CN114656526A - 一种多肽及其在骨修复中的应用 - Google Patents
一种多肽及其在骨修复中的应用 Download PDFInfo
- Publication number
- CN114656526A CN114656526A CN202210496588.9A CN202210496588A CN114656526A CN 114656526 A CN114656526 A CN 114656526A CN 202210496588 A CN202210496588 A CN 202210496588A CN 114656526 A CN114656526 A CN 114656526A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- bone
- bone repair
- scaffold
- repair
- 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.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 84
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 72
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 72
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 68
- 230000008439 repair process Effects 0.000 title claims abstract description 37
- 102000015735 Beta-catenin Human genes 0.000 claims abstract description 10
- 108060000903 Beta-catenin Proteins 0.000 claims abstract description 10
- 206010061363 Skeletal injury Diseases 0.000 claims abstract description 9
- 102000004067 Osteocalcin Human genes 0.000 claims abstract description 8
- 108090000573 Osteocalcin Proteins 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 210000001519 tissue Anatomy 0.000 claims description 24
- 108010010803 Gelatin Proteins 0.000 claims description 6
- 239000008273 gelatin Substances 0.000 claims description 6
- 229920000159 gelatin Polymers 0.000 claims description 6
- 235000019322 gelatine Nutrition 0.000 claims description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims 1
- 230000011164 ossification Effects 0.000 abstract description 7
- 210000002901 mesenchymal stem cell Anatomy 0.000 abstract description 6
- 210000001185 bone marrow Anatomy 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 3
- 102000040739 Secretory proteins Human genes 0.000 abstract description 2
- 108091058545 Secretory proteins Proteins 0.000 abstract description 2
- 230000004913 activation Effects 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- TZSMWSKOPZEMAJ-UHFFFAOYSA-N bis[(2-methoxyphenyl)methyl] carbonate Chemical compound COC1=CC=CC=C1COC(=O)OCC1=CC=CC=C1OC TZSMWSKOPZEMAJ-UHFFFAOYSA-N 0.000 abstract 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 abstract 1
- 230000007547 defect Effects 0.000 description 40
- 239000000463 material Substances 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 9
- 230000002188 osteogenic effect Effects 0.000 description 9
- 239000012188 paraffin wax Substances 0.000 description 9
- 241000700159 Rattus Species 0.000 description 8
- 230000001737 promoting effect Effects 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 239000000499 gel Substances 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 6
- 210000003625 skull Anatomy 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 5
- 239000000017 hydrogel Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 208000006735 Periostitis Diseases 0.000 description 4
- 102000013814 Wnt Human genes 0.000 description 4
- 108050003627 Wnt Proteins 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 210000003460 periosteum Anatomy 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000003125 immunofluorescent labeling Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 229910001961 silver nitrate Inorganic materials 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- PEZMQPADLFXCJJ-ZETCQYMHSA-N 2-[[2-[[(2s)-1-(2-aminoacetyl)pyrrolidine-2-carbonyl]amino]acetyl]amino]acetic acid Chemical compound NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)NCC(O)=O PEZMQPADLFXCJJ-ZETCQYMHSA-N 0.000 description 2
- RWDVGVPHEWOZMO-GUBZILKMSA-N Arg-Cys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCNC(N)=N)C(O)=O RWDVGVPHEWOZMO-GUBZILKMSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- YVHGKXAOSVBGJV-CIUDSAMLSA-N Asp-Lys-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N YVHGKXAOSVBGJV-CIUDSAMLSA-N 0.000 description 2
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 2
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 2
- GHUVBPIYQYXXEF-SRVKXCTJSA-N Cys-Cys-Tyr Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 GHUVBPIYQYXXEF-SRVKXCTJSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 102000003982 Parathyroid hormone Human genes 0.000 description 2
- 108090000445 Parathyroid hormone Proteins 0.000 description 2
- QEFHBVDWKFFKQI-PMVMPFDFSA-N Phe-His-Trp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QEFHBVDWKFFKQI-PMVMPFDFSA-N 0.000 description 2
- 230000004156 Wnt signaling pathway Effects 0.000 description 2
- 230000033558 biomineral tissue development Effects 0.000 description 2
- 229940112869 bone morphogenetic protein Drugs 0.000 description 2
- 230000010478 bone regeneration Effects 0.000 description 2
- 230000008758 canonical signaling Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 229920006237 degradable polymer Polymers 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 108010043293 glycyl-prolyl-glycyl-glycine Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 230000000887 hydrating effect Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 125000005395 methacrylic acid group Chemical group 0.000 description 2
- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 239000000199 parathyroid hormone Substances 0.000 description 2
- 229960001319 parathyroid hormone Drugs 0.000 description 2
- 210000003455 parietal bone Anatomy 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 238000010146 3D printing Methods 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- COXMUHNBYCVVRG-DCAQKATOSA-N Arg-Leu-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O COXMUHNBYCVVRG-DCAQKATOSA-N 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- XMVZMBGFIOQONW-GARJFASQSA-N Cys-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N)C(=O)O XMVZMBGFIOQONW-GARJFASQSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 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 1
- 229940124602 FDA-approved drug Drugs 0.000 description 1
- 102000005698 Frizzled receptors Human genes 0.000 description 1
- 108010045438 Frizzled receptors Proteins 0.000 description 1
- VCBWXASUBZIFLQ-IHRRRGAJSA-N His-Pro-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O VCBWXASUBZIFLQ-IHRRRGAJSA-N 0.000 description 1
- 101001043594 Homo sapiens Low-density lipoprotein receptor-related protein 5 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102100021926 Low-density lipoprotein receptor-related protein 5 Human genes 0.000 description 1
- PBIPLDMFHAICIP-DCAQKATOSA-N Lys-Glu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PBIPLDMFHAICIP-DCAQKATOSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000003076 Osteolysis Diseases 0.000 description 1
- STTYIMSDIYISRG-UHFFFAOYSA-N Valyl-Serine Chemical compound CC(C)C(N)C(=O)NC(CO)C(O)=O STTYIMSDIYISRG-UHFFFAOYSA-N 0.000 description 1
- 102000044880 Wnt3A Human genes 0.000 description 1
- 108700013515 Wnt3A Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 238000002266 amputation Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000002639 bone cement Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 229940045110 chitosan Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000002454 frontal bone Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 208000029791 lytic metastatic bone lesion Diseases 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000537 nasal bone Anatomy 0.000 description 1
- 210000000103 occipital bone Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000009818 osteogenic differentiation Effects 0.000 description 1
- 230000002138 osteoinductive effect Effects 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- -1 polytetrafluoroethylene Polymers 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 101150068520 wnt3a gene Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/28—Bones
- A61F2/2846—Support means for bone substitute or for bone graft implants, e.g. membranes or plates for covering bone defects
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/28—Bones
- A61F2002/2835—Bone graft implants for filling a bony defect or an endoprosthesis cavity, e.g. by synthetic material or biological material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2002/30001—Additional features of subject-matter classified in A61F2/28, A61F2/30 and subgroups thereof
- A61F2002/30003—Material related properties of the prosthesis or of a coating on the prosthesis
- A61F2002/3006—Properties of materials and coating materials
- A61F2002/30062—(bio)absorbable, biodegradable, bioerodable, (bio)resorbable, resorptive
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2/3094—Designing or manufacturing processes
- A61F2002/30985—Designing or manufacturing processes using three dimensional printing [3DP]
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Transplantation (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- General Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Physical Education & Sports Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cardiology (AREA)
- Vascular Medicine (AREA)
- Heart & Thoracic Surgery (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Rheumatology (AREA)
- Materials For Medical Uses (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及一种多肽及其在骨修复中的应用，属于生物医药技术领域。本发明提供的多肽，其氨基酸序列如SEQ ID NO:1所示。本发明还公开了所述多肽GS18在骨损伤，和/或骨修复中的用途。进一步的，本发明还公开了多肽GS18在骨修复中的应用和一种用于骨修复的多肽支架。本发明多肽可显著促进激活β‑catenin入核及分泌蛋白骨钙素的表达，成骨定向的骨髓间充质干细胞BMSC形成矿化结节，从而促进骨修复。
Description
技术领域
本发明属于生物医药技术领域，涉及一种可促进骨修复的人工合成多肽GS18。
背景技术
骨缺损是一种常见的临床疾病，给公众健康带来了巨大负担。骨缺损可能由多种原因引起，包括创伤、感染、肿瘤和衰老等。虽然骨组织具有强大的自我修复和重建再生能力，但尺寸较大的缺损，往往伴随着骨不连、功能障碍、愈合延迟，甚至不愈合等结局。此时，需要特殊的治疗方法进行干预，以恢复受损骨组织的结构和功能。
自体骨移植被认为是修复临界骨缺损的金标准，然而自体骨移植物的应用具有一定的局限性，其受到供体部位发病、供体来源缺乏和感染风险增加等问题的严重限制。异体骨移植(取自其他患者)可部分弥补自体骨的不足，提供一些生长因子，并具有骨诱导特性。但这种方法也面临着来源有限、伦理等一系列问题。目前，采用无机非金属或高分子材料支架的组织工程骨受到广泛关注，通过3D打印等个性化定制的支架材料能够很好的匹配缺损区域，利用其疏松多孔的结构引导细胞血管生成从而实现骨再生。然而组织工程骨高度依赖其搭载的种子细胞和细胞因子，以在体内募集和诱导修复细胞(如骨髓间充质干细胞BMSC)增殖和分化，从而形成新骨组织。目前FDA 批准的具有促进新骨形成的药物中，甲状旁腺素(PTH)在高剂量摄入时可致骨肉瘤形成，骨形成蛋白(BMP2)的半衰期短，可引起异位骨形成、骨溶解和局部炎症反应。因而有效且安全的促进骨形成的因子仍有待开发。
发明内容
本发明的目的在于克服上述现有技术的不足之处，提供一种可促进骨修复的人工合成多肽。
为实现上述目的，本发明采用的技术方案为：
本发明提供了一种多肽，所述多肽的氨基酸序列如SEQ ID NO:1所示。
本发明的多肽含有18个氨基酸，氨基酸序列为GPGGDKCRCVFHWCCYVS，即 Gly ProGly Gly Asp Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr Val Ser，发明人将多肽命名为GS18，以下多肽均用此名。
本发明的多肽，其分子量为2017.33Da。
本发明多肽GS18可采用常规的合成方法，如液相分段合成法、固相合成、生物合成法等，作为本发明所述多肽的优选实施方式，所述多肽采用采取固相多肽合成工艺合成多肽。进一步的，为确保生物安全性，本发明多肽的纯度≥95％。可使用HPLC 对产物进行纯化。
本发明多肽可用于骨损伤和/或骨修复。进一步的，适用于广泛的骨缺损适应症，如截断性骨缺损、骨相关创伤、肿瘤骨缺损等，可促进骨缺损修复和/或用于修复骨缺损。
作为优选方案，本发明多肽GS18可与组织工程上可接受的载体联用来治疗骨损伤和/或骨修复。进一步的，本发明所述多肽GS18可以以任何形式负载在骨修复材料上，植入骨缺损部位。例如可以负载到骨水泥、骨支架中，也可以与骨修复水凝胶联用采用注射到骨损伤部位的方式。
GS18能够以组织工程支架搭载的作用方式，加速骨缺损区域的修复，促进骨再生。所述组织工程上可接受的载体通常意义上是指组织工程支架，进一步的，可适用于现有所有组织工程支架，包括可生物降解的骨组织工程支架材料和非生物降解型的骨组织工程支架材料。
本发明还公开了上述多肽在激活β-catenin入核中的用途以及在促进骨钙素的表达中的用途。
体外细胞实验表明，多肽GS18可以促进成骨定向的骨髓间充质干细胞BMSC形成矿化结节，从而促进骨修复。
在大鼠颅骨缺损模型中，将GS18修饰的GelMA支架植入骨缺损区，材料可以非常贴合地嵌入缺损区内。4周后取颅骨组织，通过组织切片HE染色观察到该多肽能够吸引募集细胞进入支架材料，加速缺损区骨修复和再生。通过Goldner染色观察，GS18 修饰的GelMA支架可显著促进缺损区纤维组织形成完成缺损连接，且观察到新生骨形成。
GS18的设计来源于WNT3A配体蛋白上与细胞膜Frizzled受体及LRP5/6共受体结合的识别区段，该蛋白可激活经典Wnt信号通路，激活膜内β-catenin入核，从而启动下游功能基因的转录。
进一步地，通过组织切片及抗原修复免疫荧光染色实验发现，该多肽可激活β -catenin入核，同时见大量分泌蛋白骨钙素(Osteocalcin，Ocn)的表达。这表明，GS18 可在缺损区激活Wnt经典信号通路所引发的骨修复过程。
即，GS18可激活β-catenin入核，同时，也可促进骨钙素(Osteocalcin，Ocn)的表达，从而实现缺损骨骼结构和功能的恢复。
作为本发明所述多肽应用的优选实施方式，所述多肽应用的浓度为25～150μ g/mL，优选100～150μg/mL。
进一步的，本发明还公开了一种骨修复组合物，所述骨修复组合物含有治疗有效剂量的多肽GS18和组织工程上可接受的载体。
所述组织工程上可接受的载体通常意义上是指组织工程支架，进一步的，可适用于现有所有组织工程支架，包括可生物降解的骨组织工程支架材料和非生物降解型的骨组织工程支架材料。
作为优选方案，本发明还公开了一种多肽支架，其包含多肽GS18。优选地，所述多肽支架为生物陶瓷、金属、碳基和可降解高分子复合材料等。
进一步的，所述可降解高分子复合明胶支架优选为：海藻酸钠、壳聚糖、透明质酸及甲基丙烯酸酐化明胶(GelMA)支架。
作为优选方案，本发明多肽支架中，GS18均匀分散在甲基丙烯酸酐化明胶(GelMA)支架中。
作为优选方案，在多肽支架中，多肽GS18的浓度为25～150μg/mL，优选为100～150μg/mL。
甲基丙烯酰化明胶(GelMA)是一种光敏性生物材料，其粉末溶于水等液体时，可做到与功能因子均匀混合。GelMA具有极好的操作性,可在光引发剂的作用下迅速交联形成三维结构。GelMA具有良好的生物相容性，其结构上具有细胞黏附位点，可促进细胞的增殖迁移。
GS18修饰的GelMA支架还可在模具辅助下或通过3d打印制备成任意外形，以贴合缺损区形态的。通过改变GelMA的取代度和浓度，可以灵活调整固化后的力学性能，使其具备一定的弹性、强度和支撑性，以恢复缺损骨的结构和部分功能。
优选地，本发明多肽GS18可以在制备骨损伤和/或骨修复组合物中的应用。
本发明的有益效果:
1)本发明的多肽GS18可以促进成骨定向的骨髓间充质干细胞BMSC形成矿化结节，从而促进骨修复。
2)本发明提供的多肽类药物GS18能够吸引并募集细胞进入骨修复材料(如支架等)，具有加速骨缺损的修复和再生的作用，可作为骨组织工程的功能因子。
3)本发明的多肽可以激活β-catenin入核，同时，也可促进骨钙素(Osteocalcin，Ocn)的表达，从而促进Wnt经典信号通路所对应的骨损伤修复过程。
4)本发明多肽生理活性强、局部组织吸收快且高效、免疫原性低，可更好地替代成骨蛋白的功能。
5)本发明提供的多肽GS18属小分子多肽，制备工艺简单，成本低，产量高，具有较高的转化价值和临床应用前景。
附图说明：
图1为GS18体外促进成骨向分化的骨髓间充质干细(BMSC)矿化结节形成的硝酸银染色图。
图2为GS18多肽修饰GelMA凝胶支架材料形态图。
图3为支架作用于大鼠颅骨缺损中4周后HE染色结果。
图4为支架作用于大鼠颅骨缺损中4周后Goldner染色结果。
图5为支架作用于大鼠颅骨缺损中4周后组织切片免疫荧光染色结果。
具体实施方式
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实例范围之中。
实施例1体外细胞实验
1.1细胞培养：选用提取自3周龄C57BL/6雄性小鼠股骨原代骨髓间充质干细胞(BMSC)，在37℃、5％CO2条件下培养，以30000/孔接种到24孔板中。向含5％血清、1％双抗的α-MEM培养基中添加50μg/mL抗坏血酸、10mMβ-甘油磷酸钠、100nM 地塞米松配制成成骨诱导培养基用于成骨诱导。
1.2细胞模型：成骨诱导培养基作用于BMSC 5天使其发生成骨定向。然后实验组使用添加有150μg/mL GS18的成骨诱导培养基，对照组使用添加有150μg/mL 无效肽(Controlpeptide)的成骨诱导培养基，继续培养14天；其中，GS18采取固相多肽合成工艺合成多肽，使用HPLC对产物进行纯化，合成的多肽纯度为96.59％；无效肽的氨基酸序列为SEQ ID NO:2所示，即CKPLRLSKEEHPLK，无效肽同样采取固相多肽合成工艺合成多肽，使用HPLC对产物进行纯化，合成的多肽纯度为96.25％ (下述实施例中，如无特别声明，无效肽均为此氨基酸序列)。
1.3结果验证：在诱导完成后，使用4％多聚甲醛固定细胞，硝酸银染液(碧云天)避光染色20min，紫外照射显色。使用体式显微镜(奥林巴斯)进行整孔拍摄、光学显微镜(奥林巴斯)进行局部放大拍摄。每孔任取五个视野，使用imageJ(Vol 6.0) 对矿化结节的面积(Area)和总灰度值(IntDen)进行统计分析，**p＜0.01。
Von kossa染色结果显示(图1)：GS18作用于成骨定向BMSC后，相比于无效肽，其矿化结节的数目和成熟程度都明显升高(A)，结节面积(B)和总灰度值(C) 均有明显增加。
即，GS18作用于成骨定向的骨髓间充质干细胞(BMSC)，硝酸银染色实验证实其体外有良好的促矿化作用。
综上，本实施例说明,多肽GS18可促进成骨定向BMSC形成矿化结节，从而促进骨修复。
实施例2制备多肽修饰的GelMA水凝胶支架
2.1GelMA制备：2g明胶于60℃溶于10mL PBS后，加入125μL甲基丙烯酸酐 (MA)搅拌2小时，加入40mL PBS终止反应，将反应液倒入12-14kDa透析袋，去离子水透析，冻干机冻干，所得粉末即为GelMA。
