CN115282065B - Freeze-dried powder containing mesenchymal stem cell exosomes and preparation method and application thereof - Google Patents
Freeze-dried powder containing mesenchymal stem cell exosomes and preparation method and application thereof Download PDFInfo
- Publication number
- CN115282065B CN115282065B CN202210964131.6A CN202210964131A CN115282065B CN 115282065 B CN115282065 B CN 115282065B CN 202210964131 A CN202210964131 A CN 202210964131A CN 115282065 B CN115282065 B CN 115282065B
- Authority
- CN
- China
- Prior art keywords
- stem cell
- mesenchymal stem
- skin
- adipose
- peptide
- 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
- 210000001808 exosome Anatomy 0.000 title claims abstract description 144
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 98
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000000843 powder Substances 0.000 title abstract description 18
- 239000000203 mixture Substances 0.000 claims abstract description 53
- 239000004005 microsphere Substances 0.000 claims abstract description 50
- 210000000130 stem cell Anatomy 0.000 claims abstract description 38
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 34
- 239000002244 precipitate Substances 0.000 claims abstract description 22
- 239000002158 endotoxin Substances 0.000 claims abstract description 19
- 229920006008 lipopolysaccharide Polymers 0.000 claims abstract description 19
- 230000037394 skin elasticity Effects 0.000 claims abstract description 19
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000706 filtrate Substances 0.000 claims abstract description 14
- 210000002966 serum Anatomy 0.000 claims abstract description 14
- 239000000661 sodium alginate Substances 0.000 claims abstract description 14
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 14
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 14
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 10
- 239000003995 emulsifying agent Substances 0.000 claims abstract description 10
- 238000005530 etching Methods 0.000 claims abstract description 9
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 claims abstract description 8
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 claims abstract description 8
- 239000008157 edible vegetable oil Substances 0.000 claims abstract description 8
- 235000021283 resveratrol Nutrition 0.000 claims abstract description 8
- 229940016667 resveratrol Drugs 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 45
- 238000013268 sustained release Methods 0.000 claims description 37
- 239000012730 sustained-release form Substances 0.000 claims description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 25
- 238000002156 mixing Methods 0.000 claims description 24
- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 claims description 18
- 239000003921 oil Substances 0.000 claims description 17
- 235000019198 oils Nutrition 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 108010081589 Becaplermin Proteins 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 14
- 108010017843 platelet-derived growth factor A Proteins 0.000 claims description 14
- 239000012528 membrane Substances 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- 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 12
- 229920002674 hyaluronan Polymers 0.000 claims description 12
- 229960003160 hyaluronic acid Drugs 0.000 claims description 12
- 239000008176 lyophilized powder Substances 0.000 claims description 11
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 claims description 10
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 10
- 108010016626 Dipeptides Proteins 0.000 claims description 10
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 10
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 10
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 claims description 10
- 229940106189 ceramide Drugs 0.000 claims description 10
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 claims description 10
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 claims description 10
- 229960000271 arbutin Drugs 0.000 claims description 9
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- 238000011282 treatment Methods 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 229930003270 Vitamin B Natural products 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 7
- 235000019156 vitamin B Nutrition 0.000 claims description 7
- 239000011720 vitamin B Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 230000001640 apoptogenic effect Effects 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- 238000004945 emulsification Methods 0.000 claims description 6
- 239000000839 emulsion Substances 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 5
- 238000001723 curing Methods 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 5
- -1 sucrose fatty acid ester Chemical class 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 5
- 238000009461 vacuum packaging Methods 0.000 claims description 5
- 230000006378 damage Effects 0.000 claims description 4
- 230000004054 inflammatory process Effects 0.000 claims description 4
- DNISEZBAYYIQFB-PHDIDXHHSA-N (2r,3r)-2,3-diacetyloxybutanedioic acid Chemical compound CC(=O)O[C@@H](C(O)=O)[C@H](C(O)=O)OC(C)=O DNISEZBAYYIQFB-PHDIDXHHSA-N 0.000 claims description 3
- 206010061218 Inflammation Diseases 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 230000007815 allergy Effects 0.000 claims description 3
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 3
- 239000000194 fatty acid Substances 0.000 claims description 3
- 229930195729 fatty acid Natural products 0.000 claims description 3
- ODFAPIRLUPAQCQ-UHFFFAOYSA-M sodium stearoyl lactylate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC(=O)OC(C)C(=O)OC(C)C([O-])=O ODFAPIRLUPAQCQ-UHFFFAOYSA-M 0.000 claims description 3
- 229940080352 sodium stearoyl lactylate Drugs 0.000 claims description 3
- 239000003549 soybean oil Substances 0.000 claims description 3
- 235000012424 soybean oil Nutrition 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 206010020751 Hypersensitivity Diseases 0.000 claims description 2
- 235000019483 Peanut oil Nutrition 0.000 claims description 2
- 208000026935 allergic disease Diseases 0.000 claims description 2
- 239000000312 peanut oil Substances 0.000 claims description 2
- 208000010201 Exanthema Diseases 0.000 claims 1
- 201000005884 exanthem Diseases 0.000 claims 1
- 206010037844 rash Diseases 0.000 claims 1
- 231100000046 skin rash Toxicity 0.000 claims 1
- 230000008439 repair process Effects 0.000 abstract description 13
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 8
- 208000032544 Cicatrix Diseases 0.000 abstract description 6
- 206010029113 Neovascularisation Diseases 0.000 abstract description 6
- 208000027418 Wounds and injury Diseases 0.000 abstract description 6
- 230000008595 infiltration Effects 0.000 abstract description 6
- 238000001764 infiltration Methods 0.000 abstract description 6
- 210000004969 inflammatory cell Anatomy 0.000 abstract description 6
- 208000014674 injury Diseases 0.000 abstract description 6
- 231100000241 scar Toxicity 0.000 abstract description 6
- 230000037387 scars Effects 0.000 abstract description 6
- 230000037303 wrinkles Effects 0.000 abstract description 6
- 206010070834 Sensitisation Diseases 0.000 abstract description 5
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 abstract description 4
- 229910001424 calcium ion Inorganic materials 0.000 abstract description 4
- 230000001804 emulsifying effect Effects 0.000 abstract description 2
- 238000007711 solidification Methods 0.000 abstract description 2
- 230000008023 solidification Effects 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 59
- 210000003491 skin Anatomy 0.000 description 48
- 239000008055 phosphate buffer solution Substances 0.000 description 27
- 230000000052 comparative effect Effects 0.000 description 23
- 241000700159 Rattus Species 0.000 description 19
- 230000000694 effects Effects 0.000 description 15
- 210000002950 fibroblast Anatomy 0.000 description 14
- 230000002829 reductive effect Effects 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 230000002500 effect on skin Effects 0.000 description 13
- 230000035755 proliferation Effects 0.000 description 12
- 230000008859 change Effects 0.000 description 10
- 230000001737 promoting effect Effects 0.000 description 10
- 230000001965 increasing effect Effects 0.000 description 9
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 8
- 230000012010 growth Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 102000008186 Collagen Human genes 0.000 description 6
- 108010035532 Collagen Proteins 0.000 description 6
- 230000033115 angiogenesis Effects 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 229920001436 collagen Polymers 0.000 description 6
- 230000006872 improvement Effects 0.000 description 6
- 230000005012 migration Effects 0.000 description 6
- 238000013508 migration Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 206010013786 Dry skin Diseases 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000002870 angiogenesis inducing agent Substances 0.000 description 4
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 210000004927 skin cell Anatomy 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- 230000017423 tissue regeneration Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 201000004624 Dermatitis Diseases 0.000 description 3
- 239000000232 Lipid Bilayer Substances 0.000 description 3
- 229930003571 Vitamin B5 Natural products 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 3
- 229960002079 calcium pantothenate Drugs 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000003020 moisturizing effect Effects 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 210000000434 stratum corneum Anatomy 0.000 description 3
- 235000009492 vitamin B5 Nutrition 0.000 description 3
- 239000011675 vitamin B5 Substances 0.000 description 3
- 230000002087 whitening effect Effects 0.000 description 3
- 206010015150 Erythema Diseases 0.000 description 2
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 2
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 2
- 108010016160 Matrix Metalloproteinase 3 Proteins 0.000 description 2
- 102000003945 NF-kappa B Human genes 0.000 description 2
- 108010057466 NF-kappa B Proteins 0.000 description 2
- 235000014676 Phragmites communis Nutrition 0.000 description 2
- 102000000583 SNARE Proteins Human genes 0.000 description 2
- 108010041948 SNARE Proteins Proteins 0.000 description 2
- 102000004495 STAT3 Transcription Factor Human genes 0.000 description 2
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 2
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 2
- 229960004373 acetylcholine Drugs 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 230000001772 anti-angiogenic effect Effects 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000002222 downregulating effect Effects 0.000 description 2
- 210000004177 elastic tissue Anatomy 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 210000001097 facial muscle Anatomy 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000009499 grossing Methods 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000035992 intercellular communication Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000004530 micro-emulsion Substances 0.000 description 2
- 230000011278 mitosis Effects 0.000 description 2
- 230000004118 muscle contraction Effects 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000003076 paracrine Effects 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 230000001023 pro-angiogenic effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 230000008591 skin barrier function Effects 0.000 description 2
- 230000036548 skin texture Effects 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 208000037816 tissue injury Diseases 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 230000007730 Akt signaling Effects 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100025222 CD63 antigen Human genes 0.000 description 1
- 102100027221 CD81 antigen Human genes 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102000001187 Collagen Type III Human genes 0.000 description 1
- 108010069502 Collagen Type III Proteins 0.000 description 1
- 101100317380 Danio rerio wnt4a gene Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 1
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 1
- 238000003231 Lowry assay Methods 0.000 description 1
- 238000009013 Lowry's assay Methods 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 241000927586 Marsupenaeus Species 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 101150010310 WNT-4 gene Proteins 0.000 description 1
- 102000052548 Wnt-4 Human genes 0.000 description 1
- 108700020984 Wnt-4 Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000001153 anti-wrinkle effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 210000003074 dental pulp Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 208000023589 ischemic disease Diseases 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 235000021388 linseed oil Nutrition 0.000 description 1
- 239000000944 linseed oil Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 108010000685 platelet-derived growth factor AB Proteins 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 208000037922 refractory disease Diseases 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000036573 scar formation Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 235000020238 sunflower seed Nutrition 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 210000000106 sweat gland Anatomy 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
-
- 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
-
- 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
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0241—Containing particulates characterized by their shape and/or structure
- A61K8/025—Explicitly spheroidal or spherical shape
-
- 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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/67—Vitamins
- A61K8/673—Vitamin B 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
- A61K8/68—Sphingolipids, e.g. ceramides, cerebrosides, gangliosides
-
- 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
- A61K8/99—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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/90—Polysaccharides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Birds (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Developmental Biology & Embryology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Rheumatology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Dispersion Chemistry (AREA)
- Pain & Pain Management (AREA)
- Hematology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a freeze-dried powder containing mesenchymal stem cell exosomes, and a preparation method and application thereof, and belongs to the technical field of medical cosmetology. And (3) subculturing the rat adipose-derived stem cells, adding a DMEM culture solution containing hydrogen peroxide, lipopolysaccharide, a platelet-derived factor composition and exosome-free serum for culture, then etching films through different tracks, centrifuging the filtrate, re-suspending the precipitate in PBS, adding an emulsifying agent and sodium alginate, adding edible oil containing resveratrol, emulsifying, dropwise adding a calcium ion solution for solidification, and drying to obtain the slow-release microspheres containing the adipose-derived stem cell exosome. The freeze-dried powder disclosed by the invention can reduce inflammatory cell infiltration of damaged skin, increase neovascularization, promote skin repair, restore and improve skin elasticity, inhibit generation of skin scars, pockmarks and the like, restore skin elasticity, smooth fine wrinkles, whiten skin and the like, improve the anti-injury, anti-inflammatory and anti-sensitization capabilities of the skin, and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of medical cosmetology, in particular to freeze-dried powder containing mesenchymal stem cell exosomes, and a preparation method and application thereof.
Background
Exosomes (exosomes) are small vesicles capable of being secreted by most cells, approximately 30-150nm in diameter, with a lipid bilayer membrane structure, carrying RNA and proteins, released by most cell types, as part of the intercellular communication system. Almost all cells secrete extracellular vesicles, including mesenchymal stem cells, which are derived from mesenchymal stem cells and contain components similar to those of the parent cell, such as cytokines, growth factors, lipids, mRNAs, regulatory miRNAs, etc. They are mainly involved in cell-to-cell communication, change the microenvironment of tissues, and the like, and play a role in immune regulation, tissue injury repair, and the like, which cannot be underestimated.
Mesenchymal Stem Cells (MSCs) are matrix-derived adult stem cells, which are mainly present in connective tissue such as bone marrow, dental pulp, fat, umbilical cord, and organ interstitium. The exosomes secreted by stem cells are homogeneous vesicles with the diameter of 30-150nm, are wrapped by lipid bilayer which is the same as cytoplasmic membranes, and contain bioactive substances such as proteins, RNA and the like. Mesenchymal Stem Cells (MSCs) function in a paracrine manner by a mechanism that stem cells function in tissue repair by producing large amounts of cytokines and growth factors, as well as exosomes. At present, stem cells are widely applied to the field of tissue repair and ischemic disease treatment. Research shows that the exosomes secreted by the stem cells can simulate the biological functions of the stem cells, and are also gradually proved to promote tissue repair and treat some refractory diseases, thereby having wide application prospect. Compared with stem cells, the stem cell exosomes which are paracrine substances have more stable biological properties, and researches show that the exosomes can maintain the biological functions after being stored for 2 years at-80 ℃. In vivo experiments, lipid bilayer structure of exosomes can prevent degradation of content, keep activity of internal proteins and genetic materials, and unlike soluble cytokines secreted by stem cells, exosomes can directly enter target cells, and biological changes such as proliferation, migration, vascularization and the like of the target cells are induced by transferring bioactive substances such as specific proteins, lipids, RNAs and the like to the target cells, so that local microenvironment is changed, and various biological functions are stably and permanently exerted. The carrier type signal transduction mode ensures that the exosome has higher and more stable signal transduction efficiency and cannot be diluted by extracellular medium in organisms.
More and more researches show that stem cell exosomes can reduce apoptosis, reduce inflammatory reaction, promote angiogenesis, inhibit fibrosis, improve tissue repair potential and other important biological effects. The effects of exosomes lead to good application prospects in the treatment of tissue injuries such as myocardial infarction, skin wounds and the like, and have been studied intensively. Compared with stem cell transplantation treatment, the stem cell exosome can reduce the risk of cell transplantation tumorigenesis, so that the stem cell exosome can promote angiogenesis and strengthen damaged cell repair, and is expected to become a new treatment strategy for tissue damage repair. The unique biological advantages of exosomes also open a new idea for the development of future drug carriers. Scientists believe that exosome therapy using stem cells is safer and more operable than stem cells. Because the stem cell exosomes have certain characteristics as therapeutic products that stem cell preparations do not possess, for example, the exosomes are relatively more defined in composition than live cells, and are easy to control quality; and the stability of the exosomes in an in-vitro environment is more beneficial to transportation and storage. Therefore, the characteristics lead the exosome to have great application value.
The skin is covered on the surface of the body and is rich in blood vessels, nerves, sweat glands and other tissues. The skin is used as a first natural barrier to directly contact with the outside, so that the external stimulus such as microbial invasion, ultraviolet injury and the like can be prevented. With the excessive use of cosmetics, many people have symptoms of thinning of facial skin horny layer, easy allergy, redness and swelling, easy damage and the like, however, no effective repairing product exists on the market at present. The exosomes belong to natural liposome and have good biocompatibility with human body. The exosome has a diameter of 30-100nm, is a vesicle secreted by cells, can be directly taken up by target cells, and can be widely involved in intercellular communication. However, the exosomes are various in species, and the components and action mechanisms are currently unknown. Recent studies have found that mesenchymal stem cell exosomes expressing CD63, CD81 are capable of promoting b-catenin nuclear transfer and enhancing proliferation and migration activity of skin cells, promoting skin cell proliferation and inhibiting apoptosis by Wnt 4. Meanwhile, the composition can promote angiogenesis, regulate inflammatory reaction and inhibit scar formation.
Disclosure of Invention
The invention aims to provide a freeze-dried powder containing mesenchymal stem cell exosomes, a preparation method and application thereof, which can reduce inflammatory cell infiltration of damaged skin, increase neovascularization, promote skin repair, restore and improve skin elasticity, inhibit generation of skin scars, pockmarks and the like, restore skin elasticity, smooth fine wrinkles, whiten skin and the like, improve the anti-injury, anti-inflammatory and anti-sensitization capabilities of the skin, and have wide application prospects.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of sustained release microspheres containing adipose-derived stem cell exosomes, which comprises the steps of subculturing rat adipose-derived stem cells, adding a DMEM (DMEM) culture solution containing hydrogen peroxide, lipopolysaccharide, a platelet-derived factor composition and serum without exosomes when the cells grow to 75-80% and are fused, culturing, sequentially continuously passing through a track etching film of 10 mu m, 5 mu m and 1 mu m, repeating for 3-5 times, centrifuging filtrate at a centrifugal force of 90000-110000g for 60-70min, washing the precipitate, centrifuging at a centrifugal force of 90000-110000g for 60-70min again, re-suspending the precipitate in sterile PBS (phosphate buffer solution), adding an emulsifier and sodium alginate, adding edible oil containing resveratrol after uniform mixing, emulsifying, dropwise adding a calcium ion solution for solidification, and freeze-drying to obtain the sustained release microspheres containing adipose-derived stem cell exosomes.
As a further improvement of the invention, the method comprises the following steps:
s1, preparing a culture solution: adding hydrogen peroxide, lipopolysaccharide and platelet derived factor composition into DMEM culture medium containing exosome-free serum, mixing, and sterilizing to obtain culture solution;
s2, culturing stem cells: subculturing the rat adipose-derived mesenchymal stem cells, washing with sterile PBS when the cells grow to 75-80% and are fused, adding the cells into the culture solution prepared in the step S1, culturing, and collecting the culture solution;
s3, membrane filtration: sequentially and continuously passing the culture solution collected in the step S2 through track etching films of 10 mu m, 5 mu m and 1 mu m, repeating for 3-5 times, respectively removing dead cells, cell fragments and apoptotic bodies, and collecting filtrate;
s4, separation of exosomes: centrifuging the filtrate obtained in the step S3 for 60-70min under the centrifugal force of 90000-110000g, collecting precipitate, washing with sterile PBS, centrifuging for 60-70min under the centrifugal force of 90000-110000g again, and re-suspending the precipitate in sterile PBS to obtain exosome-PBS solution;
s5, preparing sustained release microspheres containing adipose-derived mesenchymal stem cell exosomes: adding sodium alginate and an emulsifying agent into the exosome-PBS solution prepared in the step S4 to obtain a water phase; adding resveratrol into edible oil to obtain oil phase; mixing the water phase and the oil phase, performing rapid membrane emulsification by using an SPG membrane to form emulsion, dripping a calcium chloride solution, stirring and mixing, solidifying at normal temperature, and freeze-drying to obtain the slow-release microsphere containing the adipose-derived mesenchymal stem cell exosome.
As a further improvement of the invention, the content of serum without exosome in the culture solution in the step S1 is 7-12wt%, the content of hydrogen peroxide is 0.5-1wt% and the content of lipopolysaccharide is 3-5wt%; the platelet derived factor composition is present in an amount of 0.7-1.2wt%; the temperature of the culture in the step S2 is 35-38 ℃, the culture time is 36-60h, and CO 2 The content is 4-6%, O 2 The content is 27-30%, the rest is nitrogen, wherein the percentage by volume is; the solid-to-liquid ratio of the precipitation and the sterile PBS in the step S4 is 1:10-15g/mL; in the step S5, the mass ratio of the exosome-PBS solution to the sodium alginate to the emulsifier is 100:20-25:0.1-0.3; the content of Bai Luli alcohol in the edible oil is 3-5wt%; the concentration of the calcium chloride solution is 5-7wt%; the pores of the SPG film are 2-5 mu m; the emulsifier is at least one selected from sodium stearoyl lactylate, diacetyl tartaric acid monoglyceride, sucrose fatty acid ester and distilled monoglyceride; the edible oil is at least one selected from oleum Rapae, peanut oil, oleum Maydis, soybean oil, oleum Helianthi, oleum Olivarum, and oleum Lini.
As a further improvement of the invention, the platelet derived factor composition comprises at least one of PDGF-AA, PDGF-BB and PDGF-AB, preferably a compound composition of PDGF-AA and PDGF-BB, and the mass ratio is 1:3-5.
As a further improvement of the invention, the method specifically comprises the following steps:
s1, preparing a culture solution: adding hydrogen peroxide, lipopolysaccharide and platelet derived factor composition into DMEM culture medium containing exosome-free serum, mixing, and sterilizing to obtain culture solution; the content of serum without exosome in the culture solution is 7-12wt%, the content of hydrogen peroxide is 0.5-1wt% and the content of lipopolysaccharide is 3-5wt%; the platelet derived factor composition is present in an amount of 0.7-1.2wt%;
the platelet derived factor composition is a compound composition of PDGF-AA and PDGF-BB, and the mass ratio is 1:3-5;
s2, culturing stem cells: subculturing rat adipose-derived mesenchymal stem cells, washing with sterile PBS when the cells grow to 75-80% and fusing, adding into the culture solution prepared in step S1, and adding CO at 35-38deg.C 2 The content is 4-6%, O 2 The content is 27-30%, the rest is nitrogen, wherein the percentage is volume percent, culturing is carried out for 36-60h, and the culture solution is collected;
s3, membrane filtration: sequentially and continuously passing the culture solution collected in the step S2 through track etching films of 10 mu m, 5 mu m and 1 mu m, repeating for 3-5 times, respectively removing dead cells, cell fragments and apoptotic bodies, and collecting filtrate;
s4, separation of exosomes: centrifuging the filtrate obtained in the step S3 for 60-70min under the centrifugal force of 90000-110000g, collecting precipitate, washing with sterile PBS, centrifuging for 60-70min under the centrifugal force of 90000-110000g again, and re-suspending the precipitate in sterile PBS, wherein the solid-to-liquid ratio of the precipitate to the sterile PBS is 1:10-15g/mL to obtain exosome-PBS solution;
s5, preparing sustained release microspheres containing adipose-derived mesenchymal stem cell exosomes: adding 20-25 parts by weight of sodium alginate and 0.1-0.3 part by weight of an emulsifier into 100 parts by weight of the exosome-PBS solution prepared in the step S4 to obtain a water phase; adding resveratrol into edible oil to obtain oil phase containing 3-5wt% Bai Luli alcohol; mixing the water phase and the oil phase, performing rapid membrane emulsification by using an SPG membrane with pores of 2-5 mu m to form emulsion, dropwise adding 5-7wt% of calcium chloride solution, stirring and mixing, curing at normal temperature, and freeze-drying to obtain the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes.
The invention further provides the sustained-release microsphere containing the adipose-derived mesenchymal stem cell exosome prepared by the preparation method.
The invention further provides a freeze-dried powder containing mesenchymal stem cell exosomes, which comprises the sustained-release microsphere containing the adipose-derived mesenchymal stem cell exosomes.
As a further improvement of the invention, the invention is prepared from the following raw materials in parts by weight: 2-5 parts of sustained-release microspheres containing adipose-derived stem cell exosomes, 15-20 parts of fucose, 12-15 parts of hyaluronic acid, 3-5 parts of peptide composition, 0.5-1 part of vitamin B, 0.1-1 part of ceramide and 0.5-1 part of arbutin; the peptide composition is at least one selected from the group consisting of a peptide, a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide and a nonapeptide, preferably a mixture of a dipeptide, a pentapeptide and a heptapeptide, and the mass ratio is 3-5:1-2:2-4.
The invention further protects a preparation method of the freeze-dried powder containing the mesenchymal stem cell exosomes, which comprises the steps of uniformly mixing the components according to a proportion, performing vacuum freeze-drying and packaging to obtain the freeze-dried powder containing the mesenchymal stem cell exosomes.
The invention further provides application of the freeze-dried powder containing the mesenchymal stem cell exosomes in preparing cosmetics for treating skin erythema, inflammation, allergy and damage and preparing cosmetics for recovering skin elasticity.
The invention has the following beneficial effects: adipose mesenchymal stem cell exosomes can promote proliferation and migration of fibroblasts and optimize collagen deposition by activating P13K/Akt signaling pathway, thereby restoring and improving skin elasticity. Besides fibroblasts, adipose-derived mesenchymal stem cell exosomes also have a regulating effect on vascular endothelial cells and keratinocytes, promote proliferation, migration and angiogenesis of the skin cells by activating Akt and ERK signal paths, up-regulate genes related to the regeneration function of the skin cells, promote cell proliferation, and remarkably improve re-epithelialization and collagen deposition of skin, thereby effectively increasing skin horny layer, improving skin texture and improving anti-sensitization, anti-inflammatory and anti-injury performances of the skin. In addition, the adipose-derived mesenchymal stem cell exosome also has the effect of inhibiting the generation of skin scars, pocks and the like, and by promoting the generation of type I collagen and type III collagen, the expression of collagen is inhibited at the skin pocks and the later stage of recovery of the damaged part, the ERK/MAPK signal pathway is activated, the expression of matrix metalloproteinase-3 and the ratio of the matrix metalloproteinase-3 to the matrix metalloproteinase inhibitor-1 are up-regulated, the extracellular matrix remodeling is regulated, the quality of skin recovery is improved, and the collagen deposition is reduced, so that the formation of scars, pocks and the like is inhibited. Adipose mesenchymal stem cell exosomes can also improve skin texture by reducing inflammatory responses.
In the preparation process of the adipose-derived mesenchymal stem cell exosome, the culture medium containing the low-concentration hydrogen peroxide solution can obviously enhance the proliferation and migration promoting effect of endothelial cells.
The fat mesenchymal stem cell exosomes treated by adding lipopolysaccharide show stronger anti-inflammatory capability, can promote the overexpression of miRNA-let-7B, and can regulate macrophage polarization, reduce inflammatory cell infiltration of damaged skin, increase neovascularization and promote skin repair by downregulating TLR 4/NF-kappaB and upregulating STAT3/Akt signal paths.
After the treatment by adding platelet derived factor PDGF, the secretion of the adipose-derived stem cell exosome can be obviously increased, the contents of the pro-angiogenic factors and the anti-angiogenic factors can be regulated, and the potential of the adipose-derived stem cell exosome for promoting the angiogenesis of the skin inner layer can be enhanced. PDGF-BB can obviously promote mitogenesis and promote stem cells to secrete a large number of exosomes, so that the yield of the exosomes is improved. PDGF-AA can obviously promote the mitosis of stem cells and can strongly stimulate the division and proliferation of adipose-derived mesenchymal stem cells and the secretion of exosomes. The addition of PDGF-BB and PDGF-AA also have a synergistic effect.
After the prepared adipose-derived mesenchymal stem cell exosomes are resuspended in sterile PBS solution, sodium alginate and an emulsifying agent are added, an oil phase containing the white reed quinol is added, and the oil phase is emulsified to form a microemulsion, and the sodium alginate is solidified to form a shell layer in the presence of calcium ions, so that the adipose-derived mesenchymal stem cell exosomes are wrapped in the shell layer, and the slow-release microsphere containing the adipose-derived mesenchymal stem cell exosomes is obtained, and the contact of the adipose-derived mesenchymal stem cell exosomes subjected to the microsphere treatment with other substances can be reduced, so that the activity of exosomes is prevented from being reduced, and meanwhile, the storage and transportation are facilitated, and the application range of the slow-release microsphere is increased.
Furthermore, the moisturizing components including hyaluronic acid, fucose, ceramide and vitamin B5 are added into the lyophilized powder containing mesenchymal stem cell exosomes, and the vitamin B5 can promote active substances to permeate into the stratum corneum, so that the content of hyaluronic acid in skin can be increased after absorption, and the lyophilized powder has a good moisturizing and repairing effect and can obviously repair skin barriers; the ceramide can strengthen skin barrier, lock moisture, and repair and moisturize; the fucose can well lock water and has good clearing effect on skin inflammation; the whitening component comprises arbutin, can inhibit the generation of melanin and lighten the formed melanin, thereby achieving the effect of uniform skin color; the anti-aging component comprises a peptide composition, so that skin lines can be effectively eliminated, and the anti-wrinkle and anti-inflammatory effects are achieved, and the skin is compacted; wherein, the five peptides and the seven peptides can inhibit the synthesis of SNARE receptors, inhibit the excessive release of catechol and acetylcholine of skin, and locally block the nerve transmission muscle contraction information, so that the facial muscles are relaxed, and the effect of smoothing dynamic lines, static lines and fine lines is achieved; promoting proliferation of collagen, elastic fiber and hyaluronic acid, and increasing skin water content; the dipeptide has a certain antioxidant effect, blocks the conduction of melanin-promoting hormone, reduces the formation of melanin and has a whitening effect. The three components cooperate to better promote skin repair, restore skin elasticity, smooth fine wrinkles, whiten skin and the like, and have the synergistic effect.
The freeze-dried powder containing mesenchymal stem cell exosomes prepared by the invention can reduce inflammatory cell infiltration of damaged skin, increase neovascularization, promote skin repair, recover and improve skin elasticity, inhibit generation of skin scars, pockmarks and the like, recover skin elasticity, smooth fine wrinkles, whiten skin and the like, improve the anti-injury, anti-inflammatory and anti-sensitization capabilities of the skin, and has wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a TEM image of the exosomes prepared in step S4 of example 1 of the present invention;
FIG. 2 is an SEM image of sustained release microspheres containing adipose-derived mesenchymal stem cell exosomes prepared in example 1 of the present invention;
FIG. 3 is a graph showing the cumulative release rate over time in test example 1 of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
DMEM medium was purchased from conjoint dak biotechnology limited in hunan; a lipopolysaccharide, which has been used as a carrier,<3% protein (Lowry assay), purchased from Aibisin (Shanghai) Biotechnology Co., ltd; PDGF-AA purchased from Shenzhen Xinbo biotechnology Co., ltd; PDGF-BB, purchased from Shenzhen Haodihua Biotechnology Co., ltd; rat adipose-derived mesenchymal stem cells were purchased from Shenzhen, haodihua Tuo biotechnology Co., ltd; PBS is phosphate buffer salt solution, pH7.3-7.5, and is sterile and free of Ca 2+ /Mg 2+ Preparing by oneself; track etching films of 10 mu m, 5 mu m and 1 mu m are purchased from Whatman company.
Example 1
The embodiment provides a sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes, which specifically comprises the following steps:
s1, preparing a culture solution: adding hydrogen peroxide, lipopolysaccharide and platelet derived factor composition into DMEM culture medium containing exosome-free serum, mixing, and ultraviolet sterilizing for 30min to obtain culture solution; the content of serum without exosome in the culture solution is 7wt%, the content of hydrogen peroxide is 0.5wt% and the content of lipopolysaccharide is 3wt%; the platelet-derived factor composition was 0.7wt%;
the platelet derived factor composition is a compound composition of PDGF-AA and PDGF-BB, and the mass ratio is 1:3;
s2, culturing stem cells: subculturing rat adipose-derived mesenchymal stem cells, and culturing at 1×10 when the cells reach the 4 th generation 6 Inoculating the density of individual cells into T25 cell bottle, washing with sterile PBS when the cells grow to 75% and fusing, adding into the culture solution prepared in step S1, 35 deg.C, CO 2 The content is 4 percent, O 2 The content is 27 percent, the balance is nitrogen, wherein the percentage is volume percent, the culture is carried out for 36 hours, and the culture solution is collected;
s3, membrane filtration: sequentially and continuously passing the culture solution collected in the step S2 through track etching films of 10 mu m, 5 mu m and 1 mu m, repeating for 3 times, respectively removing dead cells, cell fragments and apoptotic bodies, and collecting filtrate;
s4, separation of exosomes: centrifuging the filtrate obtained in the step S3 for 60min under the condition that the centrifugal force is 90000g, collecting precipitate, washing the precipitate with sterile PBS, centrifuging for 60min again under the condition that the centrifugal force is 90000g, re-suspending the precipitate in the sterile PBS, and obtaining an exosome-PBS solution according to the solid-to-liquid ratio of 1:10g/mL of the precipitate and the sterile PBS, wherein the exosome is in a form observed by a transmission electron microscope, and the exosome has a complete structure, is spherical and has the diameter of 30-100nm;
s5, preparing sustained release microspheres containing adipose-derived mesenchymal stem cell exosomes: adding 20 parts by weight of sodium alginate and 0.1 part by weight of diacetyl tartaric acid monoglyceride into 100 parts by weight of the exosome-PBS solution prepared in the step S4 to obtain a water phase; adding resveratrol into sunflower seed oil to obtain an oil phase containing 3wt% of Bai Luli alcohol; mixing 100 parts by weight of water phase and 120 parts by weight of oil phase, performing rapid membrane emulsification by using an SPG membrane with a pore size of 2 mu m to form emulsion, dripping 5wt% of calcium chloride solution, stirring and mixing for 10min, curing for 30min at normal temperature, and freeze-drying to obtain the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes, wherein fig. 2 is an SEM image of the prepared microsphere, and the particle size of the microsphere is 2-5 microns.
Example 2
The embodiment provides a sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes, which specifically comprises the following steps:
s1, preparing a culture solution: adding hydrogen peroxide, lipopolysaccharide and platelet derived factor composition into DMEM culture medium containing exosome-free serum, mixing, and ultraviolet sterilizing for 30min to obtain culture solution; the content of serum without exosome in the culture solution is 12wt%, the content of hydrogen peroxide is 1wt%, and the content of lipopolysaccharide is 5wt%; the platelet-derived factor composition was 1.2wt%;
the platelet derived factor composition is a compound composition of PDGF-AA and PDGF-BB, and the mass ratio is 1:5;
s2, culturing stem cells: subculturing rat adipose-derived mesenchymal stem cells, and culturing at 1×10 when the cells reach the 4 th generation 6 Inoculating the density of individual cells into T25 cell bottle, washing with sterile PBS when the cells grow to 80%, adding into the culture solution prepared in step S1, at 38deg.C, CO 2 The content is 6 percent, O 2 30% of the content, the balance being nitrogen, wherein the percentage is the volume percentage, culturing for 60 hours, and collecting the culture solution;
s3, membrane filtration: sequentially and continuously passing the culture solution collected in the step S2 through track etching films of 10 mu m, 5 mu m and 1 mu m, repeating for 5 times, respectively removing dead cells, cell fragments and apoptotic bodies, and collecting filtrate;
s4, separation of exosomes: centrifuging the filtrate obtained in the step S3 for 70min under the condition that the centrifugal force is 110000g, collecting precipitate, washing the precipitate with sterile PBS, centrifuging for 70min under the condition that the centrifugal force is 110000g again, and re-suspending the precipitate in the sterile PBS, wherein the solid-liquid ratio of the precipitate to the sterile PBS is 1:15g/mL, so as to obtain an exosome-PBS solution;
s5, preparing sustained release microspheres containing adipose-derived mesenchymal stem cell exosomes: adding 25 parts by weight of sodium alginate and 0.3 part by weight of sodium stearoyl lactylate into 100 parts by weight of the exosome-PBS solution prepared in the step S4 to obtain a water phase; adding resveratrol into linseed oil to obtain an oil phase containing 5wt% of Bai Luli alcohol; mixing 100 parts by weight of water phase and 120 parts by weight of oil phase, performing rapid membrane emulsification by using an SPG membrane with a pore size of 5 mu m to form emulsion, dripping 7wt% of calcium chloride solution, stirring and mixing for 10min, curing for 30min at normal temperature, and freeze-drying to obtain the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes.
Example 3
The embodiment provides a sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes, which specifically comprises the following steps:
s1, preparing a culture solution: adding hydrogen peroxide, lipopolysaccharide and platelet derived factor composition into DMEM culture medium containing exosome-free serum, mixing, and ultraviolet sterilizing for 30min to obtain culture solution; the content of serum without exosome in the culture solution is 10wt%, the content of hydrogen peroxide is 0.7wt% and the content of lipopolysaccharide is 4wt%; the platelet-derived factor composition was 1wt%;
the platelet derived factor composition is a compound composition of PDGF-AA and PDGF-BB, and the mass ratio is 1:4;
s2, culturing stem cells: subculturing rat adipose-derived mesenchymal stem cells, and culturing at 1×10 when the cells reach the 4 th generation 6 Inoculating the density of individual cells into T25 cell bottle, washing with sterile PBS when the cells grow to 77% and fusing, adding into the culture solution prepared in step S1, and adding CO at 37deg.C 2 The content is 5 percent, O 2 The content is 28 percent, the balance is nitrogen, wherein the percentage is volume percent, the culture is carried out for 56 hours, and the culture solution is collected;
s3, membrane filtration: sequentially and continuously passing the culture solution collected in the step S2 through track etching films of 10 mu m, 5 mu m and 1 mu m, repeating for 4 times, respectively removing dead cells, cell fragments and apoptotic bodies, and collecting filtrate;
s4, separation of exosomes: centrifuging the filtrate obtained in the step S3 for 65min under the centrifugal force of 100000g, collecting precipitate, washing with sterile PBS, centrifuging for 65min under the centrifugal force of 100000g again, and re-suspending the precipitate in sterile PBS, wherein the solid-liquid ratio of the precipitate to the sterile PBS is 1:1 g/mL to obtain exosome-PBS solution;
s5, preparing sustained release microspheres containing adipose-derived mesenchymal stem cell exosomes: adding 22 parts by weight of sodium alginate and 0.2 part by weight of sucrose fatty acid ester into 100 parts by weight of the exosome-PBS solution prepared in the step S4 to obtain a water phase; adding resveratrol into soybean oil to obtain oil phase containing 4wt% Bai Luli alcohol; mixing 100 parts by weight of water phase and 120 parts by weight of oil phase, performing rapid membrane emulsification by using an SPG membrane with a pore size of 3 mu m to form emulsion, dripping 6wt% of calcium chloride solution, stirring and mixing for 10min, curing for 30min at normal temperature, and freeze-drying to obtain the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes.
Example 4
In contrast to example 3, the platelet derived factor composition was single PDGF-AA, and the other conditions were unchanged.
Example 5
In contrast to example 3, the platelet derived factor composition was single PDGF-BB, and the other conditions were unchanged.
Comparative example 1
Compared with example 3, the culture solution in step S1 is not added with hydrogen peroxide, and other conditions are not changed.
Comparative example 2
In contrast to example 3, no lipopolysaccharide was added to the culture solution in step S1, and the other conditions were not changed.
Comparative example 3
In contrast to example 3, the platelet-derived factor composition was not added to the culture broth in step S1, and the other conditions were not changed.
Comparative example 4
In contrast to example 3, step S5 was not performed and the exosome-PBS solution prepared in step S4 was stored at-80 ℃.
Test example 1 in vitro Release test
The slow release microspheres containing adipose-derived mesenchymal stem cell exosomes prepared in example 4 and the exosome-PBS solution prepared in comparative example 4, which were labeled with 3,3' -dioctadecyl oxycarboncyanine perchlorate (100 mg), were placed in the upper chamber of a Transwell of a 24-well plate, respectively, and 1.5mL of a release medium (PBS solution containing 10% fetal bovine serum) was added to the exosome-PBS solution prepared in step S4. Placing in a constant temperature shaking table at 37 ℃ and 70r/min, and taking down 400 mu L of room solution (timely supplementing fresh lower room solution containing release medium) at a preset time point. The fluorescence intensity of the sample was measured with 488nm as excitation wavelength and 525nm as emission wavelength, and the cumulative release rate was calculated.
Cumulative release rate (%) = (total mass of adipose mesenchymal stem cell exosomes-mass of adipose mesenchymal stem cell exosomes released cumulatively)/total mass of adipose mesenchymal stem cell exosomes x 100%
The results are shown in FIG. 3. According to the graph, after the adipose tissue-derived stem cell exosomes are embedded in the shell of sodium alginate to obtain the microspheres by the method, the in-vitro release rate of the adipose tissue-derived stem cell exosomes is obviously reduced, and the long-acting release effect is achieved. After the prepared adipose-derived mesenchymal stem cell exosomes are resuspended in sterile PBS solution, sodium alginate and an emulsifying agent are added, an oil phase containing the white reed quinol is added, and the oil phase is emulsified to form a microemulsion, and the sodium alginate is solidified to form a shell layer in the presence of calcium ions, so that the adipose-derived mesenchymal stem cell exosomes are wrapped in the shell layer, and the slow-release microsphere containing the adipose-derived mesenchymal stem cell exosomes is obtained, and the contact of the adipose-derived mesenchymal stem cell exosomes subjected to the microsphere treatment with other substances can be reduced, so that the activity of exosomes is prevented from being reduced, and meanwhile, the storage and transportation are facilitated, and the application range of the slow-release microsphere is increased.
Test example 2 in vitro cell proliferation assay
Rat dermal fibroblasts (purchased from the marsupenaeus life technologies Co., ltd., passaged, 4 th to 6 th substitutes for the test) were used at 7X 10 per well 4 Cell density of individual cells was seeded in 12-well plates at 37℃with 5% CO 2 Incubate in incubator for 12h. The products (fat-containing mesenchymal substances) obtained in examples 1 to 5 and comparative examples 1 to 3 were added respectivelyStem cell exosomes 40 μg) were incubated 1, 3, 5d with 1 medium change every other day. PBS was used as a blank. Single cell suspensions were prepared by digesting the cells with 0.25% pancreatin, staining was performed after mixing the cell suspensions with 0.4% trypan blue staining solution at 9:1, and the growth rate (%) of dermal fibroblasts of rats was calculated by counting with a cell counting plate under a microscope.
Growth rate (%) = (number of rat dermal fibroblasts after incubation on day n-number of initial rat dermal fibroblasts)/number of initial rat dermal fibroblasts×100%
The results are shown in Table 1.
TABLE 1
From the above table, the sustained-release microspheres containing adipose-derived mesenchymal stem cell exosomes prepared in examples 1 to 3 of the present invention can significantly promote proliferation of rat dermal fibroblasts. And the invention obviously enhances the proliferation effect of exosomes on rat dermal fibroblasts due to the slow release effect of the microspheres.
In examples 4 and 5, the platelet-derived factor composition was single PDGF-AA or PDGF-BB, and the growth rate of dermal fibroblasts was decreased in rats, while in comparative example 3, the growth rate of dermal fibroblasts was significantly decreased in rats without the platelet-derived factor composition. After the treatment by adding platelet derived factor PDGF, the secretion of the adipose-derived stem cell exosome can be obviously increased, the contents of the pro-angiogenic factors and the anti-angiogenic factors can be regulated, and the potential of the adipose-derived stem cell exosome for promoting the angiogenesis of the skin inner layer can be enhanced. PDGF-BB can obviously promote mitogenesis and promote stem cells to secrete a large number of exosomes, so that the yield of the exosomes is improved. PDGF-AA can obviously promote the mitosis of stem cells and can strongly stimulate the division and proliferation of adipose-derived mesenchymal stem cells and the secretion of exosomes. The addition of PDGF-BB and PDGF-AA also have a synergistic effect.
In comparative example 1, the growth rate of dermal fibroblasts of rats was decreased without adding hydrogen peroxide to the culture solution in step S1. In the preparation process of the adipose-derived mesenchymal stem cell exosome, the culture medium containing the low-concentration hydrogen peroxide solution can obviously enhance the proliferation and migration promoting effect of endothelial cells.
In comparative example 2, the growth rate of dermal fibroblasts in rats was slightly decreased without lipopolysaccharide added to the culture solution in step S1. The fat mesenchymal stem cell exosomes treated by adding lipopolysaccharide show stronger anti-inflammatory capability, can promote the overexpression of miRNA-let-7B, and can regulate macrophage polarization, reduce inflammatory cell infiltration of damaged skin, increase neovascularization and promote skin repair by downregulating TLR 4/NF-kappaB and upregulating STAT3/Akt signal paths.
In comparative example 4, the embedding in step S5 was not performed, the exosomes were rapidly released within 3 days, the growth rate of rat dermal fibroblasts was reduced, and the contact of the adipose-derived mesenchymal stem cell exosomes with other substances was reduced after the embedding, so that the decrease in the activity of exosomes was avoided, and simultaneously, the storage and transportation were facilitated, and the application range was increased.
Example 6
The embodiment provides a freeze-dried powder containing mesenchymal stem cell exosomes.
The raw materials comprise the following components in parts by weight: 2 parts of sustained-release microspheres containing adipose-derived mesenchymal stem cell exosomes prepared in example 1, 15 parts of fucose, 12 parts of hyaluronic acid, 3 parts of peptide composition, 0.5 part of vitamin B, 0.1 part of ceramide and 0.5 part of arbutin;
the peptide composition is a mixture of dipeptide, pentapeptide and heptapeptide, and the mass ratio is 3:1:2.
The preparation method comprises the following steps: and uniformly mixing the components according to a proportion, performing vacuum freeze drying and packaging to obtain the freeze-dried powder containing mesenchymal stem cell exosomes.
Example 7
The embodiment provides a freeze-dried powder containing mesenchymal stem cell exosomes.
The raw materials comprise the following components in parts by weight: 5 parts of sustained-release microspheres containing adipose-derived mesenchymal stem cell exosomes, 20 parts of fucose, 15 parts of hyaluronic acid, 5 parts of peptide composition, 5 1 parts of vitamin B, 1 part of ceramide and 1 part of arbutin, which are prepared in example 2;
the peptide composition is a mixture of dipeptide, pentapeptide and heptapeptide, and the mass ratio is 5:2:4.
The preparation method comprises the following steps: and uniformly mixing the components according to a proportion, performing vacuum freeze drying and packaging to obtain the freeze-dried powder containing mesenchymal stem cell exosomes.
Example 8
The embodiment provides a freeze-dried powder containing mesenchymal stem cell exosomes.
The raw materials comprise the following components in parts by weight: 3 parts of sustained-release microspheres containing adipose-derived mesenchymal stem cell exosomes prepared in example 3, 17 parts of fucose, 13 parts of hyaluronic acid, 4 parts of peptide composition, 0.7 part of vitamin B, 0.5 part of ceramide and 0.7 part of arbutin;
the peptide composition is a mixture of dipeptide, pentapeptide and heptapeptide, and the mass ratio is 4:2:3.
The preparation method comprises the following steps: and uniformly mixing the components according to a proportion, performing vacuum freeze drying and packaging to obtain the freeze-dried powder containing mesenchymal stem cell exosomes.
Example 9
In comparison with example 8, the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes was prepared from example 4, with no change in other conditions.
Example 10
In comparison with example 8, the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes was prepared from example 5, with no change in other conditions.
Example 11
In contrast to example 8, the peptide composition was a single dipeptide with no other conditions changed.
Example 12
Compared with the embodiment 8, the peptide composition is composed of five peptides and seven peptides, the mass ratio is 2:3, and other conditions are not changed.
Comparative example 5
In comparison with example 8, the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes was prepared from comparative example 1, with no change in other conditions.
Comparative example 6
In comparison with example 8, the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes was prepared from comparative example 2, with no change in other conditions.
Comparative example 7
In comparison with example 8, the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes was prepared from comparative example 3, with no change in other conditions.
Comparative example 8
In comparison with example 8, the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes was prepared from comparative example 4, with no change in other conditions.
Comparative example 9
In comparison with example 8, no peptide composition was added, and other conditions were not changed.
The raw materials comprise the following components in parts by weight: the sustained-release microsphere containing adipose-derived mesenchymal stem cell exosomes prepared in example 3 was 7 parts, fucose was 17 parts, hyaluronic acid was 13 parts, vitamin B was 5.7 parts, ceramide was 0.5 part, and arbutin was 0.7 part.
Comparative example 10
Compared with example 8, the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes prepared in example 3 was not added, and the other conditions were not changed.
The raw materials comprise the following components in parts by weight: 7 parts of peptide composition, 17 parts of fucose, 13 parts of hyaluronic acid, 0.7 part of vitamin B5, 0.5 part of ceramide and 0.7 part of arbutin.
Test example 3
90 female volunteers aged 22-45 years were selected and randomized into 15 groups of 6 persons each, examples 6-12, comparative examples 5-10 and blank and control groups, respectively, with no other product smeared on the face of the subjects during the trial.
The using method comprises the following steps: the lyophilized powder containing mesenchymal stem cell exosomes prepared in examples 6-12 and comparative examples 5-10 were used for each group, deionized water was used for the blank group, and the sustained-release microsphere containing adipose-derived mesenchymal stem cell exosomes and the peptide composition-free product (comprising the following raw materials by weight parts: 17 parts of fucose, 13 parts of hyaluronic acid, 0.7 part of vitamin B, 0.5 part of ceramide and 0.7 part of arbutin) were used for the control group, and the preparation method was the same as in example 8). Each group of products is dissolved in water to prepare a solution according to the solid-to-liquid ratio of 1:5g/mL, and the solution is uniformly smeared on two sides of the cheek, and the two sides are respectively used for 15 days in the morning and at the evening.
And (3) data acquisition: the stratum corneum moisture content, skin elasticity and roughness were each tested 1 day before the product was used, and 1 day after use. Before each test, the subject was cleaned with the face, wiped dry, rested in a room with a relative humidity of 45.+ -. 5% at 25.+ -. 2 ℃ for 15min, and then tested for the moisture content of the stratum corneum of the test site using a skin moisture tester, the skin elasticity tester tested the skin elasticity of the test site, the skin roughness tester tested the skin roughness of the test site, 3 times per test, and averaged.
Calculation of the rate of change of skin moisture content, elasticity and roughness (%)
Rate of change (%) = (post-use test value-pre-use test value)/pre-use test value×100%
The results are shown in Table 2.
TABLE 2
From the above table, the freeze-dried powder containing mesenchymal stem cell exosomes prepared in examples 6-8 of the invention has obvious effects of promoting skin moisturizing, improving skin moisture content, improving skin elasticity and reducing skin roughness.
Examples 11 and 12 are compositions containing a single dipeptide or a combination of a penta-peptide and a hepta-peptide, wherein the water retention rate of the skin is reduced, the degree of increase in skin elasticity is reduced, and the degree of decrease in skin roughness is reduced, and the penta-peptide and the hepta-peptide inhibit synthesis of SNARE receptors, inhibit excessive release of catechol and acetylcholine of the skin, and locally block nerve transmission muscle contraction information, so that facial muscles are relaxed, and the effect of smoothing dynamic lines, static lines and fine lines is achieved; promoting proliferation of collagen, elastic fiber and hyaluronic acid, and increasing skin water content; the dipeptide has a certain antioxidant effect, blocks the conduction of melanin-promoting hormone, reduces the formation of melanin and has a whitening effect. The three components cooperate to better promote skin repair, restore skin elasticity, smooth fine wrinkles, whiten skin and the like, and have the synergistic effect.
Comparative examples 9 and 10 were not added with the peptide composition or the mesenchymal stem cell exosome containing fat respectively, the skin water retention rate was significantly reduced, the skin elasticity was significantly reduced, the skin roughness was significantly reduced, the lyophilized powder containing mesenchymal stem cell exosome prepared by the present invention was able to reduce inflammatory cell infiltration of damaged skin, increase neovascularization, promote skin repair, restore and improve skin elasticity, inhibit the formation of skin scars, pockmarks and the like, restore skin elasticity, smooth fine wrinkles, whiten skin and the like, and improve the anti-injury, anti-inflammatory and anti-sensitization capabilities of skin.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. The preparation method of the sustained-release microsphere containing adipose-derived mesenchymal stem cell exosomes is characterized by comprising the following steps:
s1, preparing a culture solution: adding hydrogen peroxide, lipopolysaccharide and platelet derived factor composition into DMEM culture medium containing exosome-free serum, mixing, and sterilizing to obtain culture solution; the content of serum without exosome in the culture solution is 7-12wt%, the content of hydrogen peroxide is 0.5-1wt% and the content of lipopolysaccharide is 3-5wt%; the platelet derived factor composition is present in an amount of 0.7-1.2wt%;
the platelet derived factor composition is a compound composition of PDGF-AA and PDGF-BB, and the mass ratio is 1:3-5;
s2, culturing stem cells: subculturing rat adipose-derived mesenchymal stem cells, washing with sterile PBS when the cells grow to 75-80% and fusing, adding into the culture solution prepared in step S1, and adding CO at 35-38deg.C 2 The content is 4-6%, O 2 The content is 27-30%, the rest is nitrogen, wherein the percentage is volume percent, culturing is carried out for 36-60h, and the culture solution is collected;
s3, membrane filtration: sequentially and continuously passing the culture solution collected in the step S2 through track etching films of 10 mu m, 5 mu m and 1 mu m, repeating for 3-5 times, respectively removing dead cells, cell fragments and apoptotic bodies, and collecting filtrate;
s4, separation of exosomes: centrifuging the filtrate obtained in the step S3 for 60-70min under the centrifugal force of 90000-110000g, collecting precipitate, washing with sterile PBS, centrifuging for 60-70min under the centrifugal force of 90000-110000g again, and re-suspending the precipitate in sterile PBS, wherein the solid-to-liquid ratio of the precipitate to the sterile PBS is 1:10-15g/mL to obtain exosome-PBS solution;
s5, preparing sustained release microspheres containing adipose-derived mesenchymal stem cell exosomes: adding 20-25 parts by weight of sodium alginate and 0.1-0.3 part by weight of an emulsifier into 100 parts by weight of the exosome-PBS solution prepared in the step S4 to obtain a water phase; adding resveratrol into edible oil to obtain oil phase containing 3-5wt% Bai Luli alcohol; mixing the water phase and the oil phase, performing rapid membrane emulsification by using an SPG membrane with pores of 2-5 mu m to form emulsion, dropwise adding 5-7wt% of calcium chloride solution, stirring and mixing, curing at normal temperature, and freeze-drying to obtain the sustained release microsphere containing adipose-derived mesenchymal stem cell exosomes.
2. The preparation method according to claim 1, wherein the emulsifier in step S4 is at least one selected from the group consisting of sodium stearoyl lactylate, diacetyl tartaric acid monoglyceride, sucrose fatty acid ester, distilled monoglyceride; the edible oil is at least one selected from oleum Rapae, peanut oil, oleum Maydis, soybean oil, oleum Helianthi, oleum Olivarum, and oleum Lini.
3. A sustained-release microsphere containing adipose-derived mesenchymal stem cell exosome prepared by the preparation method of claim 1 or 2.
4. A lyophilized powder comprising mesenchymal stem cell exosomes, comprising the sustained release microsphere comprising adipose-derived mesenchymal stem cell exosomes of claim 3.
5. The lyophilized powder containing mesenchymal stem cell exosomes according to claim 4, wherein the lyophilized powder is prepared from the following raw materials in parts by weight: 2-5 parts of sustained-release microspheres containing adipose-derived stem cell exosomes, 15-20 parts of fucose, 12-15 parts of hyaluronic acid, 3-5 parts of peptide, 0.5-1 part of vitamin B, 0.1-1 part of ceramide and 0.5-1 part of arbutin; the peptide is at least one selected from one of a primary peptide, a secondary peptide, a tripeptide, a tetra-peptide, a penta-peptide, a hexa-peptide, a hepta-peptide, an octa-peptide and a nona-peptide.
6. The lyophilized powder containing mesenchymal stem cell exosomes according to claim 5, wherein the peptide is a mixture of dipeptide, penta-peptide and hepta-peptide in a mass ratio of 3-5:1-2:2-4.
7. A method for preparing the lyophilized powder containing mesenchymal stem cell exosomes according to claim 5, wherein the components are uniformly mixed according to a proportion, and the lyophilized powder containing mesenchymal stem cell exosomes is obtained by vacuum freeze-drying and packaging.
8. Use of a lyophilized powder comprising mesenchymal stem cell exosomes according to any one of claims 4-6 for the preparation of a cosmetic for the treatment of skin rash, inflammation, allergy, damage, and for the preparation of a cosmetic for restoring skin elasticity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210964131.6A CN115282065B (en) | 2022-08-11 | 2022-08-11 | Freeze-dried powder containing mesenchymal stem cell exosomes and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210964131.6A CN115282065B (en) | 2022-08-11 | 2022-08-11 | Freeze-dried powder containing mesenchymal stem cell exosomes and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115282065A CN115282065A (en) | 2022-11-04 |
| CN115282065B true CN115282065B (en) | 2024-03-15 |
Family
ID=83829038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210964131.6A Active CN115282065B (en) | 2022-08-11 | 2022-08-11 | Freeze-dried powder containing mesenchymal stem cell exosomes and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115282065B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114288276B (en) * | 2021-12-29 | 2023-09-01 | 成都清科生物科技有限公司 | Anti-inflammatory amniotic membrane patch and preparation method thereof |
| CN117100716B (en) * | 2023-08-29 | 2024-03-12 | 广东龄值生物科技有限公司 | Nano microsphere containing iNKT cells and preparation method thereof |
| CN117243885B (en) * | 2023-11-15 | 2024-01-26 | 北京青藤谷禧干细胞科技研究院有限公司 | Stem cell exosome composition for improving skin and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160086253A (en) * | 2015-01-08 | 2016-07-19 | 한양대학교 에리카산학협력단 | Cosmetic composition containing exosomes extracted from stem cell for skin whitening, antiwrinkle or regeneration |
| CN112516067A (en) * | 2020-12-09 | 2021-03-19 | 广州美邦生物科技有限公司 | Composition containing umbilical cord mesenchymal stem cell exosome and application thereof |
| CN113249316A (en) * | 2021-06-16 | 2021-08-13 | 基因组诊断有限公司 | Preparation method and application of umbilical cord/placenta mesenchymal stem cell-derived exosome |
| CN113599492A (en) * | 2021-08-16 | 2021-11-05 | 北京戴域生物技术有限公司 | Anti-aging medicine or cosmetic and preparation method thereof |
| CN114727947A (en) * | 2019-09-25 | 2022-07-08 | 科迪亚克生物科学公司 | Extracellular vesicle composition |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003102171A1 (en) * | 2002-05-28 | 2003-12-11 | Becton, Dickinson And Company | Expansion and transdifferentiation of human acinar cells |
| WO2018091713A1 (en) * | 2016-11-18 | 2018-05-24 | Consumed Cvba | A method for preparing a growth factors containing platelet releasate |
-
2022
- 2022-08-11 CN CN202210964131.6A patent/CN115282065B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160086253A (en) * | 2015-01-08 | 2016-07-19 | 한양대학교 에리카산학협력단 | Cosmetic composition containing exosomes extracted from stem cell for skin whitening, antiwrinkle or regeneration |
| CN114727947A (en) * | 2019-09-25 | 2022-07-08 | 科迪亚克生物科学公司 | Extracellular vesicle composition |
| CN112516067A (en) * | 2020-12-09 | 2021-03-19 | 广州美邦生物科技有限公司 | Composition containing umbilical cord mesenchymal stem cell exosome and application thereof |
| CN113249316A (en) * | 2021-06-16 | 2021-08-13 | 基因组诊断有限公司 | Preparation method and application of umbilical cord/placenta mesenchymal stem cell-derived exosome |
| CN113599492A (en) * | 2021-08-16 | 2021-11-05 | 北京戴域生物技术有限公司 | Anti-aging medicine or cosmetic and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115282065A (en) | 2022-11-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115282065B (en) | Freeze-dried powder containing mesenchymal stem cell exosomes and preparation method and application thereof | |
| AU2017202287B2 (en) | Composition for differentiation induction of adipocyte containing stem cell-derived exosome, regeneration of adipose tissue, and skin whitening or wrinkle improvement | |
| Zhang et al. | In vivo study of silk fibroin/gelatin electrospun nanofiber dressing loaded with astragaloside IV on the effect of promoting wound healing and relieving scar | |
| US6620419B1 (en) | Cosmetic or dermopharmaceutical use of peptides for healing, hydrating and improving skin appearance during natural or induced ageing (heliodermia, pollution) | |
| CN108186548A (en) | A kind of preparation method of the stem cell factor Essence with anti-aging effects | |
| JP5896916B2 (en) | Cosmetic and / or pharmaceutical composition comprising locust bean extract as active agent to activate aquaporin expression | |
| CN113301910B (en) | Skin rejuvenation and healing mixtures of peptide components and uses thereof | |
| CN110833514B (en) | Polypeptide freeze-dried powder and application thereof in preparation of anti-aging cosmetics | |
| EP0462194B1 (en) | Fibroblast growth factor (fgf)-based stabilized compositions | |
| EP4137561A1 (en) | Method for preparing culture medium containing high levels of high-potency exosomes secreted by cord blood stem cells, and use thereof | |
| CN111202749A (en) | Preparation method of stem cell active factor composition with muscle cell repair function | |
| Li et al. | Evaluation of efficacy of aloin in treating acute trauma in vitro and in vivo | |
| KR101440383B1 (en) | Cosmetic Composition Comprising Human Adipocyte Conditioned Media Extracts Stabilized in Osmocell As Active Ingredient | |
| CN110496067A (en) | A kind of anti-wrinkle composition and its preparation method and application | |
| CN113876687A (en) | Application of active peptide and placental stem cell exosome for improving skin physiological characteristics in medicines or cosmetics | |
| Tian et al. | Co-delivery of bioactive peptides by nanoliposomes for promotion of hair growth | |
| KR20130109850A (en) | Kit comprising recombinant human bone morphogenetic protein for skin repair as active ingredient | |
| CN109966170B (en) | Flexible liposome cosmetic containing biological macromolecules and preparation method thereof | |
| JP6530392B2 (en) | Dermal matrix with microparticles having synergetic effect and tissue repair and method for producing the same | |
| EP1784423A1 (en) | Novel peptide and pharmaceutical composition containing said peptide | |
| WO2018120401A1 (en) | Cosmetic composition | |
| CA2654021C (en) | Use of adipose-tissue cell fractions for post-irradiation tissue regeneration | |
| FR3065009A1 (en) | SECRETOME BACTERIEN FOR ITS USE IN THE TREATMENT OF SKIN LESIONS | |
| CN110526958B (en) | Activity repair peptide Tvigour A and application thereof | |
| KR100490212B1 (en) | Anti-aging cosmetics composition containing amino acid complex |
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 |