CN113713175B - 制备水凝胶支架的方法及由此得到的支架的用途 - Google Patents
制备水凝胶支架的方法及由此得到的支架的用途 Download PDFInfo
- Publication number
- CN113713175B CN113713175B CN202110849059.8A CN202110849059A CN113713175B CN 113713175 B CN113713175 B CN 113713175B CN 202110849059 A CN202110849059 A CN 202110849059A CN 113713175 B CN113713175 B CN 113713175B
- Authority
- CN
- China
- Prior art keywords
- culture
- self
- scaffold
- hydrogel
- macrophages
- 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.)
- Active
Links
- 239000000017 hydrogel Substances 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 35
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 73
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 40
- 229920001184 polypeptide Polymers 0.000 claims abstract description 36
- 238000004113 cell culture Methods 0.000 claims abstract description 30
- 210000002540 macrophage Anatomy 0.000 claims abstract description 24
- 108010025020 Nerve Growth Factor Proteins 0.000 claims abstract description 19
- 102000015336 Nerve Growth Factor Human genes 0.000 claims abstract description 19
- 239000012228 culture supernatant Substances 0.000 claims abstract description 19
- 229940053128 nerve growth factor Drugs 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims abstract description 16
- 238000011069 regeneration method Methods 0.000 claims abstract description 16
- 238000001338 self-assembly Methods 0.000 claims abstract description 16
- 230000008929 regeneration Effects 0.000 claims abstract description 15
- 210000004322 M2 macrophage Anatomy 0.000 claims abstract description 12
- 230000003278 mimic effect Effects 0.000 claims abstract description 10
- 210000005036 nerve Anatomy 0.000 claims abstract description 10
- 239000011259 mixed solution Substances 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 230000001939 inductive effect Effects 0.000 claims abstract description 5
- 230000003110 anti-inflammatory effect Effects 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 26
- 239000001963 growth medium Substances 0.000 claims description 11
- 210000001519 tissue Anatomy 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 7
- 108090000978 Interleukin-4 Proteins 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- 239000000499 gel Substances 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 102000014413 Neuregulin Human genes 0.000 claims description 4
- 108050003475 Neuregulin Proteins 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 claims description 3
- 210000004698 lymphocyte Anatomy 0.000 claims description 3
