CN115120617A - Vaginal composite gel containing mesenchymal stem cells and platelet lysate and preparation method thereof - Google Patents
Vaginal composite gel containing mesenchymal stem cells and platelet lysate and preparation method thereof Download PDFInfo
- Publication number
- CN115120617A CN115120617A CN202210816292.0A CN202210816292A CN115120617A CN 115120617 A CN115120617 A CN 115120617A CN 202210816292 A CN202210816292 A CN 202210816292A CN 115120617 A CN115120617 A CN 115120617A
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- stem cells
- gel
- platelet lysate
- vaginal
- 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
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 140
- 239000006166 lysate Substances 0.000 title claims abstract description 64
- 239000002131 composite material Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 26
- 210000001215 vagina Anatomy 0.000 claims abstract description 17
- 239000000243 solution Substances 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract 2
- 230000010355 oscillation Effects 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 34
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 32
- 229920002125 Sokalan® Polymers 0.000 claims description 32
- 229960001631 carbomer Drugs 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 24
- 210000001519 tissue Anatomy 0.000 claims description 21
- 210000001691 amnion Anatomy 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 10
- 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 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 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 claims description 9
- 235000010413 sodium alginate Nutrition 0.000 claims description 9
- 239000000661 sodium alginate Substances 0.000 claims description 9
- 229940005550 sodium alginate Drugs 0.000 claims description 9
- 210000000130 stem cell Anatomy 0.000 claims description 9
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 claims description 8
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 8
- 230000003698 anagen phase Effects 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- 239000000029 vaginal gel Substances 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000001976 enzyme digestion Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 229940044950 vaginal gel Drugs 0.000 claims description 6
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 5
- 229960005322 streptomycin Drugs 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 4
- 210000004381 amniotic fluid Anatomy 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
- 210000003954 umbilical cord Anatomy 0.000 claims description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 208000007536 Thrombosis Diseases 0.000 claims description 3
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 229920002907 Guar gum Polymers 0.000 claims description 2
- 239000004354 Hydroxyethyl cellulose Substances 0.000 claims description 2
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 claims description 2
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 2
- 235000010418 carrageenan Nutrition 0.000 claims description 2
- 239000000679 carrageenan Substances 0.000 claims description 2
- 229920001525 carrageenan Polymers 0.000 claims description 2
- 229940113118 carrageenan Drugs 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 239000000665 guar gum Substances 0.000 claims description 2
- 235000010417 guar gum Nutrition 0.000 claims description 2
- 229960002154 guar gum Drugs 0.000 claims description 2
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 claims description 2
- 229940071826 hydroxyethyl cellulose Drugs 0.000 claims description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 claims description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 claims description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 claims description 2
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 claims description 2
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 2
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 2
- 239000000230 xanthan gum Substances 0.000 claims description 2
- 235000010493 xanthan gum Nutrition 0.000 claims description 2
- 229920001285 xanthan gum Polymers 0.000 claims description 2
- 229940082509 xanthan gum Drugs 0.000 claims description 2
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims description 2
- 206010003694 Atrophy Diseases 0.000 abstract description 10
- 230000037444 atrophy Effects 0.000 abstract description 10
- 238000011282 treatment Methods 0.000 abstract description 10
- 230000004083 survival effect Effects 0.000 abstract description 9
- 208000004746 Atrophic Vaginitis Diseases 0.000 abstract description 8
- 206010003693 Atrophic vulvovaginitis Diseases 0.000 abstract description 8
- 201000010808 postmenopausal atrophic vaginitis Diseases 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract 1
- 239000000499 gel Substances 0.000 description 85
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 16
- 239000002953 phosphate buffered saline Substances 0.000 description 16
- 230000004069 differentiation Effects 0.000 description 13
- 108090000695 Cytokines Proteins 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 210000000981 epithelium Anatomy 0.000 description 8
- 208000024891 symptom Diseases 0.000 description 7
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 210000001808 exosome Anatomy 0.000 description 6
- 239000005556 hormone Substances 0.000 description 6
- 229940088597 hormone Drugs 0.000 description 6
- 230000017423 tissue regeneration Effects 0.000 description 6
- 241000700159 Rattus Species 0.000 description 5
- 229940011871 estrogen Drugs 0.000 description 5
- 239000000262 estrogen Substances 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000009818 osteogenic differentiation Effects 0.000 description 4
- 230000009815 adipogenic differentiation Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000009816 chondrogenic differentiation Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000001794 hormone therapy Methods 0.000 description 3
- 239000000017 hydrogel Substances 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 210000004877 mucosa Anatomy 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 230000009885 systemic effect Effects 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000002458 cell surface marker Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000007646 directional migration Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000003349 gelling agent Substances 0.000 description 2
- 230000003054 hormonal effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000009245 menopause Effects 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 210000001539 phagocyte Anatomy 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- JKYKXTRKURYNGW-UHFFFAOYSA-N 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S(O)(=O)=O)=C2 JKYKXTRKURYNGW-UHFFFAOYSA-N 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 102000000018 Chemokine CCL2 Human genes 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 208000004483 Dyspareunia Diseases 0.000 description 1
- 206010014080 Ecchymosis Diseases 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 102100037241 Endoglin Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010063560 Excessive granulation tissue Diseases 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000868152 Homo sapiens Son of sevenless homolog 1 Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 206010054877 Mucosal discolouration Diseases 0.000 description 1
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000000491 Tendinopathy Diseases 0.000 description 1
- 208000023835 Tendon disease Diseases 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010046788 Uterine haemorrhage Diseases 0.000 description 1
- 206010046910 Vaginal haemorrhage Diseases 0.000 description 1
- 206010046914 Vaginal infection Diseases 0.000 description 1
- 201000008100 Vaginitis Diseases 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
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241001233305 Xanthisma Species 0.000 description 1
- 108010076089 accutase Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 208000015100 cartilage disease Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000011278 co-treatment Methods 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 206010013990 dysuria Diseases 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 210000001126 granulation tissue Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002071 nanotube Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 208000012307 pale mucous membrane Diseases 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 208000003265 stomatitis Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000005641 tunneling Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 230000001457 vasomotor Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K35/14—Blood; Artificial blood
- A61K35/19—Platelets; Megacaryocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- 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
- A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
-
- 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
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses a vaginal composite gel containing mesenchymal stem cells and platelet lysate and a preparation method thereof, belonging to the technical field of medicine preparation. The preparation method of the vagina composite gel containing the mesenchymal stem cells and the platelet lysate comprises the following steps: 1) dissolving the gel by using a PBS buffer solution, uniformly mixing, and uniformly mixing overnight at 37 ℃ under oscillation to obtain a first gel; 2) cracking the first gel obtained in the step 1) with plateletsMixing the solutions according to the ratio of (8-9.9) to (0.1-2); 3) adjusting the pH to 7; 4) adjustment of mesenchymal Stem cell Density to 10 Using formulated gel 6 ‑10 7 Per ml, vaginal complex gel was obtained. The invention effectively improves the survival time of the amniotic mesenchymal stem cells by adding the platelet lysate through using the gel compound mesenchymal stem cells, and can effectively improve the atrophy phenomenon of vaginal tissues when being applied to the treatment of atrophic vaginitis.
Description
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to a vaginal composite gel containing mesenchymal stem cells and platelet lysate and a preparation method thereof.
Background
Women often experience corresponding physiological and psychological changes after entering menopause, and the changes closely linked to the physiological symptoms are mainly urogenital symptoms, vasomotor symptoms, osteoporosis, psychoneural symptoms, metabolic abnormalities and cardiovascular diseases. Atrophic vaginitis, one of the diseases to which menopausal women are susceptible, is common in women after natural menopause and ovarian castration, and its main pathological change is atrophy of vaginal squamous epithelium due to estrogen reduction, often accompanied by glycogen reduction and increase of vaginal pH. The thinned epithelium has a reduced resistance to changes in vaginal flora, making it susceptible to inflammation by pathogenic bacteria invading the vagina, common pathogenic bacteria including: streptococcus, staphylococcus, escherichia coli, and the like. Most patients develop no symptoms, but the main symptoms of the disease are vaginal bleeding, itching, dysuria or dyspareunia. Vaginal atrophy is manifested by pale mucous membranes, ecchymosis and disappearance of folds. The surface and middle layer cells can be reduced or disappeared to different degrees under the mirror. The small ulcers with acute inflammation and granulation tissue spread over the intact epithelium, with infiltration of submucosal lymphocytes and plasma cells.
The current treatments for atrophic vaginitis are divided into hormonal and non-hormonal treatments depending on the severity of the condition. Hormone therapy is mainly to compensate for estrogen reduction caused by hypoovarianism by means of estrogen supplementation. The application method can be divided into systemic hormone treatment and local hormone treatment, because the use of systemic hormone can increase the incidence of hormone-related tumors, and therefore, the local hormone treatment, namely vaginal estrogen treatment, is generally adopted when no obvious contraindications exist. Vaginal estrogen therapy can effectively relieve symptoms such as vaginal atrophy, but research also shows that local hormone therapy can also cause risks of tumors, cerebral apoplexy and venous embolism, so hormone therapy is forbidden for patients with related risks.
Mesenchymal stem cells can participate in tissue repair processes by secreting proteins and hormones, by transporting mitochondria through tunneling nanotubes or microvesicles, and by means of exosomes or microvesicles containing nucleic acids. Mesenchymal stem cells are used as a rich source of cytokines, and research proves that the mesenchymal stem cells can secrete various cytokines such as FGF2, IGF-1, HGF, VEGF, PDGF and the like. These cytokines play an important role in tissue repair.
In the aspect of immune regulation, the mesenchymal stem cells can inhibit the proliferation of T cells and secrete IL-2 to regulate the immune activity, and can also induce the apoptosis of inflammatory T cells by activating Fas-FasL ligand axis, monocyte chemotactic protein-1 is an important cytokine secreted by the mesenchymal stem cells to the outside, the cytokine can stimulate the directional migration of the T cells, after the inflammatory T cells are apoptotic, cell fragments of the inflammatory T cells are phagocytized by phagocytes, and the phagocytes are stimulated to secrete TGF beta to the surrounding environment, and the process can lead the T cells induced by the mesenchymal stem cells to be converted into Treg cells for promoting the systemic immune tolerance.
Platelet lysate is a saturated solution containing growth factors, proteins, cytokines and chemokines involved in key healing processes and is used to treat various diseases such as alopecia, oral mucositis, root pain, osteoarthritis, cartilage and tendon diseases. Application in vaginal gel products is not seen at present.
Disclosure of Invention
The embodiment of the invention provides a vaginal composite gel containing mesenchymal stem cells and platelet lysate and a preparation method thereof, aiming at solving the problem of lack of medicament gel for effectively improving the atrophy phenomenon of vaginal tissues.
The embodiment of the invention is realized as follows:
a preparation method of vaginal composite gel containing mesenchymal stem cells and platelet lysate specifically comprises the following steps:
1) weighing 0.05g-0.1g of gel, dissolving and uniformly mixing with 10ml of PBS (phosphate buffered saline), and uniformly mixing at 37 ℃ for overnight shaking to obtain a first gel;
2) mixing the first gel obtained in the step 1) with a platelet lysate according to the ratio of (8-9.9) to (0.1-2);
3) after the gel is completely dissolved, adjusting the pH value to 7;
4) selecting mesenchymal stem cells, digesting by trypLETMExpress enzyme, washing by PBS, and counting trypan blue;
5) adjustment of mesenchymal Stem cell Density to 10 Using formulated gel 6 -10 7 And/ml, obtaining the vagina complex gel containing the mesenchymal stem cells and the platelet lysate.
The gel in the step 1) is any one or a mixture of more of carbomer, sodium alginate, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, xanthan gum, carrageenan and guar gum; preferably, the gelling agent is carbomer.
The preferred mixing ratio of the first gel to the platelet lysate in step 2) is 9: 1.
In the step 3), one of triethanolamine, sodium hydroxide or hydrochloric acid is preferably used for adjusting the pH.
The mesenchymal stem cells in the step 4) are any one or a mixture of several of amniotic mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells; preferably the mesenchymal stem cells are amniotic mesenchymal stem cells.
In a preferred embodiment, the mesenchymal stem cells are selected from mesenchymal stem cells in logarithmic growth phase. Further, the mesenchymal stem cells are amniotic mesenchymal stem cells in logarithmic growth phase.
The separation and culture of the amniotic mesenchymal stem cells specifically comprise the following steps:
taking amnion of parturient after cesarean section, and applying amnion containing 1% double antibodyDPBS washes the amnion to remove the coagulated blood clot and the redundant tissue; cutting amnion, adding 0.5% pancreatin, performing shake digestion at 37 deg.C for 1h, filtering the digested tissue with 200 mesh sieve, washing thoroughly with DPBS containing 1% double antibody, collecting tissue fragments, performing shake digestion at 37 deg.C for 1h with 1mg/ml collagenase I; filtering the digested tissue with 200 mesh screen, collecting filtrate, centrifuging at 1500rpm/min for 5min, and collecting cell precipitate; the cell pellet was resuspended in DMEM/F12 medium and cultured at 37 ℃ in 5% CO 2 A cell incubator; and filling stem cells into the amniotic fluid at regular intervals and changing the fluid to obtain the mesenchymal stem cells of the amniotic fluid.
The DPBS solution containing 1% double antibody contains 100U/ml of penicillin and 0.1mg/ml of streptomycin.
A vaginal composite gel containing mesenchymal stem cells and platelet lysate is prepared by the preparation method.
The vagina composite gel containing the mesenchymal stem cells and the platelet lysate is prepared into exosome gel by extracting the mesenchymal stem cells and the platelet lysate and using a gelling agent, the gel is free of pungent smell, is viscous, has good water solubility and adhesiveness, can be well adsorbed in female vagina, is neutral in pH value, slowly reduces in viscosity under the acidic environment of the vagina, gradually releases the coated substances and covers damaged vaginal tissues. When the gel is used, the gel is extruded into the deep part of the vagina and covers the cervix or the part of the xanthium, the mesenchymal stem cells attached to the gel can locally and continuously release various cytokines, and along with the dissolution of the gel, the mesenchymal stem cells can reach the surface of the vaginal mucosa and promote the growth of the vaginal mucosa epithelial cells through the contact with the vaginal mucosa epithelial cells. The gel is attached to a pathological part through the gel, thereby achieving the effect of protecting the wound surface. By the combined action of the mesenchymal stem cells, the platelet lysate and the gel, the resistance of the vaginal epithelium at the pathological change part to pathogenic microorganisms is improved, the damaged vaginal epithelium is repaired, and the stable state of the vaginal microenvironment is recovered, so that the effects of treating female vaginitis, cervical erosion and repairing the damaged vagina are achieved.
Compared with the prior art, the technical scheme of the invention is as follows:
1. patent application specification CN202111011722.3, a mesenchymal stem cell external gel for promoting wound healing, administration mode and application. By comparing the findings, the invention has the advantages that the platelet lysate is added into the gel, and the lysate can promote tissue repair, maintain the survival of mesenchymal stem cells in the gel and promote the mesenchymal stem cells to continuously release cytokines locally.
2. Patent application specification CN202111135546.4 of the prior art, a method for extracting exosomes derived from umbilical cord stem cells and preparing hydrogel. Compared with the patent, the invention has the advantages that the exosome is a cell vesicle secreted by the mesenchymal stem cell, but the mesenchymal stem cell can secrete various cell factors to the outside to play a role in promoting tissue repair, and the gel coated mesenchymal stem cell is applied to local parts to provide continuous cell factors for local damage.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
according to the vagina composite gel containing the mesenchymal stem cells and the platelet lysate, the platelet lysate and the (amniotic) mesenchymal stem cell gel are compounded, so that the survival time of the (amniotic) mesenchymal stem cells in the gel can be prolonged, and meanwhile, cytokines contained in the platelet lysate can effectively promote the repair of damaged tissues. The invention effectively improves the survival time of the amniotic mesenchymal stem cells by adding the platelet lysate through using the gel composite mesenchymal stem cells, and can effectively improve the atrophy phenomenon of vaginal tissues when being applied to the treatment of atrophic vaginitis.
Drawings
Fig. 1 is a graph showing the result of identifying an amniotic mesenchymal stem cell surface marker by using a flow cytometer in example 1 of the present invention.
FIG. 2 is a graph showing the adipose differentiation result of the amniotic mesenchymal stem cells according to example 1 of the present invention; wherein, left: adipogenic differentiation medium; and (3) right: complete culture medium.
FIG. 3 is a graph showing the result of osteogenic differentiation of the amniotic mesenchymal stem cells according to example 1 of the present invention; wherein, left: osteogenic differentiation medium; and (3) right: complete culture medium.
FIG. 4 is a graph showing the result of chondrogenic differentiation of the amniotic mesenchymal stem cells according to example 1 of the present invention; wherein, left: chondrogenic differentiation medium; and (3) right: complete culture medium.
FIG. 5 is a graph of trypan blue count cell statistics performed on the composite gel and carbomer gel of example 1 of the invention.
FIG. 6 is a graph showing the therapeutic effects of various vaginal gels of example 1 of the present invention on vaginal atrophy rats.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
At present, there is a problem of gel shortage of the agent effective for improving the atrophy phenomenon of vaginal tissue. In order to solve the technical problems, the invention provides a vaginal composite gel containing mesenchymal stem cells and platelet lysate and a preparation method thereof.
Example 1
1. Separating and culturing human amniotic mesenchymal stem cells:
get the productWashing amnion after cesarean section with 1% double antibody DPBS (DPBS solution containing 100U/ml penicillin and 0.1mg/ml streptomycin), and removing coagulated blood clot and excess tissue with tissue scissors and forceps; shearing amnion by tissue, adding 0.5% pancreatin, digesting for 1h at 37 deg.C under shaking, filtering digested tissue with 200 mesh sieve, washing thoroughly with 1% double antibody-containing DPBS, collecting tissue debris, digesting with 1mg/ml collagenase I at 37 deg.C under shaking for 1 h; filtering the digested tissue with 200 mesh screen, collecting filtrate, centrifuging at 1500rpm/min for 5min, and collecting cell precipitate; the cell pellet was resuspended in DMEM/F12 medium and cultured at 37 ℃ with 5% CO 2 A cell incubator; periodically filling stem cells into the amniotic membrane for liquid exchange.
2. And (3) identification of the amniotic mesenchymal stem cells:
identifying the surface marker of the amniotic mesenchymal stem cells by flow cytometry, identifying the differentiation capacity of the amniotic mesenchymal stem cells by trilineage differentiation, and identifying the morphology of the amniotic mesenchymal stem cells by microscopic observation.
3. Preparing the vagina composite gel containing the mesenchymal stem cells and the platelet lysate:
1) weighing 0.05g of carbomer, dissolving and uniformly mixing the carbomer with 10ml of PBS, and uniformly mixing the carbomer and the PBS overnight under the condition of 37 ℃ in a shaking way to obtain carbomer first gel;
2) mixing carbomer first gel with platelet lysate at a ratio of 9: 1;
3) regulating the pH value to 7 by using triethanolamine after the carbomer is completely dissolved;
4) selecting amnion mesenchyme stem cell in logarithmic growth phase, and using trypLE TM Express enzyme digestion, PBS washing, trypan blue counting;
5) cell density was adjusted to 10 using formulated carbomer gel 6 -10 7 And/ml, obtaining the vagina complex gel containing the mesenchymal stem cells and the platelet lysate.
4. And (3) identifying the surface marker of the amniotic mesenchymal stem cells:
1. collecting amnion with good growthMesenchymal stem cells using TrypLE TM Express enzyme digestion, washing 3 times with PBS, sucking appropriate amount of cell suspension, sucking same amount of trypan blue, mixing, counting, adjusting density to 10 7 /ml；
2. Using BD TM Accutase TM Cell separation Solution Cell washing, followed by 1 × 10 in BD Pharmingen 7 Resuspend at individual cell/ml concentration;
3. the identification result of the surface marker of the amniotic mesenchymal stem cell by using a flow cytometer is as follows: the amniotic mesenchymal stem cell surface marker was identified by flow cytometry, and the results are shown in fig. 1. CD90, CD105, CD73 and CD44 are positively expressed, and CD34, CD11B, CD19, CD45 and HLA-DR are negatively expressed.
5. Three-line differentiation of the amniotic mesenchymal stem cells:
1) taking well-grown mesenchymal stem cells of sheep membrane, and using trypLE TM Express enzyme digestion, PBS washing for 3 times, sucking a proper amount of cell suspension, sucking trypan blue with the same amount, mixing uniformly, counting, and adjusting the density to 10 5 /ml；
2) Taking a 24-pore plate, sucking 0.5mL of the uniformly mixed amnion mesenchymal stem cell suspension into each identification hole, and carrying out 5% CO treatment at 37 DEG C 2 Culturing in an incubator;
3) the amnion mesenchymal stem cell confluency reaches about 70 percent and starts to be induced, and a mesenchymal stem cell differentiation culture medium is prepared according to the instruction; discarding the culture medium in the experimental well, adding 0.5mL differentiation culture medium into the experimental well, adding complete culture medium into the control group, transferring the 24-well plate to 37 deg.C, and 5% CO 2 The culture box is internally provided with a culture box;
4) changing the fresh differentiation culture medium for 1 time every 3 days for the experimental wells, and changing the fresh complete culture medium for the control wells;
5) the differentiated mesenchymal stem cells were stained and the results were observed under a microscope.
6. Adipose differentiation of amniotic mesenchymal stem cells:
after the differentiation of the amniotic mesenchymal stem cells was induced by the adipogenic differentiation medium, the differentiation results are shown in fig. 2, and in the adipogenic differentiation medium, it takes about 21 days for the cells to form lipid droplets, but the lipid droplets are mainly tiny lipid droplets, and after oil red O staining, the lipid droplets appear orange (fig. 2, left). In the control group (right in FIG. 2), no lipid droplet was observed in the amniotic mesenchymal stem cells.
7. Osteogenic differentiation of amniotic mesenchymal stem cells:
after the amniotic mesenchymal stem cells are induced and differentiated by the osteogenic differentiation medium, the differentiation result is shown in fig. 3, about 21 days are needed for the amniotic mesenchymal stem cells to have obvious bone tissue precipitates, the amniotic mesenchymal stem cells are in the inducing process (on the left of fig. 3, granular precipitates can be seen, the granular precipitates can become blocks along with the inducing time, the precipitates can become orange red blocks under alizarin red S staining, and the orange red blocks do not appear under the same staining condition in a control group (on the right of fig. 3).
8. Chondrogenic differentiation of amniotic mesenchymal stem cells:
after the amniotic mesenchymal stem cells are induced and differentiated by the chondroblast differentiation medium, the differentiation result is shown in fig. 4, in the differentiation process, the growth of the amniotic mesenchymal stem cells can generate a directional migration phenomenon, cell clusters (shown in fig. 4 left) can be formed among the cells along with the extension of the induction time, and if one-step culture is carried out after the cell clusters are generated, the cell clusters can be separated from a culture dish and grow in a suspension manner in the culture medium. In the control group (right in FIG. 4), cells were not clustered by culturing MSC-T4 human mesenchymal stem cells in serum-free medium + 10% UltraGROTM-Advanced + penicillin-streptomycin (penicillin concentration 100U/ml, streptomycin concentration 0.1 mg/ml).
9. Determining the survival time of the amniotic mesenchymal stem cells in the platelet lysate gel:
(1) weighing 0.05g of carbomer, dissolving and uniformly mixing the carbomer with 10ml of PBS, shaking and uniformly mixing the carbomer at the temperature of 37 ℃ overnight, and adjusting the pH to 7 by using triethanolamine; preparing pure carbomer gel;
(2) weighing 0.05g of carbomer, dissolving and uniformly mixing the carbomer with 10ml of PBS, uniformly mixing the carbomer gel and the platelet lysate overnight under the condition of 37 ℃ in a shaking way, and mixing the carbomer gel and the platelet lysate in a ratio of 9: 1; after the carbomer is completely dissolved, triethanolamine is used for adjusting the pH value to 7, and carbomer gel containing platelet lysate is prepared; (ii) a
(3) Respectively using two gels to mix the amniotic mesenchymal stem cells uniformly until the cell density is 10 6 /ml；
(4) Respectively inoculating the two mesenchymal stem cell gels into a 12-pore plate, wherein each pore is inoculated with 1 ml; culturing in a cell culture box for 24 hours;
(5) counting cells by trypan blue;
(6) as shown in the results of fig. 5, the survival of the cells in the gel can be effectively promoted by adding the platelet lysate. Wherein the gel obtained in the step (2) is a composite gel; the gel obtained in step (1) is carbomer gel.
10. The repair effect of the amniotic mesenchymal stem cells on vaginal epithelium is as follows:
1) carrying out castration operation on a rat to establish an atrophic vaginitis model;
2) comparing the amniotic mesenchymal stem cell gel with carbomer gel and amniotic mesenchymal stem cell supernatant gel, and observing the curative effect of the amniotic mesenchymal stem cell gel on treating atrophic vaginitis;
3) wherein rats with atrophic vaginitis are divided into 4 groups, wherein group A uses sheep mesenchymal stem cell platelet lysate gel; group B used platelet lysate liquid gel; group C used neat carbomer gel; group D is not processed; normal rats without castration were selected as blank controls.
4) The therapeutic effect of the amniotic mesenchymal stem cells on the vaginal atrophy rat is shown in figure 6, wherein 1 in figure 6 is an amniotic mesenchymal stem cell platelet lysate gel group; fig. 6, 2 is a platelet lysate hydrogel gel set; fig. 6, 3 is carbomer gel group; FIG. 6, 4 is the castration group; in FIG. 6, 5 is a blank control group without castration. Compared with the pure castration group, the thickness of the vaginal epithelium of the amniotic mesenchymal stem cell platelet lysate gel group and the platelet lysate gel group is thickened. Of these three groups, the thickness of vaginal epithelium was greatest after treatment with the amniotic mesenchymal stem cell platelet lysate gel group.
Compared with the prior art, the technical scheme of the invention is as follows:
1. patent application specification CN202111011722.3, a mesenchymal stem cell external gel for promoting wound healing, administration mode and application. By comparing the findings, the invention has the advantages that the platelet lysate is added into the gel, and the lysate can promote tissue repair, maintain the survival of mesenchymal stem cells in the gel and promote the mesenchymal stem cells to continuously release cytokines locally.
2. Patent application specification CN202111135546.4 of the prior art, a method for extracting exosomes derived from umbilical cord stem cells and preparing hydrogel. Compared with the patent, the invention has the advantages that the exosome is a cell vesicle secreted by the mesenchymal stem cell, but the mesenchymal stem cell can secrete various cell factors to the outside to play a role in promoting tissue repair, and the gel coated mesenchymal stem cell is applied to local parts to provide continuous cell factors for local damage.
Example 2
1. Culturing adipose-derived mesenchymal stem cells:
taking well-grown adipose-derived mesenchymal stem cells, removing a cell culture medium when the growth abundance reaches 80%, adding a fresh complete culture medium, culturing the cells for 24-48h, and collecting the mesenchymal stem cells; in this example, the adipose-derived mesenchymal stem cells are used to replace the amniotic mesenchymal stem cells in example 1, so as to prepare the vaginal composite gel containing mesenchymal stem cells and platelet lysate.
Example 3
Collecting the amniotic mesenchymal stem cells according to example 1;
3. preparing the vagina composite gel containing the mesenchymal stem cells and the platelet lysate:
1) weighing 0.1g of carbomer, dissolving and uniformly mixing the carbomer with 10ml of PBS, and uniformly mixing the carbomer and the PBS under the condition of 37 ℃ for one night in a shaking way to obtain carbomer first gel;
2) mixing carbomer first gel with platelet lysate at a ratio of 9: 1;
3) after the carbomer is completely dissolved, adjusting the pH to 7 by using sodium hydroxide;
4) selecting amnion mesenchyme stem cell in logarithmic growth phase, and using trypLE TM Express enzyme digestion, PBS washing, trypan blue counting;
5) cell density was adjusted to 10 using formulated carbomer gel 6 -10 7 And/ml, obtaining the vagina complex gel containing the mesenchymal stem cells and the platelet lysate.
The other preparation and detection procedures were the same as in example 1.
Example 4:
collecting the amniotic mesenchymal stem cells according to example 1;
3. preparing the vagina composite gel containing the mesenchymal stem cells and the platelet lysate:
1) weighing 0.05g of sodium alginate, dissolving and uniformly mixing the sodium alginate with 10ml of PBS, and uniformly mixing the sodium alginate and the PBS under the condition of 37 ℃ for one night in a shaking way to obtain sodium alginate first gel;
2) mixing the sodium alginate first gel with the platelet lysate according to the ratio of 9: 1;
3) after the sodium alginate is completely dissolved, hydrochloric acid is used for adjusting the pH value to 7;
4) selecting amnion mesenchyme stem cell in logarithmic growth phase, and using trypLE TM Express enzyme digestion, PBS washing, trypan blue counting;
5) adjusting the cell density to 10 by using prepared sodium alginate gel 6 -10 7 And/ml, obtaining the vagina complex gel containing the mesenchymal stem cells and the platelet lysate.
The other preparation and detection procedures were the same as in example 1.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
according to the vagina composite gel containing the mesenchymal stem cells and the platelet lysate, the platelet lysate and the (amniotic) mesenchymal stem cell gel are compounded, so that the survival time of the (amniotic) mesenchymal stem cells in the gel can be prolonged, and meanwhile, cytokines contained in the platelet lysate can effectively promote the repair of damaged tissues. The invention effectively improves the survival time of the amniotic mesenchymal stem cells by adding the platelet lysate through using the gel compound mesenchymal stem cells, and can effectively improve the atrophy phenomenon of vaginal tissues when being applied to the treatment of atrophic vaginitis.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A preparation method of vaginal composite gel containing mesenchymal stem cells and platelet lysate is characterized in that: the method comprises the following steps:
1) weighing 0.05g-0.1g of gel, dissolving and uniformly mixing with 10ml of PBS buffer solution, and uniformly mixing under oscillation at 37 ℃ overnight to obtain first gel;
2) mixing the first gel obtained in the step 1) with the platelet lysate according to the proportion of (8-9.9) to (0.1-2);
3) after the gel is completely dissolved, adjusting the pH value to 7;
4) selecting mesenchymal stem cells, and using TrypLE TM Express enzyme digestion, PBS washing, trypan blue counting;
5) adjustment of mesenchymal Stem cell Density to 10 Using formulated gel 6 -10 7 And/ml, obtaining the vagina complex gel containing the mesenchymal stem cells and the platelet lysate.
2. The method for preparing the composite vaginal gel containing the mesenchymal stem cells and the platelet lysate according to claim 1, wherein the method comprises the following steps: the gel in the step 1) is any one or a mixture of more of carbomer, sodium alginate, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, xanthan gum, carrageenan and guar gum.
3. The method for preparing the composite vaginal gel containing the mesenchymal stem cells and the platelet lysate according to claim 1, wherein the method comprises the following steps: the mixing ratio of the first gel to the platelet lysate in the step 2) is 9: 1.
4. The method for preparing the composite vaginal gel containing the mesenchymal stem cells and the platelet lysate according to claim 1, wherein the method comprises the following steps: in the step 3), one of triethanolamine, sodium hydroxide or hydrochloric acid is adopted for adjusting the pH.
5. The method for preparing the composite vaginal gel containing the mesenchymal stem cells and the platelet lysate according to claim 1, wherein the method comprises the following steps: the mesenchymal stem cells in the step 4) are any one or a mixture of several of amniotic mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells.
6. The method for preparing the composite vaginal gel containing the mesenchymal stem cells and the platelet lysate according to claim 5, wherein the method comprises the following steps: the mesenchymal stem cells adopt mesenchymal stem cells in logarithmic growth phase.
7. The method for preparing the vaginal composite gel containing the mesenchymal stem cells and the platelet lysate according to claim 6, wherein the method comprises the following steps: the mesenchymal stem cells are amniotic mesenchymal stem cells in logarithmic growth phase.
8. The method for preparing the vaginal composite gel containing the mesenchymal stem cells and the platelet lysate according to claim 7, wherein: the separation and culture of the amniotic mesenchymal stem cells specifically comprise the following steps: taking the amnion of a parturient after cesarean section, washing the amnion by using DPBS containing 1% double antibody, and removing coagulated blood clots and redundant tissues; cutting amnion, adding 0.5% pancreatin, digesting at 37 deg.C under shaking for 1 hr, filtering digested tissue with 200 mesh sieve, washing thoroughly with DPBS containing 1% double antibody, collecting tissue debris, and shaking with 1mg/ml collagenase I at 37 deg.CDigesting for 1 h; filtering the digested tissue with 200 mesh screen, collecting filtrate, centrifuging at 1500rpm/min for 5min, and collecting cell precipitate; the cell pellet was resuspended in DMEM/F12 medium and cultured at 37 ℃ with 5% CO 2 A cell incubator; and filling stem cells into the amniotic fluid at regular intervals and changing the fluid to obtain the mesenchymal stem cells of the amniotic fluid.
9. The method for preparing the vaginal composite gel containing the mesenchymal stem cells and the platelet lysate according to claim 8, wherein: the DPBS solution containing 1% double antibody contains 100U/ml of penicillin and 0.1mg/ml of streptomycin.
10. A vaginal complex gel containing mesenchymal stem cells and platelet lysate obtained by the method of any one of claims 1 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210816292.0A CN115120617A (en) | 2022-07-12 | 2022-07-12 | Vaginal composite gel containing mesenchymal stem cells and platelet lysate and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210816292.0A CN115120617A (en) | 2022-07-12 | 2022-07-12 | Vaginal composite gel containing mesenchymal stem cells and platelet lysate and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115120617A true CN115120617A (en) | 2022-09-30 |
Family
ID=83383850
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210816292.0A Pending CN115120617A (en) | 2022-07-12 | 2022-07-12 | Vaginal composite gel containing mesenchymal stem cells and platelet lysate and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115120617A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115463154A (en) * | 2022-10-21 | 2022-12-13 | 上海君益禾生物医学科技有限公司 | Preparation method and application of platelet lysate and exosome composite gel |
| CN116196269A (en) * | 2023-04-18 | 2023-06-02 | 海南一龄医疗产业发展有限公司 | Biological protein gel for treating mixed vaginitis and preparation method thereof |
| CN116410921A (en) * | 2023-02-09 | 2023-07-11 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017096618A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and culturing mesenchymal stem cells from outer layer of amniotic membrane tissue of umbilical cord |
| CN110475563A (en) * | 2018-03-05 | 2019-11-19 | 杨芷 | A kind of stem cell medicine and preparation method thereof for anti-aging reparation |
| CN114366763A (en) * | 2022-01-10 | 2022-04-19 | 尹文君 | Gynecological gel and preparation method thereof |
-
2022
- 2022-07-12 CN CN202210816292.0A patent/CN115120617A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017096618A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and culturing mesenchymal stem cells from outer layer of amniotic membrane tissue of umbilical cord |
| CN110475563A (en) * | 2018-03-05 | 2019-11-19 | 杨芷 | A kind of stem cell medicine and preparation method thereof for anti-aging reparation |
| CN114366763A (en) * | 2022-01-10 | 2022-04-19 | 尹文君 | Gynecological gel and preparation method thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115463154A (en) * | 2022-10-21 | 2022-12-13 | 上海君益禾生物医学科技有限公司 | Preparation method and application of platelet lysate and exosome composite gel |
| CN116410921A (en) * | 2023-02-09 | 2023-07-11 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
| CN116410921B (en) * | 2023-02-09 | 2024-01-23 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
| CN116196269A (en) * | 2023-04-18 | 2023-06-02 | 海南一龄医疗产业发展有限公司 | Biological protein gel for treating mixed vaginitis and preparation method thereof |
| CN116196269B (en) * | 2023-04-18 | 2023-11-21 | 海南一龄医疗产业发展有限公司 | Biological protein gel for treating mixed vaginitis and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115120617A (en) | Vaginal composite gel containing mesenchymal stem cells and platelet lysate and preparation method thereof | |
| US20230405180A1 (en) | Mesenchymal stem cell-hydrogel-biodegradable or mesenchymal stem cell-hydrogel-undegradable support composition for skin regeneration or wound healing | |
| EP3189828B1 (en) | Composition for differentiation induction of adipocyte containing stem cell-derived exosome, regeneration of adipose tissue, and skin whitening or wrinkle improvement | |
| Yang et al. | Vitamin C plus hydrogel facilitates bone marrow stromal cell-mediated endometrium regeneration in rats | |
| KR20220094194A (en) | Pluripotent stem cells, pharmaceutical compositions, and methods for their preparation and applications thereof | |
| JP6010112B2 (en) | Pharmaceutical composition and pharmaceutical preparation comprising immortalized stem cells and products thereof as active ingredients | |
| Hou et al. | Endometrial regeneration in Asherman's syndrome: clinical and translational evidence of stem cell therapies | |
| JP2023504013A (en) | Biomaterials for prevention and treatment of tissue damage | |
| CN110507597A (en) | A kind of composition and preparation method thereof, application | |
| JP2021038268A (en) | Regenerative therapeutic composition and method for producing regenerative therapeutic composition | |
| WO2018235834A1 (en) | Treatment agent for epidermolysis bullosa | |
| JP2016210730A (en) | Pharmaceutical compositions and production methods thereof as well as pharmaceuticals | |
| WO2006134951A1 (en) | Long-term culture proliferation method for fat-derived stem cell | |
| CN108324926B (en) | Composition of stem cell extract and antibacterial peptide and application thereof | |
| CN111282018B (en) | Adipose-derived stem cell composition for wound repair | |
| CN115820548B (en) | Preparation method and application of animal tissue-derived apoptosis vesicles | |
| JP2018177687A (en) | Pharmaceutical composition used for treatment of infertility and production method thereof | |
| EP4257674A1 (en) | Method for isolating and culturing cord blood stem cells expressing gdf-3 at high level, and use of gdf-3 | |
| Eiro et al. | Non pregnant human uterus as source of mesenchymal stem cells | |
| US20190055517A1 (en) | Cell Line of M2c Macrophage and Its Applications | |
| US20220409652A1 (en) | miRNA-BASED PHARMACEUTICAL COMPOSITIONS AND USES THEREOF FOR THE PREVENTION AND THE TREATMENT OF TISSUE DISORDERS | |
| CN107468708A (en) | A kind of preparation method of Stem Cell Activity factor gel and the application in Hard agglut wound treatment | |
| Chin et al. | 2, 3, 5, 4′-tetrahydroxystilbene-2-O-β-D-glucoside-stimulated dental pulp stem cells-derived conditioned medium enhances cell activity and anti-inflammation | |
| CN111676190A (en) | Inducer for differentiation of stem cells into chondroblasts and application thereof | |
| CN111135195A (en) | Preparation method of stem cell active factor composition capable of delaying female vaginal muscle function deterioration |
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 |