include that exosomes particles are larger 10 times or more as compared to the antibody. If for adsorbing and removing exosomes using column or the like filled with high density antibody, binding points between the antibody and the exosomes will be densely present in the space. In that case, since the exosomes particle size as described above at least ten times as compared with the antibody, the space will be occupied by the exosomes particles. Therefore, in the case of adsorbing and removing exosomes using column or the like filled with high density antibodies, generated binding point of the antibody which is not involved in binding (adsorption) of exosomes particles that space, as a result exosomes particles of which (exosomes that have the potential to occur a disadvantage that the adsorption amount is lowered smaller surface area than the column particles, also have influenced to have less antigens present in that amount relative surface Conceivable).
　In addition, depending on the type of exosomes, conditions existing antigen on their surface are also mixed, are also known to result in titer and specificity of the antibody is largely. Furthermore, techniques for collecting exosomes with antibodies, because it is difficult to its extensive washing, also in that the recovery of highly purified exosomes had a room for improvement.
[0018]
　As described above, in view of realizing the system described above, the safety to a living body is assumed that it is secured, techniques and to efficiently destroy the exosomes derived cancer cells by a simple operation, technology and selective destruction of exosomes derived from a cancer cell, extracted quantitatively (separation) technique biomarkers that are contained in exosomes were circumstances has not been established yet.
[0019]
　Accordingly, the present invention is safe to a living body has been secured, and conveniently and efficiently destroy exosomes derived from a cancer cell, to extract the biomarkers are included in the exosomes efficiently and selectively What can provide destructive removal techniques exosomes.
Means for Solving the Problems
[0020]
　The present inventors have conducted extensive studies to solve the problems of the prior art described in the section of problems that the Invention is to Solve. As a result, the present inventors have found that by using a particular polypeptide, the safety to a living body has been secured, and the exosomes derived from cancer cells easily and efficiently destroyed, as contained in the exosomes It found to be able to construct a technique capable of extracting biomarkers efficient and selective that have, thereby completing the present invention.
[0021]
　According to the present invention, the steps of preparing an antimicrobial peptide,
　and the antimicrobial peptide, by the coexistence of the exosomes, a step of breaking the exosomes
　destruction method exosomes with is provided.
[0022]
　Furthermore, according to the present invention, it includes an antimicrobial peptide, and wherein the antimicrobial peptide is a satisfying peptide of the following (1) or (2), exosomes breakdown kit from cancer cells.
(1) a chain length of less than 10 to 50, wherein Net Charge is less than 15 greater than 0, and hydrophobic residues proportion is less than 65% to 25% (although, S-S bonds 3 or more and, except for those containing either one of the total amino acid lysine and valine residues in constituting the antimicrobial peptide).
(2) a chain length of less than 10 more, Net Charge is 0 or less, and with hydrophobic residues proportion is less than 25%, the following condition (2-1) or (2-2) it is intended to meet.
(2-1) said comprises a histidine residue 3 or more in the total amino acids constituting an antimicrobial peptide, and free of tryptophan residues and valine residues.
(2-2) wherein comprises one or more arginine residues in the total amino acids constituting antimicrobial peptides, it does not contain a phenylalanine residue, a tryptophan residue and a valine residue.
[0023]
　Furthermore, according to the present invention, and satisfy the antimicrobial peptide of the following (1) or (2), comprising the step of coexistence and exosomes,
　the exosomes are a exosomes of normal cells from the exosomes derived from a cancer cell mixtures, exosomes method of separating from normal cells.
(1) a chain length of less than 10 to 50, wherein Net Charge is less than 15 greater than 0, and hydrophobic residues proportion is less than 65% to 25% (although, S-S bonds 3 or more and, except for those containing either one of the total amino acid lysine and valine residues in constituting the antimicrobial peptide).
(2) a chain length of less than 10 more, Net Charge is 0 or less, and with hydrophobic residues proportion is less than 25%, the following condition (2-1) or (2-2) it is intended to meet.
(2-1) said comprises a histidine residue 3 or more in the total amino acids constituting an antimicrobial peptide, and free of tryptophan residues and valine residues.
(2-2) wherein comprises one or more arginine residues in the total amino acids constituting antimicrobial peptides, it does not contain a phenylalanine residue, a tryptophan residue and a valine residue.
Effect of the invention
[0024]
　According to the present invention, the safety to a living body has been secured, and conveniently and efficiently destroy exosomes derived from a cancer cell, to extract the biomarkers are included in the exosomes efficiently and selectively What can be, it is possible to provide a breakdown removal techniques exosomes. In particular, according to the destruction process of exosomes of the present invention, the chemical treatment safe and selective than the known method of performing, than known method using an antibody conveniently, efficiently, and selectively it is possible to destroy the cancer cell-derived exosomes.
　Further, according to the present invention, the exosomes from normal cells, from a body fluid sample and exosomes are mixed from cancer cells, it is possible to selectively destroy exosomes derived from cancer cells. Technique according to the present invention, in the field of cancer treatment in the future, screening of novel biomarkers related to cancer diseases, examination and diagnosis by efficient detection of existing cancer markers, and further, applied to dialysis technique it is considered that it is useful to.
DESCRIPTION OF THE INVENTION
[0025]
(Antimicrobial peptides)
　First, the "antimicrobial peptide" typically Gram negative bacteria, Gram positive bacteria, against fungi, some viruses such as, means a polypeptide which shows the bacteriostatic action or bactericidal action. On the other hand, the method of destruction exosomes of this embodiment (hereinafter, also referred to as the destructive methods.) Antimicrobial the peptide refers to one having a disrupting activity of exosomes. Among them, as the antimicrobial peptide which can be used in the destruction process, preference is given to those of exosomes derived from cancer cells can be selectively destroyed.
[0026]
　Peptides with a special feature called antimicrobial peptides, many types have been reported to date. However, the antimicrobial peptides has become known, for possessing an effect which can be removed by breaking the exosomes has not been reported so far. Further, in the antimicrobial peptides it has become known, some of which are referred to as "anti-cancer peptide" shows the effect of destroying the cancer cells (Non-Patent Document 13). Among such anti-cancer peptides, little effect on the normal cells, there is low hemolytic. However, the anticancer peptides, for that whether they have the effect of selectively destroying exosomes derived from a cancer cell has not yet been elucidated.
[0027]
　In the preliminary study results of the present inventors, in the anti-cancer peptide described above, the first place, it is confirmed that that there is no or low fracture ability of exosomes derived from cancer cells is scattered many there. Specifically, the present inventors have obtained the following findings,
　the first, less than Net charge of the peptide is 0, and anticancer which have a breaking capacity of cancer cells has become known as well as N was a peptide, there is a case where there is no or low depletion potential of exosomes derived from cancer cells.
　Second, as to exhibit anti-cancer effects were anticancer peptides including PR-39 which is known, there may be no or low fracture ability of exosomes derived from cancer cells.
　Third, as to have a cell membrane degradable cancer cells were anticancer peptides such Hepcidin TH2-3 and A6K which has become known, there is no depletion potential of exosomes derived from cancer cells or there is a case in low.
　Based on such circumstances, the present inventors have found that the mechanism of action against cancer cells antimicrobial peptides, between the mechanism of action for the exosomes derived cancer cells, correlation and similarity is not, both the mechanism of action of was presumed that are different from each other.
[0028]
　Further, the antimicrobial peptide has a positive charge. However, materials having a positive charge, typically, in combination with a compound having a negative charge such as a nucleic acid, to form the precipitated insoluble material is known (Non-Patent Document 14).
[0029]
　Therefore, using the antimicrobial peptide is expected is not easy to extract a nucleic acid component such as a micro RNA from exosomes. However, as a result of inventors studied intensively, method for extracting nucleic acid components of the micro RNA and the like from the exosomes by using an antimicrobial peptide is completed.
[0030]
　First, specific examples of the antimicrobial peptides according to the present embodiment, Magainin 2 (Magainin2) such magainin, Defensin HNP-1 or the like of defensins, lactoferricin, nisin, cecropin (cecropin), Andrographis pins such as cecropin B, moricin, Seratotokishin, melittin (melittin), dermaseptin, Bonbinin, Burebinin, esculetin, buforin such buforin IIb (buforin), MG2B, such Caerin1.1 Kaerin, LL37, mCRAMP, PR-39 , CAP-11, CAP-18 , RL-37, CRAMP-1 /2, rCRAMP, Prophenin, PMAP-23, PMAP-36, PMAP-37, BMAP-27, BMAP-28, BM AP-34, Bac5, Bac7 and cathelicidin-AL etc. Katerijin, Abaeshin, apidaecin, indolicidin, Burebinin, protegrin, tachyplesin, drosomycin, maximin (Maximin), Dermaseptin, Maculatin, TsAP-2, NRC-03, Ascaphin-8 , Polybia-MPI, NK-2 , Epinecidin-1, Pardaxin4, NRC-07, K6L9, Pep27,9R, mG2A, Histatin-5, Macropin, Tuftsin, various HHPHG, A3K, A6K, A9K, Hepcidin TH2-3, Alloferon1 , Sesquin, Gageostatin C, PNC- 28, EP3, BEPT II-1, TP7, (Gln 53 ) -Connexin 37, C-Reactive Protein (CRP), Damushijin such Dermcidin-1L, Hepcidin-20, Orein, Gaegurin, Shitoropin, protamine, and the like. Among them, Magainin 2, LL-37, protamine and nisin, MG2B, mCRAMP, Caerin 1.1, Maximin 1, Maximin 4, Dermaseptin, Maculatin 3.1, TsAP-2, NRC-03, Ascaphin-8, Polybia-MPI , NK-2, Epinecidin-1 , Short α-helical peptides (2) (GIIKKIIKKIIKKIIKKI), Pardaxin 4, NRC-07, K6L9, Magainin I, buforin IIb, Pep27,9R, mG2A, Short α-helical peptides (1) (GIIKKIIKKI), Defensin HNP-1 , cecropin B, Histatin-5, Ma cropin 1, Tuftsin, HHPHG, ( HHPHG) 2, (HHPHG) 3, (HHPHG) preferably contains one or more peptides selected from the group consisting of 4, magainin 2 from frog, Melittin, MG2B, mCRAMP, Caerin 1.1 and / or LL-37 from human is preferred.
[0031]
　The arrangement of the antimicrobial peptides, are described in the various databases described below.
URL: Http: //Aps.Unmc.Edu/AP/main.Php
URL: Http: //Defensins.Bii.A-star.Edu.Sg/
URL: Http: //Crdd.Osdd.Net/raghava/cancerppd /
URL: Http: //Peptaibol.Cryst.Bbk.Ac.Uk/home.Shtml
URL: Http: //Www.Cybase.Org.Au/
URL: Http: //Bactibase.Pfba-lab-tun.Org/ Main.Php
URL: Http: //Phytamp.Pfba-lab-tun.Org/main.Php
URL: Http: //Www.Camp.Bicnirrh.Res.In/
URL: Http: //Yadamp.Unisa.It/
URL: Http: //Split4.Pmfst.Hr/dadp/
URL: Http: //Db-mml.Sjtu.Edu.Cn/THIOBASE/
URL: Http: //Biotechlab.Fudan.Edu.Cn/database/EnzyBase/ Home.Php
URL: Http: //Biotechlab.Fudan.Edu.Cn/database/lamp/
URL: Http: //Milkampdb.Org/home.Php
URL: Http: //Dbaasp.Org/home.Xhtml
URL: Http : //www.baamps.it/
[0032]
　The antimicrobial peptide according to the present embodiment may be those extracted from organisms, including antimicrobial peptides may be those artificially synthesized by bacteria by gene recombination techniques, solid phase synthesis the organic synthetic techniques the law or the like may be those artificially total synthesis using. Further, as long as it does not impair the destruction activity of exosomes, to a portion of the amino acid residues constituting the antimicrobial peptide may be substituted with another amino acid residue, of the amino acid residues constituting the antimicrobial peptides one part is may be deleted, may be those other amino acid residue is inserted at amino acid residues constituting the antimicrobial peptide. Also, the antimicrobial peptide according to the present embodiment may be one of two or more peptides are linked through a spacer. Also, the antimicrobial peptide according to the present embodiment, C-terminal, modified (amino, etc.), N-terminal modified (acetylated, etc.), side chain modifications, be those undergoing cyclization post-translational modification of glycosylation, etc. it may be. Thus, the amino acids constituting the antimicrobial peptide according to the present embodiment is not limited to 20 kinds of essential amino acids constituting the living body, it may be a special amino acid that will be described later.
[0033]
　N-terminal modification method (side chain NH 2 group-modified side chain SH group modification) The, Acetylation, 5-FAM, BSA , Hexanoic acid, PEN, 5-FAM-Ahx, CBZ, HYNIC, Stearic acid, Abz, Dansyl , KLH, Succinylation, Dansyl-Ahx , Lauric acid, TMR, Acryl Decanoic acid, Lipoic acid, Alloc, DTPA, Maleimide, Benzoyl, various Fatty Acid, MCA, Biotin, FITC , Myristoyl, Biotin-Ahx, FITC-Ahx, Octanoic acid, BOC, Fmoc, OVA, Br-Ac-, Formylation, Palmitoyl, Dab syl ,, DHA, Nicotine, ATTO488 / 550/633 / 647N / 655, Rhodamine Green, rhodamine B, TAMRA, Texas Red, fluorescein, and the like.
[0034]
　The method for modifying the C-terminus (side chain carboxyl group), Amidation, AFC, AMC, MAPS Asymmetric 2 branches, MAPS Asymmetric 4 branches, MAPS Asymmetric 8 branches, BSA, KLH, Bzl, NHEt, Cysteamide, NHisopen, NHMe, OSU , Ester (OEt), Ester (OMe), Ester (OtBu), Ester (OTBzl), OVA, p-nitroanilide, tBu, and the like,
[0035]
　The special amino acids described above, 6-Aminocaproic acid, Aminobutyric acid, Citrulline, Cysteine ​​(Acm protected), various D-Amino acid, Dimethy-Lysine, Hydroxy-Proline, Methyl-Lysine, Norleucine, Ornithine, Pyroglutamic acid, Trimethyl- Lysine, β-Alanine, ε-Acetyl-Lysine, 3-Nitro-Tyrosine, phosphorylated Tyrosine, Gamma-GLU, Cysteine ​​(tBu), Penicillamine, Penicillamine, N-methylated Le cine, Homocysteine, N-methylated Valine, Homoserine, Isoleucine, Biotin Lysine, Cysteine ​​(Acm), N-methylated ALA, N-methylated Isoleucine, (L) 2-PAL, (L) 4-CL-PHE, (L) 1-NAL, N-methylated Phenylalanine, N-methylated Threonine, N-methylated Serine, N-methylated Tyrosine, Alpha Amino-Butyric Acid, Beta-ASP, (L) -4-Pal, Lys (5-FAM), Methionine sulfone, NMe-Asp, Aib, bu, Mpa, Hydroxy Proline, Acetylated amino acid, Mini-PEG1, Methionine sulfoxide, NH2- (PEG) 11-CH2COOH, Cyclopentylglycine, Selenocysteine, Azido-Lysine, NH2- (PEG) 2-CH2COOH, NH2- (PEG) 6 -CH2CH2COOH, NH2- (PEG) 12-CH2CH2COOH, Propargylglycine, 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, Lys (TMR), Lys (ivdde), NMe-Nle, NMe-Glu, NMe-Nva , Phg, Ser (octanoic-ac d), Dab (Dnp), Selenomethionine, Carboxyamidomethy, Beta-HomoLeucinelated, Cysteine, Methylated Arginine, Arg (Me) 2 asymmetrical, Arg (Me) 2 symmetrical, Lys (Dde) or the like.
[0036]
　Also, if the antimicrobial peptide according to the present embodiment is one that was circularized, ligated between peptide, S-S bond, the N-terminal (or side chain (carboxyl group or side chain) C-terminal cysteine between NH 2 group, can be carried out by circularization by ligation of the side chain SH group). Also, the antimicrobial peptide according to the present embodiment, may be linked via a spacer as described above.
[0037]
　Antimicrobial peptides according to the present embodiment, all amino acid chain lengths constituting the peptide (hereinafter referred to as chain length), Net charge and the percentage of the hydrophobic residue in the amino acid sequence (hereinafter, hydrophobic residues proportion both it is preferable shown) or the like is one that is controlled. Here, the chain length, represents the number of monomers such as amino acids constituting the antimicrobial peptide. The net Non charge, represents a net charge in the peptide. As the method of calculating the Net charge, various methods are known, Net charge in the present embodiment, basically, indicating the value of Net charge at neutral (pH 7.4). Also, Net charge of antimicrobial peptides to be described later in Examples, the database (URL: http: //aps.unmc.edu/AP/main.php) are described in the value or the calculated value in the database, one in which was adopted.
[0038]
　The lower limit of chain length of the antimicrobial peptide according to the present embodiment, preferably, 10 or more, more preferably 20 or more. On the other hand, the upper limit of the chain length is preferably less than 50, more preferably less than 40.
[0039]
　The lower limit of Net charge antimicrobial peptide according to the present embodiment is preferably a value greater than zero, more preferably, a value of less than 10. On the other hand, the upper limit of Net charge is preferably a value less than 15, more preferably, a value of less than 10.
[0040]
　The lower limit of the hydrophobic residues percentage of amino acid sequence that constitutes the antimicrobial peptide according to the present embodiment, preferably, not less than 0%, more preferably 25% or more. On the other hand, the upper limit of the hydrophobic residues proportion is preferably not more than 80%, more preferably less than 65%.
[0041]
　Here, the antimicrobial peptide according to the present embodiment includes the chain length, as the hydrophobic residue percentage of Net charge or amino acid sequence etc. were respectively within the above range, the specific amino acid residues or greater than a predetermined number If, depletion potential of cancer cell-derived exosomes are sometimes lowered. For example, cysteine ​​residue containing 6 or more, and, if it contains either a lysine residue or a valine residue, depletion potential of cancer cell-derived exosomes decreases. Besides, those without leucine residues include an alanine residue 6 above, be those containing one or more arginine residues free of phenylalanine residues and tryptophan residues, valine residues, any of the leucine residue for others, such as including or, there is a possibility that the breakdown potential of cancer cells derived exosomes decreases. Therefore, the number of S-S bonds present in the antimicrobial peptide according to the present embodiment is preferably less than 3.
[0042]
　Thus, the antimicrobial peptide according to the present embodiment is preferably a satisfying peptide of the following (1) or (2).
(1) a chain length of less than 10 to 50, wherein Net Charge is less than 15 greater than 0, and hydrophobic residues proportion is less than 65% to 25% (although, S-S bonds 3 or more and, except for those containing either one of the lysine residues and valine residues in the total amino acids constituting an antimicrobial peptide).
(2) a chain length of less than 10 more, Net Charge is 0 or less, and with hydrophobic residues proportion is less than 25%, the following condition (2-1) or (2-2) it is intended to meet.
(2-1) a histidine residue 3 or more in the total amino acids constituting an antimicrobial peptide, and free of tryptophan residues and valine residues.
(Including one or more arginine residues in the total amino acids constituting 2-2 Symbol antimicrobial peptides, it does not contain a phenylalanine residue, a tryptophan residue and a valine residue.
[0043]
　Also, the antimicrobial peptide according to the present embodiment, further preferably the following condition is satisfied peptide.
(Conditions) a chain length of less than 20 or more 40 comprises less than 10 Net Charge is 1 or more, and a hydrophobic residue ratio is less than 65% to 25% (although, S-S bonds to 3 or more, and, except for those containing either one of the total amino acid lysine and valine residues in constituting the antimicrobial peptide).
[0044]
　Furthermore, when classifying on the basis of the antimicrobial peptides according to the present embodiment in view that their conformation, specific examples of the classification, Magainin 2, BMAP-27, BMAP-28, cecropin, LL37, CAP-11, CAP -18, Orein, Gaegurin helix antibacterial peptides to alpha such Shitoropin and melittin, defensins, beta-sheet antimicrobial peptides such as lactoferricin and tachyplesin, helix antibacterial peptides to alpha, connecting peptide of the beta-sheet antimicrobial peptide composite antimicrobial peptides etc., include specific amino acid residues are often peptide (Histatin-5) or the like. Among them, peptides are classified into helix antibacterial peptides to α, as described later in Examples, the breaking ability of cancer cell-derived exosomes tends to be higher was confirmed.
[0045]
　The antimicrobial peptide according to the present embodiment may be used alone or in combination of two or more thereof.
[0046]
(Exosomes)
　exosomes of the present embodiment is a kind of extracellular granules organism cells release, refers to small vesicles of degree more than 100nm or less in diameter 30nm from late endosomal compartments.
　As the biomaterial comprising exosomes, (including those components separated) of blood, bone marrow fluid, lymph fluid, urine, stool, body fluids such as saliva, and the like cell debris. In the present embodiment, in response to biomaterial it can be used as long as exosomes extracted preprocesses according to a conventional method.
　Exosomes of this embodiment may be exosomes derived from any animal species. Specific examples of exosomes, human, mouse, rat, monkey, dog, cat, cow, include exosomes of warm-blooded animals from horses and birds, and the like.
[0047]
　Also, exosomes of this embodiment may be exosomes derived from any cell tumors. Specific examples of the derived tumors of exosomes, tumor cells, dendritic cells, reticulocytes, T-cells, B-cells, platelets, epithelial cells, and the like.
　Among them, exosomes according to the present embodiment exosomes derived from tumor cells involved in metastasis of cancer cells, i.e., preferably contains exosomes cancer cell-derived secreted from cancer cells. In other words, exosomes according to the present embodiment, the cancer cell-derived secreted from cancer cells may comprise only exosomes, the exosomes of normal cells from the exosomes from the cancer cells it may be a mixture, but is preferably those containing exosomes derived from cancer cells. Also, exosomes of this embodiment is a mixture of exosomes of normal cells from the exosomes from the cancer cells, as described later in Examples, selective destruction of exosomes derived from cancer cells it can.
　Then, specific examples of the cancer cells that secrete exosomes derived from a cancer cell according to the present embodiment, breast cancer cells, esophageal cancer cells, stomach cancer cells, appendix cancer cell, colon cancer cells, endometrial cancer cells, cervical cancer cells, ovarian cancer cells, brain cells, liver cancer cells, gallbladder cancer cells, bile duct cancer cells, pancreatic cancer cells, adrenal gland cancer cells, gastrointestinal stromal tumor, mesothelioma , laryngeal cancer cells, oral cancer cells (floor of mouth cancer, gum cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer), sinus cancer cells (maxillary cancer, frontal sinus cancer, ethmoid cancer, butterfly bone sinus cancer), thyroid cancer cells, kidney cancer cells, lung cancer cells, osteosarcoma, prostate cancer cells, testicular cancer, renal cell carcinoma cells, bladder cancer cells, horizontal rhabdomyosarcoma, skin cancer cells, anal cancer cell, leukemia, lymphoma, Hodgkin's disease, multiple myeloma and the like.
[0048]

　Next, a description will be given various extraction methods exosomes.
[0049]
(Ultracentrifugation)
　First, a method for extracting exosomes fraction using the ultracentrifuge.
　When extracting exosomes fraction directly from the cell, for example, by using the following method of ultracentrifugation in a manner to be described later cell culture supernatant, it is possible to extract the desired exosomes fractions. However, various processing conditions such as centrifugation, are not limited to the conditions described below.
　First, the supernatant of the target cell cultures that secrete exosomes, room temperature, after exosomes fraction was centrifuged for 15 minutes at conditions that do not precipitate as 2,000 G, to separate the insoluble fraction. Then, the supernatant fraction obtained, precipitating the exosomes by centrifugation for 70 minutes at 110,000 g. Thereafter, by removing the supernatant fraction, it is possible to recover the desired exosomes fractions.
[0050]
(Gel filtration method)
　Next, a method of extracting exosomes fraction explained by gel filtration.
　When extracting exosomes fraction from the supernatant of the cell culture, for example, it is possible to extract the desired exosomes fractions in the following method of treatment using the gel filtration method. However, various processing conditions such as centrifugation, are not limited to the conditions described below.
　First, the supernatant of the target cell cultures that secrete exosomes, room temperature, followed by centrifugation for 15 minutes at that exosomes fraction that 2,000G does not precipitate, separate the insoluble fraction. After removing the insoluble matter, at room temperature, a faster rate than centrifugation first of 12,000 G, and the supernatant fraction by centrifuging 35 minutes at that exosomes fraction does not precipitate separating the impurities partial. Then, the supernatant fraction obtained, the eluate obtained was subjected to gel filtration column and measuring the absorbance at 260nm, to recover the high absorbance fraction as the desired exosomes fractions. Further, the gel filtration column may be one prepared himself bought carrier, may be commercially available, such as Sephacryl S-400 HR (GE Healthcare Bioscience).
[0051]
(Extraction from sample)
　Next, a method of extracting exosomes fraction from the test sample such as blood.
　In order to obtain the exosomes fraction from a test sample such as blood, plasma components, or serum was separated according to known methods, subsequent to extraction with a method according to the case of extracting the cell culture supernatant exosomes fraction be able to. If the analyte sample is extracted urine, saliva, sweat, the exosomes fraction from the cerebrospinal fluid after removing the contaminants according to the known methods, since extracts with a method according to the case of cell culture supernatants be able to.
[0052]
(Antibody immunization)
　Next, a method of extracting exosomes fractions using antibodies.
　As an alternative technique to the ultracentrifugation, immunosorbent method is known. Antibodies that can be used, as long as the substance present only in the surface layer of exosomes in the blood can be recognized as antigens is not particularly limited. Specific examples of the antigen, CD9, CD63, proteins and belonging to the membrane 4 times transmembrane protein family termed tetraspanins such as CD81, Epithelial cell adhesion molecule (EpCAM ), that such human epidermal growth factor receptor type2 (HER2 ) It includes the specific proteins to the exosomes derived from the N cells. Other, CD3, CD4, CD8, CD14 , CD15, CD19, CD20, CD41, CD51, CD61, CD62e, CD66b, CD105, CD144, CD235a, Annexin V, Glycoprotein A, Valpha24 / Vbeta11, and the like. These antibodies, it is generally used in a state of being supported on a carrier.
[0053]

　exosomes method destruction according to the present embodiment includes the steps of preparing an antimicrobial peptide, and antimicrobial peptides, by the coexistence of the exosomes, a step of destroying the exosomes, the. According to this method, compared with the conventional method, it is possible to efficiently remove the exosomes in a sample. The present method is intended to be carried out in vitro, and in mild conditions with respect to components other than the exosomes in a sample is a technique capable of implementing the destruction of exosomes. In such a method, and antimicrobial peptides, after allowed to coexist and exosomes, it is preferable to incubation at a given temperature and time. Further, according to this method, the destruction of the exosomes can be simplified and the work procedure.
[0054]
　In destruction method exosomes of the present embodiment, the content of the exosomes amount and antimicrobial peptide amount in incubation solution prepared by co by mixing the antimicrobial peptide and exosomes, 1 × 10 8 to exosomes of Particles / mL in contrast, it is preferred to include the following antimicrobial peptide 100 [mu] M, more preferably as those containing the following antimicrobial peptide 10 [mu] M, more preferably as those containing the following antimicrobial peptide 1 [mu] M. In that case, it is possible to density adjustment by using a buffer such as saline or PBS.
[0055]
　Incubation temperature of antimicrobial peptides and exosomes in destruction method exosomes according to the present embodiment, an optimum value disrupting activity by the kind of the antimicrobial peptides is maximized are different, the lower limit of the incubation temperature is preferably, 0 ° C. or higher , and still more preferably at 20 ° C. or higher. On the other hand, the upper limit of the incubation temperature is preferably at 70 ° C. or less, still more preferably 40 ° C. or less.
[0056]
　The lower limit of the incubation time of the antimicrobial peptides and the exosomes in destruction method exosomes of this embodiment is preferably not less than 1 second, more preferably, at least 10 minutes. Meanwhile, the upper limit of the incubation time, preferably not more than 72 hours, still more preferably not more than 48 hours.
[0057]
　Further, in the breakdown process of exosomes of this embodiment, the incubation solution is prepared by coexistence by mixing the antimicrobial peptide and exosomes, by containing an additive component other than buffers such as saline or PBS it may be. Examples of such a specific example of the additive, and a reagent such as RNase Inhibitor stabilizing biomarkers include reagents such as protease inhibitor and BSA to stabilize the peptide. Above all, when extracting ribonucleic acid-based biomarkers contained in exosomes, it is preferable to contain RNase Inhibitor as an additive.
[0058]
　The RNase Inhibitor described above, mammalian origin (derived from human placenta, mouse-derived, porcine, etc.) RNase Inhibitor of are mainly used [15]. It said RNase Inhibitor is a protein of about 50 kDa, inactivated by non-covalent ribonuclease A, B, and C. Further, the RNase Inhibitor are known not to act to RNase T1, T2, H, U1 , U2, CL3.
　Also, sometimes oxovanadium (IV) with a stabilizing agent according complexes ribonucleoside is utilized (Non-Patent Document 16).
[0059]
　Various additives described above may be used alone or in combination of two or more thereof.
[0060]
　In destruction method exosomes of the present embodiment, the amount of the additive to be added to the incubated solution prepared by co by mixing the antimicrobial peptide and exosomes, disrupting activity of exosomes, biomarkers or biological safety aims within a range not adversely affect the sex, it can be arbitrarily selected general amount in the art. For example, if RNase Inhibitor, 0.01U ~ 20U / μL, preferably 0.1U ~ 1U / mL.
[0061]
(Carrier contact method)
　antimicrobial peptide according to the present embodiment may be one which is immobilized on a carrier. Certain peptides having a bactericidal action of antimicrobial peptides, the peptides as used in fixed to a carrier, it has been known in which can retain its function (Patent Document 3) .
[0062]
　Carriers which can be used in the destruction process of exosomes according to this embodiment, as long as it can chemically or physically binding the antimicrobial peptide or the above antibodies may be of known. Specific examples of the carrier, polyamide, polyethylene glycol, sepharose, polyvinyl alcohol, cellulose, silica, silicon, and titanium, and iron. Among them, it is preferably a sepharose and cellulose, from the viewpoint of safety in use and further preferably Sepharose.
　Further, when a sample containing exosomes is a blood containing exosomes can also be used as a carrier for immobilizing an antimicrobial peptide a dialysis membrane for hemodialysis. Examples of the dialysis membrane for hemodialysis include cellulose, cellulose acetate, polysulfone, polyether sulfone, polymethyl methacrylate, ethylene vinyl alcohol copolymer, polyacrylonitrile, polyester polymer alloy, polyarylether sulfone is .
　Also, the antimicrobial peptide may be a complex immobilized antibody of different amino acid sequence and may be one of immobilizing said complex with respect to the support. Antibody as used herein is preferably an antibody that specifically binds to exosomes. In this way, by interacting with exosomes and antibodies, while adsorbing the exosomes for the antibody, the exosomes can be destroyed by the antimicrobial peptide.
　When using immobilized antimicrobial peptide to a carrier or an immobilized the complex to a carrier, can also be used by filling the carrier with the antimicrobial peptide onto the column. Samples containing exosomes, by passing through a column packed with the carrier having the antibacterial peptide, it is possible to effectively destroy the exosomes.
[0063]
　In destruction method exosomes of this embodiment, in the case of using the antimicrobial peptide is immobilized to the antibody or carrier, as long as it does not impair the object of the present invention, between the antimicrobial peptide and the carrier, the between the antibody and the carrier or between the antimicrobial peptides and the antibodies, it may be interposed a spacer material having a predetermined length. Specific examples include polyethylene glycol, unsaturated hydrocarbons, peptides, and the like.
　The spacer material is typically via a functional group is a general term for components which can be connected by coupling the second object. Specific examples of the functional group, an amino group, a hydroxyl group, a carboxyl group, such as a thiol group. The coupling manner between the spacer, the antibody, with the carrier and the antimicrobial peptide is not particularly limited. As an example of a combination of the carrier and spacer materials include commercially available products such as HiTrap NHS-activated HP Columns (GE Healthcare Inc.).
[0064]
　Even if the support contact method, the solution applied to column extraction, it may be added to the aforementioned additives.
[0065]
　Hereinafter will be described the effect of exosomes method destruction using antimicrobial peptide according to the present embodiment.
　According to exosomes method of destruction using antimicrobial peptide according to the present embodiment, the safety to a living body and is secured, and it is possible to destroy the exosomes derived from cancer cell easily and efficiently. Further, according to this breakdown process, compared to conventional approaches, the exosomes derived from cancer cells can be selectively destroyed by a simple operation.
　Also, it exosomes method of destruction using antimicrobial peptide according to the present embodiment can be suitably used to extract biomarkers that are contained in exosomes. By using the destruction method according to the present embodiment described above, while suppressing to destroy exosomes from normal cells, it is possible to selectively destroy exosomes derived from cancer cells. Therefore, breaking method according to the present embodiment, in the field of cancer treatment in the future, screening of novel biomarkers related to cancer diseases, examination and diagnosis by efficient detection of existing cancer markers, and further, dialysis it is considered to be useful also be applied to the art.
[0066]
　Here, the extraction method of the biomarkers are contained in exosomes, solution after destruction by antimicrobial peptides can be extracted by a known method. For example, in the case of micro-RNA, it can be extracted using a commercially available RNA extraction kit. Detection of micro-RNA, for example, the extracted PCR device after obtaining cDNA using reverse transcriptase from the micro RNA were, in particular, such as measured using real-time PCR apparatus, employing the general methods known .
[0067]
　Further, according to the destruction process of exosomes of this embodiment, the exosomes in a sample, it is possible to destroy removed by substances having a safety to living body. When using this method, compared with the conventional method, exosomes and it is possible to efficiently remove the sample. Further, according to the destruction process of exosomes of the present embodiment, for example, by inhibiting the condition that different other components and exosomes in a sample is destroyed, effectively destroying removing exosomes in a sample it is possible. Therefore, as a sample containing exosomes, for example, when using blood samples taken from cancer patients living body, while suppressing the other different components to the exosomes in the blood sample is destroyed, but it is possible to destroy the exosomes derived from the N cells. In view of these circumstances, destruction method of exosomes of this embodiment can be said to be the techniques applicable to the future, cancer cell metastasis suppression system.
　Thus, for example, when a sample containing exosomes is a blood containing exosomes, a carrier having the above-mentioned antimicrobial peptides, it is possible to use as a configuration of the blood circulation system for destroying exosomes.
[0068]

　exosomes disruption kit from cancer cells according to the present embodiment is to utilize a destruction method described above is intended to include an antimicrobial peptide. The antimicrobial peptides contained in such destruction kit is for the exosomes derived from cancer cells can be selectively destroyed, a satisfying peptide of the following (1) or (2).
(1) a chain length of less than 10 to 50, wherein Net Charge is less than 15 greater than 0, and hydrophobic residues proportion is less than 65% to 25% (although, S-S bonds 3 or more and, except for those containing either one of the lysine residues and valine residues in the total amino acids constituting an antimicrobial peptide).
(2) a chain length of less than 10 more, Net Charge is 0 or less, and with hydrophobic residues proportion is less than 25%, the following condition (2-1) or (2-2) Fulfill.
(2-1) a histidine residue 3 or more in the total amino acids constituting an antimicrobial peptide, and free of tryptophan residues and valine residues.
(2-2) comprise one or more arginine residues in the total amino acids constituting antimicrobial peptides, do not contain a phenylalanine residue, a tryptophan residue and a valine residue.

[Claim 1]
　A step of preparing an antimicrobial peptide,
　and the antimicrobial peptide, by the coexistence of the exosomes, a step of breaking the exosomes
　exosomes method of destruction with.
[Claim 2]
　It said exosomes are exosomes method destruction of claim 1 which is exosomes derived from a cancer cell.
[Claim 3]
　The exosomes is a mixture of exosomes of normal cells from the exosomes derived from a cancer cell,
　in the step of destroying the exosomes, selective destruction of exosomes derived from the cancer cells, according to claim 1 exosomes method of destruction.
[Claim 4]
　Wherein the antimicrobial peptide is a satisfying peptide of the following (1) or (2), exosomes method destruction according to any one of claims 1 to 3.
(1) a chain length of less than 10 to 50, wherein Net Charge is less than 15 greater than 0, and hydrophobic residues proportion is less than 65% to 25% (although, S-S bonds 3 or more and, except for those containing either one of the total amino acid lysine and valine residues in constituting the antimicrobial peptide).
(2) a chain length of less than 10 more, Net Charge is 0 or less, and with hydrophobic residues proportion is less than 25%, the following condition (2-1) or (2-2) Fulfill.
(2-1) said comprises a histidine residue 3 or more in the total amino acids constituting an antimicrobial peptide, and free of tryptophan residues and valine residues.
(2-2) wherein comprises one or more arginine residues in the total amino acids constituting antimicrobial peptides, it does not contain a phenylalanine residue, a tryptophan residue and a valine residue.
[Claim 5]
　Wherein the antimicrobial peptide is a following condition is satisfied peptides, exosomes method destruction of claim 4.
(Conditions) a chain length of less than 20 or more 40 comprises less than 10 Net Charge is 1 or more, and a hydrophobic residue ratio is less than 65% to 25% (although, S-S bonds to 3 or more, and, except for those containing either one of the total amino acid lysine and valine residues in constituting the antimicrobial peptide).
[Claim 6]
　Contains an antimicrobial peptide, and wherein the antimicrobial peptide is a satisfying peptide of the following (1) or (2), exosomes breakdown kit from cancer cells.
(1) a chain length of less than 10 to 50, wherein Net Charge is less than 15 greater than 0, and hydrophobic residues proportion is less than 65% to 25% (although, S-S bonds 3 or more and, except for those containing either one of the total amino acid lysine and valine residues in constituting the antimicrobial peptide).
(2) a chain length of less than 10 more, Net Charge is 0 or less, and with hydrophobic residues proportion is less than 25%, the following condition (2-1) or (2-2) Fulfill.
(2-1) said comprises a histidine residue 3 or more in the total amino acids constituting an antimicrobial peptide, and free of tryptophan residues and valine residues.
(2-2) wherein comprises one or more arginine residues in the total amino acids constituting antimicrobial peptides, it does not contain a phenylalanine residue, a tryptophan residue and a valine residue.
[Claim 7]
　Further comprising a RNase Inhibitor, cancer cell-derived exosomes disruption kit of claim 6.
[8.]
　Includes a condition is satisfied the antimicrobial peptide of the following (1) or (2), a step to coexist and exosomes,
　the exosomes is a mixture of exosomes of normal cells from the exosomes from cancer cells, from a normal cell exosomes method of separation of.
(1) a chain length of less than 10 to 50, wherein Net Charge is less than 15 greater than 0, and hydrophobic residues proportion is less than 65% to 25% (although, S-S bonds 3 or more and, except for those containing either one of the total amino acid lysine and valine residues in constituting the antimicrobial peptide).
(2) a chain length of less than 10 more, Net Charge is 0 or less, and with hydrophobic residues proportion is less than 25%, the following condition (2-1) or (2-2) Fulfill.
(2-1) said comprises a histidine residue 3 or more in the total amino acids constituting an antimicrobial peptide, and free of tryptophan residues and valine residues.
(2-2) wherein comprises one or more arginine residues in the total amino acids constituting antimicrobial peptides, it does not contain a phenylalanine residue, a tryptophan residue and a valine residue.
[Claim 9]
　Step of the coexistence comprises a step of destroying the exosomes derived from the cancer cells,
　after the step of the coexistence, further comprising the step of adsorbing and removing destruction residues exosomes derived from the cancer cells, to claim 8 exosomes method of separating normal cells from the description.