- 239000002121 nanofiber Substances 0.000 claims description 3
- 210000005259 peripheral blood Anatomy 0.000 claims description 3
- 239000011886 peripheral blood Substances 0.000 claims description 3
- 210000003200 peritoneal cavity Anatomy 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 2
- 239000012737 fresh medium Substances 0.000 claims 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 210000002569 neuron Anatomy 0.000 abstract description 4
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 239000012620 biological material Substances 0.000 abstract 1
- 230000003993 interaction Effects 0.000 abstract 1
- 230000001502 supplementing effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 19
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 14
- 241000700159 Rattus Species 0.000 description 12
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 210000004116 schwann cell Anatomy 0.000 description 6
- 210000000683 abdominal cavity Anatomy 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 3
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 210000003141 lower extremity Anatomy 0.000 description 3
- 210000003497 sciatic nerve Anatomy 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 101000996663 Homo sapiens Neurotrophin-4 Proteins 0.000 description 2
- 102100033857 Neurotrophin-4 Human genes 0.000 description 2
- 208000010886 Peripheral nerve injury Diseases 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003811 finger Anatomy 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 108010043322 lysyl-tryptophyl-alpha-lysine Proteins 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 238000012604 3D cell culture Methods 0.000 description 1
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 1
- QUIGLPSHIFPEOV-CIUDSAMLSA-N Ala-Lys-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O QUIGLPSHIFPEOV-CIUDSAMLSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- 206010012455 Dermatitis exfoliative Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- MADFVRSKEIEZHZ-DCAQKATOSA-N Gln-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N MADFVRSKEIEZHZ-DCAQKATOSA-N 0.000 description 1
- BZULIEARJFRINC-IHRRRGAJSA-N Gln-Phe-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N BZULIEARJFRINC-IHRRRGAJSA-N 0.000 description 1
- LKDIBBOKUAASNP-FXQIFTODSA-N Glu-Ala-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LKDIBBOKUAASNP-FXQIFTODSA-N 0.000 description 1
- ZIYGTCDTJJCDDP-JYJNAYRXSA-N Glu-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZIYGTCDTJJCDDP-JYJNAYRXSA-N 0.000 description 1
- BPCLDCNZBUYGOD-BPUTZDHNSA-N Glu-Trp-Glu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 BPCLDCNZBUYGOD-BPUTZDHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 1
- YUTZYVTZDVZBJJ-IHPCNDPISA-N Lys-Trp-Lys Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 YUTZYVTZDVZBJJ-IHPCNDPISA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102100029268 Neurotrophin-3 Human genes 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- GDBOREPXIRKSEQ-FHWLQOOXSA-N Phe-Gln-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O GDBOREPXIRKSEQ-FHWLQOOXSA-N 0.000 description 1
- OYQBFWWQSVIHBN-FHWLQOOXSA-N Phe-Glu-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O OYQBFWWQSVIHBN-FHWLQOOXSA-N 0.000 description 1
- PEFJUUYFEGBXFA-BZSNNMDCSA-N Phe-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 PEFJUUYFEGBXFA-BZSNNMDCSA-N 0.000 description 1
- 206010039203 Road traffic accident Diseases 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 241000534944 Thia Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000003471 anti-radiation Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000000467 autonomic pathway Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- -1 but not limited to Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000000495 immunoinflammatory effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004936 left thumb Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 108010057952 lysyl-phenylalanyl-lysine Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000000508 neurotrophic effect Effects 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 210000001243 pseudopodia Anatomy 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
-
- 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3878—Nerve tissue, brain, spinal cord, nerves, dura mater
-
- 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
- A61L27/52—Hydrogels or hydrocolloids
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/48—Nerve growth factor [NGF]
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- 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
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
-
- 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
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2304—Interleukin-4 (IL-4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Dermatology (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Botany (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Wood Science & Technology (AREA)
- Medical Informatics (AREA)
- Composite Materials (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
Abstract
本发明涉及一种利用自组装多肽制备水凝胶支架的方法及由此得到的支架的用途，该制备方法包括：1)将自组装肽序列通过共价键键合神经生长因子模拟肽，得到键合功能多肽；2）分离并培养巨噬细胞，并诱导其为“替代性激活”的抗炎M2巨噬细胞，获得培养上清液并进行过滤；3）将得到的经过滤的细胞培养上清液与所述键合功能多肽混合，得到混合液并调整该混合液的浓度，所述键合功能多肽自组装形成水凝胶支架。本发明的制备方法采用M2巨噬细胞条件培养上清液构建再生微环境，结合多肽功能水凝胶支架，实现较好的整体相互作用，制备得到可以补充和调节神经再生的水凝胶支架，该支架可以促进神经细胞再生行为，为组织工程生物材料提供新的选择。
Description
技术领域
本发明涉及组织工程技术领域，更具体而言，涉及一种利用自组装多肽制备水凝胶支架的方法以及由此得到的支架的用途。
背景技术
周围神经损伤的主要原因包括交通事故、机械创伤、自然灾害和手术意外。我国周围神经损伤病例每年新增约100万例，所照成难以恢复的残疾严重影响患者的生活质量和心理健康，为家庭和社会带来了沉重的经济负担。同时，本领域已知人工神经植入物的修复效果与自体神经相比还有差距，通过局部再生微环境调控提高植入物的功能，是能否实现临床应用的关键问题。
组织工程技术的出现，为损伤神经修复提供了技术方式。组织工程包括三个要素：支架、种子细胞和信号因子。其中支架对于不仅对于再生组织起到物理连接和支持作用，而且在调控细胞再生环境发挥了重要作用。水凝胶的三维网络结构具有含水量高、适宜细胞生长的特点，通过交联生长因子模拟肽进一步提高了细胞生长环境，因此，如何成功地构建能够良好地模拟体内微环境的支架材料，对于损伤神经再生而言具有重要意义。
巨噬细胞在体内分布极广，在调节炎症过程中发挥着巨大作用，具有高度可塑性，并在机体的发育及内环境的平衡中起到重要作用。巨噬细胞发生极化为M2表型后，极化后的巨噬细胞亚型可以细致调节及回应各种不同的刺激，分泌信号因子，抗慢性炎症，促进组织修复再生，对疾病组织器官的修复程度起着关键性的作用。关于M2巨噬细胞衍生的细胞外基质构建水凝胶支架的研究还未见报道，具有重要的研究价值。
发明内容
本发明的一个方面是提供一种自组装多肽水凝胶支架制备方法，其中，所述方法包括：
（1）将自组装肽序列通过共价键键合神经生长因子模拟肽，得到键合功能多肽；
（2）分离并培养巨噬细胞，并诱导其为“替代性激活”的抗炎M2巨噬细胞，得到细胞培养上清液并进行过滤，得到经过滤的细胞培养上清液；
（3）将所述经过滤的细胞培养上清液与所述键合功能多肽混合，得到混合液并调整所述混合液的浓度，所述键合功能多肽自组装形成水凝胶支架。
优选地，神经生长因子模拟肽具有促进神经血管细胞再生的功能。
优选地，自组装肽序列与神经生长因子模拟肽通过酰胺键或二硫键共价连接。
优选地，自组装肽序列与神经生长因子模拟肽摩尔比为（1~3）：1。
优选地，巨噬细胞来自大鼠/小鼠腹腔或骨髓、或人外周血单个核淋巴细胞中的一种。
优选地，步骤（2）所述培养在30℃~40℃、3%~10% CO2的条件下进行；进一步优选地，所述培养的过程包括：将所述巨噬细胞在培养基中生长至60%~80%的汇合度后，弃去培养液并利用PBS缓冲液进行洗涤，随后向洗涤后的培养物中加入新鲜培养基并继续培养36h以上、例如36~72h、优选40~50h；
优选地，采用孔径为0.2~0.45μm的过滤材料对所述细胞培养上清液进行过滤。
优选地，M2巨噬细胞培养上清液的制备方法，其特征在于，所述细胞培养诱导物采用10ng/ml的 IL-4进行步骤（2）所述的诱导。
优选地，多肽水凝胶支架具有纳米纤维结构。
优选地，多肽水凝胶支架具有水凝胶的形态，调节后凝胶的pH为6.9~7.2，浓度为0.5wt%-4wt%。
本发明的又一方面是提供上述的制备的水凝胶支架在体外培养神经雪旺细胞中的用途，其特征在于，所述支架促进神经雪旺细胞的生长和活性。
采用本发明的任一项所述的方法制备的多水凝胶支架在组织工程再生中的用途，其特征在于，所述支架促进损伤神经的再生。
本发明的有益效果是：
1. 通过化学修饰和自组装方法制备组份更加明确，功能性更强，安全性和效率更高的多肽水凝胶支架，具有的纳米结构更适宜损伤神经的修复，增强特异性神经细胞再生速度，改善临床治疗效果。
2.合成含有不同神经生长因子模拟肽的序列，通过自组装技术制备水凝胶，进一步合成含有M2巨噬细胞条件培养条件的水凝胶支架，促进再生细胞协同治疗方向发展。
3.还可以选择不同多肽载体，不同神经生长因子模拟肽，针对不同神经细胞设计、制备个多肽水凝胶支架。
4.适宜的再生免疫环境仿生支架具有良好的神经细胞黏附和生长性能，适宜的组织相容性，纳米纤维接近于细胞外基质结构，采用本发明的方法制备的支架具有更好的再生微环境，为开发产品奠定基础。
附图说明
图1为示出自组装肽序列通过共价键键合神经生长因子模拟肽的结构模式图。
图2为示出诱导M2巨噬细胞显微镜下形态图。
图3为示出M2巨噬细胞成熟度的流式细胞检测图。其中，示出了通过流式细胞术检测到CD206的表达百分比。
图4为示出多肽水凝胶支架的透射电镜图。其中，示出了纳米纤维的结构。
图5为示出多肽水凝胶支架的倾斜实验图。
图6为原代培养神经雪旺细胞显微镜下形态图。
图7为示出经对照组培养的神经雪旺细胞和经多肽水凝胶支架培养的神经雪旺细胞的生长速度。其中，*代表P<0.05。
具体实施方式
除非另有定义，否则本文使用的科技术语具有与本发明所属领域中的普通技术人员通常所理解的相同的含义。参见例如Singleton等，Dictionary of Microbiology andMolecular Biology 2nd ed.，J. Wiley & Sons （New York，NY 1994）；Sambrook等，Molecular Cloning，A Laboratory Manual，Cold Springs Harbor Press（Cold SpringsHarbor，NY 1989）。
本领域技术人员将认识到可用于实施本发明的、与本文描述的方法和材料类似或等同的许多方法和材料。实际上，本发明并不限于本文所描述的方法和材料，而是可基于本发明的精神，进行各种常规调整或修改，并且调整或修改后的方案仍落入了本发明的保护范围之内。
除非上下文明确地指出，否则本文所使用的术语“一个”和“一种”等涵盖了复数的对象。
在本文中，通过术语“约”修饰的对象涵盖了由于测量误差等引起的误差范围内的近似值。
在本文中，除非另有定义，所使用的术语“肿瘤微环境”是指肿瘤细胞的发生及生活的内环境，具有低氧、低PH以及高压的特点，这些特点使得肿瘤微环境中存在大量的生长因子、细胞趋化因子和各种蛋白水解酶所产生的免疫炎性反应，从而有利于肿瘤的增殖、侵袭、粘附、血管生成以及抗放射化疗，促使恶性肿瘤产生。
在一个实施方式中，本发明涉及一种自组装多肽水凝胶支架制备方法，其中，所述方法包括：
（1）将自组装肽序列通过共价键键合神经生长因子模拟肽，得到键合功能多肽；
（2）分离并培养巨噬细胞，并诱导其为“替代性激活”的抗炎M2巨噬细胞，得到细胞培养上清液并进行过滤，得到经过滤的细胞培养上清液；
（3）将所述经过滤的细胞培养上清液与所述键合功能多肽混合，得到混合液并调整所述混合液的浓度，所述键合功能多肽自组装形成纳水凝胶支架。
在本文中，所述自组装肽序列由谷氨酸，赖氨酸，丙氨酸，色氨酸，精氨酸等在水溶液中自组装形成长度为10～20个氨基酸的小分子多肽。作为优选的示例，所述小分子多肽水凝胶例如包括但不限于，RADARADARADARADA(SEQ ID NO.1)、KWKAKAKAKWK(SEQ IDNO.2)、EWEAEAEAE(SEQ ID NO.3)、FEFEFKFKK(SEQ ID NO.4)以及QQKFQFQFEQQ(SEQ IDNO.5)。以上氨基酸购于西格玛奥德里奇(上海)贸易有限公司。
在一个优选的实施方式中，所述神经生长因子模拟肽可为本领域已知的任何一种，例如，来自选自如下的一种或多种，神经生长因子（NGF）、脑源性神经营养因子（BDNF）、血管内皮生长因子（VEGF）、神经营养因子-3（NT-3）和神经营养因子-4（NT-4）。
在一个优选的实施方式中，所述自组装肽序列与神经生长因子模拟肽通过化学键共价连接，摩尔比为（1～3）：1，例如，来自选自如下的一种，酰胺键或二硫键。如果选择酰胺键通过固相合成；如果选择二硫键或多肽载体通过表位肽修饰半胱氨酸，固相合成方法，然后将两条肽对接。
本文中提到的巨噬细胞可为来自大鼠/小鼠腹腔或骨髓、或人外周血单个核淋巴细胞中的一种，或者可为采用本领域已知的常规手段分离得到的巨噬细胞。
在本发明的一个优选的实施方式中，所述细胞的培养可以采用本领域已知的任何的合适的培养基（例如，可参见如下中的记载：http://www.cellbank.org.cn/peiyang.asp；https://www.atcc.org/）进行，例如但不限于含有胎牛血清的RPMI-1640培养基、含有胎牛血清的DMEM培养基、含有胎牛血清的F-12培养基、含有胎牛血清的DMEM/F-12培养基。在进一步优选的实施方式中，所述培养在30℃~40℃、3%~10% CO2的条件下进行。在更进一步优选的实施方式中，所述培养的过程包括：将所述肿瘤细胞在所述培养基中生长至60%~80%的汇合度后，弃去培养液并利用PBS缓冲液进行洗涤，随后向洗涤后的培养物中加入新鲜培养基并继续培养36h以上、例如36~72h、优选40~50h。
在一个优选的实施方式中，采用孔径为0.2~0.45μm的过滤材料对所述细胞培养上清液进行过滤。过滤得到的细胞培养上清液中主要包含：细胞分泌的蛋白质（包括作为影响免疫细胞功能的主要物质的多种细胞因子）；非编码RNA（包括小RNA和长链RNA等）；DNA等。
在一个优选的实施方式中，所述M2巨噬细胞培养上清液的制备方法，其特征在于，所述细胞培养诱导物选自IL-4，优选地，所述细胞培养刺激物为0.1-100 ng/mL，例如10ng/mL的IL-4。加入培养基培养，在本领域已知的任何的合适的培养基（例如，可参见如下中的记载：http://www.cellbank.org.cn/peiyang.asp；https://www.atcc.org/）中进行所述培养，所述培养基例如但不限于含有胎牛血清的RPMI-1640培养基、含有胎牛血清的DMEM培养基、含有胎牛血清的F-12培养基、含有胎牛血清的DMEM/F-12培养基。在进一步优选的实施方式中，所述培养在30℃~40℃、3%~10% CO2的条件下进行。在更进一步优选的实施方式中，所述培养进行36h以上、例如36~72h、优选40~50h。
在一个优选的实施方式中，所述的多肽水凝胶支架，其特征在于，所述支架具有纳米纤维结构。
在一个优选的实施方式中，所述的多肽水凝胶支架，其特征在于，所述支架具有水凝胶的形态，调节后凝胶的pH为6.9~7.2，浓度为0.5 wt %-4wt%例如1wt%。
在一个优选的实施方式中，每mL所述含有水凝胶的细胞悬液中，具有1×104~1×107个、优选5×104~1×106个、例如1×105个所述神经雪旺细胞。
在一个优选的实施方式中，所述预定形状的支持物可为三维细胞培养支架、细胞培养皿、细胞培养瓶、细胞培养微孔板、双层细胞培养板或本领域已知的任何适用于三位细胞培养的系统，例如6孔细胞培养微孔板、12孔细胞培养微孔板、24孔细胞培养微孔板、96孔细胞培养微孔板、24孔双层细胞培养板或本领域可商购的任何细胞培养微孔板，例如由Thermofisher、FlexCell等提供的商业化的三维细胞培养系统。
在一个优选的实施方式中，所述3D细胞培养在30℃~40℃、3%~10% CO2的条件下进行2-72h、例如3-72h、6-72h。
在一个实施方式中，本发明涉及上述的多肽水凝胶支架在开发损伤神经修复中的用途。例如，通过将经常规的物理、化学或生物方法处理后的多肽水凝胶支架注射、涂层等方式到神经修复导管内，促进损伤的神经再生。
以下通过实施例对本发明的方案进行进一步说明，但本发明的保护范围并不仅限于此。
实施例
如下的实施例仅用于说明的目的，而并非旨在限定本申请的保护范围。除非另有说明，否则如下的实施例中使用的所有试剂、材料和设备均为可商购的，或者可根据本领域公知的现有技术进行配制或获得。除非另有说明，否则如下的实施例中涉及的具体实验手段均为本领域的现有技术（例如，《分子克隆实验指南》（第4版），J. 萨姆布鲁克等编著，贺福初主译，科学出版社，2017年；《医学免疫学》（第7版），曹雪涛主编，人民卫生出版社，2018年）中记载的常规手段。
实施例1 自组装肽序列键合神经生长因子模拟肽的制备
将自组装肽序列与神经生长因子模拟肽通过化学键共价连接，摩尔比为1：1，共价连接选自酰胺键，如图1所示，通过固相合成，将两条肽对接（自组装肽序列RADARADARADARADA和脑源性神经营养因子模拟肽GGGIDKRHWNS）。
实施例2 大鼠腹腔巨噬细胞培养
大鼠腹腔巨噻细胞的提取分离：将2～3月龄（体重约300ｇ）的SD大鼠用脱颈法处死，置于75vol%乙醇中浸泡10 min，然后把大鼠倒置提起，用无菌的注射器往其腹腔内注射10 mL DMEM高糖基础培养基。用手指按摩大鼠腹部2 min后，将大鼠仰卧静置7 min，然后无菌打开大鼠腹腔，用另一无菌注射器抽出腹腔液体大约8 mL，400 g离心10 min，弃去上清液，用6 mL高糖DMEM完全培养基重悬细胞并接种到T25培养瓶中，在37℃、5% CO2细胞培养箱中孵育，4h后换液。贴壁细胞即是提取的大鼠腹腔巨噬细胞。
实施例3 M2巨噬细胞培养上清液的制备
分离所得的巨噬细胞并在37℃、5% CO2下用高糖DMEM完全培养基培养3天后，弃去原培养基，更换为10ng/ml IL-4、10％FBS、1％双抗的RMPI 1640培养基，继续培养24h后得到M2型巨噬细胞。培养过程中观察细胞生长状态良好。如图2所示，经共聚焦显微镜拍照后观察细胞伪足明显，形态正常。收集培养获得的M2型巨噬细胞，以CD206抗体作为标记，经流式细胞术检测培养所得的M2型巨噬细胞的纯度。如图3所示，采用含有10 ng/mL的IL-4的高糖DMEM完全培养基刺激3天后，CD206抗体双阳性表达率可到70％以上，M2型巨噬细胞纯度理想。将M2型巨噬细胞培养基换成无血清的高糖培养基培养，24h后收集上清液并用0.22μm过滤器滤去细胞和碎片备用。
实施例4 自组装水凝胶支架的制备和结构表征
将实施例1所得到的多肽序列溶解在实施例3所得的M2型巨噬细胞上清溶液中，调整浓度，通过多肽自发性自组装形成水凝胶，当组装体的质量浓度在10mg/mL及以上时，如图4-5所示，表现为水凝胶，呈交联的三维网状结构。通过透射电子显微镜，结果表明多肽链段长度和组份对聚合物纳米组装形貌和溶液-凝胶转变温度有较大影响。
实施例5 大鼠原代雪旺细胞分离培养方法和3D细胞培养
取红皮鼠于超净台，用酒精喷，将小鼠尾全身按拭干净，剪去头部。用右手调节小鼠后肢，爪心朝上，左手拇指和食指分别按住小鼠的后肢和尾部。剪去小鼠后肢及尾部上方的皮肤。用弯镊子在后肢凹陷区夹开个口子，延脊柱剪两下，将肉与脊柱分开，剔去上方肉，暴露坐骨神经等要取的组织。取SD红皮细胞1天的坐骨神经置于高糖DMEM完全培养基的培养皿中。轻轻吸掉上清，将坐骨神经取出放入EP管中（5mL）。加胶原酶1mL（20只）消化30min，用眼科剪快速剪碎组织，轻轻吹打混匀放入培养箱（37℃、5% CO2）。30min后加等量1mL0.25%胰酶（Try）轻轻吹打混匀，放培养箱5min。5min后，加至少三倍高糖DMEM完全培养基，终止消化（3-4倍），吹匀，1200rpm离心5min，弃上清。加入新鲜高糖DMEM完全培养基，吹匀，过滤，接种细胞到培养皿中。第二日，在接种16h后换液，用含有阿糖胞苷（1:1000）的高糖DMEM完全培养基培养。隔四日换液，用高DMEM完全培养基培养，直到长满为止。将收集的雪旺细胞与上述的水凝胶支架分别按照1:3的体积比混合均匀，调整其中的细胞浓度为1×105个/mL，于37℃、5% CO2下孵育30分钟，得到包含凝胶的各细胞悬液。
实施例6雪旺细胞分的活性检测
CFSE标记后，将雪旺细胞于37℃、5% CO2下在高糖DMEM完全培养基中培养3天后，流式细胞仪检测雪旺细胞的增殖情况。结果在图6中示出。观察细胞呈现为球状，更多的细胞显示出突起，因而能够更好的模拟再生微环境。通流式细胞仪检测，与对照组相比较，水凝胶支架组雪旺细胞的生长速度显著提高。
序列表
<110> 南通大学
<120> 制备水凝胶支架的方法及由此得到的支架的用途
<130> 20210726
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala
1 5 10 15
<210> 2
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Lys Trp Lys Ala Lys Ala Lys Ala Lys Trp Lys
1 5 10
<210> 3
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Glu Trp Glu Ala Glu Ala Glu Ala Glu
1 5
<210> 4
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Phe Glu Phe Glu Phe Lys Phe Lys Lys
1 5
<210> 5
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Gln Lys Phe Gln Phe Gln Phe Glu Gln Gln
1 5 10
Claims (14)
1.一种利用自组装多肽制备水凝胶支架的方法，其中，所述方法包括：
（1）将自组装肽序列通过共价键键合神经生长因子模拟肽，得到键合功能多肽，其中，所述自组装肽序列为RADARADARADARADA，所述神经生长因子模拟肽为GGGIDKRHWNS；
（2）分离并培养巨噬细胞，并诱导其为“替代性激活”的抗炎M2巨噬细胞，得到细胞培养上清液并进行过滤，得到经过滤的细胞培养上清液；
（3）将所述经过滤的细胞培养上清液与所述键合功能多肽混合，得到混合液并调整所述混合液的浓度，所述键合功能多肽自组装形成水凝胶支架。
2.如权利要求1所述的方法，其特征在于，所述自组装肽序列与神经生长因子模拟肽通过酰胺键共价连接。
3.如权利要求1或2所述的方法，其特征在于，所述自组装肽序列与神经生长因子模拟肽的摩尔比为（1~3）：1。
4.如权利要求1或2所述的方法，其特征在于，所述巨噬细胞来自大鼠/小鼠腹腔或骨髓、或人外周血单个核淋巴细胞中的一种。
5.如权利要求1或2所述的方法，其特征在于，步骤（2）所述的培养在30℃~40℃、3%~10%CO2的条件下进行。
6.如权利要求5所述的方法，其特征在于，所述培养的过程包括：将所述巨噬细胞在培养基中生长至60%~80%的汇合度后，弃去培养液并利用PBS缓冲液进行洗涤，随后向洗涤后的培养物中加入新鲜培养基并继续培养36h以上。
7.如权利要求6所述的方法，其特征在于，向洗涤后的培养物中加入新鲜培养基并继续培养36~72h。
8.如权利要求7所述的方法，其特征在于，向洗涤后的培养物中加入新鲜培养基并继续培养40~50h。
9.如权利要求1或2所述的方法，其特征在于，采用孔径为0.2~0.45μm的过滤材料对所述细胞培养上清液进行过滤。
10.如权利要求1或2所述的方法，其特征在于，采用10ng/ml的IL-4进行步骤（2）所述的诱导。
11.如权利要求1或2所述的方法，其特征在于，所述支架具有纳米纤维结构。
12.如权利要求1或2所述的方法，其特征在于，所述支架具有水凝胶的形态，调节后凝胶的pH为6.9~7.2，浓度为0.5wt%-4wt%。
13.权利要求1-12中任一项所述的方法制备的水凝胶支架在体外培养神经雪旺细胞中的用途，其特征在于，所述支架促进神经雪旺细胞的生长和活性。
14.权利要求1-12中任一项所述的方法制备的多水凝胶支架在制备用于组织工程再生的试剂中的用途，其特征在于，所述支架促进损伤神经的再生。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210998757.9A CN115160444A (zh) | 2021-07-27 | 2021-07-27 | 制备水凝胶支架的方法及由此得到的支架的用途 |
| CN202110849059.8A CN113713175B (zh) | 2021-07-27 | 2021-07-27 | 制备水凝胶支架的方法及由此得到的支架的用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110849059.8A CN113713175B (zh) | 2021-07-27 | 2021-07-27 | 制备水凝胶支架的方法及由此得到的支架的用途 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210998757.9A Division CN115160444A (zh) | 2021-07-27 | 2021-07-27 | 制备水凝胶支架的方法及由此得到的支架的用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113713175A CN113713175A (zh) | 2021-11-30 |
| CN113713175B true CN113713175B (zh) | 2022-09-16 |
Family
ID=78674033
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210998757.9A Pending CN115160444A (zh) | 2021-07-27 | 2021-07-27 | 制备水凝胶支架的方法及由此得到的支架的用途 |
| CN202110849059.8A Active CN113713175B (zh) | 2021-07-27 | 2021-07-27 | 制备水凝胶支架的方法及由此得到的支架的用途 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210998757.9A Pending CN115160444A (zh) | 2021-07-27 | 2021-07-27 | 制备水凝胶支架的方法及由此得到的支架的用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN115160444A (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114230640A (zh) * | 2021-12-21 | 2022-03-25 | 西安交通大学 | 一种具有自组装能力的氨基酸缩合物 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9526762B1 (en) * | 2013-08-09 | 2016-12-27 | William Marsh Rice University | Multidomain peptides for promoting angiogenesis |
| CN104356402B (zh) * | 2014-10-10 | 2016-08-31 | 孙念峰 | 功能性自组装纳米多肽水凝胶 |
| WO2020023251A1 (en) * | 2018-07-16 | 2020-01-30 | The Board Trustees Of University Of Illinois | Engineered exosomes for medical applications |
| CN109771694A (zh) * | 2019-03-20 | 2019-05-21 | 江苏瑞思坦生物科技有限公司 | 自组装多肽纳米纤维水凝胶支架材料的制备方法及应用 |
| CN113046316B (zh) * | 2021-04-07 | 2022-11-22 | 中南大学湘雅医院 | M2型骨髓巨噬细胞外泌体及其应用、脊髓损伤治疗制剂 |
-
2021
- 2021-07-27 CN CN202210998757.9A patent/CN115160444A/zh active Pending
- 2021-07-27 CN CN202110849059.8A patent/CN113713175B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN115160444A (zh) | 2022-10-11 |
| CN113713175A (zh) | 2021-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1617854B1 (en) | Biologically active native biomatrix composition | |
| Lander et al. | Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan. | |
| US7906110B2 (en) | Rapid preparation of stem cell matrices for use in tissue and organ treatment and repair | |
| CN108546674B (zh) | 预刺激干细胞及其制备方法和应用 | |
| RU2007114846A (ru) | Стволовые клетки и решетки, полученные из жировой ткани | |
| EP2530144A2 (en) | Composition comprising cell and biocompatible polymer | |
| WO2022110676A1 (zh) | 皮肤前体细胞诱导的施万细胞来源的囊泡在构建组织工程神经移植物中的应用 | |
| CN113713175B (zh) | 制备水凝胶支架的方法及由此得到的支架的用途 | |
| CN111534477B (zh) | 小鼠肺组织原代上皮干细胞球培养方法 | |
| CN107254431B (zh) | 一种新型组织工程皮肤制备方法 | |
| Sakai et al. | New artificial nerve conduits made with photocrosslinked hyaluronic acid for peripheral nerve regeneration | |
| CN110804607A (zh) | 一种高浓度人源间充质干细胞裂解液的制备方法 | |
| CN102220338A (zh) | 人脐带间充质干细胞表达人2.5Sβ-神经生长因子及分离纯化的方法 | |
| CN112538513B (zh) | 细胞外基质mb生物蛋白及其制备试剂盒与方法 | |
| CN103127494A (zh) | 一种神经再生生物胶及其制备方法和用途 | |
| Pan et al. | Insights into the therapeutic potential of heparinized collagen scaffolds loading human umbilical cord mesenchymal stem cells and nerve growth factor for the repair of recurrent laryngeal nerve injury | |
| CN112755250A (zh) | 一种组织工程化周围神经组织及其制备方法 | |
| Liu et al. | Bioreactor microcarrier cell culture system (Bio-MCCS) for large-scale production of autologous melanocytes | |
| Bauer et al. | Glial‐conditioned medium and attachment to ConA are essential for long‐term culture of cortical neurons | |
| CN114181902B (zh) | 一种简便、快捷的星形胶质细胞分化方法 | |
| CN116510077A (zh) | 重组人源化iii型胶原蛋白-羟基磷灰石复合骨修复材料及其应用 | |
| CN116510065A (zh) | 重组人源化iii型胶原蛋白止血海绵及其应用 | |
| CN116514956A (zh) | 重组人源化iii型胶原蛋白及其应用 | |
| CN112704728A (zh) | 一种egf高表达的干细胞温敏凝胶的制备方法及其应用 | |
| CN114832149A (zh) | 一种基于干细胞微包埋复合水凝胶的创面敷料及制备方法 |
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 | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20211130 Assignee: NANTONG EGENS BIOTECHNOLOGY Co.,Ltd. Assignor: NANTONG University Contract record no.: X2023980046726 Denomination of invention: Method for preparing hydrogel scaffold and use of the scaffold obtained therefrom Granted publication date: 20220916 License type: Common License Record date: 20231114 |
|
| EE01 | Entry into force of recordation of patent licensing contract |