CN111437244B - Preparation method and product of high-efficiency stem cell expression exosome for skin beauty - Google Patents
Preparation method and product of high-efficiency stem cell expression exosome for skin beauty Download PDFInfo
- Publication number
- CN111437244B CN111437244B CN202010296426.1A CN202010296426A CN111437244B CN 111437244 B CN111437244 B CN 111437244B CN 202010296426 A CN202010296426 A CN 202010296426A CN 111437244 B CN111437244 B CN 111437244B
- Authority
- CN
- China
- Prior art keywords
- parts
- stem cells
- cells
- exosome
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 89
- 210000001808 exosome Anatomy 0.000 title claims abstract description 55
- 230000014509 gene expression Effects 0.000 title claims abstract description 36
- 230000003796 beauty Effects 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 50
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 48
- 210000001519 tissue Anatomy 0.000 claims abstract description 33
- 239000013604 expression vector Substances 0.000 claims abstract description 31
- 238000003259 recombinant expression Methods 0.000 claims abstract description 31
- 239000006228 supernatant Substances 0.000 claims abstract description 26
- 239000000047 product Substances 0.000 claims abstract description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 239000000463 material Substances 0.000 claims abstract description 12
- 238000004108 freeze drying Methods 0.000 claims abstract description 11
- 239000000725 suspension Substances 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 82
- 210000003954 umbilical cord Anatomy 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 210000000577 adipose tissue Anatomy 0.000 claims description 17
- 108010019160 Pancreatin Proteins 0.000 claims description 16
- 229940055695 pancreatin Drugs 0.000 claims description 16
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 13
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 12
- 229930195725 Mannitol Natural products 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- POJWUDADGALRAB-UHFFFAOYSA-N allantoin Chemical compound NC(=O)NC1NC(=O)NC1=O POJWUDADGALRAB-UHFFFAOYSA-N 0.000 claims description 12
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 12
- 210000002950 fibroblast Anatomy 0.000 claims description 12
- 239000000594 mannitol Substances 0.000 claims description 12
- 235000010355 mannitol Nutrition 0.000 claims description 12
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 11
- 238000004113 cell culture Methods 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 9
- 108010031111 EBV-encoded nuclear antigen 1 Proteins 0.000 claims description 9
- 239000013078 crystal Substances 0.000 claims description 9
- 230000005059 dormancy Effects 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 102000011782 Keratins Human genes 0.000 claims description 8
- 108010076876 Keratins Proteins 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 239000011148 porous material Substances 0.000 claims description 8
- 239000006285 cell suspension Substances 0.000 claims description 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 6
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims description 6
- 102000009027 Albumins Human genes 0.000 claims description 6
- 108010088751 Albumins Proteins 0.000 claims description 6
- POJWUDADGALRAB-PVQJCKRUSA-N Allantoin Natural products NC(=O)N[C@@H]1NC(=O)NC1=O POJWUDADGALRAB-PVQJCKRUSA-N 0.000 claims description 6
- 229920002307 Dextran Polymers 0.000 claims description 6
- 102100030801 Elongation factor 1-alpha 1 Human genes 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 241000208681 Hamamelis virginiana Species 0.000 claims description 6
- 101000920078 Homo sapiens Elongation factor 1-alpha 1 Proteins 0.000 claims description 6
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims description 6
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 239000004373 Pullulan Substances 0.000 claims description 6
- 229920001218 Pullulan Polymers 0.000 claims description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- 241001506047 Tremella Species 0.000 claims description 6
- 229960000458 allantoin Drugs 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229920002674 hyaluronan Polymers 0.000 claims description 6
- 229960003160 hyaluronic acid Drugs 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 235000019423 pullulan Nutrition 0.000 claims description 6
- 239000001540 sodium lactate Substances 0.000 claims description 6
- 229940005581 sodium lactate Drugs 0.000 claims description 6
- 235000011088 sodium lactate Nutrition 0.000 claims description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 239000012679 serum free medium Substances 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 239000006143 cell culture medium Substances 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 102000008186 Collagen Human genes 0.000 claims description 3
- 108010035532 Collagen Proteins 0.000 claims description 3
- 102000029816 Collagenase Human genes 0.000 claims description 3
- 108060005980 Collagenase Proteins 0.000 claims description 3
- 102000016359 Fibronectins Human genes 0.000 claims description 3
- 108010067306 Fibronectins Proteins 0.000 claims description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 3
- 102000013275 Somatomedins Human genes 0.000 claims description 3
- 210000000601 blood cell Anatomy 0.000 claims description 3
- 229920001436 collagen Polymers 0.000 claims description 3
- 229960002424 collagenase Drugs 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 102000013370 fibrillin Human genes 0.000 claims description 3
- 108060002895 fibrillin Proteins 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000008188 pellet Substances 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 239000004480 active ingredient Substances 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 239000003826 tablet Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 13
- 230000028327 secretion Effects 0.000 description 7
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 239000012228 culture supernatant Substances 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 235000015110 jellies Nutrition 0.000 description 4
- 239000008274 jelly Substances 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000007599 discharging Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000007939 sustained release tablet Substances 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 239000003513 alkali Substances 0.000 description 2
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical group [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102100031900 Neogenin Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 108010076969 neogenin Proteins 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0216—Solid or semisolid forms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
- A61K8/20—Halogens; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
- A61K8/345—Alcohols containing more than one hydroxy group
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/36—Carboxylic acids; Salts or anhydrides thereof
- A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
- A61K8/4946—Imidazoles or their condensed derivatives, e.g. benzimidazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9728—Fungi, e.g. yeasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4741—Keratin; Cytokeratin
-
- 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
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
-
- 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
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Dermatology (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Emergency Medicine (AREA)
- Rheumatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Inorganic Chemistry (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method and a product of a stem cell high-efficiency expression exosome for skin beauty, wherein the preparation method comprises the following steps: separating stem cells from human tissues and culturing; constructing a recombinant expression vector, adding the recombinant expression vector into a culture suspension of stem cells, obtaining the stem cells capable of expressing target proteins by adopting an electrotransfection mode, extracting exosomes from a supernatant of the stem cells, adding auxiliary materials according to parts by weight, carrying out instant freeze-drying by utilizing liquid nitrogen, and drying the obtained tablets in a vacuum environment. Through the technical scheme of the invention, the recombinant expression vector can be transferred to stem cells to extract the target protein exosome, and the product for skin beauty is prepared by using a freeze-drying technology, so that the active ingredients of the exosome are effectively protected, and the exosome can be stored for a long time at room temperature and keep activity.
Description
Technical Field
The invention relates to the technical field of stem cell exosomes, in particular to a preparation method of a stem cell high-efficiency expression exosome for skin beautifying and a product of the stem cell high-efficiency expression exosome for skin beautifying.
Background
Stem cell exosomes have been studied abroad for many years, and are increasingly used in the market, while domestic application to stem cell exosomes is also increased, and the types of products for beauty skin care are also increased, but the expression efficiency of the stem cell exosomes is low, the protein expression is low, the effect of beauty skin care is general, and the stem cell exosomes can cause the defects of insufficient directional secretion, reduced activity and the like after long-term storage.
Disclosure of Invention
Aiming at least one of the problems, the invention provides a preparation method and a product for skin beauty by using a stem cell high-efficiency expression exosome, wherein a recombinant expression vector is transferred into a stem cell in an electrotransfection mode, so that a target protein exosome is extracted, and on the basis, a product for skin beauty is prepared by using a freeze-drying technology, so that active ingredients of the stem cell high-efficiency expression exosome are effectively protected, and the prepared exosome freeze-dried sustained-release tablet can be stored at room temperature for a long time, and overcomes the defects of directional secretion, low long-time storage activity and the like of the stem cell exosome. In addition, a human promoter EF1A is adopted, CD33 signal peptide is fused at the N ends of oligopeptide-1 and basic fibroblast or other proteins, protein secretion is facilitated, the synergistic effect of two cytokines can be increased by adopting an IRES expression element, the two cytokines are expressed simultaneously, the transferred recombinant expression vector plasmid can exist in stem cells for a long time and efficiently express target proteins by adopting an episome system, and cells with high expression can be quickly obtained by adopting a PURO sequence screening marker, so that the required target proteins are directionally and efficiently expressed.
In order to achieve the above object, the present invention provides a method for preparing exosome efficiently expressed by stem cells for skin beauty, comprising: separating stem cells from human tissues and culturing; combining a PB transposon, an EF1A promoter sequence, a KOZAK sequence, a target protein sequence to be expressed 1, a T2A sequence, a target protein sequence to be expressed 2, a CD33 signal peptide sequence, an IRES sequence, a PURO sequence and an episome system to construct a recombinant expression vector; adding the recombinant expression vector into the culture suspension of the stem cells, and obtaining the stem cells capable of expressing the target protein by adopting an electrotransfection mode; aseptically extracting exosomes from the supernatant of the dry cells after electrotransfection, and adding 50 parts of the exosomes, 1-5 parts of glycerol, 1-5 parts of hyaluronic acid, 0.01-0.02 part of butanediol, 0.1-5 parts of Hamamelis virginiana extract, 0.1-2 parts of allantoin, 0.1-2 parts of tremella extract, 0.1-5 parts of mannitol, 1-5 parts of beta-glucose, 0.9 part of sodium chloride, 1-10 parts of pullulan, 1-10 parts of trehalose, 0.5-5 parts of mannitol, 1-10 parts of dextran, 1-5 parts of glucose, 0.5-10 parts of sodium lactate, 0.5-2 parts of potassium chloride, 5-10 parts of albumin and a proper amount of deionized water according to parts by weight, mixing and stirring; and (3) carrying out three-second instant freeze-drying dormancy on the mixed materials by adopting liquid nitrogen, and drying the mixed materials in a vacuum environment to sublimate and discharge the ice crystals in the freeze-dried tablets.
In the above technical solution, preferably, the recombinant expression vector includes two sequences of target proteins, and the target protein sequence 1 to be expressed and the target protein sequence 2 to be expressed are respectively disposed on the front side and the rear side of the T2A sequence.
In the above technical solution, preferably, the target protein includes oligopeptide-1, basic fibroblast, keratin, collagen, fibronectin, fibrillin, regenerated protein, transforming factor and insulin-like growth factor.
In the above technical solution, preferably, the stem cells isolated from the human tissue include mesenchymal stem cells, adipose-derived stem cells and epithelial stem cells, and the human tissue includes human umbilical cord tissue and adipose tissue.
In the above technical solution, preferably, the step of performing the electrotransfection mode comprises: 1-2 days before the electrotransfection is carried out, transferring the stem cells to a cell culture flask, so that the cell confluence of the stem cells before the electrotransfection is 50% -70%; the stem cells were slowly rinsed with PBS and aspiratedPBS, digesting the stem cells by pancreatin protease, and then adding a culture medium containing serum to neutralize pancreatin; the stem cells were collected by centrifugation and resuspended in PBS, hepes and serum-free medium to a density of 1X 10 6 ～5×10 6 cell/ml; setting electrotransfection parameters to be 2.0kV and 25 uFD; adding the plasmid of the recombinant expression vector into an electric rotating cup, and setting the initial concentration of the plasmid to be 10-50 mug/ml according to the type of the stem cells; adding the resuspended cells into an electric shock cup, turning the electric shock cup for uniformly mixing; putting the electric shock cup into an electric shock groove, and carrying out pulse electric shock for one time; transferring the shocked cells to a well containing a culture medium; and (3) shaking the pore plate to uniformly mix the cells, and carrying out electrotransformation for 24-48 hours to realize transient expression of cell genes.
In the above technical scheme, preferably, when the target protein is oligopeptide-1 and basic fibroblast, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-basic fibroblast-CD 33-IRES-PURO, episome ORI-episome EBNA 1; when the target protein is oligopeptide-1 and keratin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO and episome ORI-episome EBNA 1.
In the above technical solution, preferably, the step of isolating the mesenchymal stem cells from the human umbilical cord tissue comprises: separating to obtain human umbilical cord tissue blocks, and digesting for 5-10 minutes by using pancreatin; collecting the tissue suspension and centrifuging, discarding the supernatant, and placing the tissue block in a plate; adding a DMEM cell culture medium, and periodically changing the liquid by half; removing the tissue blocks after the cells grow over the plate; after trypsinization, transferring the primary cells on the plate to a T75 culture flask; and (5) when the cells grow over the culture bottle and passage is carried out to 2-3 generations, and the mesenchymal stem cells are obtained.
In the above technical solution, preferably, the step of isolating adipose stem cells from human adipose tissue comprises: flushing adipose tissues by using D-Hanks, removing residual blood cells and tissue fragments, and putting the adipose tissues into a centrifuge tube; adding equal volume of 0.1-0.5% type I collagenase into adipose tissue, and placing into 37 deg.C steam bath oscillatorDigesting for 30-60 min; when digestion is completed, the undigested adipose tissues are discarded, and the cell suspension is centrifuged and the supernatant is discarded; the adipose-derived stem cell pellet was obtained at the bottom of the centrifuge tube and resuspended to a density of 1X 10 5 mL -1 (ii) a Inoculating the cell suspension into a cell culture bottle, and culturing by using a DMEM (DMEM) culture medium until the cell suspension grows to the fusion degree of more than 80%; collecting culture solution, washing with PBS for three times, digesting with 0.125% -0.25% pancreatin, counting cells, inoculating into cell culture bottle, and culturing to obtain adipose-derived stem cells.
In the above technical scheme, preferably, after the mixed material is subjected to three-second instant freeze-drying dormancy by using liquid nitrogen, the material is dried for 5-7 hours in a vacuum environment by using an ultra-vacuum freeze-drying oven, so that ice crystals in the freeze-dried tablet are sublimated and discharged to obtain a loose, porous and water-disintegrable tablet, and the loose, porous and water-disintegrable tablet is packaged by using a double aluminum structure and then is stored in a dark place.
The invention also provides a product for efficiently expressing the exosome by the stem cell for skin beauty, which comprises a tablet prepared by the preparation method for efficiently expressing the exosome by the stem cell for skin beauty according to any one of the technical schemes.
Compared with the prior art, the invention has the following beneficial effects: the recombinant expression vector is transferred to the stem cell in an electrotransfection mode, so that a target protein exosome is extracted, and on the basis, a product for skin beautifying is prepared by using a freeze-drying technology, so that the active ingredients of the exosome efficiently expressed by the stem cell are effectively protected, and the prepared exosome freeze-dried sustained-release tablet can be stored for a long time at room temperature, and overcomes the defects of directional secretion, low long-time storage activity and the like of the exosome of the stem cell. In addition, a human promoter EF1A is adopted, CD33 signal peptide is fused at the N ends of oligopeptide-1 and basic fibroblast or other proteins, protein secretion is facilitated, the synergistic effect of two cytokines can be increased by adopting an IRES expression element, the two cytokines are expressed simultaneously, the transferred recombinant expression vector plasmid can exist in stem cells for a long time and efficiently express target proteins by adopting an episome system, and cells with high expression can be quickly obtained by adopting a PURO sequence screening marker, so that multiple required target proteins are directionally and efficiently expressed.
Drawings
Fig. 1 is a schematic flow chart of a preparation method for efficiently expressing exosomes by stem cells for skin beauty disclosed in an embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention is described in further detail below with reference to the attached drawing figures:
as shown in fig. 1, the preparation method of the high-efficiency stem cell expression exosome for skin beauty provided by the invention comprises the following steps: separating stem cells from human tissues and culturing; combining the PB transposon, an EF1A promoter sequence, a KOZAK sequence, a target protein sequence to be expressed, a T2A sequence, a target protein sequence to be expressed, a CD33 signal peptide sequence, an IRES sequence, a PURO sequence and an episome system to construct a recombinant expression vector; adding the recombinant expression vector into a culture suspension of the stem cells, and obtaining the stem cells capable of expressing the target protein by adopting an electrotransfection mode; the exosome is aseptically extracted from the supernatant of the stem cells after electrotransfection, and 50 parts of the exosome, 1-5 parts of glycerol, 1-5 parts of hyaluronic acid, 0.01-0.02 part of butanediol, 0.1-5 parts of hamamelis virginiana extract, 0.1-2 parts of allantoin, 0.1-2 parts of tremella extract, 0.1-5 parts of mannitol, 1-5 parts of beta-glucose, 0.9 part of sodium chloride, 1-10 parts of pullulan, 1-10 parts of trehalose, 0.5-5 parts of mannitol, 1-10 parts of dextran, 1-5 parts of glucose, 0.5-10 parts of sodium lactate, 0.5-2 parts of potassium chloride, 5-10 parts of albumin and a proper amount of deionized water are added according to the parts by weight for mixing and stirring; and (3) carrying out three-second instant freeze-drying dormancy on the mixed material by adopting liquid nitrogen, and drying in a vacuum environment to sublimate and discharge ice crystals in the freeze-dried tablets.
In the embodiment, the recombinant expression vector is transferred to the stem cell in an electrotransfection mode, so that the target protein exosome is extracted, and on the basis, a product for skin beautifying is prepared by using a freeze-drying technology, so that the active ingredients of the exosome efficiently expressed by the stem cell are effectively protected, and the prepared exosome freeze-dried sustained-release tablet can be stored at room temperature for a long time, and the defects of directional secretion, low long-time storage activity and the like of the stem cell exosome are overcome. In addition, a human promoter EF1A is adopted, CD33 signal peptide is fused at the N ends of oligopeptide-1 and basic fibroblast or other proteins, protein secretion is facilitated, the synergistic effect of two cytokines can be increased by adopting an IRES expression element, the two cytokines are expressed simultaneously, the transferred recombinant expression vector plasmid can exist in stem cells for a long time and efficiently express target proteins by adopting an episome system, and cells with high expression can be quickly obtained by adopting a PURO sequence screening marker, so that the required target proteins are directionally and efficiently expressed.
In the above embodiment, preferably, the recombinant expression vector includes two sequences of the target protein, and the sequence 1 of the target protein to be expressed and the sequence 2 of the target protein to be expressed are respectively disposed on the front side and the back side of the T2A sequence.
In the above embodiments, preferably, the protein of interest includes oligopeptide-1, basic fibroblast, keratin, collagen, fibronectin, fibrillin, neogenin, transforming factor and insulin-like growth factor.
In the above examples, preferably, when the target protein is oligopeptide-1 and basic fibrin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-basic fibrin-CD 33-IRES-PURO, episome ORI-episome EBNA 1.
In the above examples, preferably, when the target protein is oligopeptide-1 and keratin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO, episome ORI-episome EBNA 1.
In the above embodiment, preferably, the stem cells isolated from human tissue include mesenchymal stem cells, adipose stem cells and epithelial stem cells, and the human tissue includes human umbilical cord tissue and adipose tissue.
In the above embodiment, preferably, after the mixed material is subjected to three-second instant freeze-drying dormancy by using liquid nitrogen, the material is dried for 5 to 7 hours in a vacuum environment by using an ultra-vacuum freeze-drying oven, so that ice crystals in the freeze-dried tablet are sublimated and discharged, and the loose, porous and water-disintegrable tablet is obtained, and is packaged by using a double aluminum structure and then is stored in a dark place.
The product for skin beauty with the high-efficiency stem cell expression exosomes provided by the invention comprises the tablet prepared by the preparation method for skin beauty with the high-efficiency stem cell expression exosomes provided by any one of the embodiments.
The preparation method and the product of the high-efficiency expression exosome of the stem cell for skin beauty provided by the above embodiment are provided, and three embodiments are provided below to explain the above method in detail.
The first embodiment is as follows:
the invention provides a preparation method of high-efficiency expression oligopeptide-1 and basic fibroblast of human umbilical cord mesenchymal stem cells for skin beauty, which comprises the following steps:
1. isolating human umbilical cord mesenchymal stem cells from a human umbilical cord;
1) separating umbilical cord Wharton's jelly, and shearing the jelly into pieces;
2) digesting for 5-10 minutes by using 0.25% pancreatin;
3) collecting the tissue suspension, centrifuging for 2000 r and 10 min;
4) discarding the supernatant, and placing the tissue block in a 90mm plate;
5) adding 2-3 ml of DMEM cell culture medium, and changing the medium half every 2-3 days;
6) removing tissue blocks when the cells grow over the plate;
7) digesting with 0.25% pancreatin, and transferring the primary cells on the plate to a T75 culture flask;
8) after the cells grow over the culture bottle, passage is carried out to 2-3 generations;
2. constructing a recombinant expression vector PB-EF 1A-KOZAK-oligopeptide-1-T2A-alkali fibrin-CD 33-IRES-PURO, an episome ORI-episome EBNA 1;
3. and (3) performing electrotransfection on the human umbilical cord mesenchymal stem cells obtained in the step (1) and the recombinant expression vector obtained in the step (2) to obtain the human umbilical cord mesenchymal stem cells capable of efficiently expressing oligopeptide-1 and basic fibroblast:
suspension of cells:
1) 1-2 days before electroporation, cells were transferred to 75cm 2 Cell culture flasks with a cell confluency of about 50-70% before transformation, at which time the number of cells is about 2-10X 10 6 And each electrical revolution is about 1-10X 10 5 (ii) individual cells;
2) slowly washing cells with 12ml of PBS, sucking out the PBS, digesting the cells with 0.4ml of trypsin protease, adding 10ml of culture medium containing serum, and neutralizing the trypsin;
3) centrifuging to collect cells, resuspending the cells in PBS, Hepes, serum-free medium, etc. to density of 1-5 × 10 6 cell/ml；
And (3) electric conversion:
1) setting a power conversion program: 2.0KV, 25 uFD;
2) adding the linearized recombinant plasmid into an electric rotating cup, wherein the required plasmid is determined according to different cells, and the initial concentration is generally 10-50 ug/ml;
3) adding cells into the electric shock cup, and turning the electric shock cup to uniformly mix the cells;
4) putting the electric shock cup into an electric shock groove, and performing pulse electric shock once;
5) transferring the cells to wells already containing 0.5ml of medium;
6) gently shaking the pore plate, uniformly mixing the cells, and performing electric transformation for 24-48h to realize instantaneous gene expression;
7) detecting the activity of the cells after 24-48 h;
wherein, the HEPES buffer solution is: 10mm Hepes; pH 7.3; 40mm NaCl.
4. Sterile extraction of the culture supernatant obtained after electrotransfection:
1) centrifuging the culture supernatant at 300-400 g for 10-20 min, collecting the supernatant, and discarding the precipitate;
2) centrifuging the supernatant collected in the step a for 40-60 min at 2000-3000 g, continuously collecting the supernatant, and removing the precipitate;
3) filtering the supernatant collected in the step b by using a filter with the pore diameter of 0.22 mu m, and collecting filtrate;
4) ultrafiltering the supernatant with filter membrane of 30-60nm, adding PBS and ultrafiltering again, and repeating for 3-5 times;
5. adding 50 weight parts of the high-efficiency expression exosome extracted in the step 4, 1-5 weight parts of glycerol, 1-5 weight parts of hyaluronic acid, 0.01-0.02 weight part of butanediol, 0.1-5 weight parts of hamamelis virginiana extract, 0.1-2 weight parts of allantoin, 0.1-2 weight parts of tremella extract, 0.1-5 weight parts of mannitol and 1-5 weight parts of beta-glucose, 0.9 weight part of sodium chloride, 1-10 weight parts of pullulan, 1-10 weight parts of trehalose, 0.5-5 weight parts of mannitol, 1-10 weight parts of dextran, 1-5 weight parts of glucose, 0.5-10 weight parts of sodium lactate, 0.5-2 weight parts of potassium chloride and 5-10 weight parts of albumin, and deionized water is supplemented to the required amount.
6. After subpackaging, instantly freezing and dormancy at the temperature of liquid nitrogen-196 ℃ within three seconds, then drying the active substance for 6 hours in an ultra-vacuum freeze drying box under a vacuum environment, sublimating and discharging ice crystals contained in the freeze-dried tablets to finally obtain loose and porous tablets which can be disintegrated when meeting water, then packaging with a double-aluminum structure, and keeping out of the sun.
Example two:
the invention provides a preparation method for efficiently expressing oligopeptide-1 and basic fibroblast by adipose-derived stem cells and used for skin beauty, which comprises the following specific steps:
1. separating and extracting adipose stem cells from adipose tissues;
1) flushing adipose tissues by using D-Hanks, removing residual blood cells and tissue fragments, and putting the adipose tissues into a centrifuge tube;
2) adding equal volume of type I collagenase of 0.1-0.5% into the tissue, and sterilizing in a 37 deg.C steam bath oscillator for 30-60 min;
3) when digestion is completed, discarding upper undigested adipose tissues, centrifuging the lower cell suspension for 10min at a speed of 300-400 g, and discarding the supernatant;
4) obtaining the adipose-derived stem cell mass at the bottom of the centrifuge tube, resuspending the adipose-derived stem cell mass, and adjusting the density to 1X 10 5 mL -1 ；
5) Inoculating to culture area of 25cm 2 Culturing the cells in the cell culture bottle by using a DMEM medium until the cells grow to the fusion degree of more than 80%;
6) collecting culture solution, washing with PBS for three times, digesting with 0.125% -0.25% pancreatin, counting cells, inoculating to 3 culture areas of 75cm 2 The cell culture flask of (1);
2. constructing a recombinant expression vector PB-EF 1A-KOZAK-oligopeptide-1-T2A-alkali fibrin-CD 33-IRES-PURO, an episome ORI-episome EBNA 1;
3. and (3) performing electrotransfection on the adipose-derived stem cells obtained in the step (1) and the recombinant expression vector obtained in the step (2) to obtain adipose-derived stem cells capable of efficiently expressing oligopeptide-1 and basic fibroblast:
suspension of cells:
1) 1-2 days before electroporation, cells were transferred to 75cm 2 Cell culture flasks, with a confluency of cells of about 50-70% before transformation. The number of cells in this case is about 2 to 10X 10 6 And each electrical revolution is about 1-10X 10 5 (ii) individual cells;
2) slowly washing cells with 12ml PBS, sucking out PBS, digesting cells with 0.4ml pancreatin protease, adding 10ml culture medium containing serum, and neutralizing pancreatin;
3) centrifuging to collect cells, resuspending the cells with PBS, Hepes, serum-free medium, etc. to density of 1-5 × 10 6 cell/ml。
And (3) electric conversion:
1) setting a power conversion program: 2.0KV, 25 uFD;
2) adding the linearized recombinant plasmid into an electric rotating cup, wherein the required plasmid is determined according to different cells, and the initial concentration is 10-50ug/ml generally;
3) adding cells into the electric shock cup, and turning the electric shock cup to uniformly mix the cells;
4) putting the electric shock cup into an electric shock groove, and performing pulse electric shock once;
5) transferring the cells to wells already containing 0.5ml of medium;
6) gently shaking the pore plate, uniformly mixing the cells, and performing electric transformation for 24-48h to realize instantaneous gene expression;
7) detecting the activity of the cells after 24-48 h;
wherein the HEPES buffer solution is: 10mm Hepes; pH 7.3; 40mm NaCl.
4. Sterile extraction of the culture supernatant obtained after electrotransfection:
5) centrifuging the culture supernatant at 300-400 g for 10-20 min, collecting the supernatant, and removing the precipitate;
6) centrifuging the supernatant collected in the step a for 40-60 min at 2000-3000 g, continuously collecting the supernatant, and discarding the precipitate;
7) filtering the supernatant collected in the step b by using a filter with the pore diameter of 0.22 mu m, and collecting filtrate;
8) ultrafiltering the supernatant with filter membrane of 30-60nm, adding PBS and ultrafiltering again, and repeating for 3-5 times;
5. adding 50 weight parts of the high-efficiency expression exosome extracted in the step 4, 1-5 weight parts of glycerol, 1-5 weight parts of hyaluronic acid, 0.01-0.02 weight part of butanediol, 0.1-5 weight parts of hamamelis virginiana extract, 0.1-2 weight parts of allantoin, 0.1-2 weight parts of tremella extract, 0.1-5 weight parts of mannitol and 1-5 weight parts of beta-glucose, 0.9 part by weight of sodium chloride, 1-10 parts by weight of pullulan, 1-10 parts by weight of trehalose, 0.5-5 parts by weight of mannitol, 1-10 parts by weight of dextran, 1-5 parts by weight of glucose, 0.5-10 parts by weight of sodium lactate, 0.5-2 parts by weight of potassium chloride and 5-10 parts by weight of albumin, and deionized water is supplemented to the required amount.
6. After subpackaging, instantly freezing and dormancy at the temperature of liquid nitrogen-196 ℃ within three seconds, then drying the active substance for 6 hours in an ultra-vacuum freeze drying box under a vacuum environment, sublimating and discharging ice crystals contained in the freeze-dried tablets to finally obtain loose and porous tablets which can be disintegrated when meeting water, then packaging with a double-aluminum structure, and keeping out of the sun.
Example three:
the invention provides a preparation method for efficiently expressing oligopeptide-1 and keratin by human umbilical cord mesenchymal stem cells for skin beauty, which comprises the following specific steps:
1. isolating human umbilical cord mesenchymal stem cells from human umbilical cord;
1) separating umbilical cord Wharton's jelly, and shearing the jelly into pieces;
2) digesting for 5-10 minutes by using 0.25% pancreatin;
3) collecting the tissue suspension, centrifuging for 2000 r and 10 min;
4) discarding the supernatant, and placing the tissue block in a 90mm dish;
5) adding 2-3 ml of DMEM cell culture medium, and changing the culture medium half every 2-3 days;
6) removing tissue blocks when the cells grow over the plate;
7) digesting with 0.25% trypsin, and transferring the primary cells on the plate to a T75 culture flask;
8) after the cells grow over the culture bottle, passage is carried out to 2-3 generations;
2. constructing a recombinant expression vector PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO, an episome ORI-episome EBNA 1;
3. and (3) performing electrotransfection on the human umbilical cord mesenchymal stem cells obtained in the step (1) and the recombinant expression vector obtained in the step (2) to obtain the human umbilical cord mesenchymal stem cells capable of efficiently expressing oligopeptide-1 and keratin:
suspension of cells:
1) 1-2 days before electroporation, cells were transferred to 75cm 2 Cell culture flasks with a cell confluency of about 50-70% before transformation, at which time the number of cells is about 2-10X 10 6 And each electrical revolution is about 1-10X 10 5 (ii) individual cells;
2) slowly washing cells with 12ml PBS, sucking out PBS, digesting cells with 0.4ml pancreatin protease, adding 10ml culture medium containing serum, and neutralizing pancreatin;
3) centrifuging to collect cells, resuspending the cells with PBS, Hepes, serum-free medium, etcSo that it has a density of 1-5 x 10 6 cell/ml。
And (3) electric conversion:
1) setting a power conversion program: 2.0KV, 25 uFD;
2) adding the linearized recombinant plasmid into an electric rotating cup, wherein the required plasmid is determined according to different cells, and the initial concentration is generally 10-50 ug/ml;
3) adding cells into the electric shock cup, and turning the electric shock cup to uniformly mix the cells;
4) putting the electric shock cup into an electric shock groove, and performing pulse electric shock once;
5) transferring the cells to wells already containing 0.5ml of medium;
6) gently shaking the pore plate, uniformly mixing the cells, and performing electric transformation for 24-48h to realize instantaneous gene expression;
7) detecting the activity of the cells after 24-48 h;
wherein, the HEPES buffer solution is: 10mm Hepes; pH 7.3; 40mm NaCl.
4. And (3) performing sterile extraction on the culture supernatant obtained after electrotransfection:
1) centrifuging the culture supernatant at 300-400 g for 10-20 min, collecting the supernatant, and discarding the precipitate;
2) centrifuging the supernatant collected in the step a for 40-60 min at 2000-3000 g, continuously collecting the supernatant, and discarding the precipitate;
3) filtering the supernatant collected in the step b by using a filter with the pore diameter of 0.22 mu m, and collecting filtrate;
4) ultrafiltering the supernatant with filter membrane of 30-60nm, adding PBS and ultrafiltering again, and repeating for 3-5 times.
5. Adding 50 weight parts of the high-efficiency expression exosome extracted in the step 4, 1-5 weight parts of glycerol, 1-5 weight parts of hyaluronic acid, 0.01-0.02 weight part of butanediol, 0.1-5 weight parts of hamamelis virginiana extract, 0.1-2 weight parts of allantoin, 0.1-2 weight parts of tremella extract, 0.1-5 weight parts of mannitol and 1-5 weight parts of beta-glucose, 0.9 part by weight of sodium chloride, 1-10 parts by weight of pullulan, 1-10 parts by weight of trehalose, 0.5-5 parts by weight of mannitol, 1-10 parts by weight of dextran, 1-5 parts by weight of glucose, 0.5-10 parts by weight of sodium lactate, 0.5-2 parts by weight of potassium chloride and 5-10 parts by weight of albumin, and deionized water is supplemented to the required amount.
6. After subpackaging, instantly freezing and dormancy at the temperature of liquid nitrogen-196 ℃ within three seconds, then drying the active substance for 6 hours in an ultra-vacuum freeze drying box under a vacuum environment, sublimating and discharging ice crystals contained in the freeze-dried tablets to finally obtain loose and porous tablets which can be disintegrated when meeting water, then packaging with a double-aluminum structure, and keeping out of the sun.
According to the extraction and preparation method of the stem cell high-efficiency expression exosome for skin beauty provided by the embodiment, the extract is also suitable for liquid skin care products.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that the present invention can be applied to simultaneously express one target protein and a plurality of target proteins, and the constructed vector can be applied to expression of a plurality of stem cells. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Beijing Benzhen workshop Biotechnology Co., Ltd
<120> preparation method and product of high-efficiency expression exosome of stem cell for skin beauty
<160>11
<210> 1
<211>1182
<212>DNA
<213> unknown
<400> 1
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60
gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240
tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300
cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg 360
tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 420
tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct 480
gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 540
tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg 600
gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 660
cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 720
cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 780
ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 840
aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 900
ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 960
gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1020
atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1080
taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1140
acagtggttc aaagtttttt tcttccattt caggtgtcgt ga 1182
<210>2
<211>48
<212>DNA
<213> unknown
<400>2
atgcccctgc tgctgctgct gcccctgctg tgggccggcg ccctggcc 48
<210> 3
<211>6
<212>DNA
<213> unknown
<400> 3
gccacc 6
<210> 4
<211>465
<212>DNA
<213> Artificial sequence
<400> 4
atggcagccg ggagcatcac cacgctgccc gccttgcccg aggatggcgg cagcggcgcc 60
ttcccgcccg gccacttcaa ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120
ctgcgcatcc accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc 180
aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt gtgtgctaac 240
cgttacctgg ctatgaagga agatggaaga ttactggctt ctaaatgtgt tacggatgag 300
tgtttctttt ttgaacgatt ggaatctaat aactacaata cttaccggtc aaggaaatac 360
accagttggt atgtggcact gaaacgaact gggcagtata aacttggatc caaaacagga 420
cctgggcaga aagctatact ttttcttcca atgtctgcta agagc 465
<210>5
<211>54
<212>DNA
<213> unknown
<400>5
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54
<210>6
<211>574
<212>DNA
<213> unknown
<400>6
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataa 574
<210>7
<211>600
<212>DNA
<213> unknown
<400>7
atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 60
cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 120
cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180
atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 240
agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300
tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360
cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 420
agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 480
gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 540
gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 600
<210>8
<211>1790
<212>DNA
<213> unknown
<400>8
aacgggtagc atatgcttcc cgggtagtag tatatactat ccagactaac cctaattcaa 60
tagcatatgt tacccaacgg gaagcatatg ctatcgaatt agggttagta aaagggtcct 120
aaggaacagc gatatctccc accccatgag ctgtcacggt tttatttaca tggggtcagg 180
attccacgag ggtagtgaac cattttagtc acaagggcag tggctgaaga tcaaggagcg 240
ggcagtgaac tctcctgaat cttcgcctgc ttcttcattc tccttcgttt agctaataga 300
ataactgctg agttgtgaac agtaaggtgt atgtgaggtg ctcgaaaaca aggtttcagg 360
tgacgccccc agaataaaat ttggacgggg ggttcagtgg tggcattgtg ctatgacacc 420
aatataaccc tcacaaaccc cttgggcaat aaatactagt gtaggaatga aacattctga 480
atatctttaa caatagaaat ccatggggtg gggacaagcc gtaaagactg gatgtccatc 540
tcacacgaat ttatggctat gggcaacaca taatcctagt gcaatatgat actggggtta 600
ttaagatgtg tcccaggcag ggaccaagac aggtgaacca tgttgttaca ctctatttgt 660
aacaagggga aagagagtgg acgccgacag cagcggactc cactggttgt ctctaacacc 720
cccgaaaatt aaacggggct ccacgccaat ggggcccata aacaaagaca agtggccact 780
cttttttttg aaattgtgga gtgggggcac gcgtcagccc ccacacgccg ccctgcggtt 840
ttggactgta aaataagggt gtaataactt ggctgattgt aaccccgcta accactgcgg 900
tcaaaccact tgcccacaaa accactaatg gcaccccggg gaatacctgc ataagtaggt 960
gggcgggcca agataggggc gcgattgctg cgatctggag gacaaattac acacacttgc 1020
gcctgagcgc caagcacagg gttgttggtc ctcatattca cgaggtcgct gagagcacgg 1080
tgggctaatg ttgccatggg tagcatatac tacccaaata tctggatagc atatgctatc 1140
ctaatctata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc 1200
ctaatctata tctgggtagt atatgctatc ctaatttata tctgggtagc ataggctatc 1260
ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc 1320
ctaatctgta tccgggtagc atatgctatc ctaatagaga ttagggtagt atatgctatc 1380
ctaatttata tctgggtagc atatactacc caaatatctg gatagcatat gctatcctaa 1440
tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagcatag gctatcctaa 1500
tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa 1560
tttatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa 1620
tctatatctg ggtagtatat gctatcctaa tctgtatccg ggtagcatat gctatcctca 1680
tgcatataca gtcagcatat gatacccagt agtagagtgg gagtgctatc ctttgcatat 1740
gccgccacct cccaaggggg cgtgaatttt cgctgcttgt ccttttcctg 1790
<210>9
<211>1926
<212>DNA
<213> unknown
<400>9
tcactcctgc ccttcctcac cctcatctcc atcacctcct tcatctccgt catctccgtc 60
atcaccctcc gcggcagccc cttccaccat aggtggaaac cagggaggca aatctactcc 120
atcgtcaaag ctgcacacag tcaccctgat attgcaggta ggagcgggct ttgtcataac 180
aaggtcctta atcgcatcct tcaaaacctc agcaaatata tgagtttgta aaaagaccat 240
gaaataacag acaatggact cccttagcgg gccaggttgt gggccgggtc caggggccat 300
tccaaagggg agacgactca atggtgtaag acgacattgt ggaatagcaa gggcagttcc 360
tcgccttagg ttgtaaaggg aggtcttact acctccatat acgaacacac cggcgaccca 420
agttccttcg tcggtagtcc tttctacgtg actcctagcc aggagagctc ttaaaccttc 480
tgcaatgttc tcaaatttcg ggttggaacc tccttgacca cgatgctttc caaaccaccc 540
tccttttttg cgcctgcctc catcaccctg accccggggt ccagtgcttg ggccttctcc 600
tgggtcatct gcggggccct gctctatcgc tcccgggggc acgtcaggct caccatctgg 660
gccaccttct tggtggtatt caaaataatc ggcttcccct acagggtgga aaaatggcct 720
tctacctgga gggggcctgc gcggtggaga cccggatgat gatgactgac tactgggact 780
cctgggcctc ttttctccac gtccacgacc tctccccctg gctctttcac gacttccccc 840
cctggctctt tcacgtcctc taccccggcg gcctccacta cctcctcgac cccggcctcc 900
actacctcct cgaccccggc ctccactgcc tcctcgaccc cggcctccac ctcctgctcc 960
tgcccctcct gctcctgccc ctcctcctgc tcctgcccct cctgcccctc ctgctcctgc 1020
ccctcctgcc cctcctgctc ctgcccctcc tgcccctcct gctcctgccc ctcctgcccc 1080
tcctcctgct cctgcccctc ctgcccctcc tcctgctcct gcccctcctg cccctcctgc 1140
tcctgcccct cctgcccctc ctgctcctgc ccctcctgcc cctcctgctc ctgcccctcc 1200
tgctcctgcc cctcctgctc ctgcccctcc tgctcctgcc cctcctgccc ctcctgcccc 1260
tcctcctgct cctgcccctc ctgctcctgc ccctcctgcc cctcctgccc ctcctgctcc 1320
tgcccctcct cctgctcctg cccctcctgc ccctcctgcc cctcctcctg ctcctgcccc 1380
tcctgcccct cctcctgctc ctgcccctcc tcctgctcct gcccctcctg cccctcctgc 1440
ccctcctcct gctcctgccc ctcctgcccc tcctcctgct cctgcccctc ctcctgctcc 1500
tgcccctcct gcccctcctg cccctcctcc tgctcctgcc cctcctcctg ctcctgcccc 1560
tcctgcccct cctgcccctc ctgcccctcc tcctgctcct gcccctcctc ctgctcctgc 1620
ccctcctgct cctgcccctc ccgctcctgc tcctgctcct gttccaccgt gggtcccttt 1680
gcagccaatg caacttggac gtttttgggg tctccggaca ccatctctat gtcttggccc 1740
tgatcctgag ccgcccgggg ctcctggtct tccgcctcct cgtcctcgtc ctcttccccg 1800
tcctcgtcca tggttatcac cccctcttct ttgaggtcca ctgccgccgg agccttctgg 1860
tccagatgtg tctcccttct ctcctaggcc atttccaggt cctgtacctg gcccctcgtc 1920
agacat 1926
<210>10
<211>468
<212>DNA
<213> Artificial sequence
<400>10
atggcagccg ggagcatcac cacgctgccc gccttgcccg aggatggcgg cagcggcgcc 60
ttcccgcccg gccacttcaa ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120
ctgcgcatcc accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc 180
aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt gtgtgctaac 240
cgttacctgg ctatgaagga agatggaaga ttactggctt ctaaatgtgt tacggatgag 300
tgtttctttt ttgaacgatt ggaatctaat aactacaata cttaccggtc aaggaaatac 360
accagttggt atgtggcact gaaacgaact gggcagtata aacttggatc caaaacagga 420
cctgggcaga aagctatact ttttcttcca atgtctgcta agagctga 468
<210>11
<211>546
<212>DNA
<213> Artificial sequence
<400> 11
atggggaaaa tcagcagtct tccaactcaa ttatttaaga tctgcctctg tgacttcttg 60
aagataaaga tacacatcat gtcgtcttca catctcttct acctggcact ctgcttgctc 120
acctttacca gctcggccac agccggacca gagacccttt gcggggctga gctggtggac 180
gctcttcagt tcgtgtgtgg accaaggggc ttttacttca acaagcccac aggctatggc 240
tccagcattc ggagggcacc acagacgggc attgtggatg agtgttgctt ccggagctgt 300
gatctgagga ggctggagat gtactgtgct ccgctgaagc ctacaaagtc agctcgttcc 360
atccgggccc agcgccacac tgacatgccc aagactcaga agtcccagcc cctatcgaca 420
cacaagaaaa ggaagctgca aaggagaagg aaaggtgagt caaaggcaca cccaggaggg 480
gaacaggagg agggagcaga ggcaactcag aaaatcagag gtgacagaga aaggaggccg 540
agctag 546
Claims (8)
1. A preparation method of a stem cell high-efficiency expression exosome for skin beauty is characterized by comprising the following steps:
separating stem cells from human tissues and culturing;
combining the PB transposon, an EF1A promoter sequence, a KOZAK sequence, a target protein sequence to be expressed, a T2A sequence, a target protein sequence to be expressed, a CD33 signal peptide sequence, an IRES sequence, a PURO sequence and an episome system to construct a recombinant expression vector; the target protein sequence 1 to be expressed and the target protein sequence 2 to be expressed are respectively arranged on the front side and the rear side of the T2A sequence, and the target proteins comprise oligopeptide-1, basic fibroblast, keratin, collagen, fibronectin, fibrillin, regenerated protein, transforming factor and insulin-like growth factor;
adding the recombinant expression vector into the culture suspension of the stem cells, and obtaining the stem cells capable of expressing the target protein by adopting an electrotransfection mode;
aseptically extracting exosomes from the supernatant of the dry cells after electrotransfection, adding 50 parts of the exosomes, 1-5 parts of glycerol, 1-5 parts of hyaluronic acid, 0.01-0.02 part of butanediol, 0.1-5 parts of Hamamelis virginiana extract, 0.1-2 parts of allantoin, 0.1-2 parts of tremella extract, 0.1-5 parts of mannitol, 1-5 parts of beta-glucose, 0.9 part of sodium chloride, 1-10 parts of pullulan, 1-10 parts of trehalose, 0.5-5 parts of mannitol, 1-10 parts of dextran, 1-5 parts of glucose, 0.5-10 parts of sodium lactate, 0.5-2 parts of potassium chloride, 5-10 parts of albumin and a proper amount of deionized water according to parts by weight, mixing and stirring;
and (3) carrying out three-second instant freeze-drying dormancy on the mixed materials by adopting liquid nitrogen, and drying the mixed materials in a vacuum environment to sublimate and discharge the ice crystals in the freeze-dried tablets.
2. The method for preparing the exosome with high stem cell expression efficiency for skin beauty according to claim 1, wherein the stem cells separated from the human tissue comprise mesenchymal stem cells, adipose-derived stem cells and epithelial stem cells, and the human tissue comprises human umbilical cord tissue and adipose tissue.
3. The method for preparing the high-expression exosome for skin beauty according to claim 1, wherein the step of implementing the electrotransfection mode comprises the following steps:
1-2 days before the electrotransfection is carried out, transferring the stem cells to a cell culture flask, so that the cell confluence of the stem cells before the electrotransfection is 50% -70%;
slowly flushing the stem cells by using PBS, sucking out the PBS, digesting the stem cells by using pancreatin protease, and adding a culture medium containing serum to neutralize pancreatin;
the stem cells were harvested by centrifugation and resuspended at a density of 1X 10 using PBS, hepes and serum-free medium 6 ～5×10 6 cell/ml；
Setting electrotransfection parameters to be 2.0kV and 25 uFD;
adding the linearized plasmid of the recombinant expression vector into an electric rotor, and setting the initial concentration of the plasmid to be 10-50 mu g/ml according to the type of the stem cells;
adding the resuspended cells into an electric shock cup, turning the electric shock cup upside down and mixing uniformly;
putting the electric shock cup into an electric shock groove, and carrying out primary pulse electric shock;
transferring the shocked cells to a well containing a culture medium;
and (4) shaking the pore plate to uniformly mix the cells, and performing electrotransformation for 24-48 hours to realize instantaneous expression of cell genes.
4. The method for preparing the exosome for skin beauty according to claim 1, wherein when the target protein is oligopeptide-1 and basic fibroblast, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-basic fibroblast-CD 33-IRES-PURO, episome ORI-episome EBNA 1;
when the target protein is oligopeptide-1 and keratin, the recombinant expression vector is PB-EF 1A-KOZAK-oligopeptide-1-T2A-keratin-CD 33-IRES-PURO, and episome ORI-episome EBNA 1.
5. The method for preparing the exosome with high expression stem cell for skin beauty according to claim 2, wherein the step of separating the mesenchymal stem cell from the human umbilical cord tissue comprises the following steps:
separating to obtain human umbilical cord tissue blocks, and digesting for 5-10 minutes by using pancreatin;
collecting the tissue suspension, centrifuging, removing supernatant, and placing the tissue block in a plate;
adding a DMEM cell culture medium, and periodically changing the liquid by half;
removing the tissue mass after the cells grow over the plate;
after trypsinization, transferring the primary cells on the plate to a T75 culture flask;
and (5) when the cells grow over the culture bottle and passage is carried out to 2-3 generations, and the mesenchymal stem cells are obtained.
6. The method for preparing exosome efficiently expressed by stem cells for skin beauty according to claim 2, wherein the step of separating adipose stem cells from human adipose tissue comprises:
flushing adipose tissues by using D-Hanks, removing residual blood cells and tissue fragments, and putting the adipose tissues into a centrifuge tube;
adding equal volume of type I collagenase of 0.1-0.5% into adipose tissue, and digesting in 37 deg.C steam bath oscillator for 30-60 min;
when digestion is completed, the undigested adipose tissues are discarded, and the cell suspension is centrifuged and the supernatant is discarded;
the adipose-derived stem cell pellet was obtained at the bottom of the centrifuge tube and resuspended to a density of 1X 10 5 mL -1 ；
Inoculating the cell suspension into a cell culture bottle, and culturing by using a DMEM (DMEM) culture medium until the cell suspension grows to the fusion degree of more than 80%;
collecting culture solution, washing with PBS for three times, digesting with 0.125% -0.25% pancreatin, counting cells, inoculating into cell culture bottle, and culturing to obtain adipose-derived stem cells.
7. The method for preparing the exosome with the high expression stem cell for skin beauty according to claim 1, is characterized in that after the mixed material is subjected to three-second instant freeze-drying dormancy by using liquid nitrogen, the material is dried for 5-7 hours in a vacuum environment by using an ultra-vacuum freeze-drying box, so that ice crystals in the freeze-dried tablet are sublimated and discharged to obtain a loose and porous tablet disintegrating in water, and the loose and porous tablet is packaged by using a double-aluminum structure and then is stored in a dark place.
8. A product for beautifying skin by using the stem cell high-efficiency expression exosome, which comprises a tablet prepared by the preparation method for beautifying skin by using the stem cell high-efficiency expression exosome according to any one of claims 1 to 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010296426.1A CN111437244B (en) | 2020-04-15 | 2020-04-15 | Preparation method and product of high-efficiency stem cell expression exosome for skin beauty |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010296426.1A CN111437244B (en) | 2020-04-15 | 2020-04-15 | Preparation method and product of high-efficiency stem cell expression exosome for skin beauty |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111437244A CN111437244A (en) | 2020-07-24 |
| CN111437244B true CN111437244B (en) | 2022-08-23 |
Family
ID=71653064
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010296426.1A Active CN111437244B (en) | 2020-04-15 | 2020-04-15 | Preparation method and product of high-efficiency stem cell expression exosome for skin beauty |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111437244B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029317A (en) * | 2021-02-23 | 2022-09-09 | 赛浦生物科技(长春)有限公司 | Nasal spray for allergic rhinitis and preparation method thereof |
| CN113304097A (en) * | 2021-05-27 | 2021-08-27 | 上海南滨江细胞生物科技有限公司 | Essence containing stem cell exosomes and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107854418A (en) * | 2017-11-10 | 2018-03-30 | 南京九圣生物科技股份有限公司 | A kind of human umbilical cord mesenchymal stem cells excretion external use creams and preparation method thereof |
| CN108633877A (en) * | 2018-05-17 | 2018-10-12 | 广东芙金干细胞再生医学有限公司 | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation |
| CN108721200A (en) * | 2018-06-07 | 2018-11-02 | 武汉赛云博生物科技有限公司 | A kind of preparation method and application of the excretion body cosmetic formulation in human mesenchymal stem cell source |
| CN109097336A (en) * | 2018-09-07 | 2018-12-28 | 深圳市新仑生物科技有限公司 | A kind of stem cell excretion body, preparation method and application |
| KR20190069277A (en) * | 2017-12-11 | 2019-06-19 | 주식회사 엑소코바이오 | A composition comprising an exosome and/or extracellular vesicle derived from stem cell as an active ingredient and its application for preventing, suppressing, alleviating or improving sensitive skin |
| CN110548001A (en) * | 2019-09-06 | 2019-12-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes |
-
2020
- 2020-04-15 CN CN202010296426.1A patent/CN111437244B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107854418A (en) * | 2017-11-10 | 2018-03-30 | 南京九圣生物科技股份有限公司 | A kind of human umbilical cord mesenchymal stem cells excretion external use creams and preparation method thereof |
| KR20190069277A (en) * | 2017-12-11 | 2019-06-19 | 주식회사 엑소코바이오 | A composition comprising an exosome and/or extracellular vesicle derived from stem cell as an active ingredient and its application for preventing, suppressing, alleviating or improving sensitive skin |
| CN108633877A (en) * | 2018-05-17 | 2018-10-12 | 广东芙金干细胞再生医学有限公司 | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation |
| CN108721200A (en) * | 2018-06-07 | 2018-11-02 | 武汉赛云博生物科技有限公司 | A kind of preparation method and application of the excretion body cosmetic formulation in human mesenchymal stem cell source |
| CN109097336A (en) * | 2018-09-07 | 2018-12-28 | 深圳市新仑生物科技有限公司 | A kind of stem cell excretion body, preparation method and application |
| CN110548001A (en) * | 2019-09-06 | 2019-12-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111437244A (en) | 2020-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111437244B (en) | Preparation method and product of high-efficiency stem cell expression exosome for skin beauty | |
| WO2010040262A1 (en) | Methods for isolating animal embryonic mesenchymal stem cells and extracting secretion substance thereof | |
| CN111826345A (en) | Human umbilical cord mesenchymal stem cell source exosome preparation | |
| CN113088487B (en) | Mesenchymal stem cell adipogenic transformation inhibitor | |
| CN108220230A (en) | A kind of separation of human adipose-derived stem cell and cultural method | |
| CN112410304A (en) | Gene-modified exosome and preparation method and application thereof | |
| CN104587447A (en) | Application of IFITM3 (interferon induced transmembrane protein 3) packaging exosome to preparation of dengue virus infection prevention medicine | |
| CN110684681B (en) | Engineering bacterium for producing chicken alpha-interferon and application thereof | |
| CN113171332A (en) | Exosome freeze-dried powder, preparation method thereof and skin care product | |
| CN116790490A (en) | Collagen-combined exosome, exosome freeze-dried powder and preparation method | |
| CN110241128B (en) | Fusion gene containing CBD, cell line, liquid ECM and application | |
| CN106606512B (en) | Mixed cell preparation for treating myocardial infarction and preparation method and application thereof | |
| CN111518774B (en) | Method and reagent for improving stress tolerance of synovial membrane mesenchymal stem cells | |
| CN116082494A (en) | Recombinant human III type collagen polypeptide, expression vector, expression strain and construction method thereof | |
| CN111440821A (en) | Preparation method and application method of stem cell for efficiently expressing target protein | |
| CN111484978A (en) | miR-140-5p overexpression modified human umbilical cord mesenchymal stem cell, and treatment preparation, preparation method and application thereof | |
| CN111718898B (en) | Method and reagent for improving stress tolerance of synovial membrane mesenchymal stem cells | |
| CN112941106B (en) | Method for delaying mesenchymal stem cell senescence through FOXP1 gene editing and mutation | |
| CN110218699B (en) | Rapid culture and differentiation method of adipose-derived stem cells | |
| CN113632784A (en) | Stem cell cryopreservation protective agent and application method thereof | |
| CN113925877A (en) | Application of gene-enhanced immune cells in lung cancer | |
| CN114164178A (en) | Traceable umbilical cord mesenchymal stem cells and preparation method and application thereof | |
| CN111500607A (en) | Fusion gene for peri-implant anti-inflammation, exosome, biological coating, preparation method and application | |
| CN101914488B (en) | Method for induced differentiation of human amniotic mesenchymal cells into insulin secreting cells | |
| CN117886922B (en) | Recombinant human fibronectin and expression system 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |