CN109400676A - (dopa)4-g5-rgds活性肽及其应用 - Google Patents
(dopa)4-g5-rgds活性肽及其应用 Download PDFInfo
- Publication number
- CN109400676A CN109400676A CN201811238308.4A CN201811238308A CN109400676A CN 109400676 A CN109400676 A CN 109400676A CN 201811238308 A CN201811238308 A CN 201811238308A CN 109400676 A CN109400676 A CN 109400676A
- Authority
- CN
- China
- Prior art keywords
- dopa
- rgds
- bone
- active peptide
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 71
- 101000829980 Homo sapiens Ral guanine nucleotide dissociation stimulator Proteins 0.000 title claims abstract description 26
- 102100023320 Ral guanine nucleotide dissociation stimulator Human genes 0.000 title claims abstract description 26
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 title claims description 93
- 229960004502 levodopa Drugs 0.000 title claims description 92
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 title claims description 88
- 239000010936 titanium Substances 0.000 claims abstract description 72
- 229910052719 titanium Inorganic materials 0.000 claims abstract description 70
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims abstract description 65
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 50
- 239000000463 material Substances 0.000 claims abstract description 37
- 238000004090 dissolution Methods 0.000 claims abstract description 27
- 230000002757 inflammatory effect Effects 0.000 claims abstract description 14
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims description 6
- 210000002997 osteoclast Anatomy 0.000 abstract description 38
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 37
- 229920001184 polypeptide Polymers 0.000 abstract description 35
- 238000000034 method Methods 0.000 abstract description 22
- 239000002245 particle Substances 0.000 abstract description 21
- 230000020411 cell activation Effects 0.000 abstract description 14
- 230000015572 biosynthetic process Effects 0.000 abstract description 9
- 239000012620 biological material Substances 0.000 abstract description 7
- 238000005314 correlation function Methods 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 230000007812 deficiency Effects 0.000 abstract description 3
- 238000004393 prognosis Methods 0.000 abstract description 3
- 210000000689 upper leg Anatomy 0.000 description 46
- 210000004027 cell Anatomy 0.000 description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 30
- 239000000243 solution Substances 0.000 description 29
- 241000700159 Rattus Species 0.000 description 25
- 239000003292 glue Substances 0.000 description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 102100036698 Golgi reassembly-stacking protein 1 Human genes 0.000 description 23
- 101001072488 Homo sapiens Golgi reassembly-stacking protein 1 Proteins 0.000 description 23
- 101001000691 Medicago sativa Pectinesterase Proteins 0.000 description 23
- 101000907437 Mycoplasma hyopneumoniae (strain 232) Chaperone protein DnaK Proteins 0.000 description 23
- 108010025832 RANK Ligand Proteins 0.000 description 22
- 102000014128 RANK Ligand Human genes 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 20
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 19
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 19
- 239000011521 glass Substances 0.000 description 19
- 239000000523 sample Substances 0.000 description 19
- 229910001868 water Inorganic materials 0.000 description 17
- 239000007788 liquid Substances 0.000 description 16
- 230000002829 reductive effect Effects 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 230000009194 climbing Effects 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 12
- 210000002540 macrophage Anatomy 0.000 description 12
- 108010047852 Integrin alphaVbeta3 Proteins 0.000 description 11
- 230000001939 inductive effect Effects 0.000 description 11
- 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 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 10
- 108010002352 Interleukin-1 Proteins 0.000 description 10
- 108090001005 Interleukin-6 Proteins 0.000 description 10
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 10
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 10
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 10
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 10
- 239000000975 dye Substances 0.000 description 10
- 235000019441 ethanol Nutrition 0.000 description 10
- 238000010603 microCT Methods 0.000 description 10
- 102000006495 integrins Human genes 0.000 description 9
- 108010044426 integrins Proteins 0.000 description 9
- 238000012545 processing Methods 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 238000010186 staining Methods 0.000 description 9
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 8
- 230000037182 bone density Effects 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 230000013011 mating Effects 0.000 description 8
- 230000026731 phosphorylation Effects 0.000 description 8
- 238000006366 phosphorylation reaction Methods 0.000 description 8
- 239000008399 tap water Substances 0.000 description 8
- 235000020679 tap water Nutrition 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 210000000170 cell membrane Anatomy 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 210000003275 diaphysis Anatomy 0.000 description 7
- 238000004043 dyeing Methods 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 229960004756 ethanol Drugs 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 150000003608 titanium Chemical class 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 6
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 6
- 230000018678 bone mineralization Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 239000012188 paraffin wax Substances 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 230000002980 postoperative effect Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- -1 DOPA amino acid Chemical class 0.000 description 5
- 102100022337 Integrin alpha-V Human genes 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 238000001994 activation Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000011010 flushing procedure Methods 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 239000003755 preservative agent Substances 0.000 description 5
- 230000002335 preservative effect Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 229950003937 tolonium Drugs 0.000 description 5
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 5
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 108010040765 Integrin alphaV Proteins 0.000 description 4
- 102000008607 Integrin beta3 Human genes 0.000 description 4
- 108010020950 Integrin beta3 Proteins 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 238000004042 decolorization Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- NNRFRJQMBSBXGO-CIUDSAMLSA-N (3s)-3-[[2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NNRFRJQMBSBXGO-CIUDSAMLSA-N 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- JUQPZRLQQYSMEQ-UHFFFAOYSA-N CI Basic red 9 Chemical compound [Cl-].C1=CC(N)=CC=C1C(C=1C=CC(N)=CC=1)=C1C=CC(=[NH2+])C=C1 JUQPZRLQQYSMEQ-UHFFFAOYSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 3
- 208000003076 Osteolysis Diseases 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 108010009711 Phalloidine Proteins 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 239000000956 alloy Substances 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 210000002805 bone matrix Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013312 flour Nutrition 0.000 description 3
- 229920001821 foam rubber Polymers 0.000 description 3
- 244000144993 groups of animals Species 0.000 description 3
- 238000007654 immersion Methods 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 210000000629 knee joint Anatomy 0.000 description 3
- 208000029791 lytic metastatic bone lesion Diseases 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000001543 one-way ANOVA Methods 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 238000004088 simulation Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 208000006386 Bone Resorption Diseases 0.000 description 2
- 206010065687 Bone loss Diseases 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 241000237536 Mytilus edulis Species 0.000 description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 244000061458 Solanum melongena Species 0.000 description 2
- 229910001069 Ti alloy Inorganic materials 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000035578 autophosphorylation Effects 0.000 description 2
- YTFJQDNGSQJFNA-UHFFFAOYSA-N benzyl dihydrogen phosphate Chemical compound OP(O)(=O)OCC1=CC=CC=C1 YTFJQDNGSQJFNA-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 230000024279 bone resorption Effects 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 102000006240 membrane receptors Human genes 0.000 description 2
- 108020004084 membrane receptors Proteins 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000000116 mitigating effect Effects 0.000 description 2
- 235000020638 mussel Nutrition 0.000 description 2
- 150000004780 naphthols Chemical class 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 210000004409 osteocyte Anatomy 0.000 description 2
- 230000002188 osteogenic effect Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 235000010288 sodium nitrite Nutrition 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000003746 surface roughness Effects 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 238000010408 sweeping Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229940095064 tartrate Drugs 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 1
- SHZLOTJPHMTVDI-QFIPXVFZSA-N (2s)-3-(2,2-dimethyl-1,3-benzodioxol-5-yl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CC1=CC=C2OC(C)(C)OC2=C1 SHZLOTJPHMTVDI-QFIPXVFZSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- ZLRFPQPVXRIBCQ-UHFFFAOYSA-N 2-$l^{1}-oxidanyl-2-methylpropane Chemical compound CC(C)(C)[O] ZLRFPQPVXRIBCQ-UHFFFAOYSA-N 0.000 description 1
- VNTJGCYVIRTGMZ-PXGLAOGESA-N 2-[[2-[[(2s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s,3r)-2-[[(2s)-2-[[2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s)-2-aminopropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-oxopentanoyl]amino]acety Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC(C)C)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)NCC(O)=O)C1=CC=C(O)C=C1 VNTJGCYVIRTGMZ-PXGLAOGESA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- PFRYFZZSECNQOL-UHFFFAOYSA-N 2-methyl-4-[(2-methylphenyl)diazenyl]aniline Chemical compound C1=C(N)C(C)=CC(N=NC=2C(=CC=CC=2)C)=C1 PFRYFZZSECNQOL-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 102100033897 Ankyrin repeat and SOCS box protein 1 Human genes 0.000 description 1
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000925496 Homo sapiens Ankyrin repeat and SOCS box protein 1 Proteins 0.000 description 1
- 244000283207 Indigofera tinctoria Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028347 Muscle twitching Diseases 0.000 description 1
- 239000004218 Orcein Substances 0.000 description 1
- 241000906034 Orthops Species 0.000 description 1
- 101800003595 Osteogenic growth peptide Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical group 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
- 208000006735 Periostitis Diseases 0.000 description 1
- 206010052306 Periprosthetic osteolysis Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- HUXIAXQSTATULQ-UHFFFAOYSA-N [6-bromo-3-[(2-methoxyphenyl)carbamoyl]naphthalen-2-yl] dihydrogen phosphate Chemical compound COC1=CC=CC=C1NC(=O)C1=CC2=CC(Br)=CC=C2C=C1OP(O)(O)=O HUXIAXQSTATULQ-UHFFFAOYSA-N 0.000 description 1
- 102000011759 adducin Human genes 0.000 description 1
- 108010076723 adducin Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000002376 aorta thoracic Anatomy 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 108010089975 arginyl-glycyl-aspartyl-serine Proteins 0.000 description 1
- 230000002917 arthritic effect Effects 0.000 description 1
- 238000011882 arthroplasty Methods 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 238000004630 atomic force microscopy Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 102000008395 cell adhesion mediator activity proteins Human genes 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 210000003298 dental enamel Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000002695 general anesthesia Methods 0.000 description 1
- 210000004349 growth plate Anatomy 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000004394 hip joint Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000012308 immunohistochemistry method Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 108091005450 macrophage integrins Proteins 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 208000026721 nail disease Diseases 0.000 description 1
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 1
- 235000019248 orcein Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000001599 osteoclastic effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000004417 patella Anatomy 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000013139 quantization Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 239000003507 refrigerant Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 235000015096 spirit Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000004018 waxing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
- A61L27/06—Titanium or titanium alloys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1019—Tetrapeptides with the first amino acid being basic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Physical Education & Sports Medicine (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rheumatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Pain & Pain Management (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供了一种(DOPA)4‑G5‑RGDS活性肽及其应用,其可以应用于制备治疗炎症性假体周围骨溶解的药物组合物,所述的药物组合物包含改性医用钛基材料的(DOPA)4‑G5‑RGDS活性肽。本发明中,(DOPA)4‑G5‑RGDS多肽改性医用钛基材料,具有简单高效性、灵活性以及生物活性的可选择性的优势,并且可以极大地改进目前钛基医用生物材料的表面改性方法存在的不足。该活性肽可以有效抑制磨损颗粒导致的破骨细胞活化及其相关功能,从而预防PPO的形成,对于TJA的预后有深远影响,使其极其有利于在临床的推广使用。
Description
技术领域
本发明属于活性肽应用技术领域,具体涉及(DOPA)4-G5-RGDS活性肽及其在制备治疗炎症性假体周围骨溶解的药物组合物中的应用。
背景技术
医用钛或其合金材料具有高机械强度、抗疲劳性能、抗腐蚀性和抗应变原性,是临床应用最广泛的承力植入材料。作为钛合金最成功的临床应用之一,关节置换术(totaljoint arthroplasty, TJA)成为了治疗严重关节炎疾病的最重要手段。然而,假体周围骨溶解(Peri-prosthetic osteolysis, PPO)是TJA后出现的重要并发症,往往是导致置换术后失败及翻修的主要原因。据报道,在美国每年有大约40000个关节置换术后的病人需要做翻修手术,并且在未来的25年内,髋关节的翻修量将会增长1.37倍,膝关节翻修量将会增加6倍,这也给病人带来巨大病痛和沉重的经济损失,而PPO作为导致置换术后失败及翻修的主要原因,其治疗显得尤为重要。目前认为假体长期磨损产生无菌性细小颗粒引起的生物学反应导致破骨细胞活化、骨吸收增加及新骨生成的减少是引起PPO的主要原因,因此研究如何有效地抑制破骨细胞活化,减少骨丢失,对于预防PPO具有重要意义。
研究表明,通过对钛基合金进行表面活化处理,可以显著改善钛基材料的表面性能,改性技术不仅可提高金属表面的稳定性和耐磨性,而且可赋予生物活性,即可使新骨组织直接沉积于金属表面,而无纤维结缔组织的中间隔层。因此,近年来医用钛合金材料的表面改性研究已成为医用生物材料的研究热点之一。
通过生物模拟多肽合成技术,设计制备出一端含有多重DOPA氨基酸序列、一端含有生物活性肽分子的生物模拟活性肽。DOPA氨基酸,即3,4-L-二羟基苯丙氨酸(3,4-dihydroxy-L-phenylalanine),广泛存在于贻贝类生物所分泌的粘附蛋白中。DOPA中的儿茶酚(即,邻苯二酚)可以和多种材料表面材料共价和非共价方式结合,是贻贝类生物附着于礁石船只等表面的重要手段。研究证明,DOPA基团和二氧化钛表面在水中简单接触的情况下很快形成稳定的配位结合作用。L-Asparticacid,L-arginylglycyl-(RGD)多肽序列为应用于涉及结合到细胞表面蛋白质的首选序列,作为整合素和其配体相互作用的识别位点,介导细胞与细胞外基质及细胞之间的相互作用。相关研究表明,整合素作为细胞膜受体,主要调控细胞膜相关功能(粘附、迁移、增值)。而且,通过对整合素及其下游相关靶点的调控,可以对破骨细胞的活化与功能产生影响。基于此,我们设计的生物模拟活性肽将DOPA与RGD肽有效整合,合成多肽( DOPA )4-G5-RGDS式1所示:
式(I)。
研究已经证实,使用( DOPA )4-G5-RGDS活性肽可有效粘附于钛基医用生物材料,对其表面进行改性,具有简单高效性、灵活性以及生物活性的可选择性(如细胞贴附因子和成骨生长因子)的优势,并且可以极大地改进目前钛基医用生物材料的表面改性方法存在的不足。
综上,( DOPA )4-G5-RGDS改性医用钛基材料可有效预防PPO的形成,对于TJA的预后有深远影响,使其极其有利于在临床的推广使用。
发明内容
本发明的目的在于研究( DOPA )4-G5-RGDS活性肽新用途技术领域的五大特性:1、通过改性,可使医用钛基材料表面粘附( DOPA )4-G5-RGDS生物模拟活性肽,从而使医用钛基材料具有生物学活性;2、可逆转磨损颗粒导致的模拟假体周围骨溶解;3、可以明显减轻磨损颗粒所产生的炎症效应;4、可有效抑制由RANKL诱导的破骨细胞生成;5、可通过影响细胞膜功能从而干预破骨细胞相关功能。
本发明的技术方案如下:
一种(DOPA)4-G5-RGDS活性肽,所述活性肽的化学结构式如式(Ⅰ)所示:
式(Ⅰ)。
本发明所述的活性肽在制备治疗炎症性假体周围骨溶解的药物组合物中的应用。
本发明所述的药物组合物包含改性医用钛基材料的(DOPA)4-G5-RGDS活性肽。
本发明技术方案通过表面改性医用钛基材料模拟假体植入物来研究( DOPA )4-G5-RGDS对磨损颗粒诱导的模拟假体周围骨溶解治疗作用,同时测定整合素(integrinαVβ3)以及经典信号通路核转录因子(Nf-κB)信号通路的相关因子P65的表达来研究这种作用与破骨细胞活化的关系,探讨( DOPA )4-G5-RGDS治疗关节置换术后骨溶解的机制及理论依据。
首先,本发明通过浸泡医用钛基材料,利用X射线光电子能谱法(XPS)和原子力显微技术(AFM)测试改性后,钛基材料表面的各元素含量变化和粗糙度变化,从而证实( DOPA )4-G5-RGDS多肽对医用钛基材料的改性作用,并证实其简便性。
随后,63只SD大鼠被随机分为Control组、Vehicle组和( DOPA )4-G5-RGDS组,每组21只。各组动物均在全麻下进行常规手术操作,由股骨远端髁间窝处向平行股骨干方向植入直径1.5mm,长1.5cm医用钛基材料钉。( DOPA )4-G5-RGDS组钛基材料钉对其表面改性。改性的步骤如下:将( DOPA )4-G5-RGDS多肽配置成一定的浓度,溶剂为去离子水、PBS缓冲液、乙醇或甲醇等常用溶剂,浓度在0.01-10mg/mL之间。将医用钛基生物材料钉浸泡于上述溶液中,浸泡时间为30min至72h之间,即可得到表面改性有细胞粘附分子RGS的TiO2表面。Vehicle组和( DOPA )4-G5-RGDS组的动物均在髓腔内置入40µL已配制的40%Ti颗粒PBS液(16mg/只),而Control组髓腔给予40µL的PBS。各组于术后28天处死动物取股骨行micro-CT及组织学检测,测定股骨骨密度、骨体积分数、骨溶解面积、骨膜厚度以及成熟破骨细胞数量等分析模拟假体周围骨溶解程度;同时应用免疫组化方法检测IntegrinαVβ3和P65的表达量,予以研究( DOPA )4-G5-RGDS多肽对于破骨细胞活化相关因子在假体周围骨质标本中表达量;再检测IL-1、IL-6和TNF-α的表达量,予以研究( DOPA )4-G5-RGDS多肽对于相关炎症因子的影响作用。采用单因素方差分析对各组数据进行统计学分析。
为更进一步证实( DOPA )4-G5-RGDS多肽对破骨细胞活化的抑制作用,本发明通过分别培养小鼠股骨巨噬细胞于经改性医用医用钛细胞爬片和普通医用钛细胞爬片之上,并以RANKL与CSF加以诱导3天、5天、7天。然后,通过抗酒石酸染色(TRAP),鬼笔环肽与细胞免疫荧光染色,以及相关量化数据分析来研究( DOPA )4-G5-RGDS多肽对于抑制由RANKL诱导的破骨细胞生成以及对破骨细胞相关功能干预。最后再以Western blot法测定整合素相关通路和NF-κB相关通路的蛋白因子表达量。
有益效果:本发明结果表明,XPS扫描结果显示,与普通PBS浸泡的钛基材料相比,改性后的钛基材料,“N”元素含量明显增加;AFM扫面结果显示 ( DOPA )4-G5-RGDS多肽改性钛基材料后,其表面粗糙度增加,有较多类蛋白团块物质粘附。
Micro-CT扫描结果显示,Vehicle组与Control组相比,假体周围骨小梁明显减少,骨密度、骨体积、骨体积分数明显减少(**p<0.01);假体周围有明显的虫蚀样变化,骨溶解面积显著增加(**p<0.01);成熟破骨细胞数量增多(**p<0.01);( DOPA )4-G5-RGDS组中较Vehicle组骨小梁明显增加,骨密度、骨体积分数明显增加,骨溶解程度明显减轻,成熟破骨细胞数量减少,比较差异 有统计学意义(*p<0.01)。
免疫组化结果显示,在Control组中和TNF-α等相关炎症因子,以及integrinαVβ3和P65的浓度较低;在Vehicle组,IL-1、IL-6和TNF-α等相关炎症因子以及integrinαVβ3及P65的含量急剧增加,与Control组相比,*p<0.05;在( DOPA )4-G5-RGDS组,IL-1、IL-6、TNF-α、integrinαVβ3及P65的含量随( DOPA )4-G5-RGDS多肽改性钛钉后浓度的增加显著下降。
RANKL及M-CSF诱导小鼠股骨巨噬细胞于医用钛细胞爬片,结果显示,经( DOPA )4-G5-RGDS多肽改性医用钛细胞爬片后, RANKL诱导生成破骨细胞生成数量分别在3天,5天,7天较正常钛片表面上明显减少,二者计数具有统计学意义(*p<0.01)。相关研究证实NF-κB通路中重要因子P65在经RANKL诱导后,可经自身磷酸化进入细胞核内,从而激活经典NF-κB信号通路,诱导破骨细胞生成。本发明中,细胞免疫荧光染色显示,经( DOPA )4-G5-RGDS多肽改性医用钛细胞爬片后, RANKL诱导小鼠巨噬细胞中P65表达入核率较普通爬片明显下降,二者计数具有统计学意义(*p<0.01)。WB结果显示经RANKL及M-CSF诱导在15min,30min,45min和60min小鼠股骨巨噬细胞P65与IKB-α磷酸化水平不同程度的升高,而经( DOPA )4-G5-RGDS多肽“一步法”处理后,各时间点P65与IKB-α磷酸化水平明显降低。细胞免疫荧光染色显示, integrinαVβ3在成熟破骨细胞膜中的表达量较高,研究证实,integrin为细胞膜受体,可调控细胞增殖,迁移,粘附等重要功能。而经( DOPA )4-G5-RGDS多肽“一步法”改性医用钛细胞爬片后,integrinαVβ3表达量减少明显减少。 同样,在WB结果中,RANKL及M-CSF诱导在1d、2d和3d小鼠股骨巨噬细胞integrinαVβ3的表达量及其下游的相关因子FAK与Src的磷酸化水平明显增高,而经( DOPA )4-G5-RGDS多肽改性医用钛细胞爬片后,这些蛋白的表达量减少明显减少。
本实验证实( DOPA )4-G5-RGDS多肽改性医用钛基材料对人工假体置换术后假体周围骨溶解有一定的防治作用,可使磨损颗粒诱导的破骨细胞活化得到抑制,并且可以抑制RANKL诱导的破骨细胞生成和破骨细胞功能。此可作为人工假体置换术后假体周围骨溶解的药物干预的一种新手段。
本发明中,( DOPA )4-G5-RGDS多肽改性医用钛基材料,具有简单高效性、灵活性以及生物活性的可选择性(如细胞贴附因子和成骨生长因子)的优势,并且可以极大地改进目前钛基医用生物材料的表面改性方法存在的不足。本发明在于研究( DOPA )4-G5-RGDS活性肽可以有效抑制磨损颗粒导致的破骨细胞活化及其相关功能,从而预防PPO的形成,对于TJA的预后有深远影响,使其极其有利于在临床的推广使用。
相对于现有技术中的方案,本发明有五大优点:
1、通过( DOPA )4-G5-RGDS活性肽浸泡医用钛基材料,可明显改性医用钛基材料表面,使其具有生物学活性,操作简便,高效;1、可逆转磨损颗粒导致的模拟假体周围骨溶解,减轻骨量丢失,增加骨密度;3、可以明显减轻磨损颗粒所产生的炎症效应,减少IL-1、IL-6和TNF-α相关炎症因子的表达;4、可有效抑制由RANKL诱导的破骨细胞生成,其机制可能与(DOPA )4-G5-RGDS多肽可抑制P65自身磷酸化后入核激活经典信号通路NF-κB有关;5、可通过影响细胞膜受体整合素及其相关信号通路的表达,从而干预破骨细胞膜的功能相关功能。综上,( DOPA )4-G5-RGDS活性肽改性医用钛基材料可有效预防PPO的形成,对于TJA的预后有深远影响,使其极其有利于在临床的推广使用。
附图说明
下面结合附图及实施例对本发明作进一步描述。
图1为( DOPA )4-G5-RGDS的分子式。
图2为XPS和AFM检测钛基材料表面的结果图。
图3为micro-CT扫描各实验组大鼠股骨的三维模型图。
图4为各实验组大鼠股骨骨密度(BMD)与大鼠股骨骨体积分数(BV/TV)的检测结果图。
图5为各实验组生物力学实验最大拔出力的检测结果图。
图6为各实验组大鼠股骨甲苯胺蓝染色图与骨假体周围骨盐沉积率(BIC%)的柱状图。
图7为各实验组大鼠股骨HE染色结果图及各实验组大鼠股骨假体周围骨溶解率(ES/BS%)的柱状图。
图8为各实验组大鼠股骨TRAP染色结果图及各实验组大鼠股骨TRAP染色阳性细胞计数结果图。
图9为各实验组大鼠股骨TNF-α、IL-1和IL-6免疫组织化学染色结果图。
图10为RANKL及M-CSF诱导小鼠股骨巨噬细胞TRAP染色结果图及TRAP染色阳性细胞计数结果图。
图11为RANKL及M-CSF诱导小鼠股骨巨噬细胞鬼笔环肽与整合素免疫荧光共染结果图。
图12为RANKL及M-CSF诱导小鼠股骨巨噬细胞1d、2d和3d后, integrinαvβ3及其下游FAK和Src的蛋白磷酸化水平变化图。
图13为各实验组大鼠股骨P65免疫组化荧光染色结果图。
图14为0min、15min、30min、45min和60min RANKL及M-CSF诱导小鼠股骨巨噬细胞P65和IKB-α蛋白磷酸化水平变化。
图15为RANKL及M-CSF诱导小鼠股骨巨噬细胞P65入核细胞免疫荧光染色结果。
图16为各实验组大鼠股骨integrinαvβ3免疫组化荧光染色结果。
具体实施方式
下面结合具体实施例来进一步描述本发明,但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
一、材料与方法
1、材料
1.1 试剂及实验设备
1.1.1 主要药品及试剂
TRAP染色试剂盒,购自Sigma,美国;多聚甲醛、PBS、DAB显色剂、苏木素、伊红、无水乙醇、蒸馏水、10%水合氯醛。钛颗粒购自美国Johnson Matthey chemicals公司( catalog #00681; Ward Hill, Massachusetts );抗体购自:integinαVβ3、P65、抗体购自Abcam公司,英国;钛钉购自美国Goodfellow公司。
( DOPA )4-G5-RGDS多肽合成简述如下:
首先将磷酸苄酯保护的丝氨酸( Fmoc-O-( benzylphospho )-Serine-OH,1 mmol ) 、2-氯三苯甲基氯树脂( 1.6 g,1.6 mmol )和N ,N-二异丙基乙胺( DIPEA , 4 mmol ) 溶解在15 mL二氯甲烷( DCM )中,反应4小时后,用甲醇( MeOH )将树脂表面没有反应的基团消耗,树脂表面则被引入磷酸苄酯保护丝氨酸。用含有20%哌啶的N-甲基吡咯烷酮( NMP )溶液除去Fmoc基团,露出氨基。随后将树脂加入含有Fmoc-Asp(OtBu)-OH/BOP/DIPEA (1.5/1.5/1.5,mmol )的NMP溶液中,以此接入叔丁基氧( OtBu )保护的天门冬氨酸。除去Fmoc基团,重复上一步骤分别接上甘氨酸和五甲基苯并呋喃-5-磺酰基( Pbf )保护的精氨酸,进而得到活性基团被保护的F m o c - R G D S - R e s i n。重复上述操作接入五个甘氨酸序列作为链接肽,得到F m o c -GGGGGRGDS-Resin。随后,使用Fmoc-DOPA(Acetonide )-OH为DOPA氨基酸原料,分步接入四重DOPA氨基酸,得到丙酮保护的Fmoc-(DOPA)4-GGGGGRGDS-Resin,用乙酸酐( Ac2O )/DIPEA/NMP( 1:4:40 )脱去Fmoc基团,同时乙酰化氨基。用三氟乙酸( TFA,2% )的二氯甲烷溶液将多肽从树脂表面断裂,随后用2 mL的TFA/MeOH/H2O/DCM (体积比,30/2.5/2.5/65 )水解3h,用高效液相色谱分离纯化即得到脱保护基团的生物模拟细胞粘附肽:( DOPA )4-GGGGGRGDS。
1.1.2 主要仪器
X射线光电子能谱机(X-ray photoelectron spectroscopy,美国)、原子力显微镜(atomic force microscopy,美国)、Micro-CT(SkyScan 1176,比利时)、石蜡切片机(Leica2135,德国)、烤片机(Leica 1120,德国)、石蜡包埋机(Leica 1150,德国)、Axiovert 40C光学显微镜(Zeiss,德国)、手术器械一套等。
1.2 实验动物
健康SD大鼠63只,雄性,体重300g,8~10周龄,清洁级,由苏州大学动物实验中心提供。喂养条件如下:4只一笼,室温18~20℃,湿度50~60%,通风良好,自由摄食进水。
2、实验方法
2.1 Ti颗粒的处理及钛基材料钉与细胞爬片
95%的颗粒直径<4μm。为去除内毒素,将颗粒溶于体积分数75%乙醇,常温下振荡1h,共4次,100%乙醇浸泡过夜,等渗盐水洗涤3次,180℃的烘箱里烘干6小时,4℃保存备用。钛基材料处理方法与其相同。
2.2 XPS与AFM扫描
将( DOPA )4-G5-YGFGG配置成一定的浓度,溶剂为去离子水、PBS缓冲液、乙醇或甲醇等常用溶剂,浓度在0.01-10mg/mL之间。将医用自攻皮质骨钛钉浸泡于上述溶液中,浸泡时间为30min至72h之间,即可得到表面改性有成骨生长肽YGFGG的钛钉与钛细胞爬片。之后将此钛基材料经干燥处理,以便XPS和AFM扫描,检测其表面元素变化与粗糙程度变化。
2.3 实验动物分组
SD大鼠63只,随机分为以下3组:
(1)Control组:21只,由股骨远端髁间窝处向平行股骨干方向植入直径1.5mm,长1.5cm医用钛钉,并在髓腔给予40µL的PBS,4周后处死;
(2)Vehicle组:21只,手术操作与Control组相同,髓腔内置入40µL 已配制的40%Ti颗粒PBS液(16mg/只)4周后处死;
(3)(DOPA )4-G5-RGDS组:21只,植入医用钛钉前,应用( DOPA )4-G5-RGDS多肽对钛钉表面改性,其余操作与Vehicle组相同,4周后处死;
2.4 大鼠股骨骨溶解模型的制备
本发明采用钛颗粒(Ti)诱导的大鼠股骨骨溶解模型来模拟TJA术后假体周围骨溶解发生的病理过程(Kaar SG, et al. Rapid repair of titanium particle-inducedosteolysis is dramatically reduced in aged mice. J Orthop Res. 2001; 19(2):171-8.)。实验大鼠用10%水合氯醛5mL/kg腹腔内注射麻醉。膝关节内侧皮肤去毛、安尔碘消毒3遍后, 在膝关节内侧处作一约1cm正中矢状切口,暴露髌骨并脱位, 由股骨远端髁间窝处向平行股骨干方向以克氏针打孔,深度1.5cm,之后植入准备好的40µL 已配制的40%Ti颗粒PBS液(16mg/只),最后植入1.5cm长,1.5mm直径的医用肽钉。皮肤以4-0缝线缝合。所有手术均在在同一天完成, 术后均已20万单位青霉素肌注。
2.5 标本采集
各组动物均于术后4周,腹腔注射10%水合氯醛麻醉,仰卧外固定于小鼠操作架上,开胸暴露心脏,经心尖向左心室插管灌注,剪开右心耳,结扎降主动脉后开放生理盐水冲洗至右心耳流出清凉液体,再以4%中性多聚甲醛200~300mL灌注,至动物四肢抽搐、变硬。灌注完毕后迅速取出股骨,剔除附着软组织。每只大鼠两根股骨置于4%多聚甲醛固定24h后,每对股骨先行micro-CT检测,后每组随机抽取7对行硬组织切片;再随机抽取7对行生物力学实验,测量髓内钉最大拔出力;剩余7对再以10%EDTA脱钙3周,除去钛钉,石蜡包埋,行组织学检测。
micro-CT检测
大鼠股骨固定24h后,行micro-CT扫描。扫描参数:分辨率9μm,电压80kV;电流100µA;每次曝光时间为100ms;0.9°/8 images。采用Wedemeyer C方法(Wedemeyer C, et al.Particle-induced osteolysis in three-dimensional micro-computed tomography.Calcif Tissue Int. 2007; 81(5): 394-402.),选定以钛钉为中心,其周围一空心圆柱体感兴趣区域(ROI;外径3mm,内径1.5mm,高度1.5cm),应用Micro-CT图像分析软件对图像进行3D分析,记录ROI股骨的骨密度(BMD, mg/mm2),骨体积与组织体积比值(BV/TV)。
2.6生物力学实验
每组标本随机选取7对股骨,行生物力学实验。由股骨远端下1/3出截断,固定股骨髁。由远端将植入钛钉徐徐推出,测试最大拔出力(N),并记录。
2.7组织学染色
部份股骨标本行硬组织切片后,以甲苯胺蓝染色。其余股骨经10%EDTA脱钙后,移除钛钉,常规石蜡包埋。取股骨水平位,于股骨远端生长板以上处连续切片,片厚5µm。分别行HE和TRAP染色。
2.7.1甲苯胺蓝染色步骤:
组织行硬组织切片过后,用水稀释甲苯胺蓝染液(按所期望染色程度,凭实验经验决定稀释度,由1:100稀释起始)。在稀释好的染色液中短暂浸泡玻片,然后在水中浸泡数次,按选定方法进行压片固定。
2.7.2 HE染色步骤:
(1)石蜡切片经二甲苯(10min×3次)脱蜡后,依次经过100%、100%、95%、90%,85%的乙醇至水,每道5min;
(2)蒸馏水冲洗3min,苏木素溶液染色5min,自来水冲洗5min;
(3)1%盐酸酒精溶液分化60s,自来水冲洗1min;
(4)10%氨水溶液中反蓝60s,自来水冲洗1min;
(5)l%伊红溶液复染3min,自来水冲洗1min;
(6)常规脱水、透明、封片。
2.7.3 抗酒石酸酸性磷酸酶染色:
抗酒石酸酸性磷酸酶(TRAP)为破骨细胞所特有,分布于破骨细胞胞浆。在含酒石酸盐的酸性条件下,TRAP能将萘酚ASBI磷酸盐水解,产生萘酚ASB1,后者立即与染液中的六偶氮副品红结合,在酶活性部位形成不溶性红色染料。通过观察这种染料可间接了解酸性磷酸酶活性。TRAP染色被用来鉴别破骨样细胞。染色采用TRAP染色试剂盒(Sigma 387A)。
2.7.3.1试剂配制:
备2个试管,一个加0.5mL fast Garnet GBC Base Solution(副品红),另一个加0.5mLSodium Nitrite Solution(亚硝酸钠),混合30s,静置2min;备2个100mL烧杯,标记A,B,配制TRAP染液(pH5.2):
A mL | B mL | |
37度蒸馏水 | 45 | 45 |
重氮化的副品红 | 1.0 | 1.0 |
Naphthol AS-BI Phosphate Solution | 0.5 | 0.5 |
Acetate solution | 2.0 | 2.0 |
Tartrate solution | - | 1.0 |
2.7.2.2 染色步骤:
(1)石蜡切片脱蜡和水化后,用PBS冲洗3次,每次3min
(2)将准备好的标本切片在丙酮溶液中固定30s;
(3)蒸馏水冲洗,不让其干;
(4)TRAP染液37度孵育1h,避光;
(5)蒸馏水冲洗3次,苏木素复染2min,PBS冲洗反蓝。
TRAP染色阳性结果为紫红色点、片状区域,参照Nich C法(Nich C, et al. Roleof direct estrogen receptor signaling in wear particle-induced osteolysis.Biomaterials. 2013; 34(3): 641-50.),以颅骨矢状缝为中心,20×光镜视野下计数成熟破骨细胞数量。
2.8 免疫组化检测IL-1、IL-6、TNF-α、integrinαVβ3及P65 1、脱蜡和水化 脱蜡前,将切片在室温中放置60分钟或60℃恒温箱中烘烤30分钟。
1)切片置于二甲苯中浸泡10分钟,更换二甲苯后再浸泡10分钟; 2)无水乙醇中浸泡5分钟; 3)95%乙醇中浸泡5分钟; 4)70%乙醇中浸泡5分钟; 2、抗原修复
酶消化方法:常用0.1%胰蛋白酶,胰蛋白酶使用前预热至37℃,切片也预热至37℃,每张切片滴加0.2mL消化液覆盖完全组织,于37℃温箱内消化约5~30分钟,避光。 3、免疫组织化学染色 1)PBS洗2~3次各5分钟; 2)滴加正常山羊血清封闭液,室温20分钟。甩去多余液体。 3)滴加Ⅰ抗100μL,室温静置1小时或者4℃过夜或者37℃1小时。 4)4℃过夜后需在37℃复温45分钟。 5)PBS洗3次各5分钟; 6)滴加Ⅱ抗40~50μL,室温静置,或37℃1小时; 7)PBS洗3次各5分钟; 8)DAB显色5~10分钟,在显微镜下掌握染色程度; 9)PBS或自来水冲洗10分钟; 10)苏木精复染3分钟,盐酸酒精分化; 11)自来水冲洗10~15分钟; 12)脱水、透明、封片、镜检。 2.9蛋白免疫印迹实验(western blot)检测
2.9.1试剂准备
1.SDS-PAGE试剂:见聚丙烯酰胺凝胶电泳实验。
2.匀浆缓冲液:1.0 M Tris-HCl(pH 6.8) 1.0 mL;10%SDS 6.0 mL;β-巯基乙醇0.2 mL;ddH2O 2.8 mL。
3.转膜缓冲液:甘氨酸 2.9 g;Tris 5.8 g;SDS 0.37 g;甲醇200 mL;加ddH2O定容至1000 mL。
4.0.01 M PBS(pH7.4):NaCl 8.0 g;KCl 0.2 g;Na2HPO4 1.44 g;KH2PO4 0.24 g;加ddH2O至1000 mL。
5.膜染色液:考马斯亮兰 0.2 g;甲醇80 mL;乙酸2 mL;ddH2O118 mL。包被液(5%脱脂奶粉,现配):脱脂奶粉1.0 g 溶于20 mL的0.01 M PBS中。
6.显色液:DAB 6.0 mg;0.01 M PBS 10.0 mL;硫酸镍胺 0.1 mL;H202 1.0 μL。
2.9.2蛋白样品制备
单层贴壁细胞总蛋白的提取
(1) 倒掉培养液,并将瓶倒扣在吸水纸上使吸水纸吸干培养液(或将瓶直立放置一会儿使残余培养液流到瓶底然后再用移液器将其吸走)。
(2) 每瓶细胞加3 mL 4℃预冷的PBS(0.01M pH7.2~7.3)。平放轻轻摇动1 min洗涤细胞,然后弃去洗液。重复以上操作两次,共洗细胞三次以洗去培养液。将PBS弃净后把培养瓶置于冰上。
(3)按1mL裂解液加10 μL PMSF(100 mM),摇匀置于冰上。(PMSF要摇匀至无结晶时才可与裂解液混合。)
(4) 每瓶细胞加400 μL含PMSF的裂解液,于冰上裂解30 min,为使细胞充分裂解培养瓶要经常来回摇动。
(5)裂解完后,用干净的刮棒将细胞刮于培养瓶的一侧(动作要快),然后用枪将细胞碎片和裂解液移至1.5 mL离心管中。(整个操作尽量在冰上进行。)
(6)于4℃下12000 rpm离心5 min。(提前开离心机预冷)
(7) 将离心后的上清分装转移倒0.5 mL的离心管中放于-20℃保存。
2.9.3蛋白含量的测定
1.制作标准曲线
(1)从-20℃取出1 mg/mL BSA,室温融化后,备用。
(2)取18个1.5 mL离心管,3个一组,分别标记为0 mg,2.5 mg,5.0 mg,10.0 mg,20.0 mg,40.0 mg。
(3)按下表在各管中加入各种试剂
(4) 混匀后,室温放置2 min。在生物分光光度计(Bio-Photometer,Eppendorf)上比色分析。
2.检测样品蛋白含量
(1)取足量的1.5 mL离心管,每管加入4℃储存的考马斯亮蓝溶液1 mL。室温放置30min后即可用于测蛋白。
(2) 取一管考马斯亮蓝加0.15 mol/L NaCl溶液100 mL,混匀放置2分钟可做为空白样品,将空白倒入比色杯中在做好标准曲线的程序下按blank测空白样品。
(3)弃空白样品,用无水乙醇清洗比色杯2次(每次0.5 mL),再用无菌水洗一次。
(4)取一管考马斯亮蓝加95 mL 0.15 mol/L NaCl NaCl溶液和5 mL待测蛋白样品,混匀后静置2 min,倒入扣干的比色杯中按sample键测样品。
2.9.4 SDS-PAGE电泳
1. 清洗玻璃板
一只手扣紧玻璃板,另一只手蘸点洗衣粉轻轻擦洗。两面都擦洗过后用自来水冲,再用蒸馏水冲洗干净后立在筐里晾干。
2. 灌胶与上样
(1)玻璃板对齐后放入夹中卡紧。然后垂直卡在架子上准备灌胶。(操作时要使两玻璃对齐,以免漏胶。)
(2)按前面方法配10%分离胶,加入TEMED后立即摇匀即可灌胶。灌胶时,可用10 mL枪吸取5 mL胶沿玻璃放出,待胶面升到绿带中间线高度时即可。然后胶上加一层水,液封后的胶凝的更快。(灌胶时开始可快一些,胶面快到所需高度时要放慢速度。操作时胶一定要沿玻璃板流下,这样胶中才不会有气泡。加水液封时要很慢,否则胶会被冲变型。)
(3)当水和胶之间有一条折射线时,说明胶已凝了。再等3 min使胶充分凝固就可倒去胶上层水并用吸水纸将水吸干。
(4)按前面方法配4%的浓缩胶,加入TEMED后立即摇匀即可灌胶。将剩余空间灌满浓缩胶然后将梳子插入浓缩胶中。灌胶时也要使胶沿玻璃板流下以免胶中有气泡产生。插梳子时要使梳子保持水平。由于胶凝固时体积会收缩减小,从而使加样孔的上样体积减小,所以在浓缩胶凝固的过程中要经常在两边补胶。待到浓缩胶凝固后,两手分别捏住梳子的两边竖直向上轻轻将其拔出。
(5)用水冲洗一下浓缩胶,将其放入电泳槽中。(小玻璃板面向内,大玻璃板面向外。若只跑一块胶,那槽另一边要垫一块塑料板且有字的一面面向外。)
(6)测完蛋白含量后,计算含50 ng蛋白的溶液体积即为上样量。取出上样样品至0.5mL离心管中,加入5×SDS 上样缓冲液至终浓度为1×。(上样总体积一般不超过15 μL,加样孔的最大限度可加20 μL样品。)上样前要将样品于沸水中煮5 min使蛋白变性。
(7) 加足够的电泳液后开始准备上样。(电泳液至少要漫过内测的小玻璃板。)用微量进样器贴壁吸取样品,将样品吸出不要吸进气泡。将加样器针头插至加样孔中缓慢加入样品。(加样太快可使样品冲出加样孔,若有气泡也可能使样品溢出。加入下一个样品时,进样器需在外槽电泳缓冲液中洗涤3次,以免交叉污染。
3. 电泳
电泳时间一般4~5 h,电压为40 V较好,也可用60 V。电泳至溴酚兰刚跑出即可终止电泳,进行转膜。
2.9.5转膜
1. 转一张膜需准备6张7.0~8.3 cm的滤纸和1张7.3~8.6 cm的硝酸纤维素膜。切滤纸和膜时一定要戴手套,因为手上的蛋白会污染膜。将切好的硝酸纤维素膜置于水上浸2 h才可使用。(用镊子捏住膜的一边轻轻置于有超纯水的平皿里,要使膜浮于水上,只有下层才与水接触。这样由于毛细管作用可使整个膜浸湿。若膜沉入水里,膜与水之间形成一层空气膜,这样会阻止膜吸水。
2. 在加有转移液的搪瓷盘里放入转膜用的夹子、两块海绵垫、一支玻棒、滤纸和浸过的膜。
3. 将夹子打开使黑的一面保持水平。在上面垫一张海绵垫,用玻棒来回擀几遍以擀走里面的气泡。(一手擀另一手要压住垫子使其不能随便移动。)在垫子上垫三层滤纸(可三张纸先叠在一起在垫于垫子上),一手固定滤纸一手用玻棒擀去其中的气泡。
4. 要先将玻璃板撬掉才可剥胶,撬的时候动作要轻,要在两个边上轻轻的反复撬。撬一会儿玻璃板便开始松动,直到撬去玻板。(撬时一定要小心,玻板很易裂。)除去小玻璃板后,将浓缩胶轻轻刮去(浓缩胶影响操作),要避免把分离胶刮破。小心剥下分离胶盖于滤纸上,用手调整使其与滤纸对齐,轻轻用玻棒擀去气泡。将膜盖于胶上,要盖满整个胶(膜盖下后不可再移动)并除气泡。在膜上盖3张滤纸并除去气泡。最后盖上另一个海绵垫,擀几下就可合起夹子。整个操作在转移液中进行,要不断的擀去气泡。膜两边的滤纸不能相互接触,接触后会发生短路。(转移液含甲醇,操作时要戴手套,实验室要开门以使空气流通。)
5. 将夹子放入转移槽槽中,要使夹的黑面对槽的黑面,夹的白面对槽的红面。电转移时会产热,在槽的一边放一块冰来降温。一般用60 V转移2 h或40 V转移3 h。
6. 转完后将膜用1×丽春红染液染5 min(于脱色摇床上摇)。然后用水冲洗掉没染上的染液就可看到膜上的蛋白。将膜晾干备用。
2.9.6免疫反应
1. 将膜用TBS从下向上浸湿后,移至含有封闭液的平皿中,室温下脱色摇床上摇动封闭1h。
2. 将一抗用TBST稀释至适当浓度(在1.5 mL离心管中);撕下适当大小的一块儿保鲜膜铺于实验台面上,四角用水浸湿以使保鲜膜保持平整;将抗体溶液加到保鲜膜上;从封闭液中取出膜,用滤纸吸去残留液后,将膜蛋白面朝下放于抗体液面上,掀动膜四角以赶出残留气泡;室温下孵育1~2 h后,用TBST在室温下脱色摇床上洗两次,每次10 min;再用TBS洗一次,10 min。
3. 同上方法准备二抗稀释液并与膜接触,室温下孵育1~2 h后,用TBST在室温下脱色摇床上洗两次,每次10 min;再用TBS洗一次,10 min,进行化学发光反应。
2.9.7化学发光,显影,定影
1. 将A和B两种试剂在保鲜膜上等体积混合;1 min后,将膜蛋白面朝下与此混合液充分接触;1 min后,将膜移至另一保鲜膜上,去尽残液,包好,放入X-光片夹中。
2. 在暗室中,将1×显影液和定影液分别到入塑料盘中;在红灯下取出X-光片,用切纸刀剪裁适当大小(比膜的长和宽均需大1 cm);打开X-光片夹,把X-光片放在膜上,一旦放上,便不能移动,关上X-光片夹,开始计时;根据信号的强弱适当调整曝光时间,一般为1 min或5 min,也可选择不同时间多次压片,以达最佳效果;曝光完成后,打开X-光片夹,取出X-光片,迅速浸入显影液中显影,待出现明显条带后,即刻终止显影。显影时间一般为1~2 min(20~25℃),温度过低时(低于16℃)需适当延长显影时间;显影结束后,马上把X-光片浸入定影液中,定影时间一般为5~10 min,以胶片透明为止;用自来水冲去残留的定影液后,室温下晾干。
2.10 统计学分析
结果数据采用SPSS11.0统计软件分析,数据用均数±标准差()表示,多组比较选用单因素方差分析(one-way ANOVA 检验),两两比较在总体方差齐的条件下,选用LSD及Dunnett-t法进行分析。p<0.05为差异有统计学意义。
二、结果
2.1 合成的( DOPA )4-G5-RGDS生物活性肽与钛基材料紧密结合
2.1.1 生物模拟细胞贴附肽( DOPA )4-G5-RGDS的分子结构如图1,其中:支撑部分含有四重的DOPA氨基酸序列,间隔部分含有五重的甘氨酸序列,活性部分则接有细胞贴附性能的RGDS活性序列。图2显示的( DOPA )4-G5-RGDS的质谱图。
2.1.2 XPS和AFM检测
XPS扫描结果显示,与普通PBS浸泡的钛基材料相比,改性后的钛基材料,“N”元素含量明显增加;AFM扫面结果显示 ( DOPA )4-G5-RGDS多肽改性钛基材料后,其表面粗糙度增加,有较多类蛋白团块物质粘附,如图2。
2.2 ( DOPA )4-G5-RGDS生物活性肽能减轻磨损颗粒引起的骨溶解
2.2.1 实验动物一般情况
各组动物均在术后30~60min内苏醒,可在笼内自由活动,正常进食,精神状态无明显变化。无切口无红肿、渗液等炎性反应,均一期愈合。实验过程中无动物死亡。
2.2.2 micro-CT检测
运用micro-CT扫描实验大鼠股骨并进行三维图像重建及定量分析,可以较为准确的描述骨量和骨微结构,从而判定骨溶解的程度。其中,分为Control组, Vehicle组, ( DOPA )4-G5-RGDS组。三维图像显示,与Control组比较,Vehicle组股骨骨量减少,假体周围骨溶解明显,给予经( DOPA )4-G5-RGDS处理后,股骨骨量增加,骨溶解减轻(图3)。
骨密度(BMD)变化:Vehicle组大鼠股骨骨密度明显降低,与Control组比较,差异有统计学意义(**p<0.01);与Vehicle组比较,( DOPA )4-G5-RGDS组大鼠股骨骨密度显著增加*p<0.05。骨体积分数(BV/TV)Vehicle组骨体积分数明显降低,与Control组比较,**p<0.01。而( DOPA )4-G5-RGDS多肽处理钛钉后,骨体积分数明显增加,**p<0.01,见图4。
2.2.3 生物力学检测
最大拔出力变化:Vehicle组拔出钛钉所用力较小,与Control组比较,差异有统计学意义(**p<0.01)。与Vehicle组比较,拔出( DOPA )4-G5-RGDS组大鼠股骨钛钉力值明显增加,*p<0.05见图5。
2.2.4 组织学检测
2.2.4.1 甲苯胺蓝染色结果:
经硬组织切片过后,光镜下,Vehicle组较Control组钛钉周围骨盐沉积明显减少,并且出现了明显骨基质缺陷。给予经( DOPA )4-G5-RGDS处理后,钛钉周围骨基质缺陷明显改善,骨盐沉积率增加。骨盐沉积率(BIC%):与Control组(27.56 ± 4.25 %)比较,Vehicle组(19.88 ± 3.09 %)骨盐沉积率明显减少,差异有统计学意义(**p<0.01)。( DOPA )4-G5-RGDS处理后,骨盐沉积率为(62.30 ± 5.18 %),与Vehicle组相比,差异有统计学意义(#p<0.05),见图6。
2.2.4.2 HE染色结果:
光镜下,与Control相比,Vehicle组骨组织有明显虫蚀样变化,骨基质明显缺损,假体周围细胞数量增多,且多为炎性细胞;( DOPA )4-G5-RGDS组,假体周围骨组织有破坏,但程度较轻有少量炎性细胞,成纤维细胞排列尚规则。骨溶解率(ES/BS):与Control组(27 ±2.287)比较,Vehicle组(44.74 ± 5.246)骨基质明显缺损,差异有统计学意义(**p<0.01)。( DOPA )4-G5-RGDS处理后,ES/BS(12.11 ± 1.436),与Vehicle组相比,差异有统计学意义(*p<0.05),见图7。
2.2.4.3 TRAP染色结果
TRAP染色阳性区域为紫红色,Control组可见点状的阳性改变,且主要集中于假体周围;Vehicle组骨溶解侧可见大片紫红色区域,表明有大量的成熟的破骨细胞存在;(DOPA)4-G5-RGDS组仅有少量阳性区域。光镜下计数结果显示Vehicle组TRAP阳性细胞为119± 10,与Control组(62 ± 7)相比, **p<0.01;而( DOPA )4-G5-RGDS组TRAP细胞数分别为41 ± 6。( DOPA )4-G5-RGDS组与Vehicle组相比,差异有统计学意义(**p<0.01),见图8。
2.3 ( DOPA )4-G5-RGDS生物活性肽能抑制磨损颗粒引起的炎症因子的表达
免疫组织化学染色的结果显示,在Control组中IL-1、IL-6和TNF-α等相关炎症因子的表达浓度较低;在Vehicle组,IL-1、IL-6和TNF-α等相关炎症因子的含量急剧增加;在(DOPA )4-G5-RGDS组,IL-1、IL-6、TNF-α的含量随( DOPA )4-G5-RGDS多肽改性钛钉后浓度的增加显著下降,如图9。
2.4 ( DOPA )4-G5-RGDS生物活性肽抑制RANKL诱导的破骨细胞活化
RANKL及M-CSF诱导小鼠股骨巨噬细胞于医用钛细胞爬片,结果显示,经( DOPA )4-G5-RGDS多肽改性医用钛细胞爬片后, RANKL诱导生成破骨细胞生成数量分别在3天、5天、7天较正常钛片表面上明显减少,二者计数具有统计学意义(*p<0.01),见图10。
2.5 ( DOPA )4-G5-RGDS生物活性肽通过integrinαVβ3/ NF-κB信号通路调控破骨细胞活化
特征性F-肌动蛋白(F-actin)环形成是破骨细胞发挥骨吸收功能的前提条件。因此,我们进行鬼笔环肽染色,以测试( DOPA )4-G5-RGDS生物活性肽对F-actin环形成的影响。 如所预期的,我们的结果表明RANKL干预组中可以清楚地观察到了分化良好的F-actin环。然而,( DOPA )4-G5-RGDS生物活性肽处理组, F-actin环的大小和数量均明显减少。
IntegrinαVβ3在成熟破骨细胞膜中的表达量较高,研究证实,integrin β3为细胞膜受体,可调控细胞增殖,迁移,粘附等重要功能。而经( DOPA )4-G5-RGDS多肽改性医用钛细胞爬片后,免疫荧光染色的结果显示,integrin β3表达量减少明显减少,如图11。Western blot结果显示,integrin β3的表达量及其下游的相关因子FAK与Src的磷酸化水平明显增高,而经( DOPA )4-G5-RGDS多肽改性医用钛细胞爬片后,这些蛋白的表达量减少明显减少,见图12。体内染色结果显示,integrin β3 在Vehicle组的表达量明显增加,而(DOPA )4-G5-RGDS多肽处理组,integrin β3的阳性细胞数量显著减少,见图13。
进一步的研究结果显示,经RANKL及M-CSF诱导在15min、30min、45min和60min小鼠股骨巨噬细胞P65与IKB-α磷酸化水平不同程度的升高,而经( DOPA )4-G5-RGDS多肽处理后,各时间点P65与IKB-α磷酸化水平明显降低,见图14。细胞免疫荧光染色显示,经( DOPA )4-G5-RGDS多肽改性医用钛细胞爬片后, RANKL诱导小鼠巨噬细胞中P65表达入核率较普通爬片明显下降(见图15),提示( DOPA )4-G5-RGDS多肽通过NF-κB信号通路调控破骨细胞活化。体内的染色结果显示,在Control组中P65的表达浓度较低;在Vehicle组, P65含量显著增加;而在( DOPA )4-G5-RGDS组P65的阳性细胞数量明显减少,表明( DOPA )4-G5-RGDS多肽通过NF-κB通路调控破骨细胞活化,减轻磨损颗粒引起的骨溶解。
本发明通过用( DOPA )4-G5-RGDS多肽改性医用钛基材料可以抑制磨损颗粒引起骨溶解的作用机制可能是多样的,其主要有以下几点:
首先,炎症反应在骨溶解机制中起到关键作用,而本实验中在本实验中,( DOPA )4-G5-RGDS多肽可明显降低IL-1、IL-6和TNF-α等相关炎症因子表达,这与本实验中( DOPA )4-G5-RGDS组大鼠骨组织中骨溶解程度减轻相符合。其次,近年来NF-κB通路的激活被认为对骨溶解的发生发展起主要作用,其中P65的磷酸化对于破骨细胞活化起重要作用,是调控破骨细胞活化的关键因子。本实验中大鼠骨组织P65含量减少,与Vehicle组比较,( DOPA )4-G5-RGDS组中股骨骨组织成熟破骨细胞数明显减少,说明( DOPA )4-G5-RGDS通过干预NF-κB通路抑制破骨细胞活化。最后,integrinαvβ3作为细胞膜受体可调控的细胞功能是多样的,而( DOPA )4-G5-RGDS多肽可使成熟破骨细胞的integrinαvβ3及其下游通路因子的表达明显减少,从而影响破骨细胞相关功能。
综上所述,( DOPA )4-G5-RGDS多肽改性医用钛基材料治疗骨溶解的机制可能是通过减轻假体周围炎症反应,干预其下游通路从而抑制NF-κB通路活化,并抑制integrinαvβ3的表达,从而抑制破骨细胞活化及功能,减轻骨溶解起到治疗作用。
Claims (3)
1.一种(DOPA)4-G5-RGDS活性肽,其特征在于,所述活性肽的化学结构式如式(Ⅰ)所示:
式(Ⅰ)。
2.权利要求1所述的活性肽在制备治疗炎症性假体周围骨溶解的药物组合物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的药物组合物包含改性医用钛基材料的(DOPA)4-G5-RGDS活性肽。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811238308.4A CN109400676A (zh) | 2018-10-23 | 2018-10-23 | (dopa)4-g5-rgds活性肽及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811238308.4A CN109400676A (zh) | 2018-10-23 | 2018-10-23 | (dopa)4-g5-rgds活性肽及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN109400676A true CN109400676A (zh) | 2019-03-01 |
Family
ID=65469018
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811238308.4A Pending CN109400676A (zh) | 2018-10-23 | 2018-10-23 | (dopa)4-g5-rgds活性肽及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109400676A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113144290A (zh) * | 2020-09-09 | 2021-07-23 | 苏州大学附属第一医院 | 一种促成骨和免疫调节的骨科材料表面涂层及其制备方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101412773B1 (ko) * | 2011-04-06 | 2014-06-30 | 포항공과대학교 산학협력단 | 티로시나아제 동시발현에 의해 제조된 홍합 접착 단백질 |
CN105949322A (zh) * | 2016-05-12 | 2016-09-21 | 苏州大学附属第医院 | 一种适用于“一步法”改性医用钛基材料的生物模拟活性肽 |
WO2017042805A1 (en) * | 2015-09-08 | 2017-03-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd | Novel compounds with dual activity |
CN106620845A (zh) * | 2017-01-07 | 2017-05-10 | 深圳清华大学研究院 | 可注射骨材料及其制备方法 |
CN107028932A (zh) * | 2017-05-12 | 2017-08-11 | 苏州大学附属第医院 | 冈田酸在治疗假体周围骨溶解中的药物用途 |
-
2018
- 2018-10-23 CN CN201811238308.4A patent/CN109400676A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101412773B1 (ko) * | 2011-04-06 | 2014-06-30 | 포항공과대학교 산학협력단 | 티로시나아제 동시발현에 의해 제조된 홍합 접착 단백질 |
WO2017042805A1 (en) * | 2015-09-08 | 2017-03-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd | Novel compounds with dual activity |
CN105949322A (zh) * | 2016-05-12 | 2016-09-21 | 苏州大学附属第医院 | 一种适用于“一步法”改性医用钛基材料的生物模拟活性肽 |
CN106620845A (zh) * | 2017-01-07 | 2017-05-10 | 深圳清华大学研究院 | 可注射骨材料及其制备方法 |
CN107028932A (zh) * | 2017-05-12 | 2017-08-11 | 苏州大学附属第医院 | 冈田酸在治疗假体周围骨溶解中的药物用途 |
Non-Patent Citations (3)
Title |
---|
ANTONINO G. SECCHI: "RGDS peptides immobilized on titanium alloy stimulate bone cell attachment, differentiation and confer resistance to apoptosis", 《JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, PART A》 * |
GUOQING PAN等: ""Biomimetic Design of Mussel-Derived Bioactive Peptides for Dual-Functionalization of Titanium-Based Biomaterials", 《J. AM. CHEM. SOC.》 * |
李宏斌等: "药物预防和治疗关节假体周围骨溶解研究进展", 《国外医学骨科学分册》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113144290A (zh) * | 2020-09-09 | 2021-07-23 | 苏州大学附属第一医院 | 一种促成骨和免疫调节的骨科材料表面涂层及其制备方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101385870B (zh) | 一种改良去细胞组织工程瓣膜/血管支架的方法 | |
Schultzberg et al. | Dopamine and cholecystokinin immunoreactive neurones in mesencephalic grafts reinnervating the neostriatum: evidence for selective growth regulation | |
CN1678734B (zh) | 分化调节试剂及其应用 | |
CN104225667B (zh) | 一种促血管生成的温敏性水凝胶粉及用其制备的温敏性水凝胶 | |
CN103285429A (zh) | 一种组织工程关节双相支架及其制备方法和应用 | |
Mabilleau et al. | Efficacy of targeting bone-specific GIP receptor in ovariectomy-induced bone loss | |
CN109400676A (zh) | (dopa)4-g5-rgds活性肽及其应用 | |
CN110857434A (zh) | 促进细胞生长和组织修复的方法及组合物 | |
Röhlich et al. | Binding pattern of anti-rhodopsin monoclonal antibodies to photoreceptor cells: an immunocytochemical study | |
CN106563173B (zh) | 一种脱细胞生物真皮材料及其制备方法和应用 | |
CN105749348A (zh) | 一种PRP/MSCs/牡蛎壳骨组织支架材料的制备方法 | |
Deviche et al. | Identification, partial characterization, and hypothalamic distribution of κ, μ, and δ opioid receptors in a passerine songbird (Junco hyemalis) | |
KR101719870B1 (ko) | 적색 파장대의 빛을 이용한 중간엽 줄기세포의 분화유도 방법 | |
CN102971019A (zh) | 用于复杂组织工程化的方法 | |
Xu et al. | Effect of carbodiimide cross-linking of decellularized porcine pulmonary artery valvular leaflets | |
CN107028932A (zh) | 冈田酸在治疗假体周围骨溶解中的药物用途 | |
Grounds et al. | The expression of extracellular matrix during adult skeletal muscle regeneration: how the basement membrane, interstitium and myogenic cells collaborate | |
Simpson | Morphological studies of possible neuroendocrine structures in Helisoma tenue (Gastropoda: Pulmonata) | |
Aljitawi et al. | Generating CK19-positive cells with hair-like structures from Wharton's jelly mesenchymal stromal cells | |
CN109959787A (zh) | 一种类风湿性关节炎辅助诊断试剂盒及检测方法 | |
CN110368496A (zh) | 成纤维细胞激活蛋白抑制剂在制备药物中的用途 | |
CN112694581B (zh) | 一种负载锂的三维微孔磺化聚醚醚酮、制备方法及其应用 | |
Fritsch et al. | Ossification in the human calcaneus: a model for spatial bone development and ossification | |
CN108949705A (zh) | 过氧化物还原酶1结合蛋白及其应用 | |
Pazzaglia et al. | The role of macrophages and giant cells in loosening of joint replacement |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190301 |