KR102122885B1 - Nanovesicles derived from Exiguobacterium bacteria and Use thereof - Google Patents
Nanovesicles derived from Exiguobacterium bacteria and Use thereof Download PDFInfo
- Publication number
- KR102122885B1 KR102122885B1 KR1020190019741A KR20190019741A KR102122885B1 KR 102122885 B1 KR102122885 B1 KR 102122885B1 KR 1020190019741 A KR1020190019741 A KR 1020190019741A KR 20190019741 A KR20190019741 A KR 20190019741A KR 102122885 B1 KR102122885 B1 KR 102122885B1
- Authority
- KR
- South Korea
- Prior art keywords
- cancer
- vesicles
- disease
- derived
- bacterial
- Prior art date
Links
- 241001468125 Exiguobacterium Species 0.000 title claims description 3
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 33
- 206010004593 Bile duct cancer Diseases 0.000 claims abstract description 32
- 206010005003 Bladder cancer Diseases 0.000 claims abstract description 32
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 32
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 32
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 32
- 206010033128 Ovarian cancer Diseases 0.000 claims abstract description 32
- 206010061535 Ovarian neoplasm Diseases 0.000 claims abstract description 32
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims abstract description 32
- 206010060862 Prostate cancer Diseases 0.000 claims abstract description 32
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims abstract description 32
- 208000006011 Stroke Diseases 0.000 claims abstract description 32
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims abstract description 32
- 208000026900 bile duct neoplasm Diseases 0.000 claims abstract description 32
- 208000006990 cholangiocarcinoma Diseases 0.000 claims abstract description 32
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims abstract description 32
- 208000010125 myocardial infarction Diseases 0.000 claims abstract description 32
- 201000002528 pancreatic cancer Diseases 0.000 claims abstract description 32
- 208000008443 pancreatic carcinoma Diseases 0.000 claims abstract description 32
- 201000005112 urinary bladder cancer Diseases 0.000 claims abstract description 32
- 208000018737 Parkinson disease Diseases 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 26
- 201000010099 disease Diseases 0.000 claims abstract description 20
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 20
- 210000004369 blood Anatomy 0.000 claims description 72
- 239000008280 blood Substances 0.000 claims description 72
- 241000894006 Bacteria Species 0.000 claims description 68
- 210000002700 urine Anatomy 0.000 claims description 35
- 238000003752 polymerase chain reaction Methods 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 10
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 8
- 238000004445 quantitative analysis Methods 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 abstract description 72
- 208000005718 Stomach Neoplasms Diseases 0.000 abstract description 29
- 206010017758 gastric cancer Diseases 0.000 abstract description 29
- 201000011549 stomach cancer Diseases 0.000 abstract description 29
- 239000000203 mixture Substances 0.000 abstract description 25
- 208000029742 colonic neoplasm Diseases 0.000 abstract description 22
- 241000588724 Escherichia coli Species 0.000 abstract description 12
- 230000028327 secretion Effects 0.000 abstract description 10
- 238000003745 diagnosis Methods 0.000 abstract description 7
- 230000002757 inflammatory effect Effects 0.000 abstract description 7
- 230000001717 pathogenic effect Effects 0.000 abstract description 7
- 230000002265 prevention Effects 0.000 abstract description 6
- 230000006872 improvement Effects 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 description 42
- 238000009826 distribution Methods 0.000 description 28
- 108020004414 DNA Proteins 0.000 description 14
- 238000000692 Student's t-test Methods 0.000 description 12
- 238000012353 t test Methods 0.000 description 12
- 235000013305 food Nutrition 0.000 description 10
- 241000894007 species Species 0.000 description 9
- 239000008194 pharmaceutical composition Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 239000004480 active ingredient Substances 0.000 description 7
- 239000000796 flavoring agent Substances 0.000 description 7
- 235000013355 food flavoring agent Nutrition 0.000 description 7
- 230000000968 intestinal effect Effects 0.000 description 7
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 210000003734 kidney Anatomy 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 210000004400 mucous membrane Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 230000030429 T-helper 17 type immune response Effects 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 208000017667 Chronic Disease Diseases 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 229920000858 Cyclodextrin Polymers 0.000 description 2
- 241001468127 Exiguobacterium aurantiacum Species 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 206010022489 Insulin Resistance Diseases 0.000 description 2
- 241000736262 Microbiota Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000007975 buffered saline Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013376 functional food Nutrition 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 108020004513 Bacterial RNA Proteins 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 239000001512 FEMA 4601 Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- HELXLJCILKEWJH-SEAGSNCFSA-N Rebaudioside A Natural products O=C(O[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@@]1(C)[C@@H]2[C@](C)([C@H]3[C@@]4(CC(=C)[C@@](O[C@H]5[C@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@H](O)[C@@H](CO)O5)(C4)CC3)CC2)CCC1 HELXLJCILKEWJH-SEAGSNCFSA-N 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000014171 carbonated beverage Nutrition 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 229940126523 co-drug Drugs 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- HELXLJCILKEWJH-UHFFFAOYSA-N entered according to Sigma 01432 Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC(C1OC2C(C(O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O HELXLJCILKEWJH-UHFFFAOYSA-N 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- -1 for example Substances 0.000 description 1
- 238000001997 free-flow electrophoresis Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000009390 immune abnormality Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000000713 mesentery Anatomy 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 229940041678 oral spray Drugs 0.000 description 1
- 239000000668 oral spray Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000019203 rebaudioside A Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
본 발명은 엑시구오박테리움 속 세균 유래 소포 및 이의 용도에 관한 것으로, 본 발명자들은 정상인에 비하여 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병 환자의 임상샘플에서 상기 소포가 유의하게 감소되어 있었고, 상기 균주에서 분리한 소포를 투여하였을 때 대장균 유래 소포와 같은 병원성 소포에 의한 염증매개체 분비를 현저히 억제함을 실험적으로 확인하였는바, 본 발명에 따른 엑시구오박테리움 속 세균 유래 소포는 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 진단방법, 및 상기 질환에 대한 예방, 개선 또는 치료용 조성물을 개발하기 위한 목적으로 유용하게 이용될 수 있을 것이다. The present invention relates to bacterial-derived vesicles of the genus Excigubacterium and uses thereof, and the present inventors compared to normal people with stomach cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, And in the clinical sample of Parkinson's disease patients, the vesicles were significantly reduced, and when the vesicles isolated from the strain were administered, it was experimentally confirmed that the inflammatory mediator secretion by pathogenic vesicles such as E. coli-derived vesicles was significantly suppressed. The bacterial-derived vesicles of the genus Excigubacterium according to the present invention include gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease diagnosis method, and the disease It may be usefully used for the purpose of developing a composition for prevention, improvement or treatment.
Description
본 발명은 엑시구오박테리움 속 세균 유래 나노소포 및 이의 용도에 관한 것으로, 보다 구체적으로 엑시구오박테리움 속 세균에서 유래하는 나노소포를 이용한 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병 또는 파킨슨병 등의 진단방법, 및 상기 소포를 포함하는 상기 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to nanovesicles derived from bacteria of the genus Excigubacterium and uses thereof, more specifically gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer using nanovesicles derived from bacteria of the genus Excigubacterium. , Prostate cancer, myocardial infarction, stroke, diabetes or Parkinson's disease diagnostic methods, and the prevention, amelioration or treatment of the disease comprising the vesicles.
21세기에 들어서면서 과거 전염병으로 인식되던 급성 감염성질환의 중요성이 덜해지는 반면, 인간과 마이크로바이옴과의 부조화에 의해 발생하는 면역기능 이상을 동반한 만성질환이 삶의 질과 인간 수명을 결정하는 주요 질환으로 질병패턴이 바뀌었다. 21세기 난치성 만성질환으로서, 암, 심혈관질환, 만성폐질환, 대사질환, 및 신경-정신질환이 인간 수명과 삶의 질을 결정하는 주요 질환으로서 국민보건에 큰 문제가 되고 있으며, 상기 난치성 만성질환은 원인인자에 의한 면역기능 이상에 따른 만성 염증을 특징으로 한다.In the 21st century, while the importance of acute infectious diseases, which were previously recognized as infectious diseases, has become less important, chronic diseases accompanied by immune dysfunction caused by mismatch between humans and microbiomes determine the quality of life and human life. The disease pattern has changed to a major disease. As a refractory chronic disease in the 21st century, cancer, cardiovascular disease, chronic lung disease, metabolic disease, and neuro-psychiatric disease are major diseases that determine human lifespan and quality of life, and have become a major problem in public health. Is characterized by chronic inflammation caused by abnormal immune function caused by causal factors.
한편, 인체에 공생하는 미생물은 100조에 이르러 인간 세포보다 10배 많고, 미생물의 유전자수는 인간 유전자수의 100배가 넘는 것으로 알려지고 있다. 미생물총(microbiota 혹은 microbiome)은 주어진 거주지에 존재하는 진정세균(bacteria), 고세균(archaea), 진핵생물(eukarya)을 포함한 미생물 군집(microbial community)을 말한다. 우리 몸에 공생하는 세균 및 주변 환경에 존재하는 세균은 다른 세포로의 유전자, 저분자화합물, 단백질 등의 정보를 교환하기 위하여 나노미터 크기의 소포(vesicle)를 분비한다. 점막은 200 나노미터(nm) 크기 이상의 입자는 통과할 수 없는 물리적인 방어막을 형성하여 점막에 공생하는 세균인 경우에는 점막을 통과하지 못하지만, 세균 유래 소포는 크기가 100 나노미터 크기 이하라서 비교적 자유롭게 점막을 통하여 상피세포를 통과하여 우리 몸에 흡수된다. 국소적으로 분비된 세균 유래 소포는 점막의 상피세포를 통해 흡수되어 국소 염증반응을 유도할 뿐만 아니라, 상피세포를 통과한 소포는 림프관을 통해 전신적으로 흡수되어 각 장기로 분포하고, 분포된 장기에서 면역 및 염증반응을 조절한다. 예를 들어, 대장균(Eshcherichia coli)와 같은 병원성 그람음성세균에서 유래하는 소포는 국소적으로 대장염을 일으키고, 혈관으로 흡수된 경우에 혈관 내피세포 염증반응을 통해 전신적인 염증반응 및 혈액응고를 촉진시키고, 또한 인슐린이 작용하는 근육세포 등에 흡수되어선 인슐린저항성과 당뇨병을 유발한다. 반면, 유익한 세균에서 유래하는 소포는 병원성 소포에 의한 면역기능 및 대사기능 이상을 조절하여 질병을 조절할 수 있다. On the other hand, it is known that the microorganisms that symbiotic with the human body reach 100 trillion times, 10 times more than human cells, and the number of genes in the microorganism is more than 100 times the number of human genes. Microbiota (microbiota) refers to a microbial community (microbial community), including bacteria, archae, and eukarya. Bacteria that live in our bodies and bacteria that exist in the surrounding environment secrete nanometer-sized vesicles to exchange information such as genes, low-molecular compounds, and proteins into other cells. The mucous membrane forms a physical barrier that cannot pass particles over 200 nanometers (nm) in size, and in the case of bacteria that symbiotic with the mucous membrane, it does not pass through the mucous membrane, but the bacterial-derived vesicles are less than 100 nanometers in size. It passes through epithelial cells through the mucous membrane and is absorbed by our body. Locally secreted bacterial-derived vesicles are absorbed through epithelial cells of the mucous membrane to induce a local inflammatory reaction. In addition, vesicles that have passed through the epithelial cells are absorbed systemically through the lymphatic vessels and distributed to each organ. It regulates immune and inflammatory responses. For example, vesicles derived from pathogenic Gram-negative bacteria such as E. coli ( Eshcherichia coli ) locally develop colitis, and when absorbed into blood vessels, promote systemic inflammatory and blood coagulation through vascular endothelial inflammatory responses. In addition, insulin is absorbed by muscle cells, etc., which act, and causes insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can control disease by controlling immune and metabolic abnormalities caused by pathogenic vesicles.
세균에서 유래하는 소포 등의 인자에 대한 면역반응은 인터루킨(Interleukin, 이하 IL)-17 사이토카인 분비를 특징으로 하는 Th17 면역반응이 발생하는데, 이는 세균 유래 소포에 노출 시 IL-6가 분비되고, 이는 Th17 면역반응을 유도한다. Th17 면역반응에 의한 염증은 호중구 침윤을 특징으로 하고, 염증이 발생하는 과정에서 대식세포 등과 같은 염증세포에서 분비되는 종양괴사인자-알파(tumor necrosis factor-alpha, 이하 TNF-α)가 중요한 역할을 담당한다. The immune response to factors such as bacterial-derived vesicles produces a Th17 immune response characterized by the secretion of interleukin-17 cytokines, which secrete IL-6 upon exposure to bacterial-derived vesicles, This induces a Th17 immune response. Inflammation due to Th17 immune response is characterized by neutrophil infiltration, and tumor necrosis factor-alpha (TNF-α) secreted by inflammatory cells such as macrophages in the process of inflammation plays an important role. In charge.
엑시구오박테리움 속 세균은 다양한 환경에 서식하는 식물에서 분리되는 그람양성세균으로서 다양한 온도에서 자랄 수 있고, 지구상의 다양한 환경에서 발견된다. 그러나 아직까지 엑시구오박테리움 속 세균이 세포밖으로 소포를 분비한다는 사실이 보고되지 않았고, 특히 암 또는 심혈관-뇌질환 등과 같은 난치성 질환의 진단 및 치료에 응용한 사례는 보고된 바가 없다.Bacteria in the genus Exiggubacterium are Gram-positive bacteria isolated from plants inhabiting various environments and can grow at various temperatures and are found in various environments on Earth. However, it has not been reported that bacteria of the genus Excigubacterium secrete vesicles out of the cells, and there have been no reports of applications in the diagnosis and treatment of incurable diseases such as cancer or cardiovascular-brain disease.
본 발명자들은 상기와 같은 종래의 문제점을 해결하기 위해 예의 연구한 결과, 메타게놈 분석을 통해 정상인에 비하여 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병 환자 유래 샘플에서 엑시구오박테리움 속 세균 유래 소포의 함량이 유의하게 감소되어 있음 확인하였다. 또한, 엑시구오박테리움 속 세균에 속하는 엑시구오박테리움 오란티아쿰(Exiguobacterium aurantiacum ) 균에서 소포를 분리하여 대식세포에 처리하였을 때, 병원성 소포에 의한 IL-6 및 TNF-α 분비를 현저히 억제함을 확인한 바, 이에 기초하여 본 발명을 완성하였다. The present inventors, as a result of diligent research to solve the above-mentioned conventional problems, meta-genomic analysis, compared to the normal human stomach cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, It was confirmed that the content of bacteria-derived vesicles was significantly reduced in samples derived from diabetes and Parkinson's disease patients. Further, eksi Guo tumefaciens eksi Guo bacterium belonging to bacteria belonging to the genus Solarium Oran thiazol glutamicum (Exiguobacterium aurantiacum) also when separating the vesicles from the bacteria were treated macrophages, significantly inhibited the IL-6 and TNF-α secretion by pathogenic parcel Upon confirmation, the present invention was completed based on this.
이에, 본 발명은 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 진단을 위한 정보제공방법을 제공하는 것을 목적으로 한다. Accordingly, an object of the present invention is to provide an information providing method for the diagnosis of gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease.
또한, 본 발명은 엑시구오박테리움 속 세균 유래 소포를 유효성분으로 포함하는 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 예방, 개선 또는 치료용 조성물을 제공하는 것을 다른 목적으로 한다. In addition, the present invention prevents gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease, which includes vesicles derived from bacteria of the genus Excigubacterium as an active ingredient. Another object is to provide a composition for improvement or treatment.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 하기의 단계를 포함하는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 진단을 위한 정보제공방법을 제공한다:In order to achieve the object of the present invention as described above, the present invention includes the following steps, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's Provides informational methods for diagnosis of illness:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 엑시구오박테리움 속 세균 유래 세포밖 소포의 함량이 낮을 경우 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병으로 분류하는 단계.(c) Gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction when the content of extracellular vesicles derived from bacteria in the excigubacterium is lower than that of normal persons through quantitative analysis of the PCR products Classified as stroke, diabetes, or Parkinson's disease.
또한, 본 발명은 하기의 단계를 포함하는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 진단 방법을 제공한다:In addition, the present invention provides a method for diagnosing gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease, comprising the following steps:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 엑시구오박테리움 속 세균 유래 세포밖 소포의 함량이 낮을 경우 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병으로 분류하는 단계.(c) Gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction when the content of extracellular vesicles derived from bacteria in the excigubacterium is lower than that of normal persons through quantitative analysis of the PCR products Classified as stroke, diabetes, or Parkinson's disease.
본 발명의 일 구현예로, (a) 단계에서의 샘플은 혈액 또는 소변일 수 있다. In one embodiment of the present invention, the sample in step (a) may be blood or urine.
본 발명의 다른 구현예로, 상기 (b) 단계에서의 프라이머쌍은 서열번호 1 및 서열번호 2의 프라이머 일 수 있다. In another embodiment of the present invention, the primer pair in step (b) may be primers of SEQ ID NO: 1 and SEQ ID NO: 2.
또한, 본 발명은 엑시구오박테리움 속 세균 유래 소포를 유효성분으로 포함하는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention is a gastrointestinal cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease, comprising vesicles derived from bacteria of the genus Excigubacterium as an active ingredient. Provided is a pharmaceutical composition for prevention or treatment.
또한, 본 발명은 엑시구오박테리움 속 세균 유래 소포를 유효성분으로 포함하는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 예방 또는 개선용 식품 조성물을 제공한다. In addition, the present invention is a gastrointestinal cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease, comprising vesicles derived from bacteria of the genus Excigubacterium as an active ingredient. Provides a food composition for prevention or improvement.
또한, 본 발명은 엑시구오박테리움 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 예방 또는 치료 방법을 제공한다. In addition, the present invention comprises the step of administering to a subject a pharmaceutical composition comprising a bacterial-derived vesicle of the genus Excigubacterium as an active ingredient, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer , Myocardial infarction, stroke, diabetes, or Parkinson's disease.
또한, 본 발명은 엑시구오박테리움 속 세균 유래 소포의, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 예방 또는 치료 용도를 제공한다. In addition, the present invention is to prevent or treat the use of bacterial-derived vesicles of the genus Excigubacterium, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease. to provide.
본 발명의 일 구현예로, 상기 소포는 평균 직경이 10 내지 200 nm인 것일 수 있다. In one embodiment of the present invention, the vesicle may have an average diameter of 10 to 200 nm.
본 발명의 다른 구현예로, 상기 소포는 엑시구오박테리움 속 세균에서 자연적 또는 인공적으로 분비되는 것일 수 있다. In another embodiment of the present invention, the vesicle may be naturally or artificially secreted from bacteria of the genus Excigubacterium.
본 발명의 또 다른 구현예로, 상기 엑시구오박테리움 속 세균 유래 소포는 엑시구오박테리움 오란티아쿰 유래 소포일 수 있다.In another embodiment of the present invention, the bacterial-derived vesicles of the genus Excigubacterium may be vesicles derived from Excigubacterium orantiacum.
본 발명자들은 장내 세균인 경우에는 체내에 흡수되지 않지만, 세균 유래 소포인 경우에는 상피세포를 통해 체내에 흡수되어, 전신적으로 분포하고, 신장, 간, 폐를 통해 체외로 배설됨을 확인하였고, 환자 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 통해 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병 환자의 혈액 또는 소변에 존재하는 엑시구오박테리움 속 세균 유래 소포가 정상인에 비하여 유의하게 감소되어 있음을 확인하였다. 또한, 엑시구오박테리움 속 세균의 한 종인 엑시구오박테리움 오란티아쿰을 체외에서 배양하여 소포를 분리하여, 체외에서 염증세포에 투여하였을 때, 병원성 소포에 의한 염증매개체 분비를 유의하게 억제함을 관찰하였는 바, 본 발명에 따른 엑시구오박테리움 속 세균 유래 소포는 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병에 대한 진단방법, 및 상기 질환에 대한 예방용 혹은 치료용 조성물에 유용하게 이용될 수 있을 것으로 기대된다.The present inventors confirmed that in the case of intestinal bacteria, they are not absorbed into the body, but in the case of bacterial-derived vesicles, they are absorbed into the body through epithelial cells, distributed systemically, and excreted outside the body through the kidneys, liver, and lungs. Analysis of bacterial-derived vesicle metagenome present in gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, and exciguo in blood or urine of Parkinson's disease patients It was confirmed that the bacteria-derived vesicles of the genus Bacterium were significantly reduced compared to normal persons. In addition, when the excipiobacterium oriantiacum, a species of bacteria of the genus Excigubacterium, was cultured in vitro to separate vesicles and administered to inflammatory cells in vitro, it significantly inhibited the secretion of inflammatory mediators by pathogenic vesicles. As observed, the bacterial-derived vesicles of the genus Excigubacterium according to the present invention are gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, and Parkinson's disease diagnostic method And, it is expected that it may be usefully used in a composition for preventing or treating the disease.
도 1a는 마우스에 세균과 세균 유래 소포 (EV)를 구강으로 투여한 후, 시간별로 세균과 소포의 분포양상을 촬영한 사진이고, 도 1b는 구강으로 투여한 후 12시간째에, 혈액, 신장, 간, 및 여러 장기를 적출하여, 세균과 소포의 체내 분포양상을 평가한 그림이다.
도 2는 위암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 3은 대장암 환자 및 정상인 소변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 4는 췌장암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 5는 담관암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 6은 유방암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 7은 난소암 환자 및 정상인 소변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 8은 방광암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 9는 전립선암 환자 및 정상인 소변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 10은 심근경색 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 11은 뇌졸중 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 12는 당뇨병 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 13은 파킨슨병 환자 및 정상인 소변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 14는 엑시구오박테리움 오란티아쿰 유래 소포의 항염증 및 면역조절 효과를 평가하기 위하여, 병원성 소포인 대장균 소포 (E. coli EV) 처리 전에 엑시구오박테리움균 유래 소포를 전처리하여, 대장균 소포에 의한 염증매개체인 IL-6 및 TNF-α 분비에 미치는 영향을 평가한 결과이다(NC: negative control; PC: positive control, E. coli EV 1 ㎍/㎖; LP_1.0: Lactobacillus plantarum EV 1.0 ㎍/㎖; EA_0.1, 1.0, 10: Exiguobacterium aurantiacum EV 0.1, 1.0, 10 ㎍/㎖).1A is a photograph of bacteria and bacterial-derived vesicles (EV) administered orally to mice, and photographs of distribution patterns of bacteria and vesicles over time. FIG. 1B shows blood and
2 is a result of comparing the distribution of bacterial-derived vesicles in the genus Exiggubacterium after performing metagenome analysis of bacterial-derived vesicles present in gastric cancer patients and normal blood.
3 is a result of comparing the distribution of bacterial-derived vesicles in the genus Exiggubacterium after performing metagenome analysis of bacterial-derived vesicles present in patients with colon cancer and normal urine.
Figure 4 is a result of comparing the distribution of bacteria-derived vesicles in the genus Excigubacterium after performing metagenome analysis of bacterial-derived vesicles present in pancreatic cancer patients and normal blood.
5 is a result of comparing the distribution of bacterial-derived vesicles in the genus Excigubacterium after performing metagenome analysis of bacterial-derived vesicles present in patients with bile duct cancer and normal blood.
Figure 6 is a result of comparing the distribution of bacterial-derived vesicles in the genus Exiggubacterium after performing metagenome analysis of bacterial-derived vesicles present in breast cancer patients and normal blood.
7 is a result of comparing the distribution of bacterial-derived vesicles in the genus Excigubacterium after performing metagenome analysis of bacterial-derived vesicles present in ovarian cancer patients and normal urine.
8 is a result of comparing the distribution of bacterial-derived vesicles in the genus Exiggubacterium after performing metagenome analysis of bacterial-derived vesicles present in patients with bladder cancer and normal blood.
9 is a result of comparing the distribution of bacterial-derived vesicles in the genus Excigubacterium after performing metagenome analysis of bacterial-derived vesicles present in patients with prostate cancer and normal urine.
Figure 10 is a result of comparing the distribution of bacterial-derived vesicles in the genus Excigubacterium after performing a metagenome analysis of bacterial-derived vesicles present in patients with myocardial infarction and normal blood.
11 is a result of comparing the distribution of bacterial-derived vesicles in the genus Exiggubacterium after performing a metagenome analysis of bacterial-derived vesicles present in stroke patients and normal blood.
12 is a result of comparing the distribution of bacterial-derived vesicles in the genus Exiggubacterium after performing metagenome analysis of bacterial-derived vesicles present in diabetic patients and normal blood.
13 is a result of comparing the distribution of bacterial-derived vesicles in the genus Excigubacterium after performing metagenome analysis of bacterial-derived vesicles in Parkinson's disease patients and normal urine.
Figure 14 is to evaluate the anti-inflammatory and immunomodulatory effects of the excipient bacterium orantiacum-derived vesicles, E. coli vesicles ( E. coli EV) pre-treatment of vesicles derived from excigubacterium bacteria before treatment, E. coli vesicles It is the result of evaluating the effects on the inflammatory mediators IL-6 and TNF-α secretion (NC: negative control; PC: positive control, E. coli EV 1 ㎍ / ㎖; LP_1.0: Lactobacillus plantarum EV 1.0 ㎍ /Ml; EA_0.1, 1.0, 10: Exiguobacterium aurantiacum EV 0.1, 1.0, 10 μg/ml).
본 발명은 엑시구오박테리움 속 세균 유래 소포 및 이의 용도에 관한 것이다. 본 발명자들은 메타게놈 분석을 통해 엑시구오박테리움 속 세균 유래 소포가 정상인에 비하여 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병 환자의 임상 샘플에 유의하게 감소되어 있음을 확인하여 상기 질병을 진단할 수 있음을 확인하였다. 또한, 엑시구오박테리움 속 세균에 속하는 엑시구오박테리움 오란티아쿰으로 부터 소포를 분리하고 특성을 분석한 결과, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병 등의 질환에 대한 예방, 개선 또는 치료용 조성물로 이용할 수 있음을 확인하였다.The present invention relates to bacterial-derived vesicles of the genus Excigubacterium and uses thereof. According to the present inventors, metagenome analysis shows that bacterial-derived vesicles from the genus Excigubacterium have gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, and Parkinson's disease. It was confirmed that the disease can be diagnosed by confirming that it is significantly reduced in the clinical sample. In addition, as a result of separating and analyzing the vesicles from Exygubacterium orantiacum belonging to the bacteria of the genus Excigubacterium, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction , It was confirmed that it can be used as a composition for preventing, improving or treating diseases such as stroke, diabetes, and Parkinson's disease.
이에, 본 발명은 하기의 단계를 포함하는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 진단을 위한 정보제공방법을 제공한다:Thus, the present invention provides a method for providing information for diagnosis of gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, or Parkinson's disease, including the following steps: do:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 엑시구오박테리움 속 세균 유래 세포밖 소포의 함량이 낮을 경우 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병으로 분류하는 단계.(c) Gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction when the content of extracellular vesicles derived from bacteria in the excigubacterium is lower than that of normal persons through quantitative analysis of the PCR products Classified as stroke, diabetes, or Parkinson's disease.
본 발명에서 사용되는 용어, “진단”이란 넓은 의미로는 환자의 병의 실태를 모든 면에 걸쳐서 판단하는 것을 의미한다. 판단의 내용은 병명, 병인, 병형, 경중, 병상의 상세한 양태, 합병증의 유무, 및 예후 등이다. 본 발명에서 진단은 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및/또는 파킨슨병 등의 발병 여부 및 질환의 수준 등을 판단하는 것이다. As used in the present invention, the term “diagnosis” broadly means judging the condition of a patient's illness across all aspects. The content of the judgment is disease name, etiology, type, severity, detailed aspects of the bed, presence of complications, and prognosis. Diagnosis in the present invention is to determine whether the onset and disease level, such as gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, and/or Parkinson's disease.
본 발명에서 사용되는 용어, “나노소포(Nanovesicle)”혹은 “소포(Vesicle)”란, 다양한 세균에서 분비되는 나노크기의 막으로 된 구조물을 의미한다. 그람음성균(gram-negative bacteria) 유래 소포, 또는 외막 소포체(outer membrane vesicles, OMVs)는 내독소(lipopolysaccharide) 뿐만 아니라 독성 단백질 및 세균 DNA와 RNA도 가지고 있고, 그람양성균(gram-positive bacteria) 유래 소포는 단백질과 핵산 외에도 세균의 세포벽 구성성분인 펩티도글리칸(peptidoglycan)과 리포테이코산(lipoteichoic acid)도 가지고 있다. 본 발명에 있어서, 나노소포 혹은 소포는 엑시구오박테리움 속 세균에서 자연적으로 분비되거나 또는 인공적으로 생산하는 것으로, 구형의 형태이며, 10 내지 200 nm의 평균 직경을 가지고 있다.As used in the present invention, the term "nanovesicle (Nanovesicle)" or "vesicle (Vesicle)" refers to a structure made of nano-sized membranes secreted by various bacteria. Gram-negative bacteria-derived vesicles, or outer membrane vesicles (OMVs), contain not only endotoxins but also toxic proteins and bacterial DNA and RNA, and gram-positive bacteria-derived vesicles In addition to proteins and nucleic acids, it also contains peptidoglycan and lipoteichoic acid, which are components of the cell wall of bacteria. In the present invention, the nanovesicles or vesicles are naturally secreted or artificially produced by bacteria of the genus Excigubacterium, and have a spherical shape and have an average diameter of 10 to 200 nm.
본 발명에서 사용되는 용어, “메타게놈”이란 “군유전체”라고도 하며, 흙, 동물의 장 등 고립된 지역 내의 모든 바이러스, 세균, 곰팡이 등을 포함하는 유전체의 총합을 의미하는 것으로, 주로 배양이 되지 않는 미생물을 분석하기 위해서 서열분석기를 사용하여 한꺼번에 많은 미생물을 동정하는 것을 설명하는 유전체의 개념으로 쓰인다. 특히, 메타게놈은 한 종의 게놈, 유전체를 말하는 것이 아니라, 한 환경단위의 모든 종의 유전체로서 일종의 혼합유전체를 말한다. 이는 오믹스적으로 생물학이 발전하는 과정에서 한 종을 정의할 때 기능적으로 기존의 한 종뿐만 아니라, 다양한 종이 서로 상호작용하여 완전한 종을 만든다는 관점에서 나온 용어이다. 기술적으로는 빠른 서열분석법을 이용해서, 종에 관계없이 모든 DNA, RNA를 분석하여, 한 환경 내에서의 모든 종을 동정하고, 상호작용, 대사작용을 규명하는 기법의 대상이다.As used in the present invention, the term “metagenome” is also called “military genome” and refers to the sum of genomes including all viruses, bacteria, and fungi in isolated areas such as soil and animal intestines. It is used as a concept of genome to identify many microorganisms at once using a sequencer to analyze microorganisms that are not. In particular, metagenome does not refer to a genome or genome of one species, but a genome of all species of one environmental unit. This is a term that comes from the viewpoint that when defining a species in the course of biological development, it is functionally not only one existing species, but also various species interacting with each other to make a complete species. Technically, using rapid sequencing, it analyzes all DNA and RNA regardless of species, and is a target for techniques to identify all species in one environment and to identify interactions and metabolism.
본 발명에 있어서, 상기 샘플은 혈액 또는 소변일 수 있으나 이에 제한되는 것은 아니다.In the present invention, the sample may be blood or urine, but is not limited thereto.
본 발명의 다른 양태로서, 본 발명은 엑시구오박테리움 속 세균 유래 소포를 유효성분으로 포함하는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 또는 파킨슨병의 예방, 치료 또는 개선용 조성물을 제공한다. 상기 조성물은 식품 조성물 및 약학적 조성물을 포함하며, 본 발명에서 식품 조성물은 건강기능식품 조성물을 포함한다. 본 발명의 조성물은 구강분무제 또는 흡입제의 제형일 수 있다. As another aspect of the present invention, the present invention includes bacterial-derived vesicles from the genus Excigubacterium, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes Or, to provide a composition for preventing, treating or improving Parkinson's disease. The composition includes a food composition and a pharmaceutical composition, and in the present invention, the food composition includes a health functional food composition. The composition of the present invention may be a formulation of an oral spray or inhalant.
본 발명에서 사용되는 용어, “예방” 이란 본 발명에 따른 조성물의 투여에 의해 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및/또는 파킨슨병 등을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term “prevention” as used in the present invention means gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, and/or Parkinson's by administration of the composition according to the present invention. It means any action that suppresses illness or delays the onset.
본 발명에서 사용되는 용어, “치료”란 본 발명에 따른 조성물의 투여에 의해 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및/또는 파킨슨병 등에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. The term “treatment” used in the present invention means gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, and/or Parkinson's by administration of the composition according to the present invention. It means all actions that improve symptoms or change beneficially for illness.
본 발명에서 사용되는 용어, “개선”이란 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다. As used herein, the term "improvement" refers to any action that at least reduces the severity of the parameters associated with the condition being treated, such as symptoms.
상기 소포는 엑시구오박테리움 속 세균을 포함하는 배양액을 원심분리, 초고속 원심분리, 고압처리, 압출, 초음파분해, 세포 용해, 균질화, 냉동-해동, 전기천공, 기계적 분해, 화학물질 처리, 필터에 의한 여과, 겔 여과 크로마토그래피, 프리-플로우 전기영동, 및 모세관 전기영동으로 이루어진 군에서 선택된 하나 이상의 방법을 사용하여 분리할 수 있다. 또한, 불순물의 제거를 위한 세척, 수득된 소포의 농축 등의 과정을 추가로 포함할 수 있다. The vesicles are centrifuged, ultra-high-speed centrifugation, high-pressure treatment, extrusion, ultrasonic decomposition, cell lysis, homogenization, freeze-thaw, electroporation, mechanical decomposition, chemical treatment, filter to the culture medium containing bacteria of the genus Excigubacterium Filtration, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for removing impurities, concentration of the obtained vesicles, and the like.
본 발명에 따른 약학적 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제 시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제, 또는 경구 섭취제 등으로 제제화할 수 있다. The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in preparation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc. If necessary, it may further contain other conventional additives such as antioxidants, buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, and the like can be additionally added to prepare formulations for injection, pills, capsules, granules, or tablets, such as aqueous solutions, suspensions, and emulsions. Regarding suitable pharmaceutically acceptable carriers and formulations, the formulations described in Remington's literature can be used to formulate according to each component. The pharmaceutical composition of the present invention is not particularly limited in the formulation, but may be formulated as an injection, an inhalant, an external preparation for skin, or an oral intake.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 피부, 비강, 기도에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, skin, nasal cavity, and airways) according to a desired method, and the dosage is the patient's condition and weight, disease Depending on the degree, drug type, route of administration and time, it can be appropriately selected by those skilled in the art.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/ 위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the pharmaceutically effective amount means an amount sufficient to treat the disease at a ratio of rational benefit/risk applicable to medical treatment, and the effective dose level depends on the type, severity, drug activity, and drug of the patient. Sensitivity to administration, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명에 따른 약학적 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.001 내지 150 mg, 바람직하게는 0.01 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, and body weight, and in general, 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day Alternatively, it may be administered in 1 to 3 times a day. However, since the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, and age, the above dosage does not limit the scope of the present invention in any way.
본 발명의 식품 조성물은 건강기능식품 조성물을 포함한다. 본 발명에 따른식품 조성물은 유효성분을 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.The food composition of the present invention includes a health functional food composition. The food composition according to the present invention may be added as an active ingredient to the food or used with other food or food ingredients, it may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (for prevention or improvement). Generally, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less with respect to the raw materials, in the manufacture of a food or beverage. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be below the above range.
본 발명의 식품 조성물은 지시된 비율로 필수 성분으로서 상기 유효성분을 함유하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물, 예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The food composition of the present invention is an essential ingredient in the indicated proportions, and other ingredients than the above-mentioned active ingredient are not particularly limited, and may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, as in conventional beverages. Examples of the natural carbohydrates described above include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract, for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다. In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and It may contain salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. These ingredients can be used independently or in combination. The proportions of these additives can also be appropriately selected by those skilled in the art.
본 발명의 일 실시예에서는 세균 및 세균 유래 소포를 마우스 경구로 투여하여 세균 및 소포의 체내 흡수, 분포, 및 배설 양상을 관찰한 바, 세균인 경우에는 장점막을 통해 흡수되지 않는데 비해 소포는 투여 5분 이내에 흡수되어 전신적으로 분포하고, 신장, 간 등을 통해 배설됨을 확인하였다(실시예 1 참조).In one embodiment of the present invention, the bacteria and bacterial-derived vesicles are administered orally to the mouse to observe the absorption, distribution, and excretion of the bacteria and vesicles. In the case of bacteria, the vesicles are not absorbed through the mesentery membrane, whereas the vesicles are administered 5 It was confirmed that it was absorbed within minutes, distributed systemically, and excreted through the kidneys, liver, and the like (see Example 1).
본 발명의 다른 실시예에서는, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병 환자에 연령과 성별을 매칭한 정상인의 혈액 또는 소변에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 위암, 대장암, 췌장암, 담관암, 유방암, 난소암, 방광암, 전립선암, 심근경색, 뇌졸중, 당뇨병, 및 파킨슨병 환자의 임상샘플에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 3 내지 14 참조).In another embodiment of the present invention, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes, and Parkinson's disease patients with age and gender matching blood or urine Bacterial metagenome analysis was performed using the vesicles isolated from. As a result, compared to the normal sample, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, myocardial infarction, stroke, diabetes mellitus, and Parkinson's disease patients in clinical samples of bacteria from the genus Excigubacterium Was confirmed to be significantly reduced (see Examples 3 to 14).
본 발명의 또 다른 실시예에서는, 엑시구오박테리움 오란티아쿰 균주를 배양하여 이로부터 분비된 소포가 면역조절 및 항염증 효과를 나타내는지를 평가하였는데, 다양한 농도의 엑시구오박테리움 오란티아쿰 유래 소포를 대식세포에 처리한 후, 염증질환 원인인자인 대장균 유래 소포를 처리하여 염증매개체 분비를 평가한 결과, 대장균 유래 소포에 의한 IL-6 및 TNF-α 분비를 엑시구오박테리움 오란티아쿰 유래 소포가 효율적으로 억제함을 확인하였다(실시예 15 참조). In another embodiment of the present invention, it was evaluated whether the vesicles secreted therefrom by culturing the strain Excigubacterium orantiacum exhibiting immunomodulatory and anti-inflammatory effects, vesicles derived from various concentrations of excigubacterium orantiacum After treatment with macrophages, the treatment of inflammatory disease-causing E. coli-derived vesicles to evaluate inflammatory mediator secretion revealed that IL-6 and TNF-α secretion by E. coli-derived vesicles was derived from Excigubacterium orantiacum. Was confirmed to effectively inhibit (see Example 15).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1. 장내 세균 및 세균 유래 소포의 체내 흡수, 분포, 및 배설 양상 분석Example 1 Analysis of intestinal bacteria and bacterial-derived vesicles in body absorption, distribution, and excretion
장내 세균과 세균 유래 소포가 위장관을 통해 전신적으로 흡수되는 지를 평가하기 위하여 하기와 같은 방법으로 실험을 수행하였다. 마우스의 위장에 형광으로 표지한 장내세균과 장내 세균 유래 소포를 각각 50 μg의 용량으로 위장관으로 투여하고 0분, 5분, 3시간, 6시간, 12시간 후에 형광을 측정하였다. 마우스 전체 이미지를 관찰한 결과, 도 1a에 나타낸 바와 같이, 세균인 경우에는 전신적으로 흡수되지 않았지만, 세균 유래 소포인 경우에는, 투여 후 5분에 전신적으로 흡수되었고, 투여 3시간 후에는 방광에 형광이 진하게 관찰되어, 소포가 비뇨기계로 배설됨을 알 수 있었다. 또한, 소포는 투여 12시간까지 체내에 존재함을 알 수 있었다. To evaluate whether intestinal bacteria and bacterial-derived vesicles are systemically absorbed through the gastrointestinal tract, experiments were performed as follows. Fluorescence-labeled intestinal bacteria and intestinal bacteria-derived vesicles were administered to the gastrointestinal tract at a dose of 50 μg to the gastrointestinal tract of mice, and fluorescence was measured after 0 minutes, 5 minutes, 3 hours, 6 hours, and 12 hours. As a result of observing the whole mouse image, as shown in Fig. 1A, in the case of bacteria, it was not absorbed systemically, but in the case of bacterial-derived vesicles, it was absorbed systemically 5 minutes after administration, and fluorescence in the bladder 3 hours after administration. It was observed that the vesicle was excreted by the urinary system. In addition, it was found that the vesicles remained in the body until 12 hours of administration.
또한, 장내세균과 장내 세균 유래 소포가 전신적으로 흡수된 후, 여러 장기로 침윤된 양상을 평가하기 위하여, 형광으로 표지한 50 μg의 세균과 세균 유래 소포를 상기의 방법과 같이 투여한 후, 투여 12시간 후에 혈액, 심장, 폐, 간, 신장, 비장, 지방, 근육을 채취하였다. 채취한 조직에서 형광을 관찰한 결과, 도 1b에 나타낸 바와 같이, 세균 유래 소포가 혈액, 심장, 폐, 간, 비장, 지방, 근육, 신장에 분포하였으나, 세균은 흡수되지 않음을 알 수 있었다.In addition, after evaluating intestinal bacteria and intestinal bacteria-derived vesicles systemically and infiltrating into various organs, 50 μg of bacteria and bacterial-derived vesicles labeled with fluorescence are administered as described above, and then administered. After 12 hours, blood, heart, lung, liver, kidney, spleen, fat, and muscle were collected. As a result of fluorescence observation in the collected tissues, as shown in FIG. 1B, it was found that bacterial-derived vesicles were distributed in blood, heart, lung, liver, spleen, fat, muscle, and kidney, but bacteria were not absorbed.
실시예 2. 임상샘플에서 세균 유래 소포 메타게놈 분석Example 2. Metagenome analysis of bacterial-derived vesicles in clinical samples
혈액, 소변 등의 임상샘플을 먼저 10 ml 튜브에 넣고 원심분리법(3,500 x g, 10min, 4℃)으로 부유물을 가라앉히고 상등액만을 새로운 10 ml 튜브에 옮겼다. 0.22㎛ 필터를 사용하여 세균 및 이물질을 제거한 후, 센트리프랩튜브 (centrifugal filters 50 kD)에 옮겨서 1500 x g, 4℃에서 15분간 원심분리하여 50 kD 보다 작은 물질은 버리며 10 ml 까지 농축 시켰다. 다시 한 번 0.22㎛ 필터(filter)를 사용하여 박테리아 및 이물질을 제거한 후, Type 90ti 로터로 150,000 x g, 4℃에서 3시간동안 초고속원심분리방법을 사용하여 상등액을 버리고 덩어리진 펠렛(pellet)을 생리식염수(PBS)로 녹였다. Clinical samples such as blood and urine were first placed in a 10 ml tube, and the suspension was settled by centrifugation (3,500 x g, 10 min, 4°C), and only the supernatant was transferred to a new 10 ml tube. After removing the bacteria and foreign matter using a 0.22㎛ filter, transferred to a centrifugal filters (centrifugal filters 50 kD) and centrifuged at 1500 x g, 4 ℃ for 15 minutes, discarding substances smaller than 50 kD and concentrating to 10 ml. Once again, remove the bacteria and foreign substances using a 0.22㎛ filter, and discard the supernatant using a ultra-high-speed centrifugation method at 150,000 xg, 4℃ for 3 hours with a Type 90ti rotor, and physiologically agglomerate pellets. It was dissolved with saline (PBS).
상기 방법으로 분리한 소포 100㎕를 100℃에서 끓여서 내부의 DNA를 지질 밖으로 나오게 하고 그 후 얼음에 5분 동안 식혔다. 그리고 남은 부유물을 제거하기 위하여 10,000 x g, 4℃에서 30분간 원심분리하고 상등액만을 모았다. 그리고 Nanodrop을 이용하여 DNA 양을 정량하였다. 이후, 상기 추출된 DNA에 세균 유래 DNA가 존재하는지 확인하기 위하여 하기 표 1에 나타낸 16s rDNA 프라이머(primer)로 PCR을 수행하여 상기 추출된 유전자에 세균 유래 유전자가 존재하는 것을 확인하였다.100 μl of the vesicles separated by the above method was boiled at 100° C. to allow the DNA inside to come out of the lipid, and then cooled on ice for 5 minutes. Then, to remove the remaining suspended solids, centrifugation was performed at 10,000 x g at 4°C for 30 minutes, and only the supernatant was collected. And the amount of DNA was quantified using Nanodrop. Subsequently, in order to confirm that the bacterial-derived DNA was present in the extracted DNA, PCR was performed with the 16s rDNA primer shown in Table 1 below to confirm that the bacterial-derived gene was present in the extracted gene.
상기 방법으로 추출한 DNA를 상기의 16S rDNA 프라이머를 사용하여 증폭을 한 다음 시퀀싱을 수행하고 (Illumina MiSeq sequencer), 결과를 Standard Flowgram Format (SFF) 파일로 출력하고 GS FLX software (v2.9)를 이용하여 SFF 파일을 sequence 파일 (.fasta)과 nucleotide qualityscore파일로 변환한 다음 리드의 신용도 평가를 확인하고, window (20 bps) 평균 base call accuracy가 99% 미만 (Phred score <20)인 부분을 제거하였다. Operational Taxonomy Unit (OTU) 분석을 위해서는 UCLUST와 USEARCH를 이용하여 시퀀스 유사도에 따라 클러스터링을 수행하고, 속(genus)은 94%, 과(family)는 90%, 목(order)은 85%, 강(class)은 80%, 문(phylum)은 75% 시퀀스 유사도를 기준으로 클러스터링을 하고 각 OTU의 문(phylum), 강(class), 목(order), 과(family), 속(genus) 레벨의 분류를 수행하고, BLASTN와 GreenGenes의 16S RNA 시퀀스 데이터베이스 (108,453 시퀀스)를 이용하여 속 수준에서 97% 이상의 시퀀스 유사도 갖는 세균을 프로파일링 하였다 (QIIME).The DNA extracted by the above method is amplified using the 16S rDNA primer described above, followed by sequencing (Illumina MiSeq sequencer), the results are output to a Standard Flowgram Format (SFF) file, and GS FLX software (v2.9) is used. After converting the SFF file to a sequence file (.fasta) and a nucleotide qualityscore file, the reliability evaluation of the read was confirmed, and the part with a window (20 bps) average base call accuracy of less than 99% (Phred score <20) was removed. . For Operational Taxonomy Unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH, genus is 94%, family is 90%, order is 85%, and river ( class) 80%, phylum 75% clustering based on sequence similarity, and each OTU's phylum, class, order, family, genus levels Sorting was performed, and BLASTN and GreenGenes 16S RNA sequence database (108,453 sequences) were used to profile bacteria having sequence similarity of 97% or higher at the genus level (QIIME).
실시예 3. 위암환자의 혈액 내 세균 유래 소포 메타게놈 분석Example 3. Metagenome analysis of bacterial-derived vesicles in the blood of gastric cancer patients
실시예 2의 방법으로 위암환자 66명의 혈액과, 나이와 성별을 매칭한 정상인 198명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 위암환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 2 및 도 2 참조).In the method of Example 2, the blood of 66 patients with gastric cancer and the blood of 198 normal people with matching age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed, followed by genus Excigubacterium. Distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of gastric cancer patients compared to normal blood (see Table 2 and FIG. 2).
실시예 4. 대장암환자 소변 세균 유래 소포 메타게놈 분석Example 4. Metagenome analysis of vesicles from urine bacteria in colorectal cancer patients
실시예 2의 방법으로 대장암환자 38명의 소변과, 정상인 38명의 소변을 대상으로, 소변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 소변에 비하여 대장암환자의 소변에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 3 및 도 3 참조).The method of Example 2 targets the urine of 38 patients with colorectal cancer and the urine of 38 normal subjects, and after extracting a gene from the vesicles present in the urine, a metagenome analysis is performed, followed by distribution of bacterial-derived vesicles in the excigubacterium. Was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the urine of colon cancer patients compared to normal urine (see Table 3 and FIG. 3).
실시예 5. 췌장암환자 혈액 세균 유래 소포 메타게놈 분석Example 5. Analysis of vesicle metagenome from blood bacteria in pancreatic cancer patients
실시예 2의 방법으로 췌장암환자 176명의 혈액과, 나이와 성별을 매칭한 정상인 271명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 췌장암환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 4 및 도 4 참조).In the method of Example 2, the blood of 176 patients with pancreatic cancer and 271 normal people with matching age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles in the excigubacterium were significantly reduced in the blood of pancreatic cancer patients compared to normal blood (see Table 4 and FIG. 4).
실시예 6. 담관암환자 혈액 세균 유래 소포 메타게놈 분석Example 6 Analysis of vesicle metagenome from blood bacteria in patients with bile duct cancer
실시예 2의 방법으로 담관암환자 79명의 혈액과, 나이와 성별을 매칭한 정상인 259명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 담관암환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 5 및 도 5 참조).In the method of Example 2, the blood of 79 patients with bile duct cancer and the blood of 259 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed, followed by genus Excigubacterium. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that bacterial-derived vesicles were significantly reduced in the blood of patients with bile duct cancer compared to normal blood (see Table 5 and FIG. 5).
실시예 7. 유방암환자 혈액 세균 유래 소포 메타게놈 분석Example 7. Analysis of bacterial vesicle metagenome from breast cancer patients
실시예 2의 방법으로 유방암환자 96명의 혈액과, 나이와 성별을 매칭한 정상인 192명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 유방암환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 6 및 도 6 참조).In the method of Example 2, the blood of 96 breast cancer patients and the blood of 192 normal people who matched age and gender were extracted from the vesicles present in the blood, followed by metagenome analysis, and then genus Excigubacterium. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 6 and FIG. 6).
실시예 8. 난소암환자 소변 세균 유래 소포 메타게놈 분석Example 8. Analysis of vesicle metagenome derived from urine bacteria in ovarian cancer patients
실시예 2의 방법으로 난소암환자 135명의 소변과, 나이와 성별을 매칭한 정상인 136명의 소변을 대상으로, 소변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 소변에 비하여 난소암환자의 소변에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 7 및 도 7 참조).In the method of Example 2, the urine of 135 patients with ovarian cancer and 136 urine of normal age and gender matched subjects were extracted from the vesicles present in the urine, and then metagenome analysis was performed, followed by excigubacterium. Distribution of vesicles from genus bacteria was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the urine of ovarian cancer patients compared to normal urine (see Table 7 and FIG. 7).
실시예 9. 방광암환자 혈액 세균 유래 소포 메타게놈 분석Example 9. Analysis of vesicle metagenome from blood bacteria in bladder cancer patients
실시예 2의 방법으로 방광암환자 91명의 혈액과, 나이와 성별을 매칭한 정상인 176명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 방광암환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 8 및 도 8 참조).In the method of Example 2, the blood of 91 patients with bladder cancer and the blood of 176 normal people who matched age and gender were extracted from the vesicles in the blood, and then a metagenome analysis was performed, followed by genus Excigubacterium. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly decreased in the blood of bladder cancer patients compared to normal blood (see Table 8 and FIG. 8).
실시예 10. 전립선암환자 소변 세균 유래 소포 메타게놈 분석Example 10. Analysis of vesicle metagenome derived from urine bacteria in patients with prostate cancer
실시예 2의 방법으로 전립선암환자 55명의 소변과, 정상인 159명의 소변을 대상으로, 소변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 소변에 비하여 전립선암환자의 소변에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 9 및 도 9 참조).In the method of Example 2, the urine of 55 patients with prostate cancer and the urine of 159 normal subjects were extracted from the vesicles present in the urine, and then a metagenome analysis was performed, followed by distribution of bacterial-derived vesicles in the genus Excigubacterium. Was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in urine of prostate cancer patients compared to normal urine (see Table 9 and FIG. 9).
실시예 11. 심근경색환자 혈액 세균 유래 소포 메타게놈 분석Example 11.Metagenome analysis of vesicles derived from blood bacteria in myocardial infarction patients
실시예 2의 방법으로 심근경색 환자 57명의 혈액과, 나이와 성별을 매칭한 정상인 163명의 혈액의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 심근경색환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 10 및 도 10 참조).Using the method of Example 2, the blood of 57 patients with myocardial infarction and the blood of 163 normal people with matching age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacteria-derived vesicles in Bacterium was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of myocardial infarction patients compared to normal blood (see Table 10 and FIG. 10).
실시예 12. 뇌졸중환자 혈액 세균 유래 소포 메타게놈 분석Example 12. Stroke patient blood germ-derived vesicle metagenome analysis
실시예 2의 방법으로 뇌졸중환자 115명의 혈액과, 나이와 성별을 매칭한 정상인 109명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 뇌졸중환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 11 및 도 11 참조).In the method of Example 2, the blood of 115 patients with stroke and the blood of 109 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed, followed by excigubacterium genus. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of stroke patients compared to normal blood (see Table 11 and FIG. 11).
실시예Example 13. 당뇨병환자 혈액 세균 유래 소포 13. Blood germs from diabetic patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 당뇨병환자 61명의 혈액과, 나이와 성별을 매칭한 정상인 122명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 당뇨병환자의 혈액에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 12 및 도 12 참조).In the method of Example 2, the blood of 61 diabetic patients and the blood of 122 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed, followed by the genus Excigubacterium. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of diabetic patients compared to normal blood (see Table 12 and FIG. 12).
실시예 14. 파킨슨병환자 소변 세균 유래 소포 메타게놈 분석Example 14. Analysis of urine bacteria-derived vesicle metagenome in Parkinson's disease patients
실시예 2의 방법으로 파킨슨병환자 39명의 소변과, 나이와 성별을 매칭한 정상인 76명의 소변을 대상으로, 소변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 엑시구오박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 소변에 비하여 파킨슨병환자의 소변에 엑시구오박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 13 및 도 13 참조).In the method of Example 2, the urine of 39 patients with Parkinson's disease and the normal urine of 76 people who matched age and gender were extracted from the vesicles present in the urine to perform metagenome analysis, and then excigubacterium Distribution of vesicles from genus bacteria was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in urine of Parkinson's disease patients compared to normal urine (see Table 13 and FIG. 13).
실시예 15. 엑시구오박테리움 오란티아쿰 유래 소포의 항염증 효과Example 15. Anti-inflammatory effect of vesicles derived from Exygubacterium orantiacum
상기 실시예의 결과를 바탕으로, 엑시구오박테리움 속 세균에 속하는 엑시구오박테리움 오란티아쿰 균주를 배양한 후 이의 소포를 분리하였다. 엑시구오박테리움 오란티아쿰 균주를 37℃ 호기성 챔버에서 흡광도(OD600)가 1.0 내지 1.5가 될 때까지 BHI(brain heart infusion) 배지에서 배양한 후 서브 컬쳐(sub-culture) 하였다. 이후 균주가 포함되어 있지 않은 배지 상등액을 회수하여 10,000 g, 4 ℃에서 15분 동안 원심분리하고 0.45 μm 필터에 거른 후 거른 상등액을 100 kDa hollow 필터 맴브레인으로 QuixStand benchtop system(GE Healthcare, UK)을 이용하여 한외여과(ultrafiltration)를 통해 200 ㎖ 부피로 농축하였다. 이후 농축시킨 상등액을 다시 한번 0.22 μm 필터로 필터링 하고, 걸러진 상등액을 150,000 g, 4 ℃에서 3시간 동안 초원심분리한 후 펠렛을 DPBS로 현탁하였다. 다음으로 10 %, 40 %, 및 50 % 옵티프렙 용액(Axis-Shield PoC AS, Norway)을 이용해 밀도구배 원심분리를 수행하였고, 저밀도 용액 제조를 위해 옵티프렙 용액을 HEPES-buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4)에 희석하여 이용하였다. 200,000 g, 4 ℃ 조건으로 2시간 동안 원심분리를 수행한 후 윗층에서부터 1 ㎖의 동일한 볼륨으로 분획된 각 용액을 150,000 g, 4 ℃ 조건으로 3시간 동안 추가로 초원심분리를 실시하였다. 이후 BCA(Bicinchoninic acid) assay를 이용해 단백질을 정량하였고, 얻어진 소포에 대하여 실험을 실시하였다. Based on the results of the above example, after culturing the strain of Exygubacterium orantiacum belonging to the bacterium of the genus Exygubacterium, its vesicles were separated. The strain of Exygubacterium orantiacum was cultured in a brain heart infusion (BHI) medium until the absorbance (OD 600 ) of 1.0 to 1.5 in a 37°C aerobic chamber was subcultured. Subsequently, the culture medium supernatant that does not contain the strain was collected, centrifuged at 10,000 g, 4°C for 15 minutes, filtered through a 0.45 μm filter, and the filtered supernatant was used as a 100 kDa hollow filter membrane using the QuixStand benchtop system (GE Healthcare, UK). And concentrated to a volume of 200 ml through ultrafiltration. Thereafter, the concentrated supernatant was filtered once again with a 0.22 μm filter, and the filtered supernatant was ultracentrifuged at 150,000 g and 4° C. for 3 hours, and then the pellet was suspended with DPBS. Next, density gradient centrifugation was performed using 10%, 40%, and 50% Optiprep solution (Axis-Shield PoC AS, Norway), and the OptiPrep solution was HEPES-buffered saline (20 mM HEPES) to prepare a low-density solution. , 150 mM NaCl, pH 7.4). After centrifugation was performed for 2 hours under conditions of 200,000 g and 4°C, each solution fractionated into the same volume of 1 ml from the upper layer was further ultracentrifuged for 3 hours under conditions of 150,000 g and 4°C. Subsequently, proteins were quantified using a BCA (Bicinchoninic acid) assay, and experiments were conducted on the obtained vesicles.
엑시구오박테리움 오란티아쿰 유래 소포가 염증세포에서 염증매개체 분비에 대한 영향을 알아보기 위해, 마우스 대식세포주인 Raw 264.7 세포에 엑시구오박테리움 오란티아쿰 유래 소포를 다양한 농도(0.1, 1, 10 ㎍/㎖)로 처리한 후, 염증질환 병원성 소포인 대장균 유래 소포 (E. coli EV)를 처리하여 염증매개체 (IL-6, TNF-α 등)의 분비량을 측정하였다. 보다 구체적으로, Raw 264.7 세포를 1 x 105 개씩 24-well 세포 배양 플레이트에 분주한 후, 24시간 동안 DMEM(Dulbeco’s Modified Eagle’s Medium) 완전배지에서 배양시켰다. 이후, 배양 상층액을 1.5 ml 튜브에 모아 3000 g에서 5분간 원심분리하여 상층액을 모아 4 ℃에 보관해두었다가 ELISA 분석을 진행하였다. To investigate the effect of vesicles derived from Exygubacterium orantiacum on inflammatory mediator secretion from inflammatory cells, various concentrations (0.1, 1, 10) of vesicles derived from Exigubacterium orantiacum were applied to Raw 264.7 cells, a mouse macrophage cell line. Μg/ml), and then treated with E. coli EV ( E. coli EV), an inflammatory disease pathogenic vesicle, to measure the amount of secretion of inflammatory mediators (IL-6, TNF-α, etc.). More specifically, after dispensing Raw 264.7 cells into a 24-well cell culture plate by 1×10 5 cells, the cells were cultured in Dulbeco's Modified Eagle's Medium (DMEM) complete medium for 24 hours. Thereafter, the culture supernatant was collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, and the supernatant was collected and stored at 4°C for ELISA analysis.
그 결과, 도 14에 나타난 바와 같이 엑시구오박테리움 오란티아쿰 유래 소포를 전 처리 한 경우, 대장균 유래 소포에 의한 IL-6 및 TNF-α 분비가 현저히 억제됨을 확인하였다. 상기 결과는, 대장균 유래 소포와 같은 병원성 소포에 의해 유도되는 염증의 발생을 엑시구오박테리움 오란티아쿰 유래 소포가 효율적으로 억제할 수 있음을 의미한다.As a result, as shown in FIG. 14, when pre-treatment of vesicles derived from Exygubacterium orantiacum, IL-6 and TNF-α secretion by E. coli-derived vesicles was significantly inhibited. The above results indicate that excigubacterium orantiacum-derived vesicles can effectively suppress the development of inflammation induced by pathogenic vesicles such as E. coli-derived vesicles.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
<110> MD Healthcare Inc. <120> Nanovesicles derived from Exiguobacterium bacteria and Use thereof <130> MP19-011 <150> KR 10-2018-0022239 <151> 2018-02-23 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> 16S_V3_F <400> 1 tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50 <210> 2 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 16S_V4_R <400> 2 gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55 <110> MD Healthcare Inc. <120> Nanovesicles derived from Exiguobacterium bacteria and Use thereof <130> MP19-011 <150> KR 10-2018-0022239 <151> 2018-02-23 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> 16S_V3_F <400> 1 tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50 <210> 2 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 16S_V4_R <400> 2 gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55
Claims (10)
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 엑시구오박테리움 (Exiguobacterium) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 하나 이상의 질환을 가지고 있을 것으로 예측하는 단계를 포함하는, 질환 발병 예측을 위한 정보제공방법으로서,
상기 (a) 단계에서의 샘플은 혈액 또는 소변이고,
상기 (a) 단계에서의 샘플이 혈액일 경우, 상기 질환은 췌장암, 담관암, 유방암, 방광암, 심근경색, 뇌졸중 및 당뇨병으로 이루어진 군으로부터 선택된 하나 이상의 질환이며,
상기 (a) 단계에서의 샘플이 소변일 경우, 상기 질환은 대장암, 난소암, 전립선암 및 파킨슨병으로 이루어진 군으로부터 선택된 하나 이상의 질환인 것을 특징으로 하는, 질환 발병 예측을 위한 정보제공방법.
(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) through the quantitative analysis of the PCR product, predicting the onset of disease, comprising predicting that if the content of extracellular vesicles derived from bacteria in the genus Exiguobacterium is low compared to a normal person As a method for providing information,
The sample in step (a) is blood or urine,
When the sample in step (a) is blood, the disease is at least one disease selected from the group consisting of pancreatic cancer, bile duct cancer, breast cancer, bladder cancer, myocardial infarction, stroke and diabetes,
When the sample in step (a) is urine, the disease is at least one disease selected from the group consisting of colorectal cancer, ovarian cancer, prostate cancer and Parkinson's disease, information providing method for predicting disease outbreak.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020180022239 | 2018-02-23 | ||
| KR20180022239 | 2018-02-23 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020200063160A Division KR102122894B1 (en) | 2018-02-23 | 2020-05-26 | Nanovesicles derived from Exiguobacterium bacteria and Use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20190101885A KR20190101885A (en) | 2019-09-02 |
| KR102122885B1 true KR102122885B1 (en) | 2020-06-15 |
Family
ID=67951430
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020190019741A KR102122885B1 (en) | 2018-02-23 | 2019-02-20 | Nanovesicles derived from Exiguobacterium bacteria and Use thereof |
| KR1020200063160A KR102122894B1 (en) | 2018-02-23 | 2020-05-26 | Nanovesicles derived from Exiguobacterium bacteria and Use thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020200063160A KR102122894B1 (en) | 2018-02-23 | 2020-05-26 | Nanovesicles derived from Exiguobacterium bacteria and Use thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (2) | KR102122885B1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110229916A1 (en) | 2007-08-17 | 2011-09-22 | The University Of Akron | Nanofibers with high enzyme loading for highly sensitive biosensors |
| KR101361730B1 (en) | 2013-06-18 | 2014-02-13 | 동국대학교 산학협력단 | DNA Polymorphism Marker for Identification of Cucumis sativus L. |
| KR101833348B1 (en) * | 2016-12-26 | 2018-03-02 | 주식회사 엠디헬스케어 | Method for diagnosis of breast cancer using analysis of bacteria metagenome |
| KR101833502B1 (en) * | 2016-12-16 | 2018-03-05 | 주식회사 엠디헬스케어 | Method for diagnosis of gastric cancer using analysis of bacteria metagenome |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1782685A1 (en) * | 2005-11-04 | 2007-05-09 | De Ruiter Seeds R&D B.V. | Disease resistant cucumber plants |
| PL2382322T3 (en) * | 2009-01-26 | 2014-05-30 | Siemens Healthcare Diagnostics Inc | Device and method for detection of humidity-compromised urine test strips |
| KR101856841B1 (en) * | 2016-05-12 | 2018-05-10 | 단국대학교 산학협력단 | Enzyme immobilized glucose biosensor and the manufacturing method thereof |
-
2019
- 2019-02-20 KR KR1020190019741A patent/KR102122885B1/en active IP Right Grant
-
2020
- 2020-05-26 KR KR1020200063160A patent/KR102122894B1/en active IP Right Grant
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110229916A1 (en) | 2007-08-17 | 2011-09-22 | The University Of Akron | Nanofibers with high enzyme loading for highly sensitive biosensors |
| KR101361730B1 (en) | 2013-06-18 | 2014-02-13 | 동국대학교 산학협력단 | DNA Polymorphism Marker for Identification of Cucumis sativus L. |
| KR101833502B1 (en) * | 2016-12-16 | 2018-03-05 | 주식회사 엠디헬스케어 | Method for diagnosis of gastric cancer using analysis of bacteria metagenome |
| KR101833348B1 (en) * | 2016-12-26 | 2018-03-02 | 주식회사 엠디헬스케어 | Method for diagnosis of breast cancer using analysis of bacteria metagenome |
Also Published As
| Publication number | Publication date |
|---|---|
| KR102122894B1 (en) | 2020-06-15 |
| KR20200062143A (en) | 2020-06-03 |
| KR20190101885A (en) | 2019-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102117567B1 (en) | Nanovesicles derived from Cupriavidus bacteria and Use thereof | |
| KR102282490B1 (en) | Nanovesicles derived from Faecalibacterium prausnitzii and Use thereof | |
| KR102118987B1 (en) | Nanovesicles derived from Morganella bacteria and Use thereof | |
| KR102118996B1 (en) | Nanovesicles derived from Veillonella bacteria and Use thereof | |
| KR102122903B1 (en) | Nanovesicles derived from Blautia bacteria and Use thereof | |
| KR102250596B1 (en) | Nanovesicles derived from Bifidobacterium bacteria and Use thereof | |
| KR102122894B1 (en) | Nanovesicles derived from Exiguobacterium bacteria and Use thereof | |
| US20220186292A1 (en) | Nano-vesicle derived from catenibacterium bacteria and use thereof | |
| KR102118201B1 (en) | Nanovesicles derived from Methylobacterium bacteria and Use thereof | |
| KR102118198B1 (en) | Nanovesicles derived from Rhizobium bacteria and Use thereof | |
| KR102118989B1 (en) | Nanovesicles derived from Enhydrobacter bacteria and Use thereof | |
| KR102118993B1 (en) | Nanovesicles derived from Prevotella bacteria and Use thereof | |
| KR102194274B1 (en) | Nanovesicles derived from Catenibacterium bacteria and Use thereof | |
| KR102230479B1 (en) | Nanovesicles derived from Turicibacter bacteria and Use thereof | |
| KR102124794B1 (en) | Nanovesicles derived from Geobacillus bacteria and Use thereof | |
| KR102118199B1 (en) | Nanovesicles derived from Atopobium bacteria and Use thereof | |
| KR102185985B1 (en) | Nanovesicles derived from Alloiococcus bacteria and Use thereof | |
| KR102185982B1 (en) | Nanovesicles derived from Pseudomonas bacteria and Use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| E601 | Decision to refuse application | ||
| X091 | Application refused [patent] | ||
| A107 | Divisional application of patent | ||
| AMND | Amendment | ||
| X701 | Decision to grant (after re-examination) | ||
| GRNT | Written decision to grant |