CN116162590A - PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof - Google Patents
PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof Download PDFInfo
- Publication number
- CN116162590A CN116162590A CN202310065300.7A CN202310065300A CN116162590A CN 116162590 A CN116162590 A CN 116162590A CN 202310065300 A CN202310065300 A CN 202310065300A CN 116162590 A CN116162590 A CN 116162590A
- Authority
- CN
- China
- Prior art keywords
- plga
- hucmsc
- exosome
- mesenchymal stem
- umbilical cord
- 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
- 210000001808 exosome Anatomy 0.000 title claims abstract description 85
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 54
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 title claims abstract description 15
- 210000003954 umbilical cord Anatomy 0.000 claims abstract description 52
- 239000002245 particle Substances 0.000 claims abstract description 46
- 239000004005 microsphere Substances 0.000 claims abstract description 43
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims abstract description 30
- 210000001519 tissue Anatomy 0.000 claims abstract description 29
- 241000699670 Mus sp. Species 0.000 claims abstract description 26
- 238000002360 preparation method Methods 0.000 claims abstract description 20
- 239000003814 drug Substances 0.000 claims abstract description 18
- 241001465754 Metazoa Species 0.000 claims abstract description 8
- 238000005538 encapsulation Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 43
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 24
- 210000004027 cell Anatomy 0.000 claims description 24
- 229910052802 copper Inorganic materials 0.000 claims description 24
- 239000010949 copper Substances 0.000 claims description 24
- 239000006228 supernatant Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 18
- 230000005540 biological transmission Effects 0.000 claims description 16
- 235000015097 nutrients Nutrition 0.000 claims description 16
- 238000011282 treatment Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 239000012091 fetal bovine serum Substances 0.000 claims description 7
- 238000004113 cell culture Methods 0.000 claims description 6
- 238000011161 development Methods 0.000 claims description 6
- 235000015110 jellies Nutrition 0.000 claims description 6
- 239000008274 jelly Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000001262 western blot Methods 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 239000007850 fluorescent dye Substances 0.000 claims description 5
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 238000007405 data analysis Methods 0.000 claims description 4
- 230000002757 inflammatory effect Effects 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 239000002105 nanoparticle Substances 0.000 claims description 4
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 210000003022 colostrum Anatomy 0.000 claims description 3
- 235000021277 colostrum Nutrition 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000002552 dosage form Substances 0.000 claims description 3
- 210000000936 intestine Anatomy 0.000 claims description 3
- 210000003463 organelle Anatomy 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 210000001644 umbilical artery Anatomy 0.000 claims description 3
- 210000003606 umbilical vein Anatomy 0.000 claims description 3
- 210000004204 blood vessel Anatomy 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000012757 fluorescence staining Methods 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000011503 in vivo imaging Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 230000002441 reversible effect Effects 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 241000282412 Homo Species 0.000 claims 1
- 230000004927 fusion Effects 0.000 claims 1
- 230000004807 localization Effects 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 238000004659 sterilization and disinfection Methods 0.000 claims 1
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 abstract description 17
- 230000009266 disease activity Effects 0.000 abstract description 7
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 abstract description 6
- 230000001575 pathological effect Effects 0.000 abstract description 6
- 230000008685 targeting Effects 0.000 abstract description 4
- 210000000952 spleen Anatomy 0.000 abstract description 3
- 239000012528 membrane Substances 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 230000000968 intestinal effect Effects 0.000 description 9
- 206010061218 Inflammation Diseases 0.000 description 8
- 230000004054 inflammatory process Effects 0.000 description 8
- 239000002033 PVDF binder Substances 0.000 description 7
- 229940079593 drug Drugs 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 208000002551 irritable bowel syndrome Diseases 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000004372 Polyvinyl alcohol Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- 102100037904 CD9 antigen Human genes 0.000 description 2
- 102000034342 Calnexin Human genes 0.000 description 2
- 108010056891 Calnexin Proteins 0.000 description 2
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 2
- 208000012671 Gastrointestinal haemorrhages Diseases 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 2
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 208000037273 Pathologic Processes Diseases 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 208000035861 hematochezia Diseases 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000009054 pathological process Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 1
- 206010022699 Intestinal stenosis Diseases 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 206010038063 Rectal haemorrhage Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000112 colonic effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000003767 ileocecal valve Anatomy 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000010045 kangjia Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000009854 mucosal lesion Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 210000004739 secretory vesicle Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 230000036262 stenosis Effects 0.000 description 1
- 208000037804 stenosis Diseases 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 229940126672 traditional medicines Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
- 239000012224 working solution Substances 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
- A61K35/28—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
- A61K47/34—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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Hematology (AREA)
- Pain & Pain Management (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of biological medicine, and particularly discloses a PLGA-coated human umbilical cord mesenchymal stem cell exosome and application thereof, wherein the PLGA-coated human umbilical cord mesenchymal stem cell exosome is prepared by the following steps: 1) preparation of human umbilical mesenchymal stem cells, 2) preparation of exosome hucMC-Ex, 3) identification of exosome hucMC-Ex, 4) preparation of PLGA-hucMC-Ex microsphere particles, and 5) identification of PLGA-hucMC-Ex microsphere particles. The invention uses DSS solution to induce the inflammatory bowel disease model of mice, and detects the weight, disease activity index, colorectal tissue pathological structure, spleen pathological structure, HE staining and the like of the mice. The results show that the human umbilical mesenchymal stem cell-derived exosome can delay inflammatory bowel disease of model animals in an oral mode by PLGA encapsulation, and has the characteristics of strong targeting, convenient administration and the like. Has good application prospect in the aspect of treating inflammatory bowel disease.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly discloses a PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof.
Background
Inflammatory bowel disease (inflammatory bowel disease, IBD) is a chronic, non-specific inflammatory disease characterized by inflammation of the intestinal tract, tissue damage, abdominal pain, frequent persistent diarrhea, weight loss, rectal bleeding.
At present, the methods for treating inflammatory bowel disease clinically include traditional medicines, immunosuppressants, biological agents, antibiotics, surgical treatments and the like, but the methods still have limitations, and new treatment methods are urgently needed clinically to inhibit or delay the occurrence of inflammatory bowel disease.
Exosomes (exosomes) are secretory vesicles of 40-150 nm diameter with a lipid bilayer membrane of the same composition as the plasma membrane. The exosomes are an important carrier for information transmission among cells, and participate in a plurality of physiological and pathological processes such as cell survival, immunoregulation, angiogenesis, tissue regeneration, wound healing and the like through selectively wrapping various proteins, nucleic acids, lipids and the like of cells from which the exosomes are derived.
Polylactic acid-glycolic acid copolymer (PLGA) is formed by random polymerization of two monomers, namely lactic acid and glycolic acid, is a degradable functional polymer organic compound, has good biocompatibility, no toxicity and good vesicle forming and film forming performances, and is widely applied to the fields of pharmacy, medical engineering materials and modern industry.
Currently, the literature reports that human umbilical cord mesenchymal stem cell-derived exosomes have therapeutic effects in alleviating IBD. Oral administration is the most accepted route of administration for IBD patients, but current exosome treatment studies for IBD are mostly limited to tail vein injection only. In addition, the individual oral exosomes are easy to be rapidly decomposed by the digestive system, and the bioavailability is low.
Disclosure of Invention
In order to solve the problems, the invention discloses a PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof.
The technical scheme of the invention is as follows:
PLGA-coated human umbilical cord mesenchymal stem cell exosomes are prepared and obtained by the following steps:
1) preparation of human umbilical mesenchymal stem cells, 2) preparation of exosome hucMC-Ex, 3) identification of exosome hucMC-Ex, 4) preparation of PLGA-hucMC-Ex microsphere particles, and 5) identification of PLGA-hucMC-Ex microsphere particles;
the step 4) preparation of PLGA-hucMSC-Ex microsphere particles comprises the following steps:
mixing human umbilical cord mesenchymal stem cell exosome hucMSC-Ex with 0.5-2ml of dichloromethane solution containing PLGA, uniformly mixing at high speed in a magnetic stirrer to obtain colostrum, then pouring the mixed solution into 30-50ml of 1-3% PVA aqueous solution, magnetically stirring for 3-8min, then pouring into 50-150ml of distilled water, stirring at low speed for 3h, centrifuging, collecting microspheres, washing with PBS for 2-4 times, and freeze-drying in a freeze dryer to obtain the PLGA-coated human umbilical cord mesenchymal stem cell exosome PLGA-hucMSC-Ex.
In the scheme, the human umbilical mesenchymal stem cell-derived exosome is wrapped by PLGA, so that the inflammatory bowel disease of a model animal can be delayed in an oral mode, and the human umbilical mesenchymal stem cell-derived exosome has the characteristics of strong targeting, convenience in administration and the like, and has good application prospects in the aspect of treating the inflammatory bowel disease.
Further, the preparation of 1) the human umbilical cord mesenchymal stem cells based on PLGA coated human umbilical cord mesenchymal stem cell exosomes comprises the following steps:
taking out the fresh umbilical cord from the biosafety cabinet, putting the umbilical cord into a culture dish containing PBS, and washing residual blood as clean as possible; the umbilical cord is cut into segments and two umbilical arteries and an umbilical vein are removed. Placing the umbilical cord Wharton's jelly part with blood vessels removed into a new cell culture dish, adding PBS containing 0.05-0.2% penicillin-streptomycin until the PBS is completely immersed, and waiting for 15-45min;
adding a dropper containing 10-20% FBS alpha-MEM nutrient solution into a cell culture dish, shearing the Wharton's jelly part of the umbilical cord into tissue blocks with the size of 0.05-0.2cm < 2 >, clamping and pasting the tissue blocks into Corning culture dishes by forceps, putting each three dishes into a large dish, and culturing for 15-45min in a reverse buckling manner until the tissue blocks are pasted on the walls of the dishes; taking out the culture dishes, and dripping 10-13 drops of the nutrient solution into each small dish. Changing the liquid once every three days of all the small dishes until the liquid is 3 to 4 times, when the interstitial long fusiform cells climb out around the tissue blocks, gently picking out the tissue blocks when the cells are continuously expanded into a vortex shape, washing the tissue blocks once by PBS, washing to remove residual tissues and nutrient solution, adding a proper amount of pancreatin to digest the cells, and marking the cells as the generation P0, and selecting the generation P3 to P5 human umbilical cord mesenchymal stem cells hucMSCs for ensuring the purity of the cells.
Further, the preparation of the 2) exosome hucMSC-Ex based on PLGA coated human umbilical mesenchymal stem cell exosome comprises the following steps:
hucMSC were cultured in 5-20% fbs alpha-MEM nutrient solution, and when cells grew to 70% -80% confluence, the supernatant was discarded, washed three times with PBS, and changed to 5-20% exogenously removed fetal bovine serum + alpha-MEM nutrient solution. After culturing for 36-60h, the supernatant was collected. Centrifuging 1000-3000g of supernatant for 15-45min to remove cell debris, collecting supernatant, and centrifuging 5000-15000g for 15-45min to remove organelles. Transferring the obtained supernatant into a 50-150kDa ultrafiltration centrifuge tube, centrifuging 1000-2000g for 15-45min, and repeating for 3-4 times to obtain concentrated solution. Centrifuging 50000-150000g of the concentrated solution in an ultracentrifuge for 60-120min, discarding the supernatant, pouring into PBS, centrifuging again for 60-120min at 50000-150000g, discarding the supernatant, adding an appropriate amount of PBS for resuspension, and sterilizing with 0.22 μm filter.
Further, the identification of the human umbilical cord mesenchymal stem cell exosome based on PLGA encapsulation in the step 3) exosome hucMSC-Ex comprises the following steps: and carrying out NTA, transmission electron microscope observation and WesternBlot detection on the obtained exosome hucSC-Ex.
Further, the identification of the 5) PLGA-hucMSC-Ex microsphere particle based on PLGA coated human umbilical cord mesenchymal stem cell exosomes comprises any one of the following steps S1-S4:
s1, observing by an electron microscope;
s2, transmission electron microscope observation;
s3, NTA detection;
s4, fluorescent staining positioning observation.
The identification method can comprehensively identify the microsphere particles from multiple angles.
Further, the above-mentioned human umbilical cord mesenchymal stem cell exosome based on PLGA encapsulation, the steps S1-S4 specifically include the following steps:
s1, observing by an electron microscope: 10mg of PLGA-hucMC-Ex microspheres are weighed and mixed with PBS, 10-30 mu l of the PLGA-hucMC-Ex microsphere PBS solution is absorbed in a six-hole plate, and the morphology of the PLGA-hucMC-Ex particles is observed by using a 10X and 40X electron microscope;
s2, observation by a transmission electron microscope: and (3) sucking 10-30 mu l of PLGA-hucMSC-Ex microsphere PBS solution, dripping the solution on a copper mesh with the diameter of 1-3mm, standing for 3-8min at room temperature, then sucking residual liquid at the edge of the copper mesh by using filter paper to avoid touching the liquid on the copper mesh, reversely buckling the copper mesh on 2-4% (w/v) phosphotungstic acid liquid drops which are dripped on a carrier in advance, and standing for 3-8min at room temperature. Taking down the copper net, drying under an incandescent lamp, and finally observing the form of an exosome under a transmission electron microscope;
s3, diluting the PLGA-hucMSC-Ex microsphere with PBS according to a certain proportion, sucking the diluted PLGA-hucMSC-Ex microsphere PBS solution by a syringe, and slowly injecting the diluted PLGA-hucMSC-Ex microsphere PBS solution into a sample chamber for analysis, so that air bubbles are avoided. Finally, video capturing and data analysis are carried out by nanoparticle tracking analyzer software;
s4, fluorescence staining the PLGA material with CY3, and simultaneously staining hucMSC-Ex with PKH67, and observing through a fluorescence microscope.
Furthermore, the PLGA-coated human umbilical cord mesenchymal stem cell exosome is applied to the development of medicines for preventing or treating inflammatory bowel diseases.
The exosomes are involved in a number of physiological and pathological processes such as cell survival, immunomodulation, angiogenesis, tissue regeneration and wound healing by selectively encapsulating a variety of proteins, nucleic acids, lipids and the like from cells.
Furthermore, the application of the PLGA-coated human umbilical cord mesenchymal stem cell exosome in the development of medicines for preventing or treating inflammatory bowel disease comprises the positioning of PLGA-hucMSC-Ex microsphere particles in a mouse body, and the method comprises the following steps:
normal and inflammatory mice were lavaged with prepared fluorescent PLGA-hucMSC-Ex particles and sacrificed after 0h,12h, dissected and the intestines removed, and examined and observed using a small animal in vivo imaging technique.
The positioning method can position the particles by real-time fluorescence.
Furthermore, the application of the PLGA-coated human umbilical cord mesenchymal stem cell exosomes in the development of medicines for preventing or treating inflammatory bowel diseases comprises the establishment of a DSS-induced mouse IBD model.
Furthermore, the application of the PLGA-coated human umbilical cord mesenchymal stem cell exosome in the development of medicines for preventing or treating inflammatory bowel diseases is disclosed, wherein the medicines are oral medicines.
Further, the application of the PLGA-coated human umbilical cord mesenchymal stem cell exosome in the development of medicines for preventing or treating inflammatory bowel disease is characterized in that the medicine is used for a human body, and the medicine dosage form is 28-38ml of PLGA-hucMSC-Ex solution of 10 mg/ml; preferably, the dosage form is 30ml.
The oral administration method is adopted, so that the use of the medicine is greatly facilitated, and the compliance of taking the medicine is improved.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a PLGA-coated human umbilical cord mesenchymal stem cell exosome and application thereof, wherein mesenchymal stem cells are obtained from fresh umbilical cords, exosomes are separated and extracted, and the exosomes and PLGA are prepared into particle microspheres by an oil-in-water mechanical method. The successful preparation of PLGA exosome particles was demonstrated by fluorescent staining of the particle microspheres. The invention uses DSS solution to induce the inflammatory bowel disease model of mice, and detects the weight, disease activity index, colorectal tissue pathological structure, spleen pathological structure, HE staining and the like of the mice. The results show that the human umbilical mesenchymal stem cell-derived exosome can delay the inflammatory bowel disease of the model animal in an oral mode by PLGA encapsulation, has the characteristics of strong targeting, convenient administration and the like, and has good application prospect in the aspect of treating the inflammatory bowel disease.
Drawings
FIG. 1 shows the result of NTA measurement of human umbilical cord mesenchymal stem cell exosomes in example 1, with a diameter of about 150 nm;
FIG. 2 is a transmission electron microscope image of human umbilical cord mesenchymal stem cell exosomes of example 1, typical vesicle-like structures;
FIG. 3 shows the result of western-blot of human umbilical mesenchymal stem cell exosomes in example 1, expressing surface markers HSP70, CD9, CD81, calnexin;
FIG. 4 is an electron microscope picture of PLGA-hucMSC-Ex particles of example 2, which are round particles;
FIG. 5 is a transmission electron microscope picture of PLGA-hucMSC-Ex particles of example 2, which are spherical particles;
FIG. 6 shows the results of particle size analysis of PLGA-hucMSC-Ex particles of example 2, having a diameter of about 40. Mu.m;
FIG. 7 is the result of fluorescent staining of PLGA-hucMSC-Ex particles in example 2, showing that hucMSC-Ex is encapsulated on PLGA particles;
FIG. 8 is an in vivo distribution of PLGA-hucMSC-Ex particles in normal mice and enteritis models following intragastric administration in example 2. In inflammatory bowel disease, the retention and absorption time of PLGA-hucMSC-Ex particles is prolonged due to intestinal canal stenosis;
FIG. 9 is a schematic diagram of the construction of a mouse animal model in example 3;
FIG. 10 is a graph showing the weight change of the three mice in example 3, showing that the DSS group showed the most significant weight loss, and the trend of body weight was improved after PLGA-hucMSC-Ex particle treatment;
FIG. 11 shows the variation of Disease Activity Index (DAI) of three mice in example 3, wherein the increase of DAI index is most pronounced in DSS group, and the trend is improved after PLGA-hucMSC-Ex treatment;
FIG. 12 shows the changes in HE staining of intestinal tissue of three mice in example 3, wherein the disorder of intestinal tissue structure of DSS group is most evident, and the trend is improved after PLGA-hucMSC-Ex treatment;
FIG. 13 shows quantitative RNA changes in intestinal tissue of three mice in example 3, in which the pro-inflammatory factors IL-1β, IL-6 and TNF- α in DSS were all elevated, and the trend was improved after PLGA-hucMSC-Ex treatment.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were conventional reagent products commercially available.
Example 1
Preparation of human umbilical cord mesenchymal stem cell exosome
1. Preparation of human umbilical cord mesenchymal stem cells
Mesenchymal stem cells were obtained from fresh umbilical cord.
The specific operation is as follows: fresh umbilical cord was removed from biosafety cabinet and placed in a petri dish containing PBS, and residual blood was rinsed as clean as possible. The umbilical cord is cut into segments and two umbilical arteries and an umbilical vein are removed. The vessel removed umbilical Wharton's jelly fraction was placed in a new cell culture dish, PBS containing 0.1% penicillin-streptomycin was added until complete immersion, and the vessel was left to stand for 30min. Another cell culture dish was added with a dropper of alpha-MEM nutrient solution containing 15% FBS, and the Wharton's jelly part of the umbilical cord was cut into tissue pieces of about 0.1cm2 in a moist environment, and the pieces were held by forceps and attached to Corning culture dishes. Every three small dishes are placed in one large dish (reducing pollution risk), and the culture is performed for 30min in a reverse-buckling manner until the tissue blocks are attached to the walls of the culture dishes. Taking out the culture dishes, and dripping 10-13 drops of the nutrient solution into each small dish. After 3-4 times, interstitial long fusiform cells climb out around the tissue blocks, when the cells are continuously amplified into a vortex shape, the tissue blocks are gently removed, PBS is washed for one time to wash out residual tissues and nutrient solution, and a proper amount of pancreatin is added to digest the cells, and the cells are marked as P0 generation. In order to ensure the purity of the cells, P3-P5 generation human umbilical cord mesenchymal stem cells (hucMSCs) are generally selected.
2. Preparation of exosomes
hucMSC were cultured in 10% fbs alpha-MEM nutrient solution, and when cells grew to 70% -80% confluence, the supernatant was discarded, washed three times with PBS, and changed to 10% exosome-free fetal bovine serum + alpha-MEM nutrient solution. After 48 hours of culture, the supernatant was collected. A certain amount of supernatant was centrifuged for 30min at 2000g to remove cell debris, and the supernatant was collected and centrifuged for 30min at 10000g to remove organelles. Transferring the obtained supernatant into a 100kDa ultrafiltration centrifuge tube, centrifuging at 1500 Xg for 30min, and repeating for 3-4 times to obtain concentrated solution. The concentrate was then centrifuged in an ultracentrifuge for 100000g,90min, the supernatant was discarded, PBS was poured in, centrifuged again for 100000g,90min, the supernatant was discarded, and an appropriate amount of PBS was added for resuspension. The exosomes were finally obtained and sterilized with a 0.22 μm filter.
3. Identification of exosomes
And (3) carrying out NTA, transmission electron microscope observation and Western Blot detection on the obtained exosomes.
The NTA detection steps are as follows: diluting the exosomes with PBS in a certain proportion, sucking the diluted exosomes with a 1ml syringe, and slowly injecting the exosomes into a sample chamber for analysis, so as to avoid bubble generation. And finally, video capturing and data analysis are carried out by the NanoSight nano particle tracking analyzer software of the instrument. As shown in FIG. 1, the exosome diameter is about 150 nm.
The transmission electron microscope observation steps are as follows: 20 mu l of exosomes are sucked and dripped on a copper mesh with the diameter of 2mm, the copper mesh is kept stand at room temperature for 5min, and then residual liquid at the edge of the copper mesh is sucked by filter paper, so that the liquid on the copper mesh is prevented from being touched. Then the copper mesh is reversely buckled on 3% (w/v) phosphotungstic acid (pH6.8) liquid drops which are added on the carrier in advance, and the copper mesh is kept stand for 5min at room temperature. The copper mesh was removed, dried under an incandescent lamp, and finally placed under a transmission electron microscope to observe the morphology of the exosomes, which are typical vesicle-like structures as shown in fig. 2.
The Western Blot procedure was as follows: (1) extraction of exosome total protein: mu.l of exosomes were pipetted into the EP tube and 20. Mu.l of protein lysate and protease inhibitor (RIPA: PMSF) were added. Mixing, oscillating with vortex oscillator for 1min, standing on ice for 10min, and repeating for 5 times to obtain the total protein of exosomes. (2) Measurement of total exosome protein concentration: protein concentration was determined by BCA method. And (3) preparing BCA working solution according to the instruction of a kit (Kangjia), measuring absorbance by an enzyme-labeled instrument, and establishing a standard curve so as to analyze and calculate the protein concentration. The resulting concentration was multiplied by the dilution factor to give the final protein concentration. (3) western blot: according to the molecular weight of the target protein, preparing 12% SDS-PAGE electrophoresis gel (10 holes), sucking liquid into a new EP tube by a pipette according to the proportion of sample loading buffer (loading buffer) =3:1, fully and uniformly mixing and boiling for 8min, and placing the mixture on ice to be loaded. The electrophoresis liquid is prepared and used at present and is led into an electrophoresis tank, the sample is loaded according to the volume of 5 mu l of protein, and the protein Marker 2 mu l is added to make protein molecular weight reference. And (3) covering an electrophoresis tank cover, connecting a power supply, adjusting the starting voltage to 60V, adjusting the voltage to 80V after the protein enters the separation gel, and determining the electrophoresis termination time according to the molecular weight of the protein required by the user. Preparing a membrane transferring solution, cutting a PVDF membrane with a proper size according to an electrophoresis result, and polarizing the PVDF membrane with methanol. And (3) taking down the gel, placing the gel on filter paper wetted by the membrane transferring liquid, attaching a PVDF membrane on the filter paper for alignment, and placing the PVDF membrane and the filter paper into an electrophoresis tank, wherein no air bubbles exist between the PVDF membrane and the filter paper, so as to form a membrane transferring clamp, filter paper, gel, filter paper and membrane transferring clamp. The current was adjusted to 300mA (constant voltage) for 90min, then the membrane was removed and placed into 5% skim milk prepared first, and the shaker was closed for 1h at room temperature. Antibodies were diluted according to the antibody instructions at CD9 (1:1000), CD81 (1:1000), HSP70 (1:800), calnexin (1:1000) and covered on blocked PVDF membrane, incubated overnight at 4 ℃. The membranes were washed with 1 XTBST for 3X 5min for a second time. The membrane was then washed with HRP-labeled goat anti-rabbit secondary antibody for 3X 5min at room temperature for 1h in 1 XTBST. The exposure liquid is prepared, uniformly covered on a PVDF film, and detected by a chemiluminescence analyzer, and the surface mark of the human umbilical mesenchymal stem cell exosome is shown in figure 3.
The above results demonstrate that we successfully extract exosomes from human umbilical cord mesenchymal stem cells.
Example 2
PLGA-hucMSC-Ex microsphere particles were prepared using the exosomes prepared in example 1
Preparation of PLGA-hucMSC-Ex microsphere particles
Mu.l of hucMSC-Ex solution (30. Mu.g/. Mu.l) was mixed with 1ml of PLGA in dichloromethane (100 mg/ml) and homogenized at high speed in a magnetic stirrer to give colostrum, then the mixture was poured into 40ml of PVA (polyvinyl alcohol) aqueous solution (2%), magnetically stirred for 5 minutes, then into 100ml of distilled water, stirred at low speed for 3 hours, centrifuged, microspheres were collected, washed 3 times with PBS, lyophilized in a lyophilizer and finally stored in a refrigerator at-20 ℃.
Identification of PLGA-hucMSC-Ex microsphere particles
The electron microscope observation steps are as follows: 10mg of PLGA-hucMSC-Ex microspheres were weighed and mixed with PBS, 20 μl of the PLGA-hucMSC-Ex microsphere PBS solution was pipetted into six well plates, and the morphology of the PLGA-hucMSC-Ex particles was visualized using a 10X and 40X electron microscope. PLGA-hucMSC-Ex microspheres are round as shown in FIG. 4.
The transmission electron microscope observation steps are as follows: 20 mu l of PLGA-hucMSC-Ex microsphere PBS solution is sucked and dripped on a copper mesh with the diameter of 2mm, the copper mesh is kept stand at room temperature for 5min, and then residual liquid at the edge of the copper mesh is sucked by filter paper, so that the liquid on the copper mesh is prevented from being touched. Then the copper mesh is reversely buckled on 3% (w/v) phosphotungstic acid (pH6.8) liquid drops which are added on the carrier in advance, and the copper mesh is kept stand for 5min at room temperature. The copper mesh was removed, dried under an incandescent lamp, and finally placed under a transmission electron microscope to observe the morphology of the exosomes, as shown in fig. 5, PLGA-hucMSC-Ex microspheres were of a typical spherical structure.
The NTA detection steps are as follows: the PLGA-hucMSC-Ex microsphere is diluted by PBS according to a certain proportion, and a 1ml syringe is used for sucking the diluted PLGA-hucMSC-Ex microsphere PBS solution, and the diluted PLGA-hucMSC-Ex microsphere PBS solution is slowly injected into a sample chamber for analysis, so that air bubbles are avoided. And finally, video capturing and data analysis are carried out by the NanoSight nano particle tracking analyzer software of the instrument. As shown in FIG. 6, the PLGA-hucMSC-Ex microsphere has a diameter of about 40. Mu.m.
The fluorescent staining positioning and observing steps are as follows: PLGA material was fluorescent stained with CY3, while hucMSC-Ex was stained with PKH67 and observed by fluorescence microscopy. As shown in FIG. 7, it can be seen that exosomes are entrapped in PLGA, forming PLGA particles of hucMSC-Ex.
Positioning of PLGA-hucMSC-Ex microsphere particles in mice
Normal and inflammatory mice were gavaged with prepared fluorescent PLGA-hucMSC-Ex particles and sacrificed after 0h,12 h. After dissecting the mice and taking out the intestines, detection and observation are performed by using a living animal imaging technique. As shown in FIG. 8, after 12h of gastric lavage, fluorescence signals were generated in the ileocecal valve and upper colon of the normal group, whereas DSS mice were unable to rapidly discharge the hucMSC-Ex-PLGA particles due to the intestinal stenosis, and the fluorescence signals were still mainly distributed in the ileum region.
Example 3
Treatment of DSS-induced mouse inflammation model with PLGA-hucMSC-Ex particles prepared in example 2
As shown in FIG. 9, the DSS was used to induce a mouse IBD model, and PLGA-hucMSC-Ex particles were used for gastric lavage intervention. The experiments were divided into Normal control group (Normal group), DSS-induced inflammation group (DSS group), PLGA-hucMSC-Ex particle intervention group (PLGA-hucMSC-Ex group): male BALB/C mice were fed 6 weeks old.
In addition to Normal, the remaining two groups were filled with 100 μl of PLGA-hucMSC-Ex in PBS (10 mg/ml) starting on day 3 and starting on day 3 with the same volume of PBS. On day 10, all mice were sacrificed and tissue specimens such as colorectal were isolated for further detection and analysis. Calculated from the table of equivalent dose ratios for human and animal body surface area conversion, if the microsphere is to be applied to the human body in the future, a solution of 28-38ml,10mg/ml PLGA-hucMSC-Ex is probably required.
1. Observation index
The index was observed and recorded daily, and the weight change, stool characteristics and occult blood or hematochezia of the mice were observed daily, and the symptoms of the mice were evaluated using the weight change, stool characteristics change and presence or absence of hematochezia as disease activity index (disease activity index, DAI). The extent of colonic mucosal lesions was observed under HE staining microscope.
2. Experimental results
As shown in fig. 10, in the experiment of the mouse inflammation model, the body weight of three groups of mice changed, the NC group stably increased, the DSS group decreased most significantly, and the trend was improved from decreasing to increasing after the PLGA-hucMSC-Ex particle treatment.
As shown in fig. 11, in the experiment of the mouse inflammation model, the DAI of three mice was changed, the NC group was not significantly changed, the DSS group was most significantly increased, and the trend was improved after the PLGA-hucMSC-Ex particle treatment.
As shown in fig. 12, in the experiment of the mouse inflammation model, the intestinal tissue HE staining of the three mice was changed, the intestinal structure of NC group was normal, the intestinal tissue structure of DSS group was most disordered, and the intestinal structure was improved after PLGA-hucMSC-Ex particle treatment.
As shown in FIG. 13, in the experiment of the mouse inflammation model, the intestinal tissue RNA quantitative change of three groups of mice, the pro-inflammatory factors IL-1β, IL-6 and TNF- α in the DSS group are all increased, and the pro-inflammatory factors are decreased and the anti-inflammatory factor IL-10 is increased after PLGA-hucMSC-Ex treatment.
Experimental results show that the mouse model of DSS-induced inflammatory bowel disease has obviously improved mouse inflammation degree through various indexes such as weight change, disease activity index, inflammatory factors, tissue lesions and the like after the human umbilical cord mesenchymal stem cell-derived exosome PLGA particles are dried. Therefore, the exosome PLGA particles derived from human umbilical cord mesenchymal stem cells can be orally taken to treat inflammatory bowel disease.
The following conclusions can be drawn from the above embodiments: the invention uses DSS solution to induce the inflammatory bowel disease model of mice, and detects the weight, disease activity index, colorectal tissue pathological structure, spleen pathological structure, HE staining and the like of the mice. The results show that the human umbilical mesenchymal stem cell-derived exosome can delay the inflammatory bowel disease of the model animal in an oral mode by PLGA encapsulation, has the characteristics of strong targeting, convenient administration and the like, and has good application prospect in the aspect of treating the inflammatory bowel disease.
The foregoing is a description of only a limited number of preferred embodiments of the invention, which are described in greater detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. The human umbilical cord mesenchymal stem cell exosome based on PLGA encapsulation is characterized by being prepared by the following steps:
1) preparation of human umbilical mesenchymal stem cells, 2) preparation of exosome hucMC-Ex, 3) identification of exosome hucMC-Ex, 4) preparation of PLGA-hucMC-Ex microsphere particles, and 5) identification of PLGA-hucMC-Ex microsphere particles;
the step 4) preparation of PLGA-hucMSC-Ex microsphere particles comprises the following steps:
mixing human umbilical cord mesenchymal stem cell exosome hucMSC-Ex with 0.5-2ml of dichloromethane solution containing PLGA, uniformly mixing at high speed in a magnetic stirrer to obtain colostrum, then pouring the mixed solution into 30-50ml of 1-3% PVA aqueous solution, magnetically stirring for 3-8min, then pouring into 50-150ml of distilled water, stirring at low speed for 3h, centrifuging, collecting microspheres, washing with PBS for 2-4 times, and freeze-drying in a freeze dryer to obtain the PLGA-coated human umbilical cord mesenchymal stem cell exosome PLGA-hucMSC-Ex.
2. The PLGA-coated human umbilical cord mesenchymal stem cell exosome of claim 1, wherein the 1) preparation of human umbilical cord mesenchymal stem cells comprises the steps of:
taking out the fresh umbilical cord from the biosafety cabinet, putting the umbilical cord into a culture dish containing PBS, and washing residual blood as clean as possible; cutting umbilical cord into several sections, removing two umbilical arteries and one umbilical vein, placing the umbilical cord Wharton's jelly part with blood vessels removed into a new cell culture dish, adding PBS containing 0.05-0.2% penicillin-streptomycin until completely immersed, and waiting for 15-45min;
adding a dropper containing 10-20% FBS alpha-MEM nutrient solution into a cell culture dish, shearing the Wharton's jelly part of the umbilical cord into tissue blocks with the size of 0.05-0.2cm < 2 >, clamping and pasting the tissue blocks into Corning culture dishes by forceps, putting each three dishes into a large dish, and culturing for 15-45min in a reverse buckling manner until the tissue blocks are pasted on the walls of the dishes; taking out the culture dishes, dropwise adding 10-13 drops of the nutrient solution into each small dish, changing the nutrient solution once every three days until the number of times of culture dishes reaches 3-4, gradually removing the tissue blocks when the tissue blocks are continuously expanded into a vortex shape, washing the PBS for one time to wash out residual tissues and the nutrient solution, adding a proper amount of pancreatin to digest the cells, and marking the cells as P0 generation, wherein P3-P5 generation human umbilical cord mesenchymal stem cells hucMSCs are selected for ensuring the purity of the cells.
3. The PLGA-coated human umbilical cord mesenchymal stem cell exosome of claim 1, wherein the 2) exosome hucMSC-Ex preparation comprises the following steps:
culturing hucMSC in alpha-MEM nutrient solution of 5-20% FBS, discarding supernatant when cells grow to 70-80% fusion, washing with PBS three times, changing to 5-20% exosome-removed fetal bovine serum+alpha-MEM nutrient solution, culturing for 36-60h, collecting supernatant, centrifuging a certain amount of supernatant for 15-45min to remove cell debris, collecting supernatant, centrifuging for 15-45min by 5000-15000g to remove organelles, transferring the obtained supernatant to a 50-150KDa ultrafiltration centrifuge tube, centrifuging for 15-45min by 1000-2000g for 3-4 times to obtain concentrated solution, centrifuging for 60-120min by using PBS after 500-150000 g in an ultracentrifuge, discarding supernatant, centrifuging for 60-120min again by using 50000-150000g, discarding supernatant, adding a proper amount of PBS, and finally obtaining exosome hucMSC-Ex after sterilization by using a filter of 0.22 μm.
4. The PLGA-coated human umbilical cord mesenchymal stem cell exosome of claim 1, wherein the step 3) identification of exosome hucMSC-Ex comprises the steps of: and carrying out NTA, transmission electron microscope observation and WesternBlot detection on the obtained exosome hucSC-Ex.
5. The identification of PLGA-coated human umbilical cord mesenchymal stem cell exosomes of claim 1, wherein the 5) PLGA-hucMSC-Ex microspheroidal particle comprises any one of the following steps S1-S4:
s1, observing by an electron microscope;
s2, transmission electron microscope observation;
s3, NTA detection;
s4, fluorescent staining positioning observation.
6. The PLGA-coated human umbilical cord mesenchymal stem cell exosome of claim 5, wherein the steps S1-S4 specifically comprise the steps of:
s1, observing by an electron microscope: 10mg of PLGA-hucMC-Ex microspheres are weighed and mixed with PBS, 10-30 mu l of the PLGA-hucMC-Ex microsphere PBS solution is absorbed in a six-hole plate, and the morphology of the PLGA-hucMC-Ex particles is observed by using a 10X and 40X electron microscope;
s2, observation by a transmission electron microscope: sucking 10-30 mu l of PLGA-hucMSC-Ex microsphere PBS solution, dripping the solution on a copper mesh with the diameter of 1-3mm, standing for 3-8min at room temperature, sucking residual liquid at the edge of the copper mesh by using filter paper to avoid touching the liquid on the copper mesh, reversely buckling the copper mesh on 2-4% (w/v) phosphotungstic acid liquid drops which are dripped on a carrier in advance, standing for 3-8min at room temperature, taking down the copper mesh, drying under an incandescent lamp, and finally observing the morphology of an exosome under a transmission electron microscope;
s3, diluting the PLGA-hucMSC-Ex microsphere by PBS according to a certain proportion, sucking the diluted PLGA-hucMSC-Ex microsphere PBS solution by a syringe, slowly injecting the diluted PLGA-hucMSC-Ex microsphere PBS solution into a sample chamber for analysis, avoiding bubble generation, and finally carrying out video capturing and data analysis by nanoparticle tracking analyzer software;
s4, fluorescence staining the PLGA material with CY3, and simultaneously staining hucMSC-Ex with PKH67, and observing through a fluorescence microscope.
7. Use of PLGA-coated human umbilical cord mesenchymal stem cell exosomes as claimed in any one of claims 1-6 in the development of a medicament for the prevention or treatment of inflammatory bowel disease.
8. The use according to claim 7, comprising the localization of PLGA-hucMSC-Ex microsphere particles in mice, comprising the steps of:
normal and inflammatory mice were lavaged with prepared fluorescent PLGA-hucMSC-Ex particles and sacrificed after 0h,12h, dissected and the intestines removed, and examined and observed using a small animal in vivo imaging technique.
9. The use according to claim 7, wherein the medicament is an oral medicament.
10. The use according to claim 9, wherein the medicament is for use in humans and the medicament dosage form is 28-38ml,10mg/ml PLGA-hucMSC-Ex solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310065300.7A CN116162590A (en) | 2023-02-06 | 2023-02-06 | PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310065300.7A CN116162590A (en) | 2023-02-06 | 2023-02-06 | PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116162590A true CN116162590A (en) | 2023-05-26 |
Family
ID=86410735
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310065300.7A Pending CN116162590A (en) | 2023-02-06 | 2023-02-06 | PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116162590A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117883413A (en) * | 2023-12-25 | 2024-04-16 | 广东壹加再生医学研究院有限公司 | PLGA nano-particles for delivering mesenchymal stem cell exosomes, and preparation method and application thereof |
| CN117883413B (en) * | 2023-12-25 | 2024-06-04 | 广东壹加再生医学研究院有限公司 | PLGA nano-particles for delivering mesenchymal stem cell exosomes, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111759862A (en) * | 2020-06-04 | 2020-10-13 | 江苏大学 | Application of stem cell exosome in preparation of anti-colitis-exacerbation medicine |
-
2023
- 2023-02-06 CN CN202310065300.7A patent/CN116162590A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111759862A (en) * | 2020-06-04 | 2020-10-13 | 江苏大学 | Application of stem cell exosome in preparation of anti-colitis-exacerbation medicine |
Non-Patent Citations (4)
| Title |
|---|
| FEI MAO , YUNBING WU , XUDONG TANG , JINGJING KANG , BIN ZHANG , YONGMIN YAN , HUI QIAN , XU ZHANG , WENRONG XU: "Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Relieve Inflammatory Bowel Disease in Mice", BIOMED RESESRCH INTERNATIONAL, vol. 2017, pages 2 * |
| HUI LI , YIHE HU , MIN ZENG , JUNXIAO YANG , XIAOLEI FAN , YINAN WANG , JIE XIE: "Exosomes From Human Urine-Derived Stem Cells Encapsulated Into PLGA Nanoparticles for Therapy in Mice With Particulate Polyethylene-Induced Osteolysis", FRONT MED (LAUSANNE), vol. 8, pages 3 * |
| TYLER J DISTEFANO , KETI VASO , CHRISTOPHER J PANEBIANCO , GEORGE DANIAS , HENRY N CHIONUMA , KURIAKOSE KUNNATH , STYLIANOS Z KARO: "Hydrogel-Embedded Poly(Lactic- co-Glycolic Acid) Microspheres for the Delivery of hMSC-Derived Exosomes to Promote Bioactive Annulus Fibrosus Repair", CARTILAGE, vol. 13, no. 3, pages 3 * |
| 胡玉燕;尹磊;张晨笑;钱晖;: "脐带间充质干细胞外泌体对大鼠肾间质纤维化的抑制作用", 江苏大学学报(医学版), vol. 30, no. 04, pages 282 - 286 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117883413A (en) * | 2023-12-25 | 2024-04-16 | 广东壹加再生医学研究院有限公司 | PLGA nano-particles for delivering mesenchymal stem cell exosomes, and preparation method and application thereof |
| CN117883413B (en) * | 2023-12-25 | 2024-06-04 | 广东壹加再生医学研究院有限公司 | PLGA nano-particles for delivering mesenchymal stem cell exosomes, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Coleman et al. | Human polyomavirus (BK) infection and ureteric stenosis in renal allograft recipients. | |
| CN105535022A (en) | Application of exosome to preparing of acute-hepatic-failure treating medicine and medicine composition | |
| CN111759862A (en) | Application of stem cell exosome in preparation of anti-colitis-exacerbation medicine | |
| CN115414397B (en) | Application of teasel root-derived extracellular vesicle-like nano-particles in preparation of medicines for preventing or treating orthopedic diseases | |
| CN106492194A (en) | A kind of stem cell excretion body preparation and its preparation method and application | |
| JP2022524380A (en) | Acoustic extracellular matrix hydrogels and their use | |
| CN108697739A (en) | Cell tablet composition containing mescenchymal stem cell and the method for curing lumen organ using the cell tablet composition | |
| WO2019096114A1 (en) | Application of pharmaceutical composition in regulation of fibroblast growth | |
| CN111588704A (en) | Targeted responsive release system and preparation method and application thereof | |
| CN110693912A (en) | Application of stem cell exosome in preparation of product for promoting wound healing | |
| WO2024022079A9 (en) | Use of human mesenchymal stem cell sheet in treatment of uterine scars | |
| JP6494393B2 (en) | Cell composition for treating uterine tissue and method for producing the same | |
| WO2021148000A1 (en) | Mesenchymal stem cell sheet and use thereof | |
| CN111529505B (en) | Functional chimeric apoptotic body and preparation method and application thereof | |
| WO2024093149A1 (en) | Method for preparing tumor-derived cell microparticles by microwave treatment | |
| CN116162590A (en) | PLGA-coated human umbilical mesenchymal stem cell exosome and application thereof | |
| CN111529504A (en) | Functional chimeric apoptotic body and preparation method and application thereof | |
| CN115025246A (en) | Multifunctional vesicle for dual-targeting vascular repair and preparation method and application thereof | |
| CN105435306B (en) | The preparation method and purposes of biological nano sticking patch | |
| CN108042572A (en) | The product and its application method for the treatment of leucoderma and application | |
| AU2021106118A4 (en) | Use of microvesicle derived from umbilical cord mesenchymal stem cell in preparation of formulation for promoting regeneration of post-traumatic skin appendage | |
| CN106267406B (en) | AIDS blood purification | |
| CN106110426B (en) | AIDS immunization therapy instrument | |
| CN110433175A (en) | Application of the serum excretion body in preparation treatment myocardial infarction drug under the conditions of heart infarction | |
| CN107456471B (en) | Application of pomegranate bark in preparation of medicine for promoting migration of mesenchymal stem cells, medicine and preparation method of medicine |
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 | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Chaoyang Inventor after: Mao Fei Inventor after: Liu Ziyue Inventor before: Zhang Chaoyang |