CN107286677A - 一种诱导上皮组织分化的蛋白肽交联膜及其制备方法 - Google Patents
一种诱导上皮组织分化的蛋白肽交联膜及其制备方法 Download PDFInfo
- Publication number
- CN107286677A CN107286677A CN201710661771.9A CN201710661771A CN107286677A CN 107286677 A CN107286677 A CN 107286677A CN 201710661771 A CN201710661771 A CN 201710661771A CN 107286677 A CN107286677 A CN 107286677A
- Authority
- CN
- China
- Prior art keywords
- collagen
- skin
- cross linking
- protein peptides
- linking membrane
- 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 93
- 238000004132 cross linking Methods 0.000 title claims abstract description 74
- 239000012528 membrane Substances 0.000 title claims abstract description 69
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 56
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 56
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 52
- 210000000981 epithelium Anatomy 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims description 20
- 230000004069 differentiation Effects 0.000 title claims description 15
- 102000008186 Collagen Human genes 0.000 claims abstract description 120
- 108010035532 Collagen Proteins 0.000 claims abstract description 120
- 229920001436 collagen Polymers 0.000 claims abstract description 120
- 102000004190 Enzymes Human genes 0.000 claims abstract description 35
- 108090000790 Enzymes Proteins 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 29
- 230000008569 process Effects 0.000 claims abstract description 29
- 239000002253 acid Substances 0.000 claims abstract description 23
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 19
- 241000276701 Oreochromis mossambicus Species 0.000 claims abstract description 14
- 238000001035 drying Methods 0.000 claims abstract description 13
- 239000002994 raw material Substances 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 230000001954 sterilising effect Effects 0.000 claims abstract description 11
- 238000004140 cleaning Methods 0.000 claims abstract description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 8
- 230000001939 inductive effect Effects 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 32
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 239000011259 mixed solution Substances 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 108091005804 Peptidases Proteins 0.000 claims description 7
- 239000004365 Protease Substances 0.000 claims description 7
- 239000003513 alkali Substances 0.000 claims description 7
- 230000006698 induction Effects 0.000 claims description 7
- 238000000108 ultra-filtration Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000005238 degreasing Methods 0.000 claims description 6
- 238000010612 desalination reaction Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000005554 pickling Methods 0.000 claims description 6
- 235000019419 proteases Nutrition 0.000 claims description 6
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 5
- 150000001718 carbodiimides Chemical class 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000003292 glue Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000007605 air drying Methods 0.000 claims description 2
- 238000004042 decolorization Methods 0.000 claims description 2
- 238000011033 desalting Methods 0.000 claims description 2
- 239000002120 nanofilm Substances 0.000 claims description 2
- 238000002203 pretreatment Methods 0.000 claims description 2
- 230000006920 protein precipitation Effects 0.000 claims description 2
- 238000005185 salting out Methods 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims 1
- 210000002919 epithelial cell Anatomy 0.000 abstract description 13
- 206010072170 Skin wound Diseases 0.000 abstract description 12
- 230000035876 healing Effects 0.000 abstract description 5
- 230000001413 cellular effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 9
- 102000003970 Vinculin Human genes 0.000 description 9
- 108090000384 Vinculin Proteins 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 9
- 102000012422 Collagen Type I Human genes 0.000 description 7
- 108010022452 Collagen Type I Proteins 0.000 description 7
- 229940096422 collagen type i Drugs 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 230000008556 epithelial cell proliferation Effects 0.000 description 6
- 230000029663 wound healing Effects 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 240000003186 Stachytarpheta cayennensis Species 0.000 description 2
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 230000016446 peptide cross-linking Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 241001269178 Garrha Species 0.000 description 1
- 229920002581 Glucomannan Polymers 0.000 description 1
- MVORZMQFXBLMHM-QWRGUYRKSA-N Gly-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 MVORZMQFXBLMHM-QWRGUYRKSA-N 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 229920002752 Konjac Polymers 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical group C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002389 environmental scanning electron microscopy Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940046240 glucomannan Drugs 0.000 description 1
- 108010038983 glycyl-histidyl-lysine Proteins 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000007602 hot air drying Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000011806 microball Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
- C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
- C08L89/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin, e.g. gelatin
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/425—Porous materials, e.g. foams or sponges
-
- 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
- C08J3/246—Intercrosslinking of at least two polymers
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2201/00—Foams characterised by the foaming process
- C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
- C08J2201/048—Elimination of a frozen liquid phase
- C08J2201/0484—Elimination of a frozen liquid phase the liquid phase being aqueous
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2207/00—Foams characterised by their intended use
- C08J2207/12—Sanitary use, e.g. diapers, napkins or bandages
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
- C08J2389/04—Products derived from waste materials, e.g. horn, hoof or hair
- C08J2389/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2489/00—Characterised by the use of proteins; Derivatives thereof
- C08J2489/04—Products derived from waste materials, e.g. horn, hoof or hair
- C08J2489/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2201/00—Properties
- C08L2201/08—Stabilised against heat, light or radiation or oxydation
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2203/00—Applications
- C08L2203/02—Applications for biomedical use
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2203/00—Applications
- C08L2203/14—Applications used for foams
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2203/00—Applications
- C08L2203/16—Applications used for films
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2205/00—Polymer mixtures characterised by other features
- C08L2205/02—Polymer mixtures characterised by other features containing two or more polymers of the same C08L -group
- C08L2205/025—Polymer mixtures characterised by other features containing two or more polymers of the same C08L -group containing two or more polymers of the same hierarchy C08L, and differing only in parameters such as density, comonomer content, molecular weight, structure
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2312/00—Crosslinking
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Dermatology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种诱导上皮组织分化的蛋白肽交联膜,蛋白肽交联膜以罗非鱼皮为原料,采用酸法制备胶原,酶法制备胶原肽,将胶原、胶原肽、交联剂按照体积质量比1%:0.1%:1%~1%:0.5%:2%比例在1体积纯水中混合,室温反应1.5~3h,经过干燥、清洗、二次干燥、灭菌步骤即得到蛋白肽交联膜产品。该蛋白肽交联膜表面多孔,且分布均匀,耐水耐热,有利于上皮细胞快速粘附和生长,可应用于皮肤创口愈合和细胞组织工程培养。
Description
技术领域
本发明涉及医药及生物制品领域,具体涉及一种诱导上皮组织分化的胶原肽交联膜。
背景技术
胶原是由三条左手螺旋的肽链组成右手三股螺旋结构的大分子蛋白质,因其具备完整的三螺旋结构,故其保有生物大分子的活性。胶原是动物体含量与分布最多的蛋白,基本上占据总蛋白质的1/3~1/4,是肌腱、软骨、韧带和角膜的主要成分。胶原最主要的传统来源是陆生动物,比如牛皮和猪皮,然而这些来源会引起宗教(比如犹太教和***教禁止消费与猪来源相关的任何产品,而印度教不会消费与牛来源相关的任何产品)和安全问题(由牛引起的疯牛病和猪引起的***),因此开发能够替代这些来源的产品,比如鱼类加工废弃物,包括鱼皮,鱼骨,鱼鳞,已受到研究者的广泛关注,因其来源广,胶原含量丰富,故这些来源对于陆生动物胶原来说,都是很好的替代品。
胶原因具有低免疫性、低毒性、促进细胞生长与粘附和能维持体内平衡等性质而被广泛应用于生物材料领域。作为动物的成纤细胞合成的一种生物高分子,是***中重要的结构蛋白,是动物体内含量最丰富、分布最广的一种蛋白质,且氨基酸含量丰富并且组成合理,营养价值高,起着维持骨结构的完整和生物力学的作用。水解胶原能够为人体胶原蛋白的合成提供优质的氨基酸原料,尤其是脯氨酸和羟脯氨酸,促进胶原的合成,及时补充人体皮肤中流失的胶原,有助于维持皮肤中胶原蛋白的特殊网状结构,从而达到改善皮肤水分状况,延缓皮肤衰老的作用。在生物和医学上,因胶原是细胞外基质的主要成分,故胶原膜能为表皮细胞的迁移、增殖铺垫支架,并提供了良好的营养基础,有利于上皮细胞的增生修复、因而能促进创面的愈合。
国内外关于胶原用于细胞培养与伤口愈合有相关报道,如现有专利技术(专利号为201310099817.4的中国专利)公开了一种动物细胞培养用胶原蛋白覆层微载体及其制备方法:将魔芋葡甘聚糖基质微球中加入活化试剂与催化剂进行活化后,再加入胶原蛋白,进行偶联反应;再加入交联试剂与PBS缓冲液,搅拌使其反应,反应完毕清洗后即得微载体产品。该专利中加入多种活化剂、催化剂、交联剂等化学试剂,且胶原的来源是以牛,猪等陆生动物,在一定程度上限制微载体的应用。
现有专利技术(专利号US4994388)公开了一种胶原蛋白覆层聚苯乙烯微球,在聚苯乙烯微球表面覆层层粘连蛋白或纤连蛋白,再置于含胶原蛋白的醋酸溶液中升温蒸发,制备了一种胶原蛋白包被微载体的制备方法。该专利所使用的层粘连蛋白和纤连蛋白价格贵,且操作条件较为严格,不利于工业化生产。
本发明的交联膜以罗非鱼皮为原料,采用酸法制备胶原膜,酶法制备胶原肽,将交联剂、胶原肽、胶原膜混合,室温反应1.5~3h,反应完毕后经干燥、灭菌即成蛋白肽交联膜产品。本发明所需的原材料来源丰富,成本低廉,比陆生动物来源的胶原产品更具有潜力与优势,且该蛋白肽交联膜表面多孔,分布均匀,耐水耐热,可实现上皮细胞快速粘附和生长。