2.2多肽修饰：2g GelMA冻干粉于60℃溶于10mL PBS中，按照0.1mg/mL的浓度添加多肽，将GS18与GelMA溶液充分混匀，然后加入2.5％光引发剂LAP，再次充分混匀。用移液枪吸取20μL上诉混合物注入聚四氟乙烯定制模具上直径为5mm 的孔中，紫外灯照射1min，待材料固化后，将材料从孔板上分离下来，放置于冰上待用。
2.3结果验证：对合成材料从模具上刚分离的形态进行拍照记录。
利用上述方法，同时制备无效肽(Control peptide)修饰的GelMA水凝胶支架。
经过多肽修饰的凝胶支架可从模具上(图2A)完整分离下来，借助模具制备成直径5mm，厚度1mm，大小均匀、厚度一致的圆柱状材料(图2B)。
实施例3多肽修饰GelMA支架植入大鼠颅骨缺损后的骨修复效果
3.1样品制备：按照0.1mg/mL的浓度将GS18及无效肽分别与GelMA水凝胶支架混合，制备0.02cm3大小的骨缺损植入材料。
3.2动物模型：采用12周龄SD雄性大鼠，每只约320±20g，每组3只。使用 2％戊巴比妥(按大鼠体重300g/ml比例注射)进行腹腔注射麻醉，取俯卧位，剃毛刀行头部备皮，碘伏消毒术区，一次性无菌孔巾铺在消毒区域。从鼻骨开始沿头顶部正中线纵行皮肤切口1.5-2.0cm，手术刀柄轻轻分离皮下组织，沿颅骨矢状缝整齐切开骨膜，钝性分离骨膜，充分暴露顶骨、枕骨和部分额骨。颅顶骨中线两侧使用环钻各制备一个直径为5mm的圆形全层骨缺损，植入消毒好的材料，实验组植入含GS18的 GelMA支架，对照组植入含无效肽的GelMA支架。复位皮肤，缝合，再次消毒。
3.3组织切片：4周后处死大鼠，取颅顶骨，4％多聚甲醛固定，EDTA(12％，pH＝7.0)脱钙，脱水，石蜡包埋，切成厚度为6μm的组织切片。
3.4结果验证：对支架刚植入颅骨缺损时及其4周取样时的存留状态进行拍照记录。65℃烘烤石蜡组织切片，二甲苯及梯度乙醇脱蜡水化，HE及Goldner染色观察缺损区新生组织的组织学形态。
图2显示：GelMA凝胶支架可完美贴合5mm的颅顶缺损(图2C)且4周后仍存留(图2D)，材料周围由骨膜形成连接将材料稳固包裹其中。
HE(图3)染色结果显示：植入材料4周后，无效肽(Control peptide)组缺损周围包绕凝胶材料形成一圈纤维连接，缺损区中央仍为大块空白无细胞长入的材料。而GS18可以很好地吸引募集细胞进入支架材料，完成缺损处纤维连接，纤维矿化程度高且可观察到类似于骨组织的成分形成,说明多肽GS18在体内可以促进骨缺损修复效果。
Goldner(图4)染色结果显示：对照组仅缺损周围由骨膜形成软组织连接，中间存留大量空白凝胶，无细胞的募集和长入；实验组GS18修饰的GelMA支架材料募集和吸引了大量细胞进入缺损区，且可看到绿色新生骨的形成，提示GS18多肽有显著的加速骨缺损修复的效果。
实施案例4免疫荧光验证Wnt经典信号通路的激活
4.1抗原修复：将实施例3.3制备得到的石蜡切片于65℃烘烤，二甲苯梯度乙醇脱蜡水化，浸泡入抗原修复液中，于100℃自然降温修复40min，后放置于室温下待温度降至室温后，泡在PBS中。
4.2免疫荧光染色：0.5％PBST组织打孔15min，5％BSA封闭20min，组化笔圈出组织，孵育Non-phospho(Active)β-Catenin抗体(cell signaling#8814)以及 osteocalcin抗体(Santa Cruz sc-365797)，使用抗体生产商推荐比例稀释抗体，每样孵育30μL，于4℃过夜孵育。第二天PBS洗涤切片30min，使用含荧光二抗(使用抗体生厂商的推荐浓度)和DAPI(稀释比例1：50)的抗体稀释液放入湿盒中室温孵育2h，随后PBS漂洗3次，每次15min。组织上滴加含DAPI的抗荧光淬灭剂，放置盖玻片，涂布透明指甲油固定盖玻片两端。
4.3结果验证：免疫荧光染色片于激光共聚焦荧光显微镜(Olympus，FV3000) 下观察并采集图像。
免疫荧光结果显示(图5)：与对照组(GelMA组)相比，GS18修饰的GelMA 支架组有大量的绿色的Activeβ-catenin(绿色标记)的表达，且表达区域与DAPI(蓝色标记)定位细胞核位置一致，提示该支架成功激活β-catenin入核，从而激活经典 Wnt信号通路。同时标记成骨前体细胞定向分化致成熟阶段的分泌蛋白骨钙素(Ocn，红色标记)也大量表达，提示GS18可引发Wnt经典通路所对应的骨修复过程，从而发挥促骨缺损修复和再生的作用。
SEQUENCE LISTING
<110> 四川大学
<120> 一种多肽及其在骨修复中的应用
<130> DJ8938-06
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Pro Gly Gly Asp Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr
1 5 10 15
Val Ser
<210> 2
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Cys Lys Pro Leu Arg Leu Ser Lys Glu Glu His Pro Leu Lys
1 5 10
Claims (10)
1.一种多肽，其特征在于：所述多肽的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的多肽，其特征在于：所述多肽用于骨损伤，和/或用于骨修复。
3.权利要求1或2所述的多肽在激活β-catenin入核中的用途。
4.权利要求1或2所述的多肽在促进骨钙素的表达中的用途。
5.根据权利要求3或4所述的用途，其特征在于：所述多肽的浓度为25～150μg/mL，优选100～150μg/mL。
6.根据权利要求1或2所述的多肽，其特征在于：其与组织工程上可接受的载体联用来治疗骨损伤和/或进行骨修复。
7.一种骨修复组合物，其特征在于：所述骨修复组合物含有治疗有效剂量的如权利要求1所述的多肽和组织工程上可接受的载体。
8.一种多肽支架，其特征在于：包含权利要求1中所述的多肽。
9.根据权利要求8所述的多肽支架，其特征在于：所述支架为甲基丙烯酸酐化明胶支架；所述多肽的浓度为25～150μg/mL。
10.如权利要求1所述的多肽在制备骨损伤和/或骨修复组合物中的应用。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210496588.9A CN114656526B (zh) | 2022-05-09 | 2022-05-09 | 一种多肽及其在骨修复中的应用 |
| US18/308,695 US11912793B2 (en) | 2022-05-09 | 2023-04-28 | Polypeptide and application thereof in bone repair |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210496588.9A CN114656526B (zh) | 2022-05-09 | 2022-05-09 | 一种多肽及其在骨修复中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114656526A true CN114656526A (zh) | 2022-06-24 |
| CN114656526B CN114656526B (zh) | 2023-04-25 |
Family
ID=82036503
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210496588.9A Active CN114656526B (zh) | 2022-05-09 | 2022-05-09 | 一种多肽及其在骨修复中的应用 |
Country Status (2)
| Country | Link |
|---|---|
| US (1) | US11912793B2 (zh) |
| CN (1) | CN114656526B (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116082453A (zh) * | 2023-03-03 | 2023-05-09 | 四川大学 | 一种用于明胶酶酶切响应的多肽及含多肽的骨缺损修复支架 |
| US11912793B2 (en) * | 2022-05-09 | 2024-02-27 | Sichuan University | Polypeptide and application thereof in bone repair |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118252975B (zh) * | 2024-05-31 | 2024-08-20 | 北京鑫康辰医学科技发展有限公司 | 一种具有免疫调控功能的骨修复材料及其制备方法和应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113388005A (zh) * | 2021-06-30 | 2021-09-14 | 北京泽勤生物医药有限公司 | 增强成骨细胞活性的多肽及其在治疗骨科疾病中的应用 |
| US20220040379A1 (en) * | 2019-01-31 | 2022-02-10 | East China University Of Science And Technology | New use of stem cell generator in preparation of bone defect repair materials |
| CN114031672A (zh) * | 2021-11-17 | 2022-02-11 | 广州领晟医疗科技有限公司 | 一种多肽及其在制备软骨修复和/或骨关节炎药物中的应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114656526B (zh) * | 2022-05-09 | 2023-04-25 | 四川大学 | 一种多肽及其在骨修复中的应用 |
-
2022
- 2022-05-09 CN CN202210496588.9A patent/CN114656526B/zh active Active
-
2023
- 2023-04-28 US US18/308,695 patent/US11912793B2/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220040379A1 (en) * | 2019-01-31 | 2022-02-10 | East China University Of Science And Technology | New use of stem cell generator in preparation of bone defect repair materials |
| CN113388005A (zh) * | 2021-06-30 | 2021-09-14 | 北京泽勤生物医药有限公司 | 增强成骨细胞活性的多肽及其在治疗骨科疾病中的应用 |
| CN114031672A (zh) * | 2021-11-17 | 2022-02-11 | 广州领晟医疗科技有限公司 | 一种多肽及其在制备软骨修复和/或骨关节炎药物中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| JAMES J. ASCIOLLA: "An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine" * |
| 韩 娜: "降钙素基因相关肽与骨修复及骨重建" * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11912793B2 (en) * | 2022-05-09 | 2024-02-27 | Sichuan University | Polypeptide and application thereof in bone repair |
| CN116082453A (zh) * | 2023-03-03 | 2023-05-09 | 四川大学 | 一种用于明胶酶酶切响应的多肽及含多肽的骨缺损修复支架 |
| CN116082453B (zh) * | 2023-03-03 | 2023-11-21 | 四川大学 | 一种用于明胶酶酶切响应的多肽及含多肽的骨缺损修复支架 |
Also Published As
| Publication number | Publication date |
|---|---|
| US11912793B2 (en) | 2024-02-27 |
| US20230357321A1 (en) | 2023-11-09 |
| CN114656526B (zh) | 2023-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114656526A (zh) | 一种多肽及其在骨修复中的应用 | |
| AU628050B2 (en) | Biosynthetic osteogenic proteins and osteogenic devices containing them | |
| US10493134B2 (en) | Compositions comprising collagen and PRP for tissue regeneration | |
| AU731768B2 (en) | Composition and method for repairing neurological tissue | |
| CN114853852B (zh) | 多肽及其在促进骨修复中的用途 | |
| US7754683B2 (en) | Bioactive peptide of bone morphogenetic protein-2 | |
| JPH03504736A (ja) | 移植用骨コラーゲンマトリックス | |
| US9889233B2 (en) | Method of producing native components, such as growth factors or extracellular matrix proteins, through cell culturing of tissue samples for tissue repair | |
| CN110478528B (zh) | 一种新型的促组织修复材料的制备方法及其应用 | |
| RU2483756C1 (ru) | СПОСОБ ПОЛУЧЕНИЯ БИОДЕГРАДИРУЕМОГО КОМПОЗИТНОГО МАТРИКСА НА ОСНОВЕ РЕГЕНЕРИРОВАННОГО ФИБРОИНА ШЕЛКА Bombyx mori И ЕГО ПРИМЕНЕНИЕ | |
| US6008197A (en) | Method for inducing bone formation using an extract of human osteosarcoma cell line SAOS-2 | |
| Ghanbari et al. | Bone regeneration: current status and future prospects | |
| CN114751960B (zh) | 多肽及其在骨修复中的应用 | |
| Saitoh et al. | Effect of polylactic acid on osteoinduction of demineralized bone: preliminary study of the usefulness of polylactic acid as a carrier of bone morphogenetic protein | |
| CN114957404B (zh) | 一种多肽及其在促进骨修复中的用途 | |
| KR20180087870A (ko) | 골재생용 세포 치료제 및 이것의 제조 방법 | |
| CN113559324B (zh) | 纳米复配液在软骨细胞自体修复及再生医学美学中的应用 | |
| EP0957944B1 (de) | Knochenmatrix, deren verwendung, therapeutische zubereitung und kit | |
| CN117752857A (zh) | 一种复合材料及其制备方法和应用 | |
| RU2623171C1 (ru) | Способ получения оптимизированного твердого ген-активированного материала, способ получения твердого матрикса носителя, оптимизированный твердый ген-активированный материал для регенерации тканей | |
| Wuran et al. | Application of silk fibroin composite scaffold in bone tissue engineering | |
| Brion et al. | Cell–biomaterials interactions: the role of growth factors | |
| CN115702953A (zh) | 一种骨组织单元-支架材料复合体及其构建方法与应用 | |
| Wang et al. | Effect Of Injectable Drug-Loaded Silk Serici/Oxidated Sodium Alginate/Naringin Combined Hydrogel (SS/OSA-NRG) On Bone Formation | |
| WO1992018137A1 (en) | Method for inducing bone formation using an extract of human osteosarcoma cell line saos-2 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |