CN112704728A - 一种egf高表达的干细胞温敏凝胶的制备方法及其应用 - Google Patents
一种egf高表达的干细胞温敏凝胶的制备方法及其应用 Download PDFInfo
- Publication number
- CN112704728A CN112704728A CN202011275013.1A CN202011275013A CN112704728A CN 112704728 A CN112704728 A CN 112704728A CN 202011275013 A CN202011275013 A CN 202011275013A CN 112704728 A CN112704728 A CN 112704728A
- Authority
- CN
- China
- Prior art keywords
- egf
- cells
- wound surface
- mscs
- stem cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000009024 Epidermal Growth Factor Human genes 0.000 title claims abstract description 96
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 24
- 101800003838 Epidermal growth factor Proteins 0.000 title abstract description 82
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 title abstract description 79
- 229940116977 epidermal growth factor Drugs 0.000 title abstract description 78
- 210000004027 cell Anatomy 0.000 claims abstract description 70
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 40
- 210000003954 umbilical cord Anatomy 0.000 claims abstract description 27
- 230000035755 proliferation Effects 0.000 claims abstract description 14
- 230000035876 healing Effects 0.000 claims abstract description 12
- 230000009471 action Effects 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 210000001339 epidermal cell Anatomy 0.000 claims abstract description 7
- 230000008439 repair process Effects 0.000 claims abstract description 7
- 210000002889 endothelial cell Anatomy 0.000 claims abstract description 5
- 206010052428 Wound Diseases 0.000 claims description 53
- 208000027418 Wounds and injury Diseases 0.000 claims description 53
- 241000713666 Lentivirus Species 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 14
- 239000013598 vector Substances 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 238000002474 experimental method Methods 0.000 claims description 8
- 206010072170 Skin wound Diseases 0.000 claims description 7
- 208000015181 infectious disease Diseases 0.000 claims description 7
- 230000005012 migration Effects 0.000 claims description 6
- 238000013508 migration Methods 0.000 claims description 6
- 206010053615 Thermal burn Diseases 0.000 claims description 5
- 238000004806 packaging method and process Methods 0.000 claims description 5
- 102000004127 Cytokines Human genes 0.000 claims description 4
- 108090000695 Cytokines Proteins 0.000 claims description 4
- 230000009286 beneficial effect Effects 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000013589 supplement Substances 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 208000028006 Corneal injury Diseases 0.000 claims 1
- 206010015911 Eye burns Diseases 0.000 claims 1
- 208000002847 Surgical Wound Diseases 0.000 claims 1
- 230000001464 adherent effect Effects 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 210000003491 skin Anatomy 0.000 abstract description 23
- 210000001519 tissue Anatomy 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 15
- 238000000034 method Methods 0.000 abstract description 12
- 230000004069 differentiation Effects 0.000 abstract description 9
- 230000007547 defect Effects 0.000 abstract description 8
- 210000002615 epidermis Anatomy 0.000 abstract description 8
- 239000003795 chemical substances by application Substances 0.000 abstract description 4
- 230000002349 favourable effect Effects 0.000 abstract description 3
- 239000007787 solid Substances 0.000 abstract description 2
- 239000000499 gel Substances 0.000 description 39
- 238000001514 detection method Methods 0.000 description 15
- 238000012258 culturing Methods 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 230000004663 cell proliferation Effects 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 230000029663 wound healing Effects 0.000 description 6
- 101150084418 EGF gene Proteins 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 210000001185 bone marrow Anatomy 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000002158 endotoxin Substances 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000000017 hydrogel Substances 0.000 description 4
- 208000014674 injury Diseases 0.000 description 4
- 210000002510 keratinocyte Anatomy 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 230000008733 trauma Effects 0.000 description 4
- 230000037314 wound repair Effects 0.000 description 4
- 102000001301 EGF receptor Human genes 0.000 description 3
- 108060006698 EGF receptor Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 241000204031 Mycoplasma Species 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 210000004207 dermis Anatomy 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 230000036512 infertility Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000037311 normal skin Effects 0.000 description 3
- 230000003076 paracrine Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 206010040943 Skin Ulcer Diseases 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000004872 soft tissue Anatomy 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 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 description 1
- 238000012604 3D cell culture Methods 0.000 description 1
- 208000012260 Accidental injury Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 206010004053 Bacterial toxaemia Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000018386 EGF Family of Proteins Human genes 0.000 description 1
- 108010066486 EGF Family of Proteins Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- 101710197056 Hyaluronan synthase 2 Proteins 0.000 description 1
- 102100040206 Hyaluronan synthase 2 Human genes 0.000 description 1
- 101710197055 Hyaluronan synthase 3 Proteins 0.000 description 1
- 102100029425 Hyaluronan synthase 3 Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000003814 Interleukin-10 Human genes 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 208000013222 Toxemia Diseases 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 229940076144 interleukin-10 Drugs 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000012977 invasive surgical procedure Methods 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 208000021601 lentivirus infection Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002906 medical waste Substances 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 210000003078 multipolar neuron Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 description 1
- 210000000282 nail Anatomy 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920006267 polyester film Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 210000001732 sebaceous gland Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000002363 skeletal muscle cell Anatomy 0.000 description 1
- 230000037380 skin damage Effects 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000000438 stratum basale Anatomy 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000000106 sweat gland Anatomy 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- 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
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Developmental Biology & Embryology (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Dermatology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rheumatology (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hematology (AREA)
- Inorganic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开了一种EGF高表达的干细胞温敏凝胶的制备方法及其应用,间充质干细胞具有自我更新和多向分化能力和优异的治疗皮肤组织创伤的功效。EGF可刺激表皮细胞、内皮细胞的增殖,对治疗创面的修复和愈合具有良好的疗效。将EGF基因修饰的脐带间充质干细胞制备为液态的温敏凝胶涂抹剂,涂敷在创伤局部后,在皮肤温度的作用下形成凝胶状固态,使凝胶中的干细胞能长时间固定在创面,并且凝胶支架有利于维持干细胞的干性及多向分化潜能,大大提高干细胞定向迁移至创面并覆盖局部表皮缺损部位并分化成皮肤组织细胞的机率;定植成功的于细胞表达EGF,并持续性地分泌至创面局部以局部表皮细胞及内皮细胞增殖,在三种因素的共同作用下最终加速创面完全愈合。
Description
技术领域
本发明涉及细胞生物学领域,特别是涉及一种EGF高表达的干细胞温敏凝胶的制备方法及其应用。
背景技术
皮肤是人体最大的器官,位于人体的表面,是人体的第一道防线。皮肤由表皮、真皮和皮下组织构成,除此以外还有丰富的血管、淋巴管、神经、肌肉和皮肤附属器(包括毛发、毛囊、皮脂腺、汗腺、甲)。表皮由外胚层分化而来,属于复层鳞状上皮,主要由角质形成细胞和树突状细胞两大类细胞组成;根据角质形成细胞的分化阶段和特点,表皮在光镜下由内向外依次分为基底层、棘层、颗粒层、透明层和角质层。基底层细胞不断地增殖产生新的角质形成细胞,又称为生发层。
皮肤创伤为日常生活中普遍的意外伤害。当受伤面积过大、伤及真皮层时,除了易于发生感染、甚至发生可能危及生命的败血症等并发症外,由于大面积皮肤缺损导致大量基底细胞的缺失、造成表皮缺损愈合困难,甚至大面积皮肤经久不愈。当前临床上用于大面积皮肤缺损的治疗主要是以皮肤软组织扩张术、自体皮肤组织移植或人工皮肤为主,然而治疗价格昂贵且耗时费工,且大面积烧伤患者残留的正常皮肤量少,能够进行皮肤软组织扩张术+自体皮肤组织移植的原材料不足,且增加了患者住院时间及治疗费用和治疗时的痛苦。此外,随着糖尿病、心血管疾病和肥胖症等疾病的日益增多的趋势,这些疾病所并发的经久不愈的慢性皮肤溃疡正成为一个全球性的难题,其治疗策略有限,疗效不理想,医疗费用高昂。
理想的皮肤创面敷料应该能提供良好的微生物污染屏障、防止创面脱水、吸收创面渗出液,提供伤口愈合的最佳环境,并且易于涂敷及清除拆卸,且必须具备良好的生物相容性、无/低致敏性过敏性、经济、无毒副作用。
近年来随着干细胞基础研究和临床研究的发展,间充质干细胞(mesenchymalstem cells,MSCs)对常规治疗困难的皮肤损伤的修复逐渐成为临床治疗的研究热点,并取得了一定的研究成果。常用的MSCs有自体骨髓MSCs、自体脂肪MSCs和异体脐带间充质干细胞(umbilical cord mesenchymal stem cells,UC-MSCs)等。移植到体内的MSCs表达炎性因子受体,在损伤组织局部释放的炎性因子的作用下,趋化、归巢于损伤局部,除了作为种子细胞直接补充局部组织细胞的缺失外,在局部分泌多种生长因子和细胞因子、激素,如白介素-10、转化生长因子、基质细胞衍生因子、血管内皮生长因子、表皮生长因子、神经生长因子等,这些因子在损伤局部发挥旁分泌作用,刺激内源性细胞增殖、促进血管再生、抑制炎性反应、抑制细胞凋亡和纤维化过程。最先报道的应用于治疗烧伤后大面积皮肤组织缺损的是来自骨髓的MSCs(BMSCs),但是,采集骨髓为有创性手术操作,提供者较痛苦,且有一定的感染风险;并且严重烧伤和外伤、毒血症或败血症,药物等会导致骨髓抑制,导致体外扩增BMSCs细胞数量减少。此外,骨髓来源的MSCs随供者年龄的增加,其数量会减少、体外扩增和分化能力降低。近年来,发现UC-MSCs是BMSCs的良好替代物,它不仅更接近胚胎干细胞、增殖能力强,多次传代扩增后仍具有干细胞特性,而且免疫源性低,同种异体来源的MSCs应用后免疫排斥反应可能性极低。脐带本是医疗废弃物,其来源较多而且易得,并且UC-MSCs的临床应用无伦理问题。UC-MSCs可在体外培养较长时间,可以分化为三个胚系的多种细胞:在适当的诱导条件下,除可分化为骨、软骨和脂肪外,还可分化为神经胶质和肝细胞样细胞、骨骼肌细胞和多极神经元等各种细胞类型。人UC-MSCs在体外培养条件下可分化为上皮细胞,形成3-4层上皮类细胞,具有向皮肤表皮和真皮分化的能力,并可形成微血管,为其临床应用提供了理论支持。
聚N-异丙基丙烯酰胺和聚乙二醇的嵌段共聚物(PNIPAAm-PEG)是一种温度敏感性的水凝胶,低温时(4℃)呈溶胶状态(类似液体),而在温度较高(37℃)时则形成固态的水凝胶。由于具有良好的亲水性,水凝胶已经被广泛应用于医学和生命科学研究中,例如3D细胞培养、组织工程和药物递送等。使用温敏水凝胶作为UC-MSCs载体将其局部应用于创面,利用其在体温下凝固成凝胶状,可以显著提升UC-MSCs在局部的存留时间,有助于UC-MSCs靶向定位在创面,增加了UC-MSCs在创面受损部位的持续接触时间;并且UC-MSCs在温敏凝胶系统中,可以较长时间维持在未分化状态,维持其多能性和干性,有利于UC-MSCs在创面局部分化为皮肤组织细胞,有利于创面修复;同时凝胶状态下,其高亲脂性为细胞增殖、细胞信号转导、气体质量交换、细胞和组织抵抗剪切力提供了有效的环境,有利于UC-MSCs的增殖、活化;此外温敏凝胶的多孔结构可作为UC-MSCs的支架,可以将营养物质、氧气等的运输最大化,使细胞能够保持最大活率,有利于UC-MSCs在局部释放细胞因子促进组织修复和增殖。且该聚合物易于操作,无毒,生物相容性好,引起排斥反应的可能性极低。
表皮细胞生长因子(EGF),是由53个氨基组成的活性多肽,是一种多功能生长因子,其通过与表皮生长因子受体(EGFR)结合后,刺激受体自身磷酸化及胞内其他蛋白络氨酸磷酸化,继而活化下游信号通路引起一系列生物学变化。EGFR主要表达于上皮细胞,但成纤维细胞及血管内皮细胞的胞膜上亦有表达,因此EGF在创面修复中发挥重要作用,具有促进表皮细胞迁移、分裂、表达透明质酸合成酶2和合成酶3从而促进透明质酸(皮肤组织间质的重要组成成分)的合成分泌等功能,进而促进表皮层增殖和细胞分化,从而促进受损创面的修复愈合。人体EGF的合成与分泌受神经因素和多种激素调节,如雄激素、孕激素等;外周血中仅含有少量EGF,EGF在组织中的含量普遍较低,尤其是创伤初期,创伤皮肤组织的EGF含量低于正常皮肤组织,于创伤后第五天才开始上调其表达量,不利于早期创面修复。此外,EGF对靶细胞增殖的影响需要在连续刺激6-12小时才可观察到明显的DNA合成的变化,因此,使用常规的EGF外用型水剂涂敷于创伤表面时,由于水剂型EGF很快流失,难以保持较长时间的局部刺激作用,临床治疗效果不佳。
因此,开发一种具有消炎作用,能够快速促进创面愈合,不产生过敏反应,无毒,无菌的具有干细胞活性的材料显得尤为重要。本专利公布的EGF高表达UC-MSCs可用于创伤的治疗。本专利利用携带EGF基因的慢病毒感染UC-MSCs,使之组成性分泌EGF,外敷烧伤创面后,能在局部持续分泌EGF促进表皮细胞、内皮细胞增殖、创面修复;UC-MSCs在皮肤创面局部微环境的诱导下,可向皮肤组织分化,作为种子细胞补充创面局部的缺损,并通过旁分泌作用促进局部组织细胞增殖修复创面;同时,温敏凝胶能在创面局部的温度作用下凝固成凝胶状,作为UC-MSCs支架系统,有利于UC-MSCs在局部定植和分化,可以明显加速烧伤等难愈性创面的愈合速度。
发明内容
为解决现有技术中存在皮肤创面愈合的时间长,治疗成本高等问题,本发明提供了一种EGF修饰人脐带间充质干细胞的技术,以及所述人脐带间充质干细胞用于治疗创伤的用途。
为实现上述目的,本发明的技术方案如下:
人脐带间充质干细胞的EGF修饰方法,采用EGF慢病毒表达载体转染脐带间充质干细胞,得到所述EGF修饰人脐带间充质干细胞;
所述人脐带间充质干细胞用于治疗创伤的应用方法,通过在甲基纤维素复合培养基中加入上述EGF修饰人脐带间充质干细胞,即得EGF修饰的人脐带间充质干细胞创面涂抹剂对创面进行涂抹治疗。
所述EGF慢病毒表达载体的EGF重组基因包括信号肽和EGF基因,所述重组基因核苷酸序列为SEQID NO.1所示的核苷酸序列。
其中,EGF慢病毒表达载体以pCDH-CMV-MCS-EF1-Puro为转染工具;
上述EGF修饰人脐带间充质干细胞的制备及其创面涂抹剂制备方法,包括如下步骤:
(1)委托北京华大基因技术有限公司合成EGF基因片段,并将其连接到PUC57克隆载体上,进行测序,在NCBI上比对序列,正确后插入pCDH-CMV-MCS-EF1-Puro载体,转化到E.coli(stbl4),使用OMEGA公司的质粒纯化试剂盒提取并纯化质粒,获得各重组表达载体的高品质质粒,下表中的引物用于PCR扩增。
(2)重组慢病毒载体:本发明中使用的载体是慢病毒载体pCDH-CMV-MCS-EF1-Puro,pCDH-CMV-MCS-EF1-Puro,载体含有一个原始复制子,一个CMV启动子序列和选择性的抗性基因(抗氨苄青霉素)。经双酶切后的EGF基因,即目标DNA,在T4连接酶作用下,与线性化的慢病毒载体DNA连接,形成表达EGF的重组慢病毒载体pCDH-CMV-EGF(见图1)。
(3)重组慢病毒载体pCDH-CMV-EGF质粒与优化后的一定比例的慢病毒包装质粒混合后转染293T细胞,转染后1-3天收获慢病毒上清、浓缩、纯化后获得携带EGF基因的慢病毒;并检测慢病毒的滴度。
(4)宿主细胞:本发明所用的宿主细胞为UC-MSCs。慢病毒感染UC-MSCs的感染复数为10。
(5)经EGF修饰的P3代UC-MSCs在体外进行培养、扩增2代后,收获大量P5代EGF高表达UC-MSCs进行临床应用。
(6)EGF高表达UC-MSCs的应用方法:P5代EGF高表达UC-MSCs经过胰酶消化、中和后,计数,取5×107个细胞的悬液,离心,用2.5mL预冷(4℃)的完全培养基重悬,使细胞密度在2×107/mL,然后按1∶1的比例与预冷(4℃)的30%浓度的温敏凝胶溶液轻轻混匀,经过质控检测(包括细胞活性、无菌、内毒素、支原体、MSCs细胞表型等)、放行后,快速、均匀地涂敷于创面,使创面表面形成一薄层凝胶覆盖创面。
(7)EGF高表达UC-MSCs的应用剂量:本发明中EGF修饰UC-MSCs的数量为0.5×106~1×108/Kg。优选的,为1×106/kg,细胞总量为5×107,EGF高表达UC-MSCs的温敏凝胶总体积为5mL,其中温敏凝胶的终浓度为15%。
一种EGF高表达的干细胞温敏凝胶的制备方法及其应用,脐带间充质干细胞具有免疫调节功能,可抑制创面局部的过度炎症反应;UC-MSCs具有强大的增殖能力和多向分化潜能,除作为种子细胞接补充局部组织细胞的缺失外,还可通过旁分泌作用促进创面局部残余的基底层细胞增殖、分化,促进创面愈合;UC-MSCs组成性表达EGF,促进表皮细胞、内皮细胞增殖、创面修复,能大大加速创面愈合;UC-MSCs温敏凝胶制剂能在创面局部的温度作用下凝固成凝胶状,作为UC-MSCs支架系统,有利于UC-MSCs在局部定植和分化,可以明显加速严重皮肤创伤如大面积烧伤、糖尿病慢性皮肤溃疡、难愈性手术切口等创面的愈合。
与现有技术相比,本发明具的增益效果是:本发明采用EGF基因修饰脐带间充质干细胞,可以提高脐带间充质干细胞促进创面受损细胞的增殖、迁移能力,从而加速创面愈合。
附图说明
图1为pCDH-CMV-EGF慢病毒载体质粒图谱
图2为UC-MSCs与EGF-高表达UC-MSCs培养至P5代细胞内EGF流式检测结果
图3为UC-MSCs与EGF-高表达MSCs培养至P5代的形态图
图4为UC-MSCs与EGF-高表达MSCs培养至P5代的流式分析结果
图5为UC-MSCs与EGF-高表达MSCs培养至P5代三系分化结果
图6为UC-MSCs与EGF-高表达UC-MSCs培养上清ELISA检测EGF含量
图7为EGF-高表达UC-MSCs对HaCaT及HFF1细胞影响的细胞增殖实验
图8为EGF-高表达UC-MSCs对HaCaT细胞影响的细胞迁移实验
图9为EGF高表达UC-MSCs修复小鼠皮肤缺损动物实验
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面将结合实施例对本发明的实施方案进行详细描述,本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均可通过市购获得的常规产品。
实施例1:将基因EGF插入慢病毒载体pCDH-CMV-MCS-Puro
委托北京华大基因技术有限公司合成EGF基因片段,并将其连接到PUC57克隆载体上,PCR扩增、双酶切后插入pCDH-CMV-MCS-EF1-Puro载体(见图1),转化到E.coli(stbl4),使用OMEGA公司的质粒纯化试剂盒提取并纯化质粒。
实施例2:EGF高表达UC-MSCs的制备
(一)UC-MSCs的制备
由合作的机构东莞市滨海湾中心医院提供健康的新生儿脐带,低温无菌保存并于3个小时内送至细胞制备实验室。分离出其中的华氏胶组织,剪碎至0.5mm2大小的小块贴壁培养,用UltraCULTURE培养基培养传代,并适时观察细胞的生长状况、形态等,待生长状况、形态良好,制备一定数量及活率>90%的第三代(P3)UC-MSCs后,对其进行质量控制,包括:细菌、真菌、内毒素、活率、表面标志物等检测。
(二)慢病毒包装
1、从液氮罐中取出冻存的293T细胞,迅速放入37℃水浴锅中并快速晃动,尽量在1~3min内使细胞溶液完全溶解。将细胞悬液缓慢滴加到预先加入8mL新鲜完全培养基(DMEM/10%FBS)的50mL离心管中,离心,1500rpm,5min。去掉上清,加入1mL新鲜的完全培养基重悬细胞沉淀后,计数、转入T75培养瓶中。将培养瓶平放入37℃、5%CO2和95%相对湿度的培养箱中培养。第二天观察细胞存活率,并更换培养基。以后每天观察细胞生长情况,传代2次后、细胞状态良好时用于实验。
2、转染前将所有试剂置于室温。在无菌离心管中,用无血清的MEM稀释质粒DNA。重组慢病毒质粒:病毒包装质粒(psPAX2):病毒包膜质粒(pMD2G)的比例为4∶2∶1,PEI与总DNA(ug)的混合比率为3∶1。加入后立即涡旋混合。将DNA/PEI的混合物在室温下孵育20分钟后均匀滴加到15cm培养皿中。转染后48-72小时内收获转染细胞的病毒上清,显微镜下观察293T细胞转染后的形态变化。
3、将含有慢病毒的细胞培养上清超离浓缩后(20000g,4℃,2小时),倒掉上清,用预冷PBS重悬慢病毒颗粒,分装为100μL/管-80℃保存;留取少量慢病毒测定慢病毒滴度,检测得出感染滴度平均值为2.322×109TU/ml。
(三)慢病毒感染脐带间充质干细胞及感染后脐带间充质干细胞的扩增培养
1、P3代MSC传代、按1.5-2×104/cm2的密度接种于6孔板中(例如接种5个孔),使细胞融合度在70-80%,放入CO2培养箱中培养过夜。
2、第二天从-80℃拿出0.3-0.5ml病毒液,冰上融化后瞬离,按MOI=10/孔计算所需的慢病毒总量,将病毒液加入5毫升UltraCULTURE培养基及终浓度为8μg/ml的聚凝胺(购自Sigma公司),轻轻混匀。
3、将6孔板中的UC-MSCs的培养基上清吸弃,将上述慢病毒稀释液平均加入6孔板中,约1.1mL/孔,继续将6孔板放入37℃,5%的CO2培养箱中培养。24小时后每孔换液2ml/孔新鲜UltraCUL TURE培养基。48小时后6孔板中UC-MSCs达90%以上汇合度后,将细胞进行传代,各孔感染了慢病毒的UC-MSCs消化、中和后将细胞合并、按5×103/cm2的密度接种于T75培养瓶中,补加培养基10mL,放入37℃,5%的CO2培养箱中继续培养48-72h达90%以上汇合度后,再次传代至T175培养瓶中,放入37℃,5%的CO2培养箱中继续培养48-72h达90%以上汇合度后,收获大量高表达EGF的P5代UC-MSCs。
(四)利用流式细胞术检测EGF在UC-MSCs内的表达效率:
将培养瓶中MSC消化、中和后取2×106个感染EGF慢病毒的UC-MSCs,同时取2×106个未感染慢病毒的UC-MSCs作为对照细胞,4%的多聚甲醛固定后用0.5%saponin穿膜处理、胞内染色后、利用流式细胞仪分别检测感染EGF的UC-MSCs细胞和未感染UC-MSCs细胞的细胞内EGF表达率,结果显示病慢毒感染效率高达97.4%(见图2)。
培养至P5代的UC-MSCs和EGF高表达的UC-MSCs细胞常规进行形态学检测(见图3)及干细胞表面标志物检测(见图4)、三系分化实验(见图5),EGF高表达的UC-MSCs细胞形态正常,并未上调HLA-DR,EGF-MSC仍具有低免疫原性,并保持干细胞的表面标志物的正常表达,满足干细胞鉴定最低标准。
(五)UC-MSCs与EGF高表达的UC-MSCs细胞培养基上清中EGF含量ELISA检测
P5代UC-MSCs及EGF高表达的UC-MSCs按1×104的密度接种于6孔板中,在不同的培养时间点(12h、24h、48h)取上清进行ELISA检测,实验重复3次,每次3个复孔.检测结果用SIGMAPLOT软件统计分析,每个时间点的UC-MSCs和EGF高表达的UC-MSCs上清EGF含量的两组间比较用Mann-Whitney Rank Sum Test的方法统计分析,三个时间点的EGF高表达的UC-MSCs上清EGF含量的三组数据比较用Kruskal-Wallis One Way Analysis of Variance onRanks的方法统计分析,如图6所示,EGF高表达的UC-MSCs细胞均表达EGF,且与对照组有显著性差异。
实施例3:EGF高表达的UC-MSCs细胞的功能验证
(一)增殖实验
(1)用0.4μm孔径的聚酯膜Transwell进行共培养,观察EGF高表达的UC-MSCs分泌的细胞因子对模型细胞增殖的作用,下层接种模型细胞,上层小室接种EGF高表达的UC-MSCs,上层小室与下层培养板的培养底面积比为1∶2.1。
(2)6孔板中接种5×105个HaCaT(人正常皮肤永生化角质形成细胞株)或HFF1(人成纤维细胞株)。
(3)上层小室接种2×105个EGF高表达的UC-MSCs,将小室放入6孔板与HaCaT或HFF1进行共培养,对照组为上层小室接种UC-MSCs,6孔板中接种5×105个HaCaT或HFF1细胞。
(4)在不同的时间点(0、18、36小时)用CCK试剂盒检测六孔板中HaCaT或HFF1细胞增殖情况,每次在6孔板的培养基中HaCaT或HFF1细胞的培养基(总体积2ml)中加入CCKsolution 200μL后将培养板放在CO2培养箱4小时后,每孔取200μL上清用酶标仪读取OD450值,细胞培养板继续置于CO2培养箱中培养,直至36小时后观察终点。三个时间点的UC-MSCs与EGF高表达的UC-MSCs对HaCaT或HFF1细胞增殖的促进作用以OD450值代表,每个时间点比较两组共培养后的OD450值,用Mann-Whitney Rank Sum Test的方法统计分析,P<0.05为有统计学意义,结果如图7,EGF-高表达UC-MSCs对HaCaT及HFF1细胞增殖有促进作用。
(二)迁移实验
(1)用0.4μm孔径的聚酯膜Transwell进行共培养,下层接种模型细胞,实验组上层小室接种EGF高表达的UC-MSCs;阳性对照组为上层小室接种UC-MSCs;阴性对照组为上层小室仅放入UltraCUL TURE培养基。
(2)6孔板接种1.2×106个HaCaT细胞。
(3)第二天当HaCaT或者HFF1细胞细胞贴壁、汇合度至95%-100%时,每孔用10μL枪头进行划痕,并用PBS轻轻润洗6孔板3次。
(4)实验组将接种了5×105个高表达EGF的UC-MSCs的小室放入6孔板,使高表达EGF的UC-MSCs与划痕后的HaCaT进行共培养;阳性对照组为上层小室接种5×105个UC-MSCs与与划痕后的HaCaT进行共培养;阴性对照组为与实验组等体积的UltraCULTURE培养基与与划痕后的HaCaT进行共培养。
(5)通过0h,12h,24h显微镜成像观察并计算愈合率。实验结果如图8所示,与EGF-MSC共培养的细胞的增殖、迁移速度更快,证明本发明的EGF高表达UC-MSCs能在烧伤局部促进伤口周围表皮细胞的增殖、迁移,促进创面的愈合。
(三)体内实验
(1)取5-8周的Balb/c雌性小鼠20只,用水合氯醛麻醉后背部脱毛;
(2)100℃加热边长9mm的正方形金属片5分钟,烫伤脱毛部位15s;
(3)烫伤后24h剔除构建失败的小鼠。
(4)配制EGF高表达的UC-MSCs温敏凝胶:P5代EGF高表达UC-MSCs经过胰酶消化、中和后,计数,取5×107个细胞的悬液,离心,用2.5mL预冷(4℃)的完全培养基重悬,使细胞密度在2×107/mL,然后按1∶1的比例与预冷(4℃)的30%浓度的温敏凝胶溶液轻轻混匀,经过质控检测(包括细胞活性、无菌、内毒素、支原体、MSCs细胞表型等)、放行后全程冷链运输。
烫伤后第二天将小鼠分为3组,治疗组创面涂抹EGF高表达UC-MSCs温敏凝胶复合制剂1mL,对照一组创面涂抹普通UC-MSCs温敏凝胶1mL,对照二组创面涂抹温敏凝胶1mL;涂抹完毕后观察凝胶凝固情况,若小鼠麻醉后体温较低、凝胶难以凝固,可置于红外线灯下保温、促进凝胶凝固。
(5)每三天观察统计小鼠伤口愈合情况。
(6)烫伤后第21天,处死小鼠,取出烫伤部位皮肤进行检测。
(7)通过观察表皮再生、创面愈合情况,判断EGF高表达的UC-MSCs对皮肤创伤愈合的促进作用,结果如图9显示,EGF高表达UC-MSCs能更快促进小鼠皮肤缺损的修复。
实施例4:临床级温敏凝胶的制备方法
在生物安全柜中称取30g PNIPAAm-PEG温敏凝胶粉末放入无菌的250mL玻璃烧杯中,加入预冷的灭菌超纯水80ml及无菌磁力棒,密闭后置于4℃冰箱中,于磁力搅拌器上持续搅拌溶解3小时后,在生物安全柜中用预冷的灭菌超纯水定容至100mL,用22μm的滤器过滤除菌后分装于15毫升的灭菌离心管中,得到30%的温敏凝胶水溶液,4℃保存;需大规模配制时,则用大容器scale up制备;
实施例5:EGF高表达的UC-MSCs温敏凝胶的储存与使用方法
(一)EGF高表达的UC-MSCs制剂中的两种成分分开分别储存:
a、EGF高表达的UC-MSCs的储存按常规的细胞冻存方法:将培养的大量活率>90%、处于对数生长期的P5代EGF高表达的UC-MSCs消化、中和后,800rpm室温离心5分钟,去上清,缓慢加入GMP级别无血清细胞冻存液,调整细胞密度为1×107//mL,混匀后分装为1mL/支(留取少量细胞悬液进行质控检测),置于-80度冰箱。留样进行细胞活性、无菌、内毒素、支原体、MSCs细胞表型、细胞染色体核型、三系分化能力、成瘤性等检测合格后,将细胞置于液氮中长期储存。
b、温敏凝胶的储存:将分装于15mL灭菌离心管中的温敏凝胶水溶液于4℃密封保存,定期(3个月)取1ml样品置于37℃水浴锅中检测其凝固时间观察其稳定性,超过一年则重新配置。
(二)EGF高表达的UC-MSCs温敏凝胶快速配制方法
在临床使用前,EGF高表达的UC-MSCs采用快速复苏方法:从液氮罐中取出检测合格的EGF高表达的UC-MSCs冻存管后立即浸入37℃水浴箱振荡,当冰块溶解后,立即加入含10mL UltraCUL TURE培养基的离心管,800rpm室温离心5分钟,去上清,计数细胞,取5×107个细胞用2.5ml预冷的30%浓度的温敏凝胶溶液轻轻混匀,得到5mL细胞密度为1×107//mL的UC-MSCs温敏凝胶悬液(温敏凝胶终浓度为15%),经快速质控检测(包括微生物检测和细胞活率检测)、放行后全程冷链运输至床边,快速均匀地涂敷于创面,使创面表面形成一薄层凝胶覆盖创面,待凝胶在皮温的作用下凝固后按创面常规治疗。以后每天观察创面愈合情况,若有必要,可重复涂敷。
Claims (8)
1.一种EGF高表达的干细胞温敏凝胶的制备方法及其应用,其特征在于:将EGF的核苷酸序列合成后,构建重组慢病毒载体pCDH-CMV-EGF质粒,将所述pCDH-CMV-EGF质粒用于包装成携带有EGF编码基因的慢病毒,将所述携带EGF编码基因的慢病毒感染P3代的脐带间充质干细胞,得到EGF高表达的脐带间充质干细胞。
2.如权利要求1所述的一种EGF高表达的干细胞温敏凝胶的制备方法及其应用,其特征在于:所述的编码EGF的基因片段为序列表SEQ.ID.NO.1所示的核苷酸序列。
3.如权利要求1所述的一种EGF高表达的干细胞温敏凝胶的制备方法及其应用,其特征在于:所述P3代的脐带间充质干细胞如下制备:取新生儿的脐带,分离出其中的华氏胶组织,剪碎至0.5mm2大小的组织块贴壁培养,用UltraCUL TURE培养基培养传代,传至P3代时用于此实验。
4.如权利要求1所述的一种EGF高表达的干细胞温敏凝胶的制备方法及其应用,其特征在于:将所述携带EGF编码基因的慢病毒感染P3代的间充质干细胞如下操作:所述携带EGF编码基因的慢病毒的感染复数(MOI)为10。
5.如权利要求1所述的一种EGF高表达的干细胞温敏凝胶的制备方法及其应用,其特征在于:所述的EGF修饰脐带间充质干细胞经过传代、扩增2代后,得到大量高表达EGF的脐带间充质干细胞,在使用前将P5代EGF高表达脐带间充质干细胞与温敏凝胶混合用于制备治疗角膜损伤、烧烫伤及难愈合的手术切口等创面的修复的液态制剂。
6.如权利要求1所述的一种EGF高表达的干细胞温敏凝胶用于制备治疗创伤的药物。
7.一种治疗创伤的药物,其特征在于:含有权利要求1所述的EGF高表达脐带间充质干细胞温敏凝胶液态制剂,全程冷链运输至医院,用于治疗皮肤难愈性创伤,涂敷至创面后在局部皮温的作用下凝固为凝胶状,覆盖创面并有利于凝胶中的干细胞定植至创面局部。
8.一种治疗创伤的药物,其特征在于:含有权利要求1所述的EGF高表达的干细胞制备为温敏凝胶液态制剂后,干细胞在创面局部定植成功后,除作为种子细胞补充局部缺失的组织细胞、定向分化为创面组织细胞、分泌各种细胞因子促进创面修复外,组成性分泌EGF,促进创面局部周围的表皮细胞、内皮细胞的增殖及向创面迁移,加速创面愈合。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011275013.1A CN112704728A (zh) | 2020-11-09 | 2020-11-09 | 一种egf高表达的干细胞温敏凝胶的制备方法及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011275013.1A CN112704728A (zh) | 2020-11-09 | 2020-11-09 | 一种egf高表达的干细胞温敏凝胶的制备方法及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112704728A true CN112704728A (zh) | 2021-04-27 |
Family
ID=75543219
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011275013.1A Pending CN112704728A (zh) | 2020-11-09 | 2020-11-09 | 一种egf高表达的干细胞温敏凝胶的制备方法及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112704728A (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1834253A (zh) * | 2006-03-30 | 2006-09-20 | 中国人民解放军第二军医大学 | 人早胚来源的表皮或毛囊干细胞的制备及应用 |
CN111273030A (zh) * | 2020-02-25 | 2020-06-12 | 芜湖天明生物技术有限公司 | 一种定量检测重组人表皮生长因子直接竞争elisa法试剂盒及其应用 |
-
2020
- 2020-11-09 CN CN202011275013.1A patent/CN112704728A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1834253A (zh) * | 2006-03-30 | 2006-09-20 | 中国人民解放军第二军医大学 | 人早胚来源的表皮或毛囊干细胞的制备及应用 |
CN111273030A (zh) * | 2020-02-25 | 2020-06-12 | 芜湖天明生物技术有限公司 | 一种定量检测重组人表皮生长因子直接竞争elisa法试剂盒及其应用 |
Non-Patent Citations (3)
Title |
---|
D. H. YOU等: "Effects of human epidermal growth factor gene-transfected mesenchymal stem cells on fibroblast migration and proliferation", CELL PROLIFERATION, pages 1 - 2 * |
李敏等: "水凝胶材料和间充质干细胞在组织工程中的应用进展", 中国生物医学工程学报, vol. 39, no. 3, pages 367 * |
蔡德南;陈斐;王木盛;: "脐带间充质干细胞移植对烧伤患者新生肉芽组织EGF及VEGF表达的影响", 广东医学, vol. 39, no. 5, pages 740 - 744 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2171043B1 (en) | Treatment of diseases and disorders using self-renewing colony forming cells cultured and expanded in vitro | |
CN105154407B (zh) | 一种皮肤修复功能提高的人脂肪干细胞的制备方法及其应用 | |
JPH09505575A (ja) | イン・ビボでの治療性生産物の生産のための組成物 | |
Labus et al. | Expression of Wnt genes in early wound healing | |
CN113563452B (zh) | 一种生物活性肽及其与脂肪干细胞外泌体在皮肤增殖修复中的应用 | |
JP7116501B2 (ja) | 幹細胞材料およびその製造方法 | |
CN110898078A (zh) | 一种间充质干细胞分泌因子的制备及应用 | |
Laurent et al. | Bringing safe and standardized cell therapies to industrialized processing for burns and wounds | |
CN113144221B (zh) | 外泌体制剂及其制备方法和应用 | |
CN113274410A (zh) | 外泌体水凝胶复合物在制备修复皮肤瘢痕的药物中的应用 | |
CN112704728A (zh) | 一种egf高表达的干细胞温敏凝胶的制备方法及其应用 | |
CN109593725A (zh) | 一种重组间充质干细胞及其应用 | |
JP2014524269A (ja) | 自己移植システムの調製方法およびこれにより得られた移植システム | |
CN105435244A (zh) | Lin28a基因过表达腺相关病毒在促进创伤修复中的应用 | |
CN111733161B (zh) | circ6148及其重组载体在促进血管生成上的应用 | |
CN112316114B (zh) | 外泌体用于提高间充质干细胞组织修复功能的用途 | |
CN105861420B (zh) | 一种诱导内耳干细胞分化为毛细胞的培养基及其方法 | |
CN113713175A (zh) | 制备水凝胶支架的方法及由此得到的支架的用途 | |
JP4554940B2 (ja) | ヒト胎盤由来の間葉系細胞を含む医薬及び該細胞を用いたvegfの製造方法 | |
Barrientos et al. | Bone regeneration with autologous adipose-derived mesenchymal stem cells: A reliable experimental model in rats | |
SG185752A1 (en) | Compositions and methods of using living and non-living bioactive devices with components derived from self- renewing colony forming cells cultured and expanded in vitro | |
KR100725133B1 (ko) | 자가혈청과 태반추출물을 이용한 섬유아세포의 배양방법 및이를 이용한 피부재생용 조성물 | |
CN111840327B (zh) | 治疗糖尿病足的间质干细胞制剂及其应用 | |
CN112587718B (zh) | 利用间充质干细胞分泌的活性因子制备疤痕凝胶的方法 | |
TWI493035B (zh) | Human umbilical cord mesenchymal stem cells in vitro culture method, promote cell growth and wound healing of the relevant components of the manufacturing method and its application. |
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 |