CN107308441A - Stem cell culture supernatant gel combination and its preparation method and application - Google Patents
Stem cell culture supernatant gel combination and its preparation method and application Download PDFInfo
- Publication number
- CN107308441A CN107308441A CN201710451536.9A CN201710451536A CN107308441A CN 107308441 A CN107308441 A CN 107308441A CN 201710451536 A CN201710451536 A CN 201710451536A CN 107308441 A CN107308441 A CN 107308441A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- cell culture
- culture supernatant
- hegf
- gel combination
- 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
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 45
- 239000012228 culture supernatant Substances 0.000 title claims abstract description 31
- 238000004113 cell culture Methods 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims abstract description 10
- 230000014509 gene expression Effects 0.000 claims abstract description 10
- 210000002966 serum Anatomy 0.000 claims abstract description 8
- 206010072170 Skin wound Diseases 0.000 claims abstract description 5
- 229920000609 methyl cellulose Polymers 0.000 claims abstract description 4
- 239000001923 methylcellulose Substances 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims abstract description 3
- 239000006228 supernatant Substances 0.000 claims description 26
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 235000010981 methylcellulose Nutrition 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 238000005457 optimization Methods 0.000 claims description 8
- 108020004705 Codon Proteins 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 230000009465 prokaryotic expression Effects 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000012460 protein solution Substances 0.000 claims description 4
- 229920001503 Glucan Polymers 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 239000013613 expression plasmid Substances 0.000 claims description 3
- 210000003000 inclusion body Anatomy 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 229940116978 human epidermal growth factor Drugs 0.000 claims 1
- 101150012509 sub gene Proteins 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 231100000241 scar Toxicity 0.000 abstract description 4
- 206010052428 Wound Diseases 0.000 description 40
- 208000027418 Wounds and injury Diseases 0.000 description 39
- 210000003491 skin Anatomy 0.000 description 22
- 239000000499 gel Substances 0.000 description 21
- 230000029663 wound healing Effects 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 9
- 238000004043 dyeing Methods 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000006378 damage Effects 0.000 description 5
- 230000035876 healing Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 210000003954 umbilical cord Anatomy 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 208000025865 Ulcer Diseases 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 210000003780 hair follicle Anatomy 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000036548 skin texture Effects 0.000 description 3
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 2
- 208000028990 Skin injury Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000035508 accumulation Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 230000003328 fibroblastic effect Effects 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 235000015110 jellies Nutrition 0.000 description 2
- 239000008274 jelly Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 210000000106 sweat gland Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000397 ulcer Toxicity 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108010081589 Becaplermin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 1
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010056340 Diabetic ulcer Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 102000004890 Interleukin-8 Human genes 0.000 description 1
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 208000004210 Pressure Ulcer Diseases 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108010031374 Tissue Inhibitor of Metalloproteinase-1 Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000000558 Varicose Ulcer Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Substances N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 229940096397 interleukin-8 Drugs 0.000 description 1
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000008338 local blood flow Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000007427 paired t-test Methods 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 231100000075 skin burn Toxicity 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- 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
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Virology (AREA)
- Inorganic Chemistry (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a kind of stem cell culture supernatant gel combination and its preparation method and application, the gel combination is made up of stem cell culture supernatant, hEGF's albumen and methylcellulose, and solvent is water;The stem cell culture supernatant is to use collection purifying after the stem cell media culture without serum to obtain mescenchymal stem cell;HEGF's albumen is to obtain induced expression after human epidermal growth factor gene sequence optimisation.The protein binding that the present invention is obtained after free serum culture supernatant human epidermal growth factor gene is optimized, the gel combination of gained is used for skin wound reparation, and effect significantly, and does not stay scar.
Description
Technical field
The invention belongs to medical biomaterial technical field, and in particular to a kind of stem cell culture supernatant gel combination and
Its preparation method and application.
Background technology
The agglutination of the surface of a wound is a complicated process, and it is related to the re-epithelialization at defect of skin position, granulation group
The processes such as the remodeling of hyperplasia, inflammatory reaction, blood vessel hyperplasia and the surface of a wound knitted, one process of any of which goes wrong, and can all lead
Cause wound healing obstacle.At present, the method for wound repairing is mainly operation, will open wound and is changed into by operation stitching and be closed
Wound is closed, so that accelerate wound healing, but postoperative scar, the wound overtension left occurs function barrier occur after splitting or heal again
The complication such as hinder.For some complex surface of a wound, such as the radioactivity surface of a wound, diabetes, the surface of a wound being used for a long time after hormone
Usually occur point of local blood circulation obstacle, the reduction of fibroblastic bioactivity, mescenchymal stem cell Deng, these surface of a wound
Change and paracrine action be suppressed waits negative effect so that surface of a wound promoting epidermization is slow, inflammatory reaction reduction, the anti-infective energy of the surface of a wound
Power declines, even if the surface of a wound is smaller, closes the surface of a wound using skin-grafting or the method directly sutured, is also often difficult healing.In recent years,
There is the cell factor such as recombinant bovine or human fibroblastic growth factor (b-FGF), recombined human EGF, rhPDGF-BB through priority
The launch such as gel, spray or dressing are simultaneously applied to clinic, are shown by the clinical test results of multicenter large sample,
To surface of a wound such as the burn wound of light degree Ⅱ~III degree, varicose ulcer of lower extremity, diabetes, radiation ulcer and bedsores
The therapeutic intervention of cell factor is used alone or in combination, can accelerate wound healing, and can significantly improve the healing quality of the surface of a wound.But
It is that there are still many deficiencies:The effect of these growth factors is single;Act on after the surface of a wound, quickly by the protein kinase institute in the surface of a wound
Degraded, it is impossible to effectively continuingly act on specificity target cell, so as to cause the reduction of its bioavilability, it is impossible to obtain satisfaction
Clinical effectiveness.
In recent years research report shows mescenchymal stem cell to the repair function of histoorgan except relying on its self-replacation
Outside the ability of directed differentiation, more is to rely on a large amount of trophic factors of its secretion to adjust microenvironment in body, and activation is suffered from
The stem cell of dormancy in person's body, so that reaching improves the effect of body pathology situation.U.S. Stemnion, Inc. etc. pass through animal
The wound healing correlation factor being rich in experiment reference's amnion multipotential stem cell culture supernatant can be effectively facilitated wound
Healing, to burn, wound and diabetic ulcer etc. have preferable curative effect.There is scholar in umbilical cord Derived Stem Cells supernatant
Composition carried out careful research, its supernatant, user's growth factor antibodies chip are collected to the umbilical cord stem cells of culture
Analyzed with ELISA experiments, as a result show that supernatant contains different cell factor and chemokines, such as substantial amounts of leucocyte
Interleukin 6, interleukin 8, MCP-1, TIMP-1, GM-CSF, TGF-1 etc., these factors are in general wound
It is extremely important.
HEGF (human EGF, hEGF) is that one kind is present in human multiple tissue, with extensive biology
The polypeptide of activity, it is by the single chain polypeptide of 53 Amino acid profiles, also known as oligopeptides -1.EGF has been obtained extensively at present
Corneal transplantation, skin burn and wound, skin ulcerous, mouth after general application, such as ophthalmic cornea damage, cataract extraction
Chamber ulcer, gastrointestinal ulceration and glioma, squamous cell carcinoma etc.;The cosmeceutical containing EGF can also be made, promotes
Human epidermal cell metabolism.Currently with prokaryotic system express Urogastrone (rhEGF) major drawbacks be expression quantity compared with
Low, bioactivity is not high, and the eukaryotic expression cycle is long, cumbersome, and the shortcomings of cost is high is difficult large-scale production.
The content of the invention
The technical problem of solution:It is an object of the invention to provide a kind of stem cell culture supernatant gel combination and its preparation
Methods and applications, the stem cell culture supernatant gel combination optimizes stem cell culture supernatant and human epidermal growth factor gene
The protein binding obtained afterwards, for skin wound reparation, effect significantly, and does not stay scar.
Technical scheme:Stem cell culture supernatant gel combination, by stem cell culture supernatant, hEGF's albumen
With methylcellulose composition, solvent is water;The stem cell culture supernatant is that mescenchymal stem cell is used into doing without serum
Purifying is collected after cell culture medium culture to obtain;HEGF's albumen is by human epidermal growth factor gene sequence
Induced expression is obtained after optimization.
Further, the sequence after the human epidermal growth factor gene sequence optimisation is as shown in SEQIDNO.1.
The preparation method of the stem cell culture supernatant gel combination, comprises the following steps:
Step 1, mescenchymal stem cell is used into the stem cell media culture without serum, collects supernatant, centrifuged,
Ammonium sulfate is added in gained supernatant, stirring, standing, centrifugation take precipitation, are dissolved in water, filters, obtain albumen filtrate;
Step 2, step 1 gained albumen filtrate is purified through gel column, then freezed, obtain lyophilized supernatant;
Step 3, the gene optimization of e. coli codon preferences is carried out to the code area of human epidermal growth factor gene,
Nde I and the restriction enzyme sites of Xho I are added respectively in gained optimization N-terminal and C-terminal, and full genome conjunction is carried out to obtained composition sequence
Into, and it is connected into the prokaryotic expression plasmid pET-30a-hEGF that prokaryotic expression carrier pET-30a is recombinated;
Step 4, step 3 gained recombinant plasmid transformed is obtained with inclusion bodies into Escherichia coli after induced expression
HEGF's albumen of expression, obtains hEGF's albumen after isolating and purifying;
Step 5, step 2 obtained freeze-drying supernatant sterilized water is dissolved, obtained aqueous solution and hEGF's albumen
The aqueous solution, methylated cellulose aqueous solution mixing, obtain stem cell culture supernatant gel combination.
Further, ammonium sulfate step is added in step 1 to carry out in 0 DEG C of ice bath.
Further, static conditions are 4 DEG C, 12h-16h in step 1.
Further, the gel column used in step 2 is 0.1M-0.15M for glucan G-25 gel columns, eluant, eluent
The NaCl aqueous solution.
Further, the concentration that the supernatant aqueous solution is freezed in step 5 is 19mg/mL, and hEGF's albumen is water-soluble
The concentration of liquid is 160-640ng/mL, and the mass concentration of methylated cellulose aqueous solution is 3%.
Application of the above-mentioned stem cell culture supernatant gel combination in skin wound reparation.
Beneficial effect:The albumen knot that the present invention is obtained after free serum culture supernatant human epidermal growth factor gene is optimized
Close, worth gel combination is used for skin wound reparation, effect significantly, and does not stay scar.
Brief description of the drawings
Fig. 1 is mouse union of wounded skin dyeing in 15 days in embodiment 1;
Fig. 2 is mouse union of wounded skin Masson dyeing in 15 days in embodiment 1;
Wherein, arrow meaning is surface of a wound position in figure.
Embodiment
Invention is described in further detail below by embodiment.But it will be understood to those of skill in the art that under
Row embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
Embodiment 1
1. the preparation of lyophilized supernatant
The collection of 1.1HU-MSCs supernatants
The preparation of mescenchymal stem cell:Fresh and healthy neonatal umbilical cord is taken, is rinsed well umbilical cord with PBS, blood vessel is removed,
The Fahrenheit glue tissue of the inside is stripped out, gained tissue is fully shredded to 1mm3Size, add serum free medium, in 37 DEG C, 5%
CO2Cultivate, after cell is covered with, passed on 0.25% Trypsin Induced in incubator.When the umbilical cord Derived Stem Cells in the 3rd generation
When covering with 70% in blake bottle, it is further cultured for 48 hours, collects supernatant standby.
Serum free medium main formula:Human serum albumins:4g/L-10g/L, glycine:30.00mg/L, it is nonessential
Amino acid:1mg/L-50mg/L, rh-insulin:1mg/L-10mg/L, human transferrin:5mg/L-20mg/L.
1.2HU-MSCs supernatants are slightly purified
1.2.1 cells and supernatant salt precipitation
500mL step 1.1 gained supernatants are taken, 8000rpm, 4 DEG C of centrifugation 15min remove tissue block therein and other are miscellaneous
Matter, is placed in 1L beaker, is slowly added to the 304g ammonium sulfate solids weighed in advance (in terms of 85% saturation degree, with reference to ammonium sulfate
Precipitating reagent table), side edged magnetic stirrer, at least 1h is added, and above operation is completed in 0 DEG C of frozen water mixing bath.4
Continue to stir to being completely dissolved in DEG C chromatography cabinet, 12h is stood in chromatography cabinet, 9000rpm, 4 DEG C of centrifugation 10min collect albumen and sunk
Form sediment, be dissolved in water and be settled to 400mL, rushed with filter (plus 2 0.45 μm of filter membranes) filtrate protein solution, then with sterile deionized water
Wash several times that filter membrane is to reduce the loss of albumen, final albumen filtrate volume is about 540mL.
1.2.2 gel filtration desalination, decolouring
Glucan G-25 gel columns (5*60cm) are connected into AKTA protein purification instrument, with the deionized water of the NaCl containing 0.15M
As eluant, eluent, flow velocity 10mL/min, each sample introduction 80mL are adjusted, until ultraviolet absorption value substantially rises, starts to connect sample
Until there is no UV absorption, by the sample being collected into preservation at 4 DEG C after 0.22um membrane filtrations.
1.3 lyophilized concentrations
1) by the purifying supernatant being collected into, freeze dryer is lyophilized into powder, freezes program such as table one;
Table one freezes program
2) freeze-dried powder is dissolved into the albumen condensed liquid that concentration is 19mg/mL with sterilized water, it is standby.
The preparation of 2.hEGF albumen
GeneBank is logged in, the gene order of hEGF's hEGF active peptides is searched, its length nucleic acid is
159bp, hEGF are eucaryote peptide sequence, it is contemplated that follow-up research and development are expressed in protokaryon, therefore above-mentioned hEGF is true
The coding codon of core peptide sequence is compared analysis with Escherichia coli Preference codon.According to the degenerate of codon and
Do not change hEGF active peptide sequence of amino acid it is constant in the case of, with reference to biosoftware Vector NTI Suitor 7.0
HEGF nucleotide sequences are analyzed, genetic modification optimization are carried out to known hEGF, mainly by hEGF protogene sequences
The rare codon of Escherichia coli makes the codon of preference into, to improve high level expression of the target gene in Escherichia coli.And
Transgenosis checking is carried out, the optimization of hEGF genes is finally given as shown in SEQIDNO.1.
People hEGF gene optimization sequence N-terminals and C-terminal are added into Nde I and the restriction enzyme sites of Xho I respectively.Serve Hai Shenggong companies
Full genome synthesis is carried out, and is connected into prokaryotic expression carrier pET-30a (Military Medical Science Institute's present) Nde I and the digestions of Xho I
Between site, the prokaryotic expression plasmid pET-30a-hEGF recombinated.By recombinant plasmid transformed into Escherichia coli.IPTG is lured
The hEGF small peptides for obtaining expressing with inclusion bodies after expression are led, hEGF albumen is obtained after isolating and purifying.
3. the preparation of stem cell culture supernatant gel combination
Lyophilized supernatant sterilized water dissolves, and it is 19mg/mL to freeze protein concentration in the supernatant aqueous solution, with 160ng/mL
HEGF protein solutions are mixed in equal volume, are then that 3% methylated cellulose aqueous solution is mixed in equal volume with mass concentration, are produced solidifying
Jelly (low dosage);
Lyophilized supernatant sterilized water dissolves, and it is 19mg/mL to freeze protein concentration in the supernatant aqueous solution, with 640ng/mL
HEGF protein solutions are mixed in equal volume, are then that 3% methylated cellulose aqueous solution is mixed in equal volume with mass concentration, are produced solidifying
Jelly (high dose);
Lyophilized supernatant sterilized water dissolves, and it is 19mg/mL to freeze protein concentration in the supernatant aqueous solution, is with mass concentration
3% methylcellulose is mixed in equal volume, produces gel (no hEGF).
The skin injury of 4.ICR mouse species is healed
4.1 packet
Experimental animal is randomly divided into three big groups of blank control group (10) and experiment material group (30), wherein testing material
The sample number that material group is divided into 3 groups, every group again is 10.
1. blank control group:10, skin of back damage, wound does not give any processing during experiment;
2. experiment material group:Totally 30, it is divided into I, II, III group
I groups:10, skin of back damage, wound gives gel (low dosage) processing during experiment.
II groups:10, skin of back damage, wound gives gel (high dose) processing during experiment.
III groups:10, skin of back damage, wound gives gel (no hEGF) processing during experiment.
The foundation of 4.2 skin injury animal models
Each group ICR mouse anesthesias, prostrate is fixed on operating table, is removed back hair with shaver, is beaten using 5mm skins
Hole device prepares skin trauma model in mouse back.It is administered once at regular time and quantity daily (gel, 50 μ L/ times).
Take the tissue time:3rd, 6,8,15 days when select 5 mouse etherizations in each experiment, blank control group at random and put to death
And take pictures, leave and take skin histology of each group containing the surface of a wound and part normal structure.It is fixed in formalin, paraffin organization is made and cuts
Piece, for follow-up HE dyeing and Masson dyeing.
Wound healing rate is calculated
Using ImageJ image analysis softwares, the size of the healing surface of a wound is calculated.
Wound healing rate=(initial surface of a wound size-size for the surface of a wound that do not heal)/initial surface of a wound size × 100%
The healing rate of the surface of a wound is as shown in the table in different time points, by three groups of Wound healing rates of different time points respectively with
Blank control group carries out paired t-test, wound healing the 5th, 7 days, no hEGF groups and high dose group Wound healing rate are all higher than
Control group, the statistically significant (P of its difference<0.05);And low dose group is the 5th, the 7 days difference between control group has pole
Notable statistical significance (P<0.01).And the 3rd, 9 days, Wound healing rate each group is compared without significant difference.
The healing rate (%) of the surface of a wound of table two
Note:* represent compared with control group, the statistically significant (P of its difference<0.05);* represented compared with control group, its
Difference has extremely notable statistical significance (P<0.01).
HE is dyed
3rd, 6,8,15 days leave and take each group incision content side 1cm scope ring layers skin histology and carry out paraffin section and make
HE dyeing is carried out, to assess the general condition of different group wound healings.
As shown in figure 1, dyeing visible, the 15th day, the visible obvious epidermis homogeneous of the blank control group surface of a wound, wound through conventional HE
Still retain certain necrotic zone at mouthful, space and slight crack are still visible, skin texture is loose, and the hair follicle tissue in skin is not yet
Restoration ecosystem, and the trend of collagenous fibres is irregular.Experimental group epidermis homogeneous is then relatively thin, hair follicle, sweat gland etc. has occurred
Skin affiliated group.Wherein, no hEGF groups wound area is significantly less than blank group, and the newborn epithelium thickened begins with a small amount of skin
The differentiation of accessory structure;High dose and low dose group surface of a wound region complete epithelialization, and differentiate the attached knot of substantial amounts of skin
Collagenous fibres in structure, but high dose group corium still have a little fracture;Comparatively low dose group surface of a wound recovery effects are best, its
In order, skin texture is more close for arrangement of collagen fibers, and Skin appendages are the most clear, complete.
Masson is dyed
3rd, 6,8,15 days leave and take each group incision content side 1cm scope ring layers skin histology and carry out paraffin section and make
Masson dyeing is carried out, to assess the general condition of collagen formation during different group wound healings.
As shown in Fig. 2 visible through Masson dyeing, the 15th day, blank group neonatal dermal collagen slightly had accumulation, collagenous fibres
Arrangement is relatively unordered, and outer skin homogeneous is thicker, and incomplete epithelialization, skin level is less;It is (dark blue without hEGF group collagen accumulations
Color) clearly, skin texture is comparatively dense, it may be observed that a small amount of newborn Skin appendages;High dose and low dose group
The surface of a wound complete epithelialization, it may be observed that substantial amounts of hair follicle, sweat gland.Particularly low dose group, repairing effect preferably, its skin
Structure is rich in level, and the arrangement of collagenous fibres is clearly orderly, no fracture or mixed and disorderly phenomenon.
SEQUENCE LISTING
<110>Jiangsu Mai Jian biotechnologies Development stock Co., Ltd
<120>Stem cell culture supernatant gel combination and its preparation method and application
<130> 20170615
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 159
<212> DNA
<213>Artificial sequence
<400> 1
aacagcgact ctgaatgccc actgtcccac gatggttact gcctgcatga tggtgtgtgc 60
atgtatattg aagctctgga caagtatgca tgcaattgtg ttgtaggcta catcggcgag 120
cgttgtcaat accgtgacct gaaatggtgg gaactgcgc 159
Claims (8)
1. stem cell culture supernatant gel combination, it is characterised in that:By stem cell culture supernatant, hEGF's albumen
With methylcellulose composition, solvent is water;
The stem cell culture supernatant is to collect pure after mescenchymal stem cell is used into the stem cell media culture without serum
Change is obtained;
HEGF's albumen is that human epidermal growth factor gene sequence is optimized into rear induced expression to obtain.
2. stem cell culture supernatant gel combination according to claim 1, it is characterised in that:The human epidermal growth factor
Sub- gene order optimize after sequence as shown in SEQIDNO.1.
3. the preparation method of the stem cell culture supernatant gel combination described in claim 1 or 2, it is characterised in that:Including with
Lower step:
Step 1, mescenchymal stem cell is used into the stem cell media culture without serum, collects supernatant, centrifugation, in gained
Ammonium sulfate is added in supernatant, stirring, standing, centrifugation take precipitation, are dissolved in water, filters, obtain albumen filtrate;
Step 2, step 1 gained albumen filtrate is purified through gel column, then freezed, obtain lyophilized supernatant;
Step 3, the gene optimization of e. coli codon preferences is carried out to the code area of human epidermal growth factor gene, in institute
Obtain optimization N-terminal and C-terminal and add Nde I and the restriction enzyme sites of Xho I respectively, full genome synthesis is carried out to obtained composition sequence,
And it is connected into the prokaryotic expression plasmid pET-30a-hEGF that prokaryotic expression carrier pET-30a is recombinated;
Step 4, step 3 gained recombinant plasmid transformed is obtained expressing with inclusion bodies into Escherichia coli, after induced expression
HEGF's albumen, hEGF's albumen is obtained after isolating and purifying;
Step 5, step 2 obtained freeze-drying supernatant sterilized water is dissolved, obtained aqueous solution and hEGF's albumen are water-soluble
Liquid, methylated cellulose aqueous solution mixing, obtain stem cell culture supernatant gel combination.
4. the preparation method of stem cell culture supernatant gel combination according to claim 3, it is characterised in that:Step 1
Middle addition ammonium sulfate step is carried out in 0 DEG C of ice bath.
5. the preparation method of stem cell culture supernatant gel combination according to claim 3, it is characterised in that:Step 1
Middle static conditions are 4 DEG C, 12h-16h.
6. the preparation method of stem cell culture supernatant gel combination according to claim 3, it is characterised in that:Step 2
The middle gel column used is glucan G-25 gel column, and eluant, eluent is the 0.1M-0.15M NaCl aqueous solution.
7. the preparation method of stem cell culture supernatant gel combination according to claim 3, it is characterised in that:Step 5
In to freeze the concentration of the supernatant aqueous solution be 19mg/mL, the concentration of hEGF's protein solution is 160-640ng/mL,
The mass concentration of methylated cellulose aqueous solution is 3%.
8. application of the stem cell culture supernatant gel combination in skin wound reparation described in claim 1.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710451536.9A CN107308441A (en) | 2017-06-15 | 2017-06-15 | Stem cell culture supernatant gel combination and its preparation method and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710451536.9A CN107308441A (en) | 2017-06-15 | 2017-06-15 | Stem cell culture supernatant gel combination and its preparation method and application |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107308441A true CN107308441A (en) | 2017-11-03 |
Family
ID=60183290
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710451536.9A Pending CN107308441A (en) | 2017-06-15 | 2017-06-15 | Stem cell culture supernatant gel combination and its preparation method and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107308441A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108517002A (en) * | 2018-04-20 | 2018-09-11 | 命之本源医疗科技(北京)有限公司 | A kind of stabilizer of stem cell culture supernatant secretory protein |
CN108715832A (en) * | 2018-06-01 | 2018-10-30 | 段海峰 | A kind of mescenchymal stem cell and preparation method and application inhibiting tumour growth |
CN113813441A (en) * | 2020-06-19 | 2021-12-21 | 辽宁医学诊疗科技研发中心有限公司 | Liquid band-aid containing stem cell repair factors and preparation method thereof |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101698682A (en) * | 2009-10-26 | 2010-04-28 | 陕西省微生物研究所 | Double-functional fusion protein based on antibacterial peptide, preparation method and applicaitoin thereof |
CN101857865A (en) * | 2010-06-09 | 2010-10-13 | 黑龙江省科学院微生物研究所 | Method for constructing recombinant human epdermal growth favtur (hEGF) engineering bacteria |
CN102311503A (en) * | 2007-06-06 | 2012-01-11 | 天津溥瀛生物技术有限公司 | Recombinant human serum albumin / FGF fusion protein with continuous effect on restoration of a plurality of skin cells |
CN102993305A (en) * | 2012-11-16 | 2013-03-27 | 上海赛伦生物技术有限公司 | Humanized anti-human epidemic growth factor receptor (EGFR) antibody as well as encoding gene and application thereof |
CN103834664A (en) * | 2014-03-27 | 2014-06-04 | 中国人民解放军军事医学科学院军事兽医研究所 | Recombinant epidermal growth factor (EGF) and preparation method thereof |
CN105154407A (en) * | 2015-10-14 | 2015-12-16 | 紫程瑞生会(北京)生物技术发展有限公司 | Preparation method and application of human adipose-derived stem cells for improving skin repairing function |
CN105797205A (en) * | 2016-04-20 | 2016-07-27 | 江苏迈健生物科技发展股份有限公司 | Stem cell culture supernate gel and preparation method thereof |
CN106497975A (en) * | 2016-10-21 | 2017-03-15 | 浙江生创精准医疗科技有限公司 | The preparation method of genetic recombination stem cell medicine and its application in skin injury reparation, scar suppress |
-
2017
- 2017-06-15 CN CN201710451536.9A patent/CN107308441A/en active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102311503A (en) * | 2007-06-06 | 2012-01-11 | 天津溥瀛生物技术有限公司 | Recombinant human serum albumin / FGF fusion protein with continuous effect on restoration of a plurality of skin cells |
CN101698682A (en) * | 2009-10-26 | 2010-04-28 | 陕西省微生物研究所 | Double-functional fusion protein based on antibacterial peptide, preparation method and applicaitoin thereof |
CN101857865A (en) * | 2010-06-09 | 2010-10-13 | 黑龙江省科学院微生物研究所 | Method for constructing recombinant human epdermal growth favtur (hEGF) engineering bacteria |
CN102993305A (en) * | 2012-11-16 | 2013-03-27 | 上海赛伦生物技术有限公司 | Humanized anti-human epidemic growth factor receptor (EGFR) antibody as well as encoding gene and application thereof |
CN103834664A (en) * | 2014-03-27 | 2014-06-04 | 中国人民解放军军事医学科学院军事兽医研究所 | Recombinant epidermal growth factor (EGF) and preparation method thereof |
CN105154407A (en) * | 2015-10-14 | 2015-12-16 | 紫程瑞生会(北京)生物技术发展有限公司 | Preparation method and application of human adipose-derived stem cells for improving skin repairing function |
CN105797205A (en) * | 2016-04-20 | 2016-07-27 | 江苏迈健生物科技发展股份有限公司 | Stem cell culture supernate gel and preparation method thereof |
CN106497975A (en) * | 2016-10-21 | 2017-03-15 | 浙江生创精准医疗科技有限公司 | The preparation method of genetic recombination stem cell medicine and its application in skin injury reparation, scar suppress |
Non-Patent Citations (5)
Title |
---|
国家药典委员会: "《中华人民共和国药典 三部 注释》", 31 March 2016, 中国医药科技出版社 * |
李美娜: "人表皮生长因子在大肠杆菌中的表达及其多克隆抗体的制备", 《中国优秀硕士论文库 基础科学辑》 * |
沈萍: "《微生物遗传学》", 30 September 1995 * |
赵彦林: "重组人表皮生长因子凝胶在烧伤创面中的应用", 《中国当代医药》 * |
邹克琴: "《基因工程原理和技术》", 31 January 2009, 浙江大学出版社 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108517002A (en) * | 2018-04-20 | 2018-09-11 | 命之本源医疗科技(北京)有限公司 | A kind of stabilizer of stem cell culture supernatant secretory protein |
CN108715832A (en) * | 2018-06-01 | 2018-10-30 | 段海峰 | A kind of mescenchymal stem cell and preparation method and application inhibiting tumour growth |
CN108715832B (en) * | 2018-06-01 | 2020-11-10 | 段海峰 | Mesenchymal stem cell for inhibiting tumor growth and preparation method and application thereof |
CN113813441A (en) * | 2020-06-19 | 2021-12-21 | 辽宁医学诊疗科技研发中心有限公司 | Liquid band-aid containing stem cell repair factors and preparation method thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Fett et al. | Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells | |
CN105797205B (en) | Stem cell culture supernatant gel and preparation method thereof | |
KR20020079761A (en) | Sericin-containing material, process for producing the same and method of using the same | |
CN108578781A (en) | Air bladder source biovalve material and the preparation method and application thereof | |
CN112745394B (en) | Recombinant human-like collagen and preparation method and application thereof | |
CN107308441A (en) | Stem cell culture supernatant gel combination and its preparation method and application | |
CN105176914A (en) | Simultaneous autologous epidermic cell and fibrocyte preparation method and biological cosmetic product thereof | |
CN113462632B (en) | Balsam pear exosome, extraction method and application thereof in preparation of medicines for treating burns and scalds | |
CN103468771B (en) | Method for extracting collagens from bovine achilles tendon | |
CN111393521A (en) | Extraction method of jellyfish collagen | |
CN106497886A (en) | A kind of hair regeneration induction liquid and its preparation method and application | |
CN113563452B (en) | Biological active peptide and application of biological active peptide and adipose-derived stem cell exosome in skin proliferation repair | |
CN101773687A (en) | Preparation method of composite soft-tissue patch | |
CN104611289A (en) | Method for simultaneously preparing autologous epidermal cells and fibroblasts, and biological beauty product comprising autologous epidermal cells and fibroblasts | |
CN112852878A (en) | Method for producing recombinant humanized III-type collagen by transgenic silkworms | |
CN113274410A (en) | Application of exosome hydrogel complex in preparation of medicine for repairing skin scar | |
CN114917413B (en) | Amniotic membrane loaded with recombinant polypeptide and preparation method thereof | |
JP6383721B2 (en) | Wound healing agent | |
CN105169377A (en) | Novel application of lysozyme-antibacterial peptide fusion proteins | |
CN105411874A (en) | Chick embryo bioactive peptide, preparation method and applications | |
CN105396136B (en) | CCN1(Cyr61)Application in treatment skin injury and atrophoderma relevant disease | |
CN114716515A (en) | Polypeptide analogue and preparation method and application thereof | |
CN100334114C (en) | Novel fusion protein production and uses | |
JPWO2002102845A1 (en) | Method for producing functional polypeptide derived from silk fibroin and its use | |
CN105087482A (en) | Cell culture substrate and application and use method thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171103 |
|
RJ01 | Rejection of invention patent application after publication |