发明内容
本发明的目的是提供一种诱导上皮组织分化的蛋白肽交联膜。本发明的交联膜以罗非鱼皮为原料,采用酸法制备胶原膜、酶法制备胶原肽,将胶原、胶原肽、交联剂混合,室温反应1.5~3h,经过干燥、灭菌步骤即成蛋白肽交联膜产品。本发明所需的原材料来源丰富,成本低廉,比陆生动物来源的胶原产品更具有潜力与优势,且该蛋白肽交联膜表面多孔,且分布均匀,耐水耐热,有利于上皮细胞快速粘附和生长。
本发明的具体技术方案如下:
一种诱导上皮组织分化的蛋白肽交联膜及其制备方法,其特征在于:所述交联膜以罗非鱼皮为原料,经酸法制备的胶原、经酶法制备的胶原肽、再将得到的胶原、胶原肽与交联剂交联,交联后所得物质再经过干燥、清洗、二次干燥、灭菌步骤后,得到诱导上皮组织分化的蛋白肽交联膜,制备步骤包括:
A.酸法制备胶原步骤,以罗非鱼皮作为原料,经过酸法工艺将罗非鱼皮制备成胶原;
B.酶法制备胶原肽步骤,以罗非鱼皮为原料,通过在酸法制备得到的罗非鱼皮胶原中加入商业酶水解获得的胶原酶解液,经超滤分离,干燥得到酶法制备的胶原肽;
C.交联步骤,将经过酸法制备的胶原、酶法制备的胶原肽与交联剂混合;
D.交联后所得物质再经过干燥、清洗、二次干燥、灭菌步骤后,得到诱导上皮组织分化的蛋白肽交联膜;
所述酶法制备胶原肽步骤中的超滤是将胶原酶解液通过分子膜进行分离获得1ku~5ku组分;
所述酶法制备胶原肽步骤中的商业酶为中性蛋白酶、胶原酶和碱性蛋白酶;
所述酶法制备胶原肽步骤中的干燥采用真空冷冻干燥;
所述交联步骤中的交联剂是指将碳化二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)按质量比为0.23:0.056~1.61:0.39混合,得到EDC/NHS交联剂;
所述交联步骤中,胶原、胶原肽与交联剂混合的比例为在1体积纯水中,酸法制备的胶原、酶法制备的胶原肽、EDC/NHS交联剂按照质量百分比1%:0.1%:0.3%~1%:0.5%:2%混合;
所述的交联步骤中的混合是指将胶原、胶原肽与交联剂在混合过程中于300rpm/min匀速搅拌1.5~3h,得到混合溶液;
所述的干燥步骤,是指将交联步骤得到的混合溶液进行冻干或自然晾干,得到交联膜;
所述清洗步骤,是指经干燥得到的交联膜用纯水连续冲洗三次;
所述的二次干燥步骤,是指将清洗后的交联膜于40℃热风干燥;
所述的灭菌步骤,是指干燥后的交联膜正反两面用紫外光照灭菌30~45min,于无菌条件下保存。
进一步的:所述酸法制备胶原步骤中,胶原由以下步骤制备而成:
A.鱼皮预处理步骤,将鱼皮去鳞、清洗后,将鱼皮切成0.5~1.5mm2得到剪切后的鱼皮;
B.脱色步骤,是指将剪切后鱼皮浸泡在0.4%~0.8%NaOH溶液中,搅拌4h~24h,得到脱色后的鱼皮;
C.脱脂步骤,将脱色后的鱼皮浸泡在质量百分比为20%的乙醇中搅拌4h~24h,得到脱色脱脂后的鱼皮;
D.浸酸步骤,将脱色脱脂后的鱼皮浸泡于质量百分比为1.2%~3%乙酸中搅拌4~24h,得到浸酸后的粗胶原溶液;
E.盐析步骤,将浸酸后的粗胶原溶液中加入NaCl粉末,直至蛋白质沉淀,得到盐析后的粗胶原蛋白;
F.脱盐步骤,将盐析后的粗胶原蛋白以质量百分比为0.6%~1.2%的乙酸溶液溶解,经8ku~12ku滤膜过滤得到脱盐后的胶原溶液;
G.干燥步骤,将脱盐后的胶原溶液进行真空冷冻干燥。
进一步的:所述酶法制备胶原肽步骤中,商业酶为中性蛋白酶、碱性蛋白酶、胶原酶,其酶解温度55~65℃,中性蛋白酶的酶解pH为7.0~7.5;碱性蛋白酶的酶解pH为6~8.5;胶原酶的酶解pH为8.5~9.0,酶解时间150~240min。
进一步的:所述的超滤压力为0.1~0.2MPa。
本发明的有益效果是:
1.发明所需的原材料来源丰富,成本低廉,比陆生动物胶原在生物医学领域更具有优势。
2.本发明制备得到的蛋白肽交联膜,将胶原肽与胶原交联,稳定了胶原肽对上皮细胞黏中附因子表达的促进作用。胶原膜表面多孔,且分布均匀,耐水耐热,有利于上皮细胞快速粘附和生长。
附图说明
图1是蛋白肽交联膜对小鼠皮肤创口愈合作用图,图中的皮肤创口直径20mm,分为A、B两个对照组;A组为蛋白肽交联膜覆盖皮肤创口0-6天的效果;B组为使用某品牌创口贴覆盖皮肤创口0-6天的效果;
图2是蛋白肽交联膜的微观结构图,分为2个对照组;A为蛋白肽交联膜的微观结构;B为实验室用鼠尾胶原膜微观结构;
图3是蛋白肽交联膜对上皮细胞HaCat的细胞增殖作用图,4个柱状数值从左到右依次为上皮细胞HaCat在酸法制备胶原、酶法制备胶原肽、蛋白肽交联膜和实验室用鼠尾胶原材质上培养24小时后的细胞增殖率;
图4是蛋白肽交联膜培养上皮细胞HaCat的蛋白因子表达图,白色为黏着斑蛋白vinculin;分为2个对照组,A为蛋白肽交联膜培养的vinculin蛋白表达;B为实验室用鼠尾胶原培养的vinculin蛋白表达。
具体实施方式
为了更好的理解本发明,本发明列举实施例如下。所举实施例的目的仅仅是为了理解本发明,但不用于限制本发明的范围。
实施例1
一种诱导上皮组织分化的蛋白肽交联膜,以生产可用于皮肤创口的诱导上皮组织分化的蛋白肽交联膜为例。依次进行如下步骤:
(1)酸法制备胶原:32kg罗非鱼皮经过剪切机切成1mm2的鱼皮块;将鱼皮沥干水分,浸泡于含有900L 0.4%的NaOH溶液中,室温搅拌48h,捞出鱼皮、用冰蒸馏水冲洗至pH7,即为脱色后的鱼皮;再将脱色后的鱼皮放入900L 20%(质量百分比)的乙醇溶液室温搅拌24h,捞出鱼皮,用蒸馏水冲洗5遍,得到脱色脱脂后的鱼皮;将 脱灰脱脂后的鱼皮浸泡于900L 3%(质量百分比)的乙酸溶液中,室温搅拌,粗棉布过滤后,经10000r/min离心30min,取上清液,4℃保存,所获沉淀按上述条件重新提取一次,将二次获得的离心上清液混合后,得到浸酸后的粗胶原溶液;将浸酸后的粗胶原溶液移入离心罐,分批加入21kg NaCl粉末离心上清液中,5000r/min离心10min收集沉淀,得到盐析后的粗胶原蛋白;以0.6%乙酸溶液(质量百分比)为透析液溶解盐析后的促胶原蛋白,经12ku滤膜过滤,收集滤膜滤出的溶液,得到脱盐后的胶原溶液;将脱盐后的胶原溶液真空冷冻干燥,得到4.5kg胶原粉末,密封储存,。
(2)酶法制备胶原肽:取3.5kg由酸法制备得到的胶原粉末,加入35L磷酸缓冲溶液中,加入70g碱性蛋白酶,调溶液pH9.0,酶解温度55℃,酶解时间150min。酶解终止后,将酶解液加热至85℃保持10min,灭酶活。将灭酶后的酶解液经4000r/min离心10min,收集上清液,于1ku~5ku超滤膜分离,分离压力0.4MPa,得到胶原肽溶液。经真空浓缩和冷冻干燥得到280g胶原肽粉。
(3)蛋白肽交联膜的制备:将1kg酸法制备的胶原溶解于100Kg的1.2%(质量百分比)乙酸溶液中,加入EDC/NHS 300g、胶原肽208g。其中EDC和NHS的质量比例为1.15:0.28。混合溶液于室温下进行300rpm/min匀速搅拌30min后,将溶液平铺于5块长宽为1m×0.6m的长方形托盘中,放入真空冷冻机进行干燥。将冻干之后的膜用纯水清洗三次,于40℃热风干燥箱中风干。干燥后的膜两面经紫外照射60min后,切成0.5m×0.3m的长方形膜,于真空密封袋中无菌保存,即为胶原肽交联膜。
实施例2
一种诱导上皮组织分化的蛋白肽交联膜,以生产可用于科研细胞培养用的促进上皮细胞增殖的蛋白肽交联膜为例。
(1)胶原的制备步骤与实施例1中胶原的制备步骤相同。
(2)胶原肽的制备步骤与实施例1中胶原肽的制备步骤相同。
(3)蛋白肽交联膜的制备:其中胶原、胶原肽、交联剂混合溶液的制备与实施例1中的步骤相同。得到的混合溶液于室温下进行300rpm/min匀速搅拌2h后,将混合溶液以50mL/cm2的密度平铺于细胞培养用6孔板的每孔加入混合溶液1.6mL、12孔板的每孔加入溶液800μL、96孔板的每孔加入溶液100μL。将加入混合溶液的细胞培养板放入真空冷冻机进行干燥。将冻干之后的膜用纯水清洗三次,于40℃热风干燥箱中风干。干燥后的膜经紫外正反面照射60min后,于真空密封袋中无菌保存。即为促进上皮细胞增殖的蛋白肽交联膜。
本发明将市面上某品牌创口贴与本发明制成的可用于皮肤创口的诱导上皮组织分化的蛋白肽交联膜进行了皮肤创口愈合效果评价。本发明采用小鼠创口模型检测蛋白肽交联膜对皮肤创口的愈合作用,并以某品牌创口贴作对比。结果如图1所示。本发明制备的蛋白肽交联膜对直径为20mm的皮肤创口覆盖第4天,伤口愈合率为72%;覆盖第6天,伤口愈合率达94.5%,几乎完全愈合。而某品牌创口贴对皮肤创口盖第4天,伤口愈合率为51.2%;覆盖第6天,伤口愈合率只有81.3%。本发明制备的蛋白交联膜对皮肤创口的愈合效果要优于某品牌创口贴。
此外,本发明还做了实验室常用的鼠尾胶原与本发明制成的可用于科研细胞培养用的促进上皮细胞增殖的蛋白肽交联膜进行了结构分析、对上皮细胞增殖功效分析。结果如下:
(1)蛋白肽交联膜的微观结构
采用扫描电镜对蛋白肽交联膜的微观结构进行观察,并以实验室常用于上皮细胞培养的鼠尾胶原的微观结构进行对比。结果如图2所示。本发明制备的蛋白肽交联膜表面平整、膜形成的孔洞大小均一、分布有规律,为上皮组织的黏附提供了物理基础。而实验室常用的鼠尾胶原膜表面粗糙、膜形成的孔洞大小不一、分布不均匀。
(3)蛋白肽交联膜对上皮细胞增殖率的促进作用:
采用MTT比色法对上皮细胞HaCat的增殖率检测,结果如图3所示。与实验室常用鼠尾胶原相比,本发明制备得到的胶原、胶原肽、蛋白肽交联膜对上皮细胞HaCat的增殖均有促进作用。其中,蛋白肽交联膜的促进作用最为显著。说明本发明制备的蛋白肽交联膜适合于上皮细胞实验室培养。
(4)上皮细胞在蛋白肽交联膜上的细胞因子表达:
采用荧光探针标记、荧光显微镜观察上皮细胞HaCat在蛋白肽交联膜上的细胞因子vinculin的表达,与实验室常用鼠尾胶原相对比,结果如图4所示。Vinculin在细胞的信号转导,细胞黏附,细胞固定等过程中起至关重要的作用,调节和参与细胞的增殖与分化,更重要的是,Vinculin在创伤愈合中的作用举足轻重。Vinculin的高表达能很充分的说明膜材料对细胞生长能起到促进作用。本发明制备的蛋白肽交联膜上,Hacat细胞中vinculin表达的数量与分布密度远远高于细胞在鼠尾胶原的表达。此外,HaCat细胞的生长方向、分布沿着膜材料的方向与分布生长,细胞整体的生长趋势呈现出三维的立体结构。而细胞在鼠尾胶原材料上的生长为单纯的平面结构,并且细胞的分布较疏松,同时vinculin分子的表达量也很少。说明本发明制备的蛋白肽交联膜适合于上皮细胞实验室培养。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (4)
1.一种诱导上皮组织分化的蛋白肽交联膜及其制备方法,其特征在于:所述蛋白肽交联膜以罗非鱼皮为原料,经酸法制备的胶原、经酶法制备的胶原肽,再将得到的胶原、胶原肽与交联剂交联,交联后所得物质再经过干燥、清洗、二次干燥、灭菌步骤后,得到诱导上皮组织分化的蛋白肽交联膜,制备步骤包括:
A.酸法制备胶原步骤,以罗非鱼皮作为原料,经过酸法工艺将罗非鱼皮制备成胶原;
B.酶法制备胶原肽步骤,以罗非鱼皮为原料,通过在酸法制备得到的罗非鱼皮胶原中加入商业酶水解获得的胶原酶解液,经超滤分离,干燥得到酶法制备的胶原肽;
C.交联步骤,将经过酸法制备的胶原、酶法制备的胶原肽与交联剂混合;
D.交联后所得物质再经过干燥、清洗、二次干燥、灭菌步骤后,得到诱导上皮组织分化的蛋白肽交联膜;
所述酶法制备胶原肽步骤中的超滤是将胶原酶解液通过分子膜进行分离获得1ku~5ku组分;
所述酶法制备胶原肽步骤中的商业酶为中性蛋白酶、胶原酶和碱性蛋白酶;
所述酶法制备胶原肽步骤中的干燥采用真空冷冻干燥;
所述交联步骤中的交联剂是指将碳化二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)按质量比为0.23:0.056~1.61:0.39混合,得到EDC/NHS交联剂;
所述交联步骤中,胶原、胶原肽与交联剂混合的比例为在1体积纯水中,酸法制备的胶原、酶法制备的胶原肽、EDC/NHS交联剂按照质量百分比1%:0.1%:0.3%~1%:0.5%:2%混合;
所述的交联步骤中的混合是指将胶原、胶原肽与交联剂在混合过程中于300rpm/min匀速搅拌1.5~3h,得到混合溶液;
所述的干燥步骤,是指将交联步骤得到的混合溶液进行冻干或自然晾干,得到交联膜;
所述清洗步骤,是指经干燥得到的交联膜用纯水连续冲洗三次;
所述的二次干燥步骤,是指将清洗后的交联膜于40℃热风干燥;
所述的灭菌步骤,是指干燥后的交联膜正反两面用紫外光照灭菌30~45min,于无菌条件下保存。
2.根据权利要求1所述的诱导上皮组织分化的蛋白肽交联膜,其特征在于:所述酸法制备胶原步骤中,胶原由以下步骤制备而成:
A.鱼皮预处理步骤,将鱼皮去鳞、清洗后,将鱼皮切成0.5~1.5mm2得到剪切后的鱼皮;
B.脱色步骤,是指将剪切后鱼皮浸泡在0.4%~0.8%NaOH溶液中,搅拌4h~24h,得到脱色后的鱼皮;
C.脱脂步骤,将脱色后的鱼皮浸泡在质量百分比为20%的乙醇中搅拌4h~24h,得到脱色脱脂后的鱼皮;
D.浸酸步骤,将脱色脱脂后的鱼皮浸泡于质量百分比为1.2%~3%乙酸中搅拌4~24h,得到浸酸后的粗胶原溶液;
E.盐析步骤,将浸酸后的粗胶原溶液中加入NaCl粉末,直至蛋白质沉淀,得到盐析后的粗胶原蛋白;
F.脱盐步骤,将盐析后的粗胶原蛋白以质量百分比为0.6%~1.2%的乙酸溶液溶解,经8ku~12ku滤膜过滤得到脱盐后的胶原溶液;
G.干燥步骤,将脱盐后的胶原溶液进行真空冷冻干燥。
3.根据权利要求1所述的诱导上皮组织分化的蛋白肽交联膜,其特征在于:所述酶法制备胶原肽步骤中,商业酶为中性蛋白酶、碱性蛋白酶、胶原酶,其酶解温度55~65℃,中性蛋白酶的酶解pH为7.0~7.5;碱性蛋白酶的酶解pH为6~8.5;胶原酶的酶解pH为8.5~9.0,酶解时间150~240min。
4.根据权利要求1所述的诱导上皮组织分化的蛋白肽交联膜,其特征在于:所述的超滤压力为0.1~0.2MPa。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710661771.9A CN107286677A (zh) | 2017-08-04 | 2017-08-04 | 一种诱导上皮组织分化的蛋白肽交联膜及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710661771.9A CN107286677A (zh) | 2017-08-04 | 2017-08-04 | 一种诱导上皮组织分化的蛋白肽交联膜及其制备方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107286677A true CN107286677A (zh) | 2017-10-24 |
Family
ID=60105301
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710661771.9A Pending CN107286677A (zh) | 2017-08-04 | 2017-08-04 | 一种诱导上皮组织分化的蛋白肽交联膜及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107286677A (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109985057A (zh) * | 2019-04-15 | 2019-07-09 | 四川大学 | 四面体骨架核酸在制备皮肤修复的药物中的用途 |
CN113144283A (zh) * | 2021-04-26 | 2021-07-23 | 广东海洋大学 | 一种促进创伤愈合的TSCP-GelMA水凝胶及其制备和应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102703555A (zh) * | 2012-06-08 | 2012-10-03 | 集美大学 | 小分子鱼皮胶原蛋白肽的提取制备方法 |
CN106729984A (zh) * | 2016-12-22 | 2017-05-31 | 青岛琛蓝海洋生物工程有限公司 | 一种鱼胶原蛋白修复海绵及其制备方法 |
-
2017
- 2017-08-04 CN CN201710661771.9A patent/CN107286677A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102703555A (zh) * | 2012-06-08 | 2012-10-03 | 集美大学 | 小分子鱼皮胶原蛋白肽的提取制备方法 |
CN106729984A (zh) * | 2016-12-22 | 2017-05-31 | 青岛琛蓝海洋生物工程有限公司 | 一种鱼胶原蛋白修复海绵及其制备方法 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109985057A (zh) * | 2019-04-15 | 2019-07-09 | 四川大学 | 四面体骨架核酸在制备皮肤修复的药物中的用途 |
CN113144283A (zh) * | 2021-04-26 | 2021-07-23 | 广东海洋大学 | 一种促进创伤愈合的TSCP-GelMA水凝胶及其制备和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN100382772C (zh) | 含蚕丝丝素的医用神经移植物的制备方法 | |
CN101234216B (zh) | 适用于生物医用材料的胶原基冷冻凝胶及其制备方法 | |
CN107653291A (zh) | 多步酶法协同制备藏牦牛皮胶原蛋白和胶原多肽的方法 | |
CN101361989A (zh) | 双层膜状组织修补材料及其制备方法 | |
CN102886063A (zh) | 一种纳米微晶纤维素增强胶原复合基质的制备与应用 | |
EP2231205B1 (en) | Colloidal collagen burn wound dressing produced from jellyfish | |
CN101366976A (zh) | 人源化异种细胞外基质材料及其制备方法 | |
CN101361990A (zh) | 一种双层人工皮肤及其制备方法 | |
CN105079783B (zh) | 药物组合物及其制备方法和用途 | |
CN106421884B (zh) | 两步冷冻法制备止血海绵的方法 | |
CN107556377A (zh) | 重组人源胶原蛋白及其医用纳米纤维膜 | |
Mahboudi et al. | The study of fibroblast cell growth on the porous scaffold of gelatin–starch blend using the salt-leaching and lyophilization method | |
CN107286677A (zh) | 一种诱导上皮组织分化的蛋白肽交联膜及其制备方法 | |
CN102675473A (zh) | 基因重组人型活性碱性成纤维细胞生长因子融合蛋白及其制备方法和应用 | |
Ma et al. | Expression, characterization, and application potentiality evaluation of recombinant human-like collagen in Pichia pastoris | |
CN1197631C (zh) | 一种含有表皮生长因子的皮肤组织工程支架的构建方法 | |
CN107715181A (zh) | 一种可生物降解的组织工程皮肤支架的制备方法 | |
CN1544097A (zh) | 一种生物医用材料及其制备方法和用途 | |
CN114191609A (zh) | 胶原微纤维海绵及其制备方法 | |
CN112426402A (zh) | 一种基于成纤维外泌体的敷贴及制备方法 | |
CN105295080A (zh) | 一种利于细胞粘附的功能丝素膜的制备方法 | |
Chirita | Mechanical properties of collagen biomimetic films formed in the presence of calcium, silica and chitosan | |
CN105801920B (zh) | 化学交联组合物、含其的生物医学材料以及其用途 | |
CN101554490B (zh) | 一种制备可生物降解的组织工程用血管中间层支架材料的方法 | |
CN111407929A (zh) | 一种新型人工皮肤及其制备方法 |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171024 |
|
WD01 | Invention patent application deemed withdrawn after publication |