KR102526447B1 - A composition for preventing or treating of liver disease comprising conditioned medium of tonsil-derived mesenchymal stem cell - Google Patents
A composition for preventing or treating of liver disease comprising conditioned medium of tonsil-derived mesenchymal stem cell Download PDFInfo
- Publication number
- KR102526447B1 KR102526447B1 KR1020180037755A KR20180037755A KR102526447B1 KR 102526447 B1 KR102526447 B1 KR 102526447B1 KR 1020180037755 A KR1020180037755 A KR 1020180037755A KR 20180037755 A KR20180037755 A KR 20180037755A KR 102526447 B1 KR102526447 B1 KR 102526447B1
- Authority
- KR
- South Korea
- Prior art keywords
- mesenchymal stem
- stem cells
- derived mesenchymal
- conditioned medium
- preventing
- Prior art date
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 70
- 239000003636 conditioned culture medium Substances 0.000 title claims abstract description 59
- 239000000203 mixture Substances 0.000 title claims abstract description 27
- 210000002741 palatine tonsil Anatomy 0.000 title claims description 41
- 208000019423 liver disease Diseases 0.000 title abstract description 44
- 208000019425 cirrhosis of liver Diseases 0.000 claims abstract description 21
- 239000004480 active ingredient Substances 0.000 claims abstract description 7
- 235000013305 food Nutrition 0.000 claims description 18
- 206010016654 Fibrosis Diseases 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000012679 serum free medium Substances 0.000 claims description 6
- 239000012228 culture supernatant Substances 0.000 claims description 5
- 230000004761 fibrosis Effects 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 claims description 4
- 108010016113 Matrix Metalloproteinase 1 Proteins 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 claims description 3
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 13
- 239000003112 inhibitor Substances 0.000 abstract description 4
- 210000000130 stem cell Anatomy 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 22
- 238000000034 method Methods 0.000 description 16
- 239000002609 medium Substances 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 12
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 11
- 208000006454 hepatitis Diseases 0.000 description 10
- 239000006143 cell culture medium Substances 0.000 description 9
- 210000005228 liver tissue Anatomy 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 8
- 102000008186 Collagen Human genes 0.000 description 7
- 108010035532 Collagen Proteins 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 229920001436 collagen Polymers 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 230000007882 cirrhosis Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 230000036541 health Effects 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 101150008656 COL1A1 gene Proteins 0.000 description 5
- 101100490443 Mus musculus Acvr1 gene Proteins 0.000 description 5
- 101150000629 TGFB1 gene Proteins 0.000 description 5
- 101150077804 TIMP1 gene Proteins 0.000 description 5
- 102000005353 Tissue Inhibitor of Metalloproteinase-1 Human genes 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 235000013376 functional food Nutrition 0.000 description 5
- 231100000283 hepatitis Toxicity 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 208000004930 Fatty Liver Diseases 0.000 description 4
- 206010019708 Hepatic steatosis Diseases 0.000 description 4
- 231100000354 acute hepatitis Toxicity 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 208000010706 fatty liver disease Diseases 0.000 description 4
- 235000013373 food additive Nutrition 0.000 description 4
- 239000002778 food additive Substances 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 231100000240 steatosis hepatitis Toxicity 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000002659 cell therapy Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 239000000314 lubricant Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 208000008964 Chemical and Drug Induced Liver Injury Diseases 0.000 description 2
- 101100447432 Danio rerio gapdh-2 gene Proteins 0.000 description 2
- 101150112014 Gapdh gene Proteins 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 206010019728 Hepatitis alcoholic Diseases 0.000 description 2
- 206010019799 Hepatitis viral Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 102000046299 Transforming Growth Factor beta1 Human genes 0.000 description 2
- 101800002279 Transforming growth factor beta-1 Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 208000002353 alcoholic hepatitis Diseases 0.000 description 2
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 2
- 210000004727 amygdala Anatomy 0.000 description 2
- 229940121375 antifungal agent Drugs 0.000 description 2
- 239000003429 antifungal agent Substances 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 239000007884 disintegrant Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000007902 hard capsule Substances 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000003475 metalloproteinase inhibitor Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000013424 sirius red staining Methods 0.000 description 2
- 239000007901 soft capsule Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 229940099456 transforming growth factor beta 1 Drugs 0.000 description 2
- 201000001862 viral hepatitis Diseases 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 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-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-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
- 206010072268 Drug-induced liver injury Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229920002907 Guar gum Polymers 0.000 description 1
- 206010019637 Hepatic atrophy Diseases 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930194936 Tylosin Natural products 0.000 description 1
- 239000004182 Tylosin Substances 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 231100000359 cholestasis Toxicity 0.000 description 1
- 230000007870 cholestasis Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- -1 corrigents Substances 0.000 description 1
- 229960000913 crospovidone Drugs 0.000 description 1
- 230000002338 cryopreservative effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 201000008865 drug-induced hepatitis Diseases 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012595 freezing medium Substances 0.000 description 1
- 238000012812 general test Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 229960005150 glycerol Drugs 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000000665 guar gum Substances 0.000 description 1
- 235000010417 guar gum Nutrition 0.000 description 1
- 229960002154 guar gum Drugs 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 229940069445 licorice extract Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 235000019462 natural additive Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940068965 polysorbates Drugs 0.000 description 1
- 229920000523 polyvinylpolypyrrolidone Polymers 0.000 description 1
- 235000013809 polyvinylpolypyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 229940069328 povidone Drugs 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000007483 tonsillectomy Methods 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 description 1
- 229960004059 tylosin Drugs 0.000 description 1
- 235000019375 tylosin Nutrition 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- 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
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
Abstract
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 유효성분으로 포함하는 간질환 예방, 개선 또는 치료용 조성물에 관한 것이다.
본 발명의 간질환 예방, 개선 또는 치료용 조성물은 타유래 중간엽 줄기세포의 조정 배지와 대비하여, 조정 배지 내 면역 조절 인자, 간섬유화 억제 인자들의 상이한 프로파일을 가지고 이로 인하여 간질환의 예방, 개선 및 치료에 우수한 효과를 가진다. The present invention relates to a composition for preventing, improving or treating liver disease comprising a conditioned medium of tonsillar-derived mesenchymal stem cells as an active ingredient.
The composition for preventing, improving or treating liver disease of the present invention has a different profile of immune modulators and liver fibrosis inhibitors in the conditioned medium compared to the conditioned medium of heterologous mesenchymal stem cells, thereby preventing or improving liver disease. And it has an excellent effect on treatment.
Description
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 유효성분으로 포함하는 간질환 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing, improving or treating liver disease comprising a conditioned medium of tonsillar-derived mesenchymal stem cells as an active ingredient.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 개선용 식품 조성물에 관한 것이다.The present invention relates to a food composition for preventing or improving liver disease comprising a conditioned medium of tonsillar-derived mesenchymal stem cells.
간은 우리 몸에서 지질 등의 대사 작용, 해독, 담즙의 배설, 각종 영양소의 저장, 조혈이나 혈액 응고, 및 순환 혈액량의 조절 등 다양한 역할을 담당하고 있으며, 생명 유지를 위한 필수적인 장기 중 하나이다. 간의 기능을 보다 구체적으로 살펴보면, 우선, 에너지 대사를 관리하는 기능이 있어 음식물에서 흡수된 모든 영양소들이 간에서 에너지를 생산할 수 있는 물질로 대사되어 전신에 공급되거나 저장된다. 둘째, 간은 약 2,000 여종의 효소, 알부민, 응고 인자들의 혈청 단백질, 담즙산, 인지질, 콜레스테롤 등의 지방 등을 합성하고 저장하며 분배하는 기능이 있다. 셋째, 해독 및 분해 기능으로서 약물, 술, 독성물질 등을 해독시키는 기능, 각종 대사산물을 십이지장으로 배설하는 기능 및 면역 기능이 있어 생명 유지에 중요한 역할을 하고 있다.The liver plays various roles in our body, such as metabolism of lipids, detoxification, excretion of bile, storage of various nutrients, hematopoiesis, blood coagulation, and control of circulating blood volume, and is one of the essential organs for maintaining life. Looking at the function of the liver in more detail, first of all, it has a function of managing energy metabolism, so all nutrients absorbed from food are metabolized into substances capable of producing energy in the liver and supplied to or stored throughout the body. Second, the liver has the function of synthesizing, storing, and distributing about 2,000 kinds of enzymes, albumin, serum proteins of coagulation factors, bile acids, phospholipids, and fats such as cholesterol. Third, as a detoxification and decomposition function, it plays an important role in maintaining life by detoxifying drugs, alcohol, toxic substances, etc., excreting various metabolites into the duodenum, and immune function.
일반적으로 간에 염증이 생기는 간염이 간질환의 대부분을 차지하며, 양상에 따라 급성 간염과 만성 감염, 원인에 따라 바이러스성 간염, 알코올성 간염, 약물성 간염 등으로 나눌 수 있다. 또한, 이런 이상으로 유발되는 간질환에는 지방간, 간염, 간경변증, 간암 등이 있다. 간질환의 진행 과정의 기전은 완전히 밝혀지지는 않았으나 일차적으로 지방간이 발생된 후 이차적인 세포 손상이 수반되어 지방간 및 간경화 등의 진행성 간질환으로 발전하는 것으로 알려져 있다. 또한, 간질환은 초기에 자각 증상이 없어 상당히 진행되어서야 발견되기 때문에, 국내뿐만 아니라 세계적으로 사망 원인의 수위를 차지하고 있다.In general, hepatitis, which causes inflammation of the liver, accounts for most of the liver diseases, and can be divided into acute hepatitis and chronic infection according to the aspect, and viral hepatitis, alcoholic hepatitis, and drug-induced hepatitis according to the cause. In addition, liver diseases caused by such abnormalities include fatty liver, hepatitis, cirrhosis, and liver cancer. Although the mechanism of the progression of liver disease has not been fully elucidated, it is known that primary fatty liver is followed by secondary cell damage, leading to progressive liver disease such as fatty liver and cirrhosis. In addition, since liver disease has no subjective symptoms at an early stage and is not discovered until it is significantly progressed, it occupies the leading cause of death not only in Korea but also worldwide.
한편, 줄기세포(stem cell)는 생물 조직을 구성하는 다양한 세포들로 분화할 수 있는 세포로서 배아, 태아 및 성체의 각 조직에서 얻을 수 있는 분화되기 전 단계의 미분화 세포들을 총칭한다. On the other hand, stem cells (stem cells) are cells capable of differentiating into various cells constituting biological tissues, and collectively refer to undifferentiated cells in a pre-differentiated stage that can be obtained from each tissue of an embryo, fetus, and adult.
치료 방법으로서 최근 줄기세포 및 조직공학을 이용한 이식치료법이 개발되고 있다. 하지만, 이러한 이식 치료 기법으로는 치료효과가 뛰어나고 인체의 면역반응을 일으키지 않는 안정한 세포 치료제를 개발하기 어렵다는 문제가 있다. 그 동안 여러 가지 줄기세포를 이용하여 세포 치료제로써 활용하기 위한 시도들이 진행되고 있지만, 면역 조절 능력에 대한 확인은 아직 부분적으로만 진행되고 있다. 또한, 최근에는 줄기세포를 직접 손상된 조직에 이식하는 방법 이외에 줄기세포의 배양액에 포함된 매개성 물질의 탁월한 측분비 효과가 생체내 또는 생체외 실험에서 입증되고 있다. 줄기세포는 인체 내에서 일생 동안 세포의 성장과 재생에 관련된 다양한 성장인자와 사이토카인 등을 분비하고, 줄기세포 배양액에는 이러한 단백질 성분들이 함유되어 있으며, 세포이식 시의 부작용인 면역거부반응을 회피할 수 있으므로, 줄기세포 배양액은 치료제로 개발하기에 유리한 장점을 가지기 때문에 관련 연구개발이 진행되고 있다. As a treatment method, transplant therapy using stem cells and tissue engineering has recently been developed. However, there is a problem in that it is difficult to develop a stable cell therapy that has excellent therapeutic effect and does not cause an immune response in the human body using such a transplant therapy technique. In the meantime, attempts have been made to utilize various stem cells as cell therapy agents, but confirmation of their immune modulating ability has only partially progressed. In addition, in addition to the method of directly transplanting stem cells into damaged tissues, the excellent paracrine effect of the mediating substances contained in the culture medium of stem cells has recently been demonstrated in in vivo or in vitro experiments. Stem cells secrete various growth factors and cytokines related to cell growth and regeneration throughout life in the human body. Therefore, since stem cell culture medium has an advantageous advantage for development as a therapeutic agent, related research and development is being conducted.
상기와 같은 줄기세포 관련 연구를 진행하기 위해서는 줄기세포의 원활한 수급이 필요하다. 그러나, 중간엽 줄기세포 중에서도 몇몇 줄기세포는 그 이용에 있어서 세포를 얻는데 큰 제한이 있기 때문에 그 이용이 어렵다. 예를 들어, 제대혈, 지방조직으로부터 유래한 중간엽 줄기세포는 침습적인 방법을 이용해 얻어야만 한다. 가장 비침습적인 방법을 이용해 얻을 수 있는 세포는 골수채취를 통한 중간엽 줄기세포이지만, 골수채취는 마취가 필요하고 고통을 유발하여, 그 이용에 제한이 있다. 이에 대한 대안으로 환자 맞춤형 줄기세포를 분리하기 위해 말초혈액을 이용한 세포획득법 등이 요구되고 있으나 말초혈액만으로는 성인에서 분리할 수 있는 중간엽 줄기세포의 수가 너무 적고 분리방법이 경제적이지 못하며, 분리해낸다고 해도 세포치료에 사용가능한 양만큼 증식이 원활하지 않은 경우가 대부분이기에 좀 더 실용성을 높일 수 있는 대체 방안이 필요하다. 또한, 고령인 환자에서 얻는 성체 줄기세포는 낮은 연령에게서 얻는 세포들에 비해 세포능이 현저히 떨어지며 각종 인자들의 분비 및 줄기세포의 이동 능력 등이 떨어지기 때문에, 저연령의 환자로부터 자연스럽게 분리될 수 있거나 버려지는 조직으로부터 세포를 얻을 필요성이 있다. 또한, 이렇게 얻어진 세포는 실험에 용이한 양적 확보가 가능하고 세포의 계대 배양 시에 분화능이 잘 유지될 필요성이 있다. In order to conduct stem cell-related research as described above, a smooth supply and demand of stem cells is required. However, some stem cells among mesenchymal stem cells are difficult to use because there are great limitations in obtaining cells in their use. For example, mesenchymal stem cells derived from umbilical cord blood or adipose tissue must be obtained using an invasive method. The cells that can be obtained using the most non-invasive method are mesenchymal stem cells through bone marrow harvesting, but bone marrow harvesting requires anesthesia and causes pain, so there is a limitation in its use. As an alternative to this, a cell acquisition method using peripheral blood is required to isolate patient-specific stem cells, but the number of mesenchymal stem cells that can be isolated from adults using only peripheral blood is too small and the separation method is not economical, and the separation method is not economical. Even if it is produced, in most cases the growth is not as smooth as the amount that can be used for cell therapy, so an alternative method that can increase practicality is needed. In addition, since adult stem cells obtained from elderly patients have significantly lower cellular activity and lower secretion of various factors and lower ability of stem cells to migrate compared to cells obtained from younger patients, they can be naturally isolated from younger patients or discarded. need to obtain cells from tissue. In addition, it is necessary that the cells obtained in this way can be easily secured quantitatively for experiments and that the differentiation potential is well maintained during subculture of the cells.
또한, 줄기세포들은 그 유래에 따라 그 특성이 상이하다. In addition, stem cells have different characteristics depending on their origin.
따라서 특정 질환에 대한 치료를 위해 질환에 따라 치료에 적합한 특성을 가지는 줄기세포를 선택하고 이의 특성을 이용하여 특정 질환에 대한 치료 방법을 개발할 필요가 있다.Therefore, for the treatment of a specific disease, it is necessary to select stem cells having characteristics suitable for treatment according to the disease and to develop a treatment method for the specific disease by using the characteristics.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 유효 성분으로 포함하는 간질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating liver disease comprising a conditioned medium of tonsillar-derived mesenchymal stem cells as an active ingredient.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 개선용 식품 조성물을 제공한다.The present invention provides a food composition for preventing or improving liver disease comprising a conditioned medium of tonsillar-derived mesenchymal stem cells.
본 발명자들은 간질환을 예방 또는 치료할 수 있는 적합한 종류의 줄기세포를 확인하던 중, 편도 유래 중간엽 줄기세포의 조정배지가 IfL-1Ra, proMMP-1 및 preprostromelysin로부터 선택되는 어느 하나 이상을 높은 함량으로 조정 배지 내 함유하고 타 유래 중간엽 줄기세포의 조정배지와 다른 프로파일의 함량으로 그 조성을 달리 가짐으로써 간질환의 치료에 효과적임을 확인하여 본 발명을 완성하였다. While identifying a suitable type of stem cell capable of preventing or treating liver disease, the inventors of the present invention found that a conditioned medium of tonsillar-derived mesenchymal stem cells had a high content of any one or more selected from IfL-1Ra, proMMP-1, and preprostromelysin. The present invention was completed by confirming that it was effective in treating liver disease by containing it in the conditioned medium and having a composition different from that of the conditioned medium of other derived mesenchymal stem cells and having a different composition.
본 발명에 있어서, 편도 유래 중간엽 줄기세포란 목의 안쪽과 코의 뒷부분에 위치하여 외부에서 침입하는 세균 등의 물질로부터 일차적으로 우리 몸을 방어함과 동시에 림프상피 면역조직으로 작용을 수행하는 조직인 편도에서 유래된 자기 복제 능력을 가지면서 두 개 이상의 새로운 세포로 분화하는 능력을 가진 미분화된 줄기세포를 의미한다. In the present invention, tonsillar-derived mesenchymal stem cells are tissues that are located in the back of the neck and the back of the nose to primarily defend our body from substances such as bacteria invading from the outside and act as a lymphatic epithelial immune tissue. It refers to undifferentiated stem cells derived from the amygdala that have the ability to differentiate into two or more new cells while having the ability to self-renew.
본 발명에 있어서, "편도 유래 중간엽 줄기세포 조정 배지"는 편도 유래 중간엽 줄기세포를 배양하여 수득된 배양액으로부터 편도 유래 중간엽 줄기세포가 제거된 배양액 또는 상층액을 의미하며, "편도 유래 중간엽 줄기세포 배양 상층액", "편도 유래 중간엽 줄기세포 조건 배양액" 또는 "편도 유래 중간엽 줄기세포 배양 배지"와 호환적으로 사용될 수 있다.In the present invention, "tonsil derived mesenchymal stem cell conditioned medium" means a culture medium or supernatant from which tonsil-derived mesenchymal stem cells are removed from a culture medium obtained by culturing tonsil-derived mesenchymal stem cells, and "tonsil derived medium Mesenchymal stem cell culture supernatant", "tonsil derived mesenchymal stem cell condition culture medium" or "tonsil derived mesenchymal stem cell culture medium" may be used interchangeably.
본 발명에서 용어 “예방”이란 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지의 투여로 간 질환의 발병을 억제 또는 지연시키는 모든 행위를 말한다. 본 발명에서 용어 “치료”는 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지의 투여로 상기 질환의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다. 본 발명에서 "개선"이란 본 발명의 조성물을 개체에 투여하거나 섭취시켜 간질환의 나쁜 상태를 좋게 하는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to all activities that suppress or delay the onset of liver disease by administering the conditioned medium of tonsillar-derived mesenchymal stem cells according to the present invention. In the present invention, the term "treatment" refers to all activities that improve or beneficially change the symptoms of the disease by administration of the conditioned medium of tonsillar-derived mesenchymal stem cells according to the present invention. In the present invention, "improvement" refers to all actions that improve the poor state of liver disease by administering or ingesting the composition of the present invention to a subject.
본 발명에서 용어 “간 질환”이란 간이 수행하는 여러 가지 기능 중 한 가지 이상의 기능에 문제가 생겨 정상적으로 대사를 수행할 수 없는 것을 말한다. 간 질환에서 가장 많이 나타나는 것은 간염으로 급성 간염과 만성 간염으로 나뉘며, 일반적으로는 급성 간염이 치료하기 쉽고 양성에 속한다. 급성 간염은 원인 별로 바이러스성 간염, 알코올성 간염, 중독성 간염 등이 있다. 본 발명에서 간 질환은 편도 유래 중간엽 줄기세포의 조정 배지를 이용하여 치료될 수 있는 것이라면 제한 없이 포함될 수 있으나, 그 예로 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간성 혼수, 간위축증 및 간암으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. 본 발명에서는 편도 유래 중간엽 줄기세포의 조정 배지를 사용하여 간질환의 억제 및 치료효과를 확인하였다. 바람직하게 상기 간질환은 간경변이다. In the present invention, the term "liver disease" refers to a problem in which one or more of the various functions performed by the liver is unable to perform normal metabolism. The most common liver disease is hepatitis, which is divided into acute hepatitis and chronic hepatitis. In general, acute hepatitis is easy to treat and belongs to benign hepatitis. Acute hepatitis includes viral hepatitis, alcoholic hepatitis, and toxic hepatitis by cause. In the present invention, liver diseases may be included without limitation as long as they can be treated using the conditioned medium of tonsillar-derived mesenchymal stem cells, but examples include hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, liver ischemia, alcoholic liver disease, and hepatic disease. It may be any one or more selected from the group consisting of coma, hepatic atrophy, and liver cancer. In the present invention, the inhibitory and therapeutic effects of liver disease were confirmed using the conditioned medium of tonsillar-derived mesenchymal stem cells. Preferably, the liver disease is cirrhosis.
본 발명은 편도 유래 중간엽 줄기세포의 조정배지를 유효 성분으로 포함하는 간질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating liver disease comprising a conditioned medium of tonsillar-derived mesenchymal stem cells as an active ingredient.
본 발명에 따른 편도 유래 중간엽 줄기세포의 조정배지는 타 유래 중간엽 줄기세포의 조정 배지와 대비하여 IL-1Ra, proMMP-1 및 preprostromelysin로부터 선택되는 어느 하나 이상의 함량이 높으며, 조정 배지 내 면역 조절 인자, 간섬유화 억제 인자들의 상이한 프로파일 등을 가짐으로써 간질환의 예방 및 치료에 우수한 효과를 가진다. 특히 간의 섬유화 억제에 우수한 효과를 나타낸다.The conditioned medium of tonsillar-derived mesenchymal stem cells according to the present invention has a higher content of any one or more selected from IL-1Ra, proMMP-1, and preprostromelysin compared to the conditioned medium of other derived mesenchymal stem cells, and the immune regulation in the conditioned medium It has excellent effects in preventing and treating liver diseases by having different profiles of factors, liver fibrosis inhibitors, and the like. In particular, it shows an excellent effect on inhibiting fibrosis of the liver.
또한, 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지는 편도 유래 중간엽 줄기세포가 타 유래 줄기세포와 대비하여 기원 조직인 편도의 획득이 용이하며 수술 후 버려지는 편도 조직의 재활용이 가능하고 초기 수득율이 매우 높다는 점에서 주입해야 하는 조정 배지를 용이하게 얻을 수 있고 주입 가능한 양을 타유래 줄기세포 조정배지와 대비하여 자유롭게 조절할 수 있어 큰 장점을 가진다.In addition, the conditioned medium for tonsillar-derived mesenchymal stem cells according to the present invention is easy to obtain the tonsil, which is the tissue of origin, in comparison with other-derived stem cells, and it is possible to recycle discarded tonsil tissue after surgery. Since the yield is very high, the conditioned medium to be injected can be easily obtained, and the amount that can be injected can be freely adjusted compared to other derived stem cell conditioned media, which has a great advantage.
본 발명에 있어서, 편도 유래 중간엽 줄기세포의 조정배지는 개체로부터 분리, 배양 및 특수한 조작을 통해 제조된 조정 배지를 포함하며, 치료, 진단 및 예방의 목적으로 사용되는 의약품으로 사용되어 간질환에 사용될 수 있다. In the present invention, the conditioned medium of tonsillar-derived mesenchymal stem cells includes a conditioned medium prepared through isolation, culture, and special manipulation from an individual, and is used as a medicine for treatment, diagnosis, and prevention to treat liver disease. can be used
본 발명에 있어서 간질환은 상기 언급된 바와 같다. Liver diseases in the present invention are as mentioned above.
본 발명에 있어서, 편도 유래 중간엽 줄기세포는 당업계에 알려진 방법에 따라 편도로부터 추출 가능하다. 예컨대, 대한민국특허 제10-1508413호 등에 기재된 방법에 따라 편도로부터 추출가능하다. In the present invention, tonsil-derived mesenchymal stem cells can be extracted from tonsils according to a method known in the art. For example, it can be extracted from tonsils according to the method described in Korean Patent No. 10-1508413.
편도 유래 중간엽 줄기세포 배양액은 편도 유래 중간엽 줄기세포를 배양하고 세포를 제거한 후, 수득된 배양액, 배양 상층액 또는 이의 농축물이거나 이의 동결건조물일 수 있다.The tonsil-derived mesenchymal stem cell culture medium may be a culture medium obtained after culturing the tonsil-derived mesenchymal stem cells and removing the cells, a culture supernatant or a concentrate thereof, or a lyophilized product thereof.
본 발명에 있어서, 편도 유래 중간엽 줄기세포 배양액은 편도 유래 중간엽 줄기 세포를 혈청 배지에서 계대 배양한 후, 무혈청 배지에서 배양하여 수득될 수 있다.In the present invention, the tonsil-derived mesenchymal stem cell culture medium may be obtained by subculturing the tonsil-derived mesenchymal stem cells in a serum medium and then culturing them in a serum-free medium.
편도 유래 중간엽 줄기세포는 줄기세포 배양용 배지를 사용하여 통상적으로 배양될 수 있다. 편도 유래 중간엽 줄기세포 배양액은 편도 조직으로부터 수득된 편도 유래 중간엽 줄기세포를 혈청 배지에서 계대배양한 후, 무혈청 배지에서 계대 배양하여 수득되고, 이를 그대로 사용하거나 또는 원심분리나 필터를 이용한 여과에 의해 줄기세포 및 거대분자를 제거한 후에 수득된 상층액을 사용할 수 있다. 또한, 수득된 상층액은 그대로 사용하거나 또는 농축하여 수득된 농축물로 사용할 수 있다.Tonsil-derived mesenchymal stem cells can be conventionally cultured using a stem cell culture medium. Tonsil-derived mesenchymal stem cell culture medium is obtained by subculturing tonsil-derived mesenchymal stem cells obtained from tonsillar tissue in a serum medium and then subculturing in a serum-free medium, and is used as it is or filtered using a centrifugal separation or filter. The supernatant obtained after removing the stem cells and macromolecules by the method can be used. In addition, the obtained supernatant can be used as it is or used as a concentrate obtained by concentrating.
편도 유래 중간엽 줄기세포의 배양을 위한 배양용 배지 및 배양 조건은 본 발명이 속하는 기술분야에서 잘 알려져 있으며, 통상의 지식을 가진 자가 적절하게 선택하거나 변형하여 이용할 수 있다.Culture medium and culture conditions for culturing tonsillar-derived mesenchymal stem cells are well known in the art to which the present invention belongs, and can be appropriately selected or modified by those skilled in the art.
상기 혈청 배지는 편도 유래 중간엽 줄기세포와 동일한 세포형을 유지하고 보관하는데 적합한 배지로서, 혈청이 보충된 DMEM 배지일 수 있다. 혈청은 우태아혈청(FBS)일 수 있으나, 이에 한정되지 않으며, 혈청 배지의 총 중량에 대해 1 내지 10 중량%일 수 있다. 필요에 따라, 항생제, 항진균제 및 마이코플라스마 억제제 등을 포함할 수 있으며, 이들은 배지의 총 중량에 대해 1 중량%일 수 있다. 항생제는 페니실린-스트렙토마이신 등 세포 배양에서 통상적으로 사용되는 항생제를 포함하고, 항진균제는 암포테리신-B(fungizone) 등을 포함하며, 마이코플라스마 억제제는 겐타마이신, 시프로플록사신, 타일로신 등을 포함하나, 이에 한정되지 않는다.The serum medium is a medium suitable for maintaining and storing the same cell type as tonsillar-derived mesenchymal stem cells, and may be DMEM medium supplemented with serum. Serum may be fetal bovine serum (FBS), but is not limited thereto, and may be 1 to 10% by weight based on the total weight of the serum medium. If necessary, antibiotics, antifungal agents and mycoplasma inhibitors may be included, which may be 1% by weight relative to the total weight of the medium. Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), and the like, and mycoplasma inhibitors include gentamicin, ciprofloxacin, and tylosin. , but not limited thereto.
상기 무혈청 배지 중에서의 배양은 혈청 배지에서의 배양액을 제거하고, 세포를 인산염 완충용액으로 세척한 후에 수행될 수 있다.Culturing in the serum-free medium may be performed after removing the culture medium in the serum medium and washing the cells with a phosphate buffer solution.
바람직하게, 상기 혈청 배지는 5 내지 12% 우태아혈청 및 1%의 항생제가 보충된 DMEM 배지일 수 있다.Preferably, the serum medium may be DMEM medium supplemented with 5 to 12% fetal bovine serum and 1% antibiotics.
바람직하게, 상기 무혈청 배지는 DMEM 배지일 수 있다.Preferably, the serum-free medium may be DMEM medium.
본 발명의 일 실시양태에 따르면, 편도 유래 중간엽 줄기세포 배양액은 편도로부터 단리된 편도 조직을 잘게 자른 후, 편도 조직을 배양 플레이트의 바닥에 부착시키고, 10% 우태혈청 및 1% 페니실린-스트렙토마이신이 보충된 DMEM 배지 중에서 37℃ 및 5% CO2 상태의 배양기에서 배양하는 단계, 세포의 밀도가 80%의 포화상태 (confluence)에 도달했을 때, 세포를 분리하고 계대 배양시키는 단계, 세포를 3 내지 4일간 배양한 후, 인산염 완충용액으로 세척하는 단계, 세척된 세포를 혈청 및 항생제를 포함하지 않는 DMEM 배지에서, 37℃ 및 5% CO2 상태의 배양기에서 배양하는 단계에 의해 수득될 수 있다.According to one embodiment of the present invention, the tonsil-derived mesenchymal stem cell culture medium is prepared by cutting the tonsil tissue isolated from the tonsil into small pieces, attaching the tonsil tissue to the bottom of a culture plate, and adding 10% fetal bovine serum and 1% penicillin-streptomycin. In this supplemented DMEM medium, culturing in an incubator at 37 ° C and 5% CO 2 , separating and subculturing the cells when the cell density reaches 80% confluence,
본 발명의 일실시양태에 따르면, 편도 유래 중간엽 줄기세포 배양액은 편도 유래 중간엽 줄기세포 배양액을 원심분리나 여과시켜 세포를 제거하는 단계에 의해 수득될 수 있다.According to one embodiment of the present invention, the tonsil-derived mesenchymal stem cell culture medium may be obtained by centrifuging or filtering the tonsil-derived mesenchymal stem cell culture medium to remove cells.
본 발명의 약학 조성물은 통상의 방법에 따라 액제, 현탁액, 에멀젼, 로션, 연고, 동결건조제 등 다양한 제형으로 제제화될 수 있다. The pharmaceutical composition of the present invention may be formulated into various formulations such as solutions, suspensions, emulsions, lotions, ointments, and freeze-drying agents according to conventional methods.
본 발명의 약학 조성물은 약학적 분야의 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위투여형의 약학적 제제로 제형화시켜 투여할 수 있으며, 상기 제제는1회 또는 수회 투여에 의해 효과적인 투여량을 포함한다. 이러한 목적에 적합한 제형으로는 비경구투여 제제로서 주사제, 주입제 등이 바람직하다. 또한, 상기 간질환 예방 또는 치료용 약학 조성물은 약학적으로 허용가능한 통상의 불활성 담체 및 희석제를 포함할 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 약학적으로 허용가능한 담체 및 희석제는 전분, 당, 및 만니톨과 같은 부형제, 칼슘 포스페이트 등과 같은 충전제 및 증량제, 카르복시메틸셀룰로오스, 히드록시프로필셀룰로오스 등과 같은 셀룰로오스 유도체, 젤라틴, 알긴산염, 및 폴리비닐 피롤리돈 등과 같은 결합제, 활석, 스테아린산 칼슘, 수소화 피마자유 및 폴리에틸렌 글리콜과 같은 윤활제, 포비돈, 크로스포비돈과 같은 붕해제, 폴리소르베이트, 세틸알코올, 및 글리세롤 등과 같은 계면활성제를 포함하나, 이에 한정되지 않는다. 상기 약학적으로 허용되는 담체 및 희석제는 편도 유래 중간엽 줄기세포의 조정 배지 및 이를 투여받을 수혜자에 대해 생물학적 및 생리학적으로 친화적인 것일 수 있다. 희석제로는 이에 한정되지 않으나, 염수, 수용성 완충액, 용매 및/또는 분산제(dispersion media)를 들 수 있다. 이외에도, 예를 들어, 주사제의 경우에는 보존제, 무통화제, 가용화제 또는 안정화제 등을, 국소투여용 제제의 경우에는 기제(base), 부형제, 윤활제 또는 보존제 등을 추가로 포함할 수 있다. 본 발명의 조성물은 동결되지 않은 채 사용되거나 차후 사용을 위해 동결될 수 있다. 동결되어야 할 경우, 표준 냉동보존제 (예를 들어 DMSO, 글리세롤, 에피라이프 (Epilife®) 세포 동결 배지 (Cascade Biologics))가 동결 전 세포 집단에 첨가될 수 있다.The pharmaceutical composition of the present invention can be administered by formulating a pharmaceutical preparation in unit dosage form suitable for administration into the body of a patient according to a conventional method in the pharmaceutical field, and the preparation is effective for administration by one or several administrations. include the quantity Formulations suitable for this purpose include injections, injections, and the like as preparations for parenteral administration. In addition, the pharmaceutical composition for preventing or treating liver disease may include a conventional pharmaceutically acceptable inert carrier and diluent. Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical composition of the present invention include excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, and gelatin. binders such as , alginates, and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, interfaces such as polysorbates, cetyl alcohol, and glycerol. Including, but not limited to active agents. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the conditioned medium of tonsillar-derived mesenchymal stem cells and the recipient to receive the same. Diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media. In addition, for example, in the case of injections, preservatives, analgesics, solubilizers, or stabilizers may be further included, and in the case of preparations for topical administration, bases, excipients, lubricants, or preservatives may be further included. The composition of the present invention may be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) can be added to the cell population prior to freezing.
또한, 당업계에서 통상적으로 사용하는 투여방법을 이용하여 투여될 수 있으며, 바람직하게는 치료가 필요한 환자의 질환 부위에 직접 투여가 가능하나 이에 한정되지는 않는다. 또한, 상기 투여는 카테터를 이용한 비외과적 투여 및 질환부위 절개 후 주입 등 외과적 투여방법 모두 가능하다. 투여량은 농축 정도에 따라 그 투여량이 달라질 수 있으나 10 μl/kg 내지 1 ml/kg 체중을 1회 또는 수회로 나누어 투여할 수 있다. In addition, it can be administered using an administration method commonly used in the art, preferably directly to the diseased area of a patient in need of treatment, but is not limited thereto. In addition, the administration can be performed by both surgical administration methods such as non-surgical administration using a catheter and injection after incision in the diseased area. The dosage may vary depending on the degree of concentration, but 10 μl/kg to 1 ml/kg of body weight may be administered once or divided into several times.
그러나, 유효성분의 실제 투여량은 치료하고자 하는 질환, 질환의 중증도, 투여경로, 환자의 체중, 연령 및 성별 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the weight, age and sex of the patient, and therefore, the dosage is It does not limit the scope of the present invention in any way.
본 발명은 또한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 간질환 예방 또는 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for preventing or improving liver disease comprising a conditioned medium of tonsillar-derived mesenchymal stem cells.
본 발명은 통상적으로 이용되는 식품으로써 일반적으로 사용될 수 있다. The present invention can be generally used as a commonly used food.
본 발명의 식품 조성물은 건강기능식품으로서 사용될 수 있다. 상기 "건강기능식품"이라 함은 건강기능식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The food composition of the present invention can be used as a health functional food. The term “health functional food” refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act, and “functional” refers to food that is not related to the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.
본 발명의 식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 "식품 첨가물"로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. The food composition of the present invention may include conventional food additives, and the suitability as the "food additive" is determined according to the general rules of the food additive code approved by the Ministry of Food and Drug Safety and general test methods, etc., unless otherwise specified. It is judged according to the specifications and standards for the item.
상기 "식품 첨가물 공전"에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류들을 들 수 있다.Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
본 발명의 식품 조성물은 조성물 총 중량에 대하여 편도 유래 중간엽 줄기세포의 조정배지를 0.01 내지 95 중량%, 바람직하게는 5 내지 90 중량%로 포함할 수 있다. The food composition of the present invention may include 0.01 to 95% by weight, preferably 5 to 90% by weight of the conditioned medium of tonsillar-derived mesenchymal stem cells, based on the total weight of the composition.
또한, 본 발명의 식품 조성물은 간질환의 예방 및/또는 개선을 목적으로, 정제, 캡슐, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.In addition, the food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing and/or improving liver diseases.
예를 들어, 캡슐 형태의 건강기능식품 중 경질캡슐제는 통상의 경질캡슐에 본 발명에 따른 편도 유래 중간엽 줄기세포의 조정배지, 및 부형제 등의 첨가제와의 혼합물을 충진하여 제조할 수 있으며, 연질캡슐제는 본 발명에 따른 식품 조성물 및 부형제 등의 첨가제와의 혼합물을 젤라틴 등 캡슐기제에 충진하여 제조할 수 있다. 상기 연질캡슐제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.For example, hard capsules among capsule-type health functional foods can be prepared by filling conventional hard capsules with a mixture of the conditioned medium of tonsil-derived mesenchymal stem cells according to the present invention and additives such as excipients, Soft capsules can be prepared by filling a mixture of the food composition according to the present invention with additives such as excipients into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다 (대한약전 해설편, 문성사, 한국약학대학협의회, 제 5 개정판, p33-48, 1989).Definitions of terms for the excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in documents known in the art, and include those having the same or similar functions (Korean Pharmacopoeia Explanatory Edition, Munseongsa, Korea Association of Colleges of Pharmacy, 5th Rev., p33-48, 1989).
상기 식품의 종류에는 특별한 제한이 없으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There is no particular limitation on the type of food, and it includes all health functional foods in the usual sense.
본 발명은 편도 유래 중간엽 줄기세포의 조정 배지를 대상체에 투여하는 단계를 포함하는 간질환의 치료방법을 제공한다.The present invention provides a method for treating liver disease comprising administering a conditioned medium of tonsil-derived mesenchymal stem cells to a subject.
본 발명에 "편도 유래 중간엽 줄기세포의 조정 배지", "간질환", "투여" 등의 용어는 상기에서 설명한 바와 동일하다.In the present invention, terms such as "conditioned medium for tonsillar-derived mesenchymal stem cells", "liver disease", and "administration" are the same as those described above.
상기 대상체는 동물을 말하며, 전형적으로 본 발명의 편도 유래 중간엽 줄기세포의 조정 배지를 이용한 치료로 유익한 효과를 나타낼 수 있는 포유동물일 수 있다. 이러한 대상체의 바람직한 예로 인간과 같은 영장류가 포함될 수 있다. 또한 이와 같은 대상체들에는 간질환의 증상을 갖거나 이와 같은 증상을 가질 위험이 있는 대상체들이 모두 포함될 수 있다.The subject refers to an animal, and typically may be a mammal that can exhibit a beneficial effect by treatment using the conditioned medium of the tonsil-derived mesenchymal stem cells of the present invention. Preferred examples of such subjects may include primates such as humans. In addition, such subjects may include all subjects who have symptoms of liver disease or are at risk of having such symptoms.
본 발명은 또한 간질환의 치료를 위한 약제의 제조에서 편도 유래 중간엽 줄기세포의 조정배지의 용도를 제공한다. The present invention also provides the use of a conditioned medium of tonsillar-derived mesenchymal stem cells in the manufacture of a drug for the treatment of liver disease.
본 발명은 또한 간질환의 치료에 사용하기 위한 편도 유래 중간엽 줄기세포의 조정배지를 포함하는 조성물을 제공한다. The present invention also provides a composition comprising a conditioned medium of tonsillar-derived mesenchymal stem cells for use in the treatment of liver disease.
본 발명은 또한 간질환의 치료를 위한 편도 유래 중간엽 줄기세포의 조정배지의 용도를 제공한다. The present invention also provides a use of a conditioned medium of tonsillar-derived mesenchymal stem cells for the treatment of liver disease.
본 발명의 간질환 예방 또는 치료용 약학 조성물은 타유래 중간엽 줄기세포의 조정 배지와 대비하여, 조정 배지 내 면역 조절 인자, 간섬유화 억제 인자들의 상이한 프로파일을 가지고 이로 인하여 간질환의 예방 및 치료에 우수한 효과를 가진다. The pharmaceutical composition for preventing or treating liver disease of the present invention has a different profile of immune modulatory factors and liver fibrosis inhibitory factors in the conditioned medium compared to the conditioned medium of heterologous mesenchymal stem cells, and is therefore useful for preventing and treating liver diseases. has an excellent effect.
도 1은 간질환 모델에서 편도 유래 중간엽 줄기세포의 조정 배지(T-MSC CM)의 치료 효과 확인 실험의 스케쥴을 나타낸다.
도 2는 CCl4 전처치로 손상된 간 조직에서 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 회복 효과를 H&E 염색 및 Sirius 염색을 통해 확인한 결과를 나타낸다.
도 3은 CCl4 전처치로 손상된 간 조직에서 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 섬유화 회복 효과를 확인한 결과를 나타낸다.
도 4는 CCl4 전처치로 손상된 간 조직에서 편도 유래 중간엽 줄기세포의 조정배지 처리에 따른 collagen type 1 alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1)과 metallopeptidase inhibitor 1 (Timp1)의 발현 변화를 확인한 결과를 나타낸다. Figure 1 shows the schedule of experiments confirming the therapeutic effect of tonsil-derived mesenchymal stem cell conditioned medium (T-MSC CM) in a liver disease model.
Figure 2 shows the results of confirming the recovery effect of tonsillar-derived mesenchymal stem cells treated with conditioned medium in liver tissue damaged by CCl 4 pretreatment through H&E staining and Sirius staining.
Figure 3 shows the result of confirming the fibrosis recovery effect according to the conditioned medium treatment of tonsillar-derived mesenchymal stem cells in liver tissue damaged by CCl 4 pretreatment.
Figure 4 shows the expression of collagen type 1 alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1) and metallopeptidase inhibitor 1 (Timp1) according to treatment with conditioned medium of tonsillar-derived mesenchymal stem cells in liver tissue damaged by CCl 4 pretreatment. Indicates the result of confirming the change.
본 발명의 이해를 돕기 위하여 실시예, 제조예를 제시한다. 하기의 실시예, 제조예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예, 제조예에 의해 본 발명의 내용이 한정되는 것은 아니다.Examples and preparation examples are provided to aid understanding of the present invention. The following examples and preparation examples are provided to more easily understand the present invention, but the content of the present invention is not limited by the examples and preparation examples.
<< 실시예Example 1> 편도 유래 1> Amygdala Origin 중간엽mesenchyme 줄기세포의 조정 배지의 제조 Preparation of conditioned medium for stem cells
편도 유래 중간엽 줄기세포(Tonsil-derived mesenchymal stem cell, T-MSC) 는 이대목동병원 이비인후-두경부외과에서 편도적출술을 시행하는 환자로부터 적출된 편도조직(4-20세의 저연령층 조직, 임상윤리위원회 심의 통과: ECT 11-53-02)으로부터 얻었다. 이는 대한민국 등록 특허 제10-1508413호와 같이 제조되었으며, 편도 유래 중간엽 줄기세포 (7~8 passage)는 10% FBS, 1% 페니실린/스트렙토마이신이 보충된 DMEM 하에 배양되었다. Tonsil-derived mesenchymal stem cells (T-MSC) are tonsillar tissue extracted from patients undergoing tonsillectomy at the Department of Otorhinolaryngology-Head and Neck Surgery at Ewha Womans University Mok-dong Hospital (younger age group 4-20 years old tissue, clinical Ethics Committee review passed: ECT 11-53-02). It was prepared as described in Korean Registered Patent No. 10-1508413, and tonsil-derived mesenchymal stem cells (7-8 passages) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.
조정 배지를 제조하기 위해 편도 유래 중간엽 줄기세포는 80 % confluency까지 배양되었고, 이에 도달한 후에 FBS와 항생제가 포함되지 않은 신선한 배양 배지로 교환되었다. 세포는 48 시간 추가로 배양되었고 조정 배지가 회수되었다. 조정 배지는 Amicon Ultra centrifugal filter(3 kDa cut-off)를 이용하여 60분 간 5000g로 원심분리에 의해 20배 농축되었다. To prepare the conditioned medium, tonsil-derived mesenchymal stem cells were cultured to 80% confluency, and then exchanged with fresh culture medium without FBS and antibiotics. Cells were cultured for an additional 48 hours and conditioned medium was withdrawn. The conditioned medium was concentrated 20-fold by centrifugation at 5000g for 60 minutes using an Amicon Ultra centrifugal filter (3 kDa cut-off).
<< 실시예Example 2> 2> CCl4CCl4 간질환 모델에서 편도 유래 Tonsil derived from liver disease model 중간엽mesenchyme 줄기세포의 of stem cells 조정배지adjusted badge 투여에 의한 치료 효과 확인 Confirmation of treatment effect by administration
간경화는 사염화탄소(carbon tetrachloride, CCl4)를 이용하여 유도하였으며, 실험 스케쥴은 도 1에 나타내었다. Liver cirrhosis was induced using carbon tetrachloride (CCl 4 ), and the experimental schedule is shown in FIG. 1 .
7주령의 C57BL/6 마우스를 무작위로 미처리군과 사염화탄소 처리군으로 나누었다. 사염화탄소의 1회 투여양은 10 μl/g으로 사염화탄소를 미네랄오일에 10% 농도로 희석하여 일주일에 2차례 3주간에 걸쳐 복강내 투여하였다. 전처치를 마친 실험동물을 3그룹으로 나누어 사염화탄소 투여 직후 희생 (CCl4+No treatment, n=3), 1x106개의 편도줄기세포를 48시간 동안 DMEM에 배양하여 수득한 조정배지를 20배 농축하여 꼬리정맥을 통해 2회 투여(CCl4+T-CM, n=4)한 후, 2주 후 마우스를 희생하여 간조직을 획득하였다. 실험 및 절차는 이화여자대학교 동물 윤리위원회의 승인 하에 수행되었고, 모든 실험은 관련 구제 및 가이드라인 하에 수행되었다. Seven-week-old C57BL/6 mice were randomly divided into untreated groups and carbon tetrachloride-treated groups. Carbon tetrachloride was diluted to a concentration of 10% in mineral oil at a single dose of 10 μl/g and administered intraperitoneally twice a week for 3 weeks. The pretreated experimental animals were divided into 3 groups, sacrificed immediately after carbon tetrachloride administration (CCl 4 +No treatment, n=3), 1x10 6 tonsil stem cells were cultured in DMEM for 48 hours, and the obtained conditioned medium was concentrated 20 times. After administration twice through tail vein (CCl 4 +T-CM, n=4), mice were sacrificed 2 weeks later to obtain liver tissue. Experiments and procedures were performed under the approval of the Animal Ethics Committee of Ewha Womans University, and all experiments were performed under relevant rescue and guidelines.
1. 조직학적 분석1. Histological analysis
조직학적 분석을 위하여 5 μm 두께의 간 조직 파라핀 절편을 제작하여 헤마톡실린과 에오신 (hematoxylin & eosin, H&E) 염색을 수행하였다. 간 조직의 섬유화는 콜라겐 축적을 시리우스 레드(Sirius Red) 염색으로 확인한 후, 콜라겐 섬유의 염색 정도를 단위 면적 당 화소밀도(pixel densities)로 수치화하여 콜라겐 인덱스를 계산하였다.For histological analysis, 5 μm-thick liver tissue paraffin sections were prepared and stained with hematoxylin & eosin (H&E). Fibrosis of liver tissue was confirmed by Sirius Red staining for collagen accumulation, and then the collagen index was calculated by quantifying the degree of staining of collagen fibers in terms of pixel densities per unit area.
그 결과를 도 2에 나타내었다. H&E 염색 결과, CCl4 전처치로 손상된 간 조직이 편도줄기세포 조정배지(T-CM) 처리에 의해 회복됨을 확인할 수 있었고, 시리우스 레드 염색 결과, CCl4 주입 그룹에서 확인된 광범위한 콜라겐 축적 병변이 편도줄기세포 조정배지 처리에 의해 대조군과 같은 수준으로 감소함을 확인할 수 있었다. The results are shown in FIG. 2 . As a result of H&E staining, it was confirmed that liver tissue damaged by CCl 4 pretreatment was recovered by treatment with tonsil stem cell conditioned medium (T-CM), and as a result of Sirius Red staining, extensive collagen accumulation lesions confirmed in the CCl 4 injection group were found in the tonsil. It was confirmed that the stem cell conditioned medium treatment decreased to the same level as the control group.
이를 콜라겐 인덱스로 수치화한 결과를 도3에 나타내었다. The result of quantifying this as a collagen index is shown in FIG. 3.
도 3에서 확인되는 바와 같이 편도 유래 중간엽 줄기세포의 조정 배지(T-CM)투여에 의해 통계학적으로 유의한 간 섬유화의 감소를 확인하였다(***p<0.001). As confirmed in FIG. 3, a statistically significant reduction in liver fibrosis was confirmed by the administration of conditioned medium (T-CM) of tonsil-derived mesenchymal stem cells (***p<0.001).
2. 간질환 표지 유전자 발현 확인2. Verification of liver disease marker gene expression
CCl4 전처치 및 편도줄기세포 조정배지 주입에 의한 간조직에서의 간질환, 특히 간경변 관련 유전자 발현의 변화를 확인하기 위하여, 간 조직의 mRNA를 추출하여 cDNA로 합성 후, real-time PCR을 통하여 collagen type 1, alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1)과 metallopeptidase inhibitor 1 (Timp1)의 발현을 확인하였다. 사용한 프라이머의 서열을 다음과 같다.In order to confirm changes in gene expression related to liver disease, especially cirrhosis, in liver tissue by CCl 4 pretreatment and tonsil stem cell conditioned medium injection, mRNA was extracted from liver tissue, synthesized into cDNA, and real-time PCR was performed. The expression of collagen type 1, alpha 1 (Col1a1), transforming growth factor beta 1 (Tgfb1) and metallopeptidase inhibitor 1 (Timp1) was confirmed. The sequences of the primers used are as follows.
[표 1] 프라이머 서열 정보 [Table 1] Primer sequence information
위 분석 결과를 도 4에 나타내었다. The above analysis results are shown in FIG. 4 .
도 4에서 확인되는 바와 같이, 유전자 발현 분석 결과 CCl4 전처치 그룹에서 증가한 간경변 표지유전자(Col1a1, Tgfb1, Timp1)의 발현이 편도 유래 중간엽 줄기세포의 조정배지 처리에 의해 통계적으로 유의하게 감소한 것을 확인하였다 (*p<0.05, **p<0.01).As confirmed in FIG. 4, as a result of gene expression analysis, it was confirmed that the expression of cirrhosis marker genes (Col1a1, Tgfb1, Timp1), which increased in the CCl4 pretreatment group, was statistically significantly decreased by treatment of tonsil-derived mesenchymal stem cells with conditioned medium. (*p<0.05, **p<0.01).
상기 결과들로부터 본원 발명에 따른 편도 유래 중간엽 줄기세포의 조정 배지가 간질환의 예방 및 치료에 우수한 효과가 있음을 확인하였다. From the above results, it was confirmed that the conditioned medium of tonsillar-derived mesenchymal stem cells according to the present invention has excellent effects in preventing and treating liver diseases.
<110> EWHA UNIVERSITY - INDUSTRY COLLABORATION FOUNDATION <120> A COMPOSITION FOR PREVENTING OR TREATING OF LIVER DISEASE COMPRISING CONDITIONED MEDIUM OF TONSIL-DERIVED MESENCHYMAL STEM CELL <130> P17-147-EWHA <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 Fowrard primer <400> 1 gctcctctta ggggccact 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Col1a1 Reverse primer <400> 2 ccacgtctca ccattgggg 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tgfb1 Forward primer <400> 3 ctcccgtggc ttctagtgc 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Tgfb1 Reverse primer <400> 4 gccttagttt ggacaggatc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Timp1 Forward primer <400> 5 cttggttccc tggcgtactc 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Timp1 Reverse primer <400> 6 acctgatccg tccacaaaca g 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh Forward primer <400> 7 aggtcggtgt gaacggattt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh Reverse primer <400> 8 tgtagaccat gtagttgagg 20 <110> EWHA UNIVERSITY - INDUSTRY COLLABORATION FOUNDATION <120> A COMPOSITION FOR PREVENTING OR TREATING OF LIVER DISEASE COMPRISING CONDITIONED MEDIUM OF TONSIL-DERIVED MESENCHYMAL STEM CELL <130> P17-147-EWHA <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> artificial sequence <220> <223> Col1a1 Fowrard primer <400> 1 gctcctctta ggggccact 19 <210> 2 <211> 19 <212> DNA <213> artificial sequence <220> <223> Col1a1 Reverse primer <400> 2 ccacgtctca ccattgggg 19 <210> 3 <211> 19 <212> DNA <213> artificial sequence <220> <223> Tgfb1 Forward primer <400> 3 ctcccgtggc ttctagtgc 19 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> Tgfb1 reverse primer <400> 4 gccttagttt ggacaggatc 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> Timp1 Forward primer <400> 5 cttggttccc tggcgtactc 20 <210> 6 <211> 21 <212> DNA <213> artificial sequence <220> <223> Timp1 Reverse primer <400> 6 acctgatccg tccacaaaca g 21 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> Gapdh Forward primer <400> 7 aggtcggtgt gaacggattt 20 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> Gapdh Reverse primer <400> 8 tgtagaccat gtagttgagg 20
Claims (12)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020180037755A KR102526447B1 (en) | 2018-03-30 | 2018-03-30 | A composition for preventing or treating of liver disease comprising conditioned medium of tonsil-derived mesenchymal stem cell |
| PCT/KR2019/003626 WO2019190218A1 (en) | 2018-03-30 | 2019-03-28 | Composition, comprising tonsil-derived mesenchymal stem cell-controlling medium, for prevention or treatment of hepatic disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020180037755A KR102526447B1 (en) | 2018-03-30 | 2018-03-30 | A composition for preventing or treating of liver disease comprising conditioned medium of tonsil-derived mesenchymal stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20190114678A KR20190114678A (en) | 2019-10-10 |
| KR102526447B1 true KR102526447B1 (en) | 2023-04-26 |
Family
ID=68058465
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020180037755A KR102526447B1 (en) | 2018-03-30 | 2018-03-30 | A composition for preventing or treating of liver disease comprising conditioned medium of tonsil-derived mesenchymal stem cell |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR102526447B1 (en) |
| WO (1) | WO2019190218A1 (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9235321B2 (en) | 2012-11-14 | 2016-01-12 | Facebook, Inc. | Animation sequence associated with content item |
| KR101520536B1 (en) * | 2013-05-16 | 2015-05-14 | 이화여자대학교 산학협력단 | Differentiation method from tonsil-derived mesenchymal stem cells to hepatocytes and cell therapy composition comprising tonsil-derived mesenchymal stem cells for treatment of hepatopathy |
| KR101822153B1 (en) | 2016-04-25 | 2018-01-26 | (주) 노바렉스 | Pharmaceutical composition for preventing or treating liver disease comprising licorice extract having glycyrrhizin and liquiritin |
-
2018
- 2018-03-30 KR KR1020180037755A patent/KR102526447B1/en active IP Right Grant
-
2019
- 2019-03-28 WO PCT/KR2019/003626 patent/WO2019190218A1/en active Application Filing
Non-Patent Citations (2)
| Title |
|---|
| SCIENTIFIC REPORTS, Vol. 5, No. 8616, pp. 1-7 (2015)* |
| STEM CELLS AND DEVELOPMENT, Vol. 22, No. 6, pp. 845-854 (2013)* |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019190218A1 (en) | 2019-10-03 |
| KR20190114678A (en) | 2019-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109562129B (en) | Composition for preventing or treating pulmonary fibrosis comprising exosome isolated from adipose-derived stem cells as an active ingredient | |
| KR101629151B1 (en) | Composition including stem cell-derived exosome for inducing adipogenic differentiation and adipose tissue regeneration | |
| CN109963574B (en) | Composition for preventing or treating liver fibrosis comprising adipose stem cell-derived exosomes as active ingredient | |
| KR101775262B1 (en) | A composition for preventing or treating of skin inflammation disease comprising tonsil-derived mesenchymal stem cell or conditioned medium thereof | |
| JPWO2006041088A1 (en) | Brain transitional bone marrow progenitor cells | |
| KR20120095022A (en) | Cell therapy composition for preventing or treating graft-versus-host disease comprising mesenchymal stem cell and regulatory t cell | |
| JP5406017B2 (en) | Use of cells derived from adipose tissue for the manufacture of antitumor medicaments | |
| US11622964B2 (en) | Method for destroying cellular mechanical homeostasis and promoting regeneration and repair of tissues and organs, and use thereof | |
| KR102560912B1 (en) | Pharmaceutical composition for treating or preventing lung cancer or inhibiting cancer metastasis, comprising exosomes as an active ingredient | |
| KR102526447B1 (en) | A composition for preventing or treating of liver disease comprising conditioned medium of tonsil-derived mesenchymal stem cell | |
| JP6974892B1 (en) | Cancer cachexia improving agent and cancer cachexia improving method | |
| WO2018093233A1 (en) | Composition containing adipose stem cell-derived exosomes as active ingredient for preventing or treating liver fibrosis | |
| KR102037996B1 (en) | A composition for preventing or treating of diabetes mellitus type2 comprising tonsil-derived mesenchymal stem cell or conditioned medium thereof | |
| KR102152659B1 (en) | A composition for inhibiting cancer metastasis comprising tonsil-derived mesenchymal stem cell conditioned medium | |
| KR20200132550A (en) | A composition for preventing or treating of blood vessel disease comprising tonsil-derived mesenchymal stem cell or conditioned medium thereof | |
| US20200155608A1 (en) | Therapeutic Treatments Via Intravenous Infusion Of Mesenchymal Stem Cells | |
| KR20200092860A (en) | Surface-modified exosomes using bile acid, method for producing thereof and method for inducing proliferation of cells | |
| KR20190138244A (en) | A composition for preventing or treating of graft versus host disease comprising conditioned medium of tonsil-derived mesenchymal stem cell | |
| JP7044429B1 (en) | Composition for shrinking or eliminating tumors | |
| CN116925186B (en) | Mesenchymal stem cell treatment method for neonatal pulmonary dysplasia | |
| WO2023167484A1 (en) | Composition for preventing or treating retinal degenerative disease containing exosomes extracted from mesenchymal stem cells | |
| KR101789475B1 (en) | A composition for preventing or treating of osteoporosis comprising tonsil-derived mesenchymal stem cell or conditioned medium thereof | |
| KR20190122196A (en) | A composition for preventing or treating of diabetes mellitus type2 comprising tonsil-derived mesenchymal stem cell or conditioned medium thereof | |
| KR20170091882A (en) | A composition for preventing or treating of osteoporosis comprising tonsil-derived mesenchymal stem cell or conditioned medium thereof | |
| JP2023001294A (en) | Preventive or therapeutic agent for organ fibrosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |