CN115105637A - Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction - Google Patents
Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction Download PDFInfo
- Publication number
- CN115105637A CN115105637A CN202210899898.5A CN202210899898A CN115105637A CN 115105637 A CN115105637 A CN 115105637A CN 202210899898 A CN202210899898 A CN 202210899898A CN 115105637 A CN115105637 A CN 115105637A
- Authority
- CN
- China
- Prior art keywords
- conjunctival
- subconjunctival
- acellular matrix
- repair
- fibroblast
- 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.)
- Granted
Links
- 239000011159 matrix material Substances 0.000 title claims abstract description 97
- 210000002950 fibroblast Anatomy 0.000 title claims abstract description 92
- 230000008439 repair process Effects 0.000 claims abstract description 44
- 230000007547 defect Effects 0.000 claims abstract description 38
- 210000000130 stem cell Anatomy 0.000 claims abstract description 29
- 238000000338 in vitro Methods 0.000 claims abstract description 25
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 22
- 210000001519 tissue Anatomy 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 14
- 230000035755 proliferation Effects 0.000 claims abstract description 13
- 230000001737 promoting effect Effects 0.000 claims abstract description 13
- 210000002175 goblet cell Anatomy 0.000 claims abstract description 12
- 230000004069 differentiation Effects 0.000 claims abstract description 9
- 230000012010 growth Effects 0.000 claims abstract description 8
- 206010016654 Fibrosis Diseases 0.000 claims abstract description 6
- 230000004761 fibrosis Effects 0.000 claims abstract description 6
- 230000005764 inhibitory process Effects 0.000 claims abstract description 6
- 230000037390 scarring Effects 0.000 claims abstract description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 14
- 239000012620 biological material Substances 0.000 claims description 14
- 210000004027 cell Anatomy 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 239000012528 membrane Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- 210000004379 membrane Anatomy 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 11
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 11
- 210000002744 extracellular matrix Anatomy 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 7
- 229930003268 Vitamin C Natural products 0.000 claims description 7
- 235000019154 vitamin C Nutrition 0.000 claims description 7
- 239000011718 vitamin C Substances 0.000 claims description 7
- 108020004414 DNA Proteins 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 102000011782 Keratins Human genes 0.000 claims description 5
- 108010076876 Keratins Proteins 0.000 claims description 5
- 102000013013 Member 2 Subfamily G ATP Binding Cassette Transporter Human genes 0.000 claims description 5
- 108010090306 Member 2 Subfamily G ATP Binding Cassette Transporter Proteins 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 5
- 230000001079 digestive effect Effects 0.000 claims description 5
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 4
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 210000005081 epithelial layer Anatomy 0.000 claims description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 4
- 239000012535 impurity Substances 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 238000012423 maintenance Methods 0.000 claims description 4
- 108090000145 Bacillolysin Proteins 0.000 claims description 3
- 102000015735 Beta-catenin Human genes 0.000 claims description 3
- 108060000903 Beta-catenin Proteins 0.000 claims description 3
- 102000029816 Collagenase Human genes 0.000 claims description 3
- 108060005980 Collagenase Proteins 0.000 claims description 3
- 102000035092 Neutral proteases Human genes 0.000 claims description 3
- 108091005507 Neutral proteases Proteins 0.000 claims description 3
- 101001077376 Oryza sativa subsp. japonica UMP-CMP kinase 4 Proteins 0.000 claims description 3
- 229930182555 Penicillin Natural products 0.000 claims description 3
- 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 3
- 230000003321 amplification Effects 0.000 claims description 3
- 229960002424 collagenase Drugs 0.000 claims description 3
- 108010007093 dispase Proteins 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 229940049954 penicillin Drugs 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 3
- 102400000552 Notch 1 intracellular domain Human genes 0.000 claims description 2
- 101800001628 Notch 1 intracellular domain Proteins 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 102000014736 Notch Human genes 0.000 claims 2
- 108010070047 Notch Receptors Proteins 0.000 claims 2
- 102000013814 Wnt Human genes 0.000 claims 2
- 230000037361 pathway Effects 0.000 claims 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 abstract description 7
- 241000270923 Hesperostipa comata Species 0.000 abstract description 4
- 238000009958 sewing Methods 0.000 abstract description 3
- 210000000795 conjunctiva Anatomy 0.000 description 18
- 238000010186 staining Methods 0.000 description 9
- 102000015728 Mucins Human genes 0.000 description 8
- 108010063954 Mucins Proteins 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000004043 dyeing Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000003125 immunofluorescent labeling Methods 0.000 description 6
- 238000002054 transplantation Methods 0.000 description 5
- 208000018380 Chemical injury Diseases 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 206010053615 Thermal burn Diseases 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 208000016807 X-linked intellectual disability-macrocephaly-macroorchidism syndrome Diseases 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 239000002771 cell marker Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000017423 tissue regeneration Effects 0.000 description 3
- KZMRYBLIGYQPPP-UHFFFAOYSA-M 3-[[4-[(2-chlorophenyl)-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]cyclohexa-2,5-dien-1-ylidene]methyl]-n-ethylanilino]methyl]benzenesulfonate Chemical compound C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)Cl)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 KZMRYBLIGYQPPP-UHFFFAOYSA-M 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 208000018465 Conjunctival injury Diseases 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010067776 Ocular pemphigoid Diseases 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 201000010002 cicatricial pemphigoid Diseases 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000012757 fluorescence staining Methods 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 239000012128 staining reagent Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012353 t test Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000004130 Blepharoptosis Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 102100033601 Collagen alpha-1(I) chain Human genes 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 206010015995 Eyelid ptosis Diseases 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 108010029483 alpha 1 Chain Collagen Type I Proteins 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000010217 blepharitis Diseases 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009786 epithelial differentiation Effects 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 210000000744 eyelid Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 229960003376 levofloxacin Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 201000003004 ptosis Diseases 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000002747 voluntary effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- C12N5/0656—Adult fibroblasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Rheumatology (AREA)
- Cell Biology (AREA)
- Vascular Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention provides an application of a subconjunctival fibroblast acellular matrix in conjunctival reconstruction. The invention discloses a subconjunctival fibroblast acellular matrix for promoting the growth and proliferation of conjunctival epithelial cells; maintaining conjunctival epithelial stem cell characteristics; the activity of a notch channel and the inhibition state of a wnt channel in the in-vitro differentiation process of the conjunctival epithelial stem cells are maintained; promoting the repair of large-area conjunctival defects; promoting expression of goblet cells and mucin after conjunctival defect repair; reducing the level of fibrosis and scarring of the stromal layer following repair of the conjunctival defect. The subconjunctival fibroblast acellular matrix prepared by the method is a colorless transparent film, has certain mechanical strength and stretchability, can tolerate surgical needle and thread sewing, can construct an acellular matrix with thickness obviously superior to that of adipose mesenchymal stem cells, has Young modulus close to that of normal conjunctival tissues, and is convenient for clinical popularization.
Description
Technical Field
The invention relates to the technical field of tissue function and ophthalmic repair, in particular to application of a conjunctiva acellular matrix in conjunctiva reconstruction.
Background
The conjunctiva is an important component of the ocular surface, and its structural and functional integrity is critical to maintaining ocular surface homeostasis. A plurality of clinically common intractable eye surface diseases such as chemical injury, thermal burn and the like can cause serious blepharospherical adhesion, large-area conjunctival defects can occur after the adhesion is separated by an operation, and conjunctiva reconstruction is needed. At present, common surgical modes comprise autologous/allogenic conjunctival transplantation, orolabial mucosa transplantation, amniotic membrane transplantation and the like, but the clinical application is limited due to the problems of limited tissue sources, transplant immune rejection, poor repair effect, poor function replacement and the like, and a brand new conjunctival reconstruction mode is urgently needed to be developed.
At present, researches show that a plurality of polymer synthetic materials can be used for conjunctival defect repair, but the materials have defects in biocompatibility and functional substitution, and the tear film stability is seriously influenced by the deletion of conjunctival goblet cells (important single-cell secretory glands in conjunctiva and mucin which form a tear film mucus layer) after operation, so that an ideal repair effect cannot be achieved. With the development of tissue engineering and stem cell technology in recent years, the in vitro construction of a functional conjunctiva provides possibility for solving the above problems. The acellular matrix is used as a biological material of natural source, has excellent biocompatibility, not only can provide an adhesion surface and structural support for cell growth, but also is stored with abundant growth factors to form a tissue microenvironment beneficial to cell growth, and is an excellent scaffold carrier for tissue regeneration and repair. The acellular matrix from various cell sources is already applied to the fields of bone tissue regeneration, wound repair and the like, and the inventor has also previously constructed the acellular matrix from the adipose-derived mesenchymal stem cells for conjunctival injury repair (published articles) and has a certain effect of promoting the proliferation of conjunctival epithelial cells. However, the acellular matrix derived from the adipose-derived mesenchymal stem cells has poor mechanical strength, is difficult to apply to clinical surgical suture, and the adaptability of the acellular matrix to the growth microenvironment of conjunctival stem cells needs to be further improved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the application of the subconjunctival fibroblast acellular matrix in conjunctival reconstruction.
The technical scheme for solving the problems is as follows:
the invention provides application of a subconjunctival fibroblast acellular matrix in preparing conjunctival structure and function repair products, wherein the conjunctival structure and function repair products at least have one of the following functions:
1) promoting growth and proliferation of conjunctival epithelial cells;
2) maintaining conjunctival epithelial stem cell characteristics;
3) the activity of a notch channel and the inhibition state of a wnt channel in the in-vitro differentiation process of the conjunctival epithelial stem cells are maintained;
4) promoting the repair of large-area conjunctival defects;
5) promoting expression of goblet cells and mucin after conjunctival defect repair;
6) reducing the level of fibrosis and scarring of the stromal layer following repair of the conjunctival defect.
In some embodiments of the invention, the conjunctival structural and functional repair product comprises a functional conjunctival reconstruction biomaterial for the treatment of blepharocophere adhesion.
The functional conjunctival reconstruction biomaterial for treating the blepharospherical adhesion is a subconjunctival fibroblast acellular matrix.
The invention also provides a preparation method of the biological material, which comprises the steps of obtaining subconjunctival fibroblasts, culturing and expanding in vitro and preparing an acellular matrix.
In some embodiments of the invention, the obtaining of subconjunctival fibroblasts comprises the following specific steps: washing conjunctival tissue, incubating with protease, scraping epithelial layer, retaining subconjunctival matrix layer, digesting in digestive juice to disperse into single cell suspension, filtering to remove impurities, centrifuging, and collecting precipitate to obtain subconjunctival fibroblast.
In some embodiments of the invention, the in vitro culture expansion comprises the following specific steps: and (3) resuspending the obtained subconjunctival fibroblasts by using a culture solution, inoculating the subconjunctival fibroblasts into a culture dish for culture, inoculating the cultured subconjunctival fibroblasts into a culture plate, culturing until the cells are fused, adding vitamin C into the culture solution, and carrying out in-vitro continuous culture.
In some embodiments of the invention, the preparation of the acellular matrix comprises the following specific steps: culturing until extracellular matrix forms a membrane with proper thickness, performing decellularization treatment, removing genetic material DNA, and washing with PBS to obtain the biomaterial.
Compared with the prior art, the invention has the following beneficial effects:
1. the existing conjunctiva reconstruction biological material has the problems of poor biocompatibility and function substitution, incapability of effectively maintaining the characteristics and proliferation potential of conjunctiva stem cells, less expression of conjunctiva tissue goblet cells after repair and poor eye surface condition, and the existing adipose mesenchymal stem cell acellular matrix has the problems of low mechanical strength and intolerance to surgical suture.
2. The subconjunctival fibroblast acellular matrix prepared by the invention is a colorless transparent film, is easy to obtain, can be stably stored in vitro for a long time, has excellent biocompatibility, low immunogenicity and good mechanical strength, can bear surgical suture, can be used as a functional conjunctiva reconstruction biomaterial for treating the blepharocophere adhesion, and has good treatment and repair effects on large-area conjunctival defects, serious damage to ocular surface homeostasis and the like caused by acid-base chemical injury, thermal burn, Steven-Johnson syndrome, blepharitis pseudobullosa and the like after the blepharocophere adhesion.
Drawings
FIG. 1 shows the preparation and mechanical strength measurement of the subconjunctival fibroblast acellular matrix. Panel a is a gross photograph, transparency and tensile display of subconjunctival fibroblast acellular matrix; panel B is a display of subconjunctival fibroblast acellular matrix that is resistant to surgical needle and thread suturing; the C picture is a graph comparing HE staining and Masson staining of the conjunctival fibroblast acellular matrix and the adipose mesenchymal stem cell acellular matrix; d picture is a statistical chart of the tissue thickness of the subconjunctival fibroblast acellular matrix and the adipose tissue-derived mesenchymal stem cell acellular matrix; e is a comparison of Young's modulus of subconjunctival fibroblast acellular matrix with that of normal conjunctival tissue; and F is an immunofluorescence staining contrast diagram before and after extracellular matrix decellularization of the subconjunctival fibroblasts. Data are presented as mean ± standard deviation, using t test; indicates that there is a statistical difference between the two groups, p < 0.05; indicates that there is a statistical difference between the two groups, p < 0.01; indicates that there was a statistical difference between the two groups, p < 0.001.
FIG. 2 is a graph of the biocompatibility assay of the subconjunctival fibroblast acellular matrix. A is a scanning electron microscope detection representative picture before and after the subconjunctival fibroblast acellular matrix is inoculated with cells; b is a representative diagram of dead and alive staining before and after the conjunctival epithelial cells are inoculated on the acellular matrix of the fibroblasts under the conjunctiva; c, a dead and live staining result statistical chart; the D picture is a CCK-8 detection statistical result; e is a representative diagram of fluorescent staining in an EdU proliferation experiment; f is a statistical chart of the result of the EdU proliferation experiment; g picture is fluorescence staining representation picture of conjunctival epithelial cell dryness index p 63; h is a statistical graph of the fluorescent staining result of p 63; FIG. I is a representative diagram of the differentiation index CK4 of conjunctival epithelial cells by fluorescent staining; the J picture is a statistical picture of the result of CK4 fluorescent staining. Data are presented as mean ± standard deviation, using t test; indicates that there is a statistical difference between the two groups, p < 0.05; indicates that there is a statistical difference between the two groups, p < 0.01; indicates that there was a statistical difference between the two groups, p < 0.001.
FIG. 3 is a schematic representation of the maintenance of conjunctival epithelial stem cell characteristics by the subconjunctival fibroblast acellular matrix. A is a representative image of immunofluorescence staining of a conjunctival epithelial stem cell marker ABCG2, which is obtained by respectively inoculating conjunctival epithelial stem cells into a subconjunctival fibroblast acellular matrix and a blank culture dish for in vitro culture; b is a representative image of immunofluorescence staining of a conjunctival epithelial stem cell marker p 63; panel C is a representative image of immunofluorescence staining of the conjunctival epithelial cell differentiation marker keratin CK 4.
FIG. 4 is a graph of the in vivo repair effect of subconjunctival fibroblast acellular matrix in combination with conjunctival epithelial stem cells on large conjunctival defects. A is a schematic diagram of rabbit large-area conjunctival defect model construction and acellular matrix transplantation operation; b, a picture B is a silk amine green staining showing the effect of repairing conjunctival defects by using the subconjunctival fibroblast acellular matrix, NC is a blank control group, DM is a transplanted subconjunctival fibroblast acellular matrix group, DM-cjECs is a transplanted subconjunctival fibroblast acellular matrix composite conjunctival epithelial cell group, and DM-p75+ + is a transplanted subconjunctival fibroblast acellular matrix composite conjunctival epithelial stem cell group; c is a representative diagram of paraffin section HE, PAS and Masson staining results of tissues of each group of defect repair completion parts; and the D picture is a representative picture of the immunofluorescence staining result of the frozen section of the tissue of each group of defect repair completion parts.
Detailed Description
The invention provides application of a subconjunctival fibroblast acellular matrix in preparing conjunctival structure and function repair products, wherein the conjunctival structure and function repair products at least have one of the following functions:
1) promoting growth and proliferation of conjunctival epithelial cells;
2) maintaining conjunctival epithelial stem cell characteristics;
3) the activity of a notch channel and the inhibition state of a wnt channel in the in-vitro differentiation process of the conjunctival epithelial stem cells are maintained;
4) promoting the repair of large-area conjunctival defects;
5) promoting expression of goblet cells and mucin after conjunctival defect repair;
6) reducing the level of fibrosis and scarring of the stromal layer following repair of the conjunctival defect.
In the present invention, the conjunctival structural and functional repair product necessarily comprises the subconjunctival fibroblast acellular matrix, and the subconjunctival fibroblast acellular matrix is used as an active ingredient for the aforementioned functions. In the conjunctival structure and function repair product of the present invention, the active ingredient exerting the above function may be only a subconjunctival fibroblast acellular matrix, or may include other products having similar functions.
In some embodiments of the invention, the conjunctival structural and functional repair product comprises a functional conjunctival reconstruction biomaterial for the treatment of blepharocophere adhesion. Further, the blepharocophere adhesion includes acid-base chemical injury, thermal burn, Steven-Johnson syndrome, blepharocophere adhesion caused by ocular pemphigoid, or the like.
The functional conjunctival reconstruction biomaterial for treating the blepharospherical adhesion is a subconjunctival fibroblast acellular matrix.
The invention also provides a preparation method of the biological material, which comprises the steps of obtaining subconjunctival fibroblasts, culturing and expanding in vitro and preparing an acellular matrix.
In some embodiments of the invention, the obtaining of subconjunctival fibroblasts comprises the following specific steps: washing conjunctival tissue, incubating with protease, scraping epithelial layer, retaining subconjunctival matrix layer, digesting in digestive juice until dispersed into single cell suspension, filtering to remove impurities, centrifuging, and collecting precipitate to obtain subconjunctival fibroblast. In some embodiments of the invention, the wash medium is a PBS buffer containing penicillin. In some embodiments of the present invention, the incubation temperature is 4 ℃ and the incubation time is 10-18 h, preferably 14 h. In some embodiments of the invention, the protease is Dispase II neutral protease with a concentration of 4-6 mg/mL, preferably 5 mg/mL. In some embodiments of the invention, the digestive fluid is 0.1% to 0.2% collagenase a, preferably 0.1%. In some embodiments of the invention, the digestion temperature is 37 ℃ and the digestion time is 16-20 hours, preferably 18 hours.
In some embodiments of the invention, the in vitro culture expansion comprises the following specific steps: and (3) resuspending the obtained subconjunctival fibroblasts by using a culture solution, inoculating the subconjunctival fibroblasts into a culture dish for culture, inoculating the cultured subconjunctival fibroblasts into a culture plate, culturing until the cells are fused, adding vitamin C into the culture solution, and performing in vitro continuous culture. In some embodiments of the invention, the culture plate is a six-well culture plate. In some embodiments of the invention, the concentration of vitamin C is 10-20 μ g/mL, preferably 10 μ g/mL. Because the formula of the culture solution contains vitamin C, the culture solution can stimulate fibroblasts to secrete a large amount of extracellular matrix, so that the acellular matrix after acellular has certain thickness and toughness and can be completely uncovered. In some embodiments of the invention, the in vitro continuous culture time is 2 weeks. In some embodiments of the invention, the culture medium is replaced every several days, preferably every 2 days, during the in vitro continuous culture.
In some embodiments of the invention, the preparation of the acellular matrix comprises the following specific steps: culturing until extracellular matrix forms a membrane with proper thickness, performing decellularization treatment, removing genetic material DNA, cleaning with PBS, and storing at 4 ℃ to obtain the biomaterial. In some embodiments of the invention, the decellularization solution used in the decellularization process is a solution containing 0.5-1% Triton-100 and 15-25 mM NH 4 OH in PBS, preferably 1% Triton-100 and 20mM NH 4 OH PBS in some embodiments of the invention, the removal of genetic material DNA is specifically performed by treating the extracellular matrix membrane with 100-200U/ml DNase; preferably, extracellular matrix membrane patches are treated with PBS containing 200U/ml DNase for 2h at 37 ℃.
The subconjunctival fibroblast acellular matrix prepared by the method is a colorless transparent film (shown as A in figure 1), has certain mechanical strength and stretchability, and can resist surgical needle and thread sewing (shown as B in figure 1). The main components of the subconjunctival fibroblast acellular matrix are fibronectin and collagen, and the subconjunctival fibroblast acellular matrix can provide a growth environment which is easy to adhere and favorable for proliferation for conjunctival epithelial cells and is favorable for functional conjunctival reconstruction.
In the previous research, it was found that the adipose derived acellular matrix can be used for ocular surface injury repair. In the invention, the configurable thickness of the subconjunctival fibroblast acellular matrix is obviously superior to that of the adipose mesenchymal stem cell-derived acellular matrix (shown as C and D in figure 1).
In the present invention, the young's modulus of the subconjunctival fibroblast acellular matrix is close to that of normal conjunctival tissue (as shown in fig. 1E).
In the invention, the subconjunctival fibroblast acellular matrix can obviously promote the growth and proliferation of conjunctival epithelial cells.
In the invention, the subconjunctival fibroblast acellular matrix can effectively maintain the characteristics of conjunctival epithelial stem cells, including but not limited to maintaining the expression of conjunctival epithelial cell dryness markers p75, p63 and ABCG2 and inhibiting the expression of conjunctival differentiation keratin CK 4.
In the invention, the subconjunctival fibroblast acellular matrix can maintain the activity of a notch channel and the inhibition state of a wnt channel in the in-vitro differentiation process of the conjunctival epithelial stem cells, including but not limited to maintaining the expression of NICD and inhibiting the expression of beta-catenin/active-beta-catenin.
In the invention, the subconjunctival fibroblast acellular matrix can obviously promote the repair of large-area conjunctival defects.
In the invention, the subconjunctival fibroblast acellular matrix can obviously promote the expression of goblet cells and mucin after the conjunctival defect is repaired. Goblet cells function as single-cell mucous glands specifically expressed in the conjunctiva, and mucin secreted by the goblet cells is important for maintaining the stability of the ocular surface tear film.
In the invention, the subconjunctival fibroblast acellular matrix can obviously reduce the fibrosis and scarring level of the matrix layer after conjunctival defect repair.
Further, functional conjunctival remodeling actions of the subconjunctival fibroblast acellular matrix include, but are not limited to, blepharoptosis caused by acid-base chemical injury, thermal burn, Steven-Johnson syndrome, ocular pemphigoid, and the like. In the invention, the functional conjunctival reconstruction effect of the subconjunctival fibroblast acellular matrix is realized mainly by maintaining the stem cell characteristics of the conjunctival epithelial stem cells, and the good proliferation activity of the conjunctival epithelial cells, the normal differentiation of goblet cells and the secretion of mucin all depend on the maintenance of the characteristics of the conjunctival epithelial stem cells.
The invention simulates conjunctival defect after the eyelid bulb adhesion separation by preparing a large-area rabbit conjunctival defect model, and adopts the combination of subconjunctival fibroblast acellular matrix and conjunctival epithelial stem cells to repair the defect. Through HE dyeing, PAS dyeing, Masson dyeing and immunofluorescence dyeing detection on a defect repair completion area, the subconjunctival fibroblast acellular matrix is found to be capable of obviously accelerating the repair speed of conjunctival defects, the subconjunctival epithelium structure of conjunctival tissues after repair is complete, goblet cells and mucin are rich in expression, the subconjunctival stroma layer is low in fibrosis and scarring level, and normal stem cell distribution with high expression of a basal layer notch channel and low expression of a wnt/beta-catenin channel in the subconjunctival epithelium is maintained.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the methods of testing, methods of preparation, and methods of preparation disclosed herein employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
Example 1: preparation of subconjunctival fibroblast acellular matrix
1. Experimental methods
The preparation of the subconjunctival fibroblast acellular matrix comprises the steps of obtaining subconjunctival fibroblasts, in-vitro culture and amplification and preparation of the acellular matrix.
Obtaining subconjunctival fibroblasts: the human conjunctival tissue used was obtained from the eye tissue bank of the ninth human hospital affiliated with the Shanghai university of medicine, and was obtained from voluntary donations with informed consent of patients and was subject to ethical review. Conjunctival tissue was washed several times in PBS containing penicillin and incubated with Dispase II neutral protease (5mg/ml, Roche Diagnostics GmbH, Manheim, Germany) for 14 hours at 4 ℃. The epithelial layer was scraped off with sterile forceps for the next day, the subconjunctival matrix layer was retained, digested with collagenase a (0.1%, Roche Diagnostics GmbH) at 37 ℃ for 18 hours until dispersed as a single cell suspension, and the cell suspension was filtered with a 70 μm cell filter to remove impurities.
In vitro culture and amplification: the cell suspension was centrifuged, inoculated into a 10cm dish, and amplified in vitro in culture using DMEM (Gibco) supplemented with 10% fetal bovine serum. Cultured subconjunctival fibroblasts were seeded on a six-well culture plate, and after culturing until the cells were fused, vitamin C (Sigma-Aldrich) was added to the culture broth at a concentration of 50ug/ml to promote secretion of extracellular matrix. In vitro culture was continued for about 2 weeks with medium changes every 2 days.
Preparation of acellular matrix: after culturing until extracellular matrix forms a film with appropriate thickness, a membrane containing 1% Triton-100(Sigma-Aldrich) and 20mM NH was used 4 OH (Sigma-Aldrich) in PBS was used as a decellularization solution, decellularization treatment was performed for 5 minutes to destroy cell structures, and extracellular matrix membrane pieces were treated with 200U/ml DNase in PBS at 37 ℃ for 2 hours to remove genetic material DNA. Washed 3 times with PBS and stored at 4 ℃.
2. Analysis of results
The prepared subconjunctival fibroblast acellular matrix membrane has good transparency, certain mechanical strength and stretchability, and can resist surgical needle and thread sewing (fig. 1A-B).
Example 2: mechanical property detection
1. Test method
And (3) carrying out Young modulus detection on the prepared subconjunctival fibroblast acellular matrix membrane by adopting an atomic force microscope, and taking normal conjunctival tissue as a control group. And randomly selecting 10 positions uniformly distributed for detection on each membrane, and detecting 3 membranes cultured in different batches for statistical analysis.
2. Test results
The young's modulus of the subconjunctival fibroblast acellular matrix was close to that of normal conjunctival tissue with no statistical difference (fig. 1E).
Example 3: immunofluorescence staining
1. Dyeing method
The subconjunctival fibroblast acellular matrix or the conjunctival epithelial stem cells seeded on the subconjunctival fibroblast acellular matrix were fixed with 4% paraformaldehyde at room temperature for 20 minutes, permeabilized in PBS containing 0.3% Triton X-100(Sigma-Aldrich) for 15 minutes, and then blocked with 5% donkey serum at room temperature for 1 hour. Primary antibodies against FN (Santa Cruz), COL1A1(Abcam), COL3A (Abcam), p75(Cell Signaling Technology), p63(Abcam), ABCG2(Abcam), CK4(Abcam) were incubated overnight at 4 ℃. The next day, the primary antibody was washed off, and the sample was incubated with Alexa fluorescence-conjugated secondary antibody at room temperature for 1 hour, washed thoroughly, stained with DAPI at room temperature for 10 minutes, and photographed under a fluorescence microscope.
2. Analysis of results
The subconjunctival fibroblast acellular matrix was completely decellularized, with fibronectin and collagen as the major components (fig. 1F). Fluorescence staining of conjunctival epithelial cells seeded on the subconjunctival fibroblast acellular matrix expressing conjunctival epithelial stem cell marker p63 and specific keratin CK4 was observed and counted (fig. 2G-J). The subconjunctival fibroblast acellular matrix was able to maintain the expression of stem cell markers p63 and ABCG2, inhibiting the expression of differentiated keratin CK4 during in vitro culture (figure 3).
Example 4: dead and alive dyeing
1. Dyeing method
Conjunctival epithelial cells were inoculated into 24-well culture plates each plated with a blank and subconjunctival fibroblast decellularization matrix at a density of 10 ten thousand per well, two dead and live staining reagents (Invitrogen) were added at concentrations of 1:10000 and 1:2000, respectively, incubated at 37 ℃ for 15 minutes in the dark, washed out with PBS, photographed under a fluorescence microscope, and counted.
2. Analysis of results
Conjunctival epithelial cells survived well on the subconjunctival fibroblast acellular matrix with no statistical difference in the proportion of dead cells compared to the blank control group (fig. 2B-C).
Example 5: CCK-8 detection
1. Detection method
Conjunctival epithelial cells were seeded at a density of 1000 per well in 6-well culture plates with blank and plated subconjunctival fibroblast acellular matrix, respectively, with 6 replicates per subset set. After adherent growth of the cells, 200. mu.L of Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each well and incubated at 37 ℃ for 4h in the absence of light. Absorbance at 450nm was measured in 96-well plates by pipetting 100. mu.L of the culture solution every 24 hours.
2. Analysis of results
The conjunctival epithelial cells inoculated on the acellular matrix of the fibroblasts under the conjunctiva proliferated well, and the proliferation rate of the conjunctival epithelial cells was superior to that of the blank control group (fig. 2D).
Example 6: EdU cell proliferation staining
1. Dyeing method
The conjunctival epithelial cells are respectively inoculated into a blank 24-hole culture plate and a 24-hole culture plate paved with subconjunctival fibroblast acellular matrix for culture at the density of 5 ten thousand per hole, an EdU reagent (Biyun day) is added according to the proportion of 1:1000, the cells are fixed for 15 minutes at room temperature by 4% paraformaldehyde after being continuously cultured for 2 hours at 37 ℃, the EdU mixed staining reagent prepared according to the instruction is added after being washed by PBS, and the cells are incubated for 30 minutes at room temperature in a dark place. After washing with PBS, DAPI was added dropwise and stained at room temperature for 10 minutes, and photographed under a fluorescence microscope and counted.
2. Analysis of results
The proliferation of conjunctival epithelial cells inoculated on the acellular matrix of the fibroblasts under the conjunctiva is good, and the EdU staining positive rate is better than that of a blank control group (figures 2E-F).
Example 7: construction of rabbit conjunctival defect model and subconjunctival fibroblast acellular matrix transplantation
1. Experimental methods
New Zealand big-ear white rabbits weighing approximately 2kg were anesthetized by intramuscular injection of Shutai (approved by the ethical Committee of Experimental animals). The bulbar conjunctiva of both eyes was fully exposed, the defect area was marked with a sterile trephine of about 8mm in diameter, and the bulbar conjunctiva was completely cut off with scissors to the scleral layer. The conjunctival defect sites were marked with lissamine green stain (Sigma-Aldrich) and the original defect size was recorded by photography. The animal experiment groups are as follows: a blank control group NC, a transplanted subconjunctival fibroblast acellular matrix group DM, a transplanted subconjunctival fibroblast acellular matrix composite conjunctival epithelial cell group DM-cjECs, a transplanted subconjunctival fibroblast acellular matrix composite conjunctival epithelial stem cell group DM-p75+ +, and a positive control group PC. The 10-0 aseptic silk thread is adopted to sew the under-membrane fibroblast acellular matrix at the conjunctival defect, and the levofloxacin eye drops are given to resist infection and relieve pain after operation. And (5) performing lissamine green staining on the defect part one week and two weeks after the operation, and photographing to record the defect repair condition.
2. Analysis of results
The subconjunctival fibroblast acellular matrix can obviously promote the repair of conjunctival injury, and the DM-p75+ + group has the fastest repair speed (figures 4A-B).
Example 8: HE/PAS/Masson staining
1. Experimental methods
At the end of the second week, animals were euthanized, and the conjunctival tissues of each defect repair-completed area were fixed in 4% paraformaldehyde and paraffin-embedded tissue sections were performed. Each group of paraffin sections was stained with hematoxylin-eosin, glycogen and masson, and histological photographs were taken under an optical microscope.
2. Analysis of results
The fibroblast acellular matrix under conjunctiva has the best effect on repairing the conjunctival damage, and the morphology, the tissue integrity, the expression of goblet cells and mucin and the repair condition of the matrix layer of the repaired part of the DM-p75+ + group are all obviously superior to those of other groups (figure 4C).
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. The application of the subconjunctival fibroblast acellular matrix in preparing conjunctival structure and function repair products, wherein the conjunctival structure and function repair products have at least one of the following functions:
1) promoting growth and proliferation of conjunctival epithelial cells;
2) maintaining conjunctival epithelial stem cell characteristics;
3) the activity of a notch channel and the inhibition state of a wnt channel in the in-vitro differentiation process of the conjunctival epithelial stem cells are maintained;
4) promoting the repair of large-area conjunctival defects;
5) promoting expression of goblet cells and mucin after conjunctival defect repair;
6) reducing the level of fibrosis and scarring of the stromal layer following repair of the conjunctival defect.
2. Use according to claim 1, further comprising one or more of the following features:
(2a) the maintenance of the characteristics of the conjunctival epithelial stem cells comprises the maintenance of the expression of dry markers p75, p63 and ABCG2 and the inhibition of the expression of conjunctival differentiation keratin CK 4;
(3a) the expression of the notch pathway NICD is maintained, and the expression of the wnt pathway beta-catenin/active-beta-catenin is inhibited.
3. The use of claim 1, wherein the conjunctival structural and functional repair product is a functional conjunctival reconstruction biomaterial for the treatment of blepharon adhesion.
4. The use according to claim 3, wherein the biomaterial is prepared by a method comprising obtaining subconjunctival fibroblasts, in vitro culture expansion and preparation of an acellular matrix.
5. The use of claim 4, wherein the obtaining of subconjunctival fibroblasts comprises the specific steps of: washing conjunctival tissue, incubating with protease, scraping epithelial layer, retaining subconjunctival matrix layer, digesting in digestive juice until dispersed into single cell suspension, filtering to remove impurities, centrifuging, and collecting precipitate to obtain subconjunctival fibroblast.
6. Use according to claim 5, further comprising one or more of the following features:
a) the washing medium is a PBS buffer solution containing penicillin;
b) the incubation temperature is 4 ℃, and the incubation time is 10-18 h;
c) the protease is Dispase II neutral protease with the concentration of 4-6 mg/mL;
d) the digestive juice is 0.1 to 0.2 percent of collagenase A;
e) the digestion temperature is 37 ℃, and the digestion time is 16-20 h.
7. The use according to claim 4, wherein said in vitro culture amplification comprises the following specific steps: and (3) resuspending the obtained subconjunctival fibroblasts by using a culture solution, inoculating the subconjunctival fibroblasts into a culture dish for culture, inoculating the cultured subconjunctival fibroblasts into a culture plate, culturing until the cells are fused, adding vitamin C into the culture solution, and carrying out in-vitro continuous culture.
8. Use according to claim 7, further comprising one or more of the following features:
i) the concentration of the vitamin C is 10-20 mug/mL;
ii) changing the culture solution every several days during the in vitro continuous culture process.
9. Use according to claim 4, characterized in that the preparation of said acellular matrix comprises the following specific steps: culturing until extracellular matrix forms a membrane with proper thickness, performing decellularization treatment, removing genetic material DNA, and cleaning with PBS to obtain the biological material.
10. Use according to claim 9, further comprising one or several of the following features:
(1) the cell removing liquid used for the cell removing treatment contains 0.5-1% of Triton-100 and 15-25 mM NH 4 PBS of OH;
(2) the specific operation of removing the genetic material DNA is to treat the extracellular matrix membrane by using 100-200U/ml DNase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210899898.5A CN115105637B (en) | 2022-07-28 | 2022-07-28 | Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210899898.5A CN115105637B (en) | 2022-07-28 | 2022-07-28 | Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115105637A true CN115105637A (en) | 2022-09-27 |
| CN115105637B CN115105637B (en) | 2023-11-10 |
Family
ID=83333699
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210899898.5A Active CN115105637B (en) | 2022-07-28 | 2022-07-28 | Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115105637B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800372A (en) * | 2005-12-02 | 2006-07-12 | 王平安 | Engineered extracellular matrix preparation method |
| CN101590292A (en) * | 2009-06-11 | 2009-12-02 | 山东省眼科研究所 | The preparation method of acellular conjunctiva matrix and application |
| CN101648010A (en) * | 2008-08-13 | 2010-02-17 | 北京中科颐诺生物技术有限公司 | Preparation for promoting fibroblasts to secrete extracellular matrix components and preparation method thereof |
| CN102892880A (en) * | 2010-01-14 | 2013-01-23 | 奥加诺吉尼西斯公司 | Bioengineered tissue constructs and methods for producing and using thereof |
| WO2013028968A1 (en) * | 2011-08-25 | 2013-02-28 | Wisconsin Alumni Research Foundation | 3-dimensional cardiac fibroblast derived extracellular matrix |
| CN113425909A (en) * | 2021-06-30 | 2021-09-24 | 上海交通大学医学院附属第九人民医院 | Biological material for repairing corneal injury and preparation method and application thereof |
-
2022
- 2022-07-28 CN CN202210899898.5A patent/CN115105637B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800372A (en) * | 2005-12-02 | 2006-07-12 | 王平安 | Engineered extracellular matrix preparation method |
| CN101648010A (en) * | 2008-08-13 | 2010-02-17 | 北京中科颐诺生物技术有限公司 | Preparation for promoting fibroblasts to secrete extracellular matrix components and preparation method thereof |
| CN101590292A (en) * | 2009-06-11 | 2009-12-02 | 山东省眼科研究所 | The preparation method of acellular conjunctiva matrix and application |
| CN102892880A (en) * | 2010-01-14 | 2013-01-23 | 奥加诺吉尼西斯公司 | Bioengineered tissue constructs and methods for producing and using thereof |
| WO2013028968A1 (en) * | 2011-08-25 | 2013-02-28 | Wisconsin Alumni Research Foundation | 3-dimensional cardiac fibroblast derived extracellular matrix |
| CN113425909A (en) * | 2021-06-30 | 2021-09-24 | 上海交通大学医学院附属第九人民医院 | Biological material for repairing corneal injury and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| QI XING，ET AL.: "Decellularization of Fibroblast Cell Sheets for Natural Extracellular Matrix Scaffold Preparation" * |
| ZHAO LONG，ET AL.: "Ocular surface repair using decellularized porcine conjunctiva" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115105637B (en) | 2023-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mariappan et al. | In vitro culture and expansion of human limbal epithelial cells | |
| Rohaina et al. | Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane | |
| Basu et al. | Human limbal biopsy–derived stromal stem cells prevent corneal scarring | |
| Liu et al. | Silk fibroin as a biomaterial substrate for corneal epithelial cell sheet generation | |
| Murakami et al. | Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells | |
| US9144585B2 (en) | Isolated mesenchymal progenitor cells and extracellular matrix produced thereby | |
| JP4392855B2 (en) | Corneal epithelium-like sheet and method for producing the same | |
| Ma et al. | The graft of autologous adipose-derived stem cells in the corneal stromal after mechanic damage | |
| JPWO2006003818A1 (en) | Corneal epithelial sheet and method for producing the same | |
| Ma et al. | Corneal stroma regeneration with acellular corneal stroma sheets and keratocytes in a rabbit model | |
| CN106860919A (en) | De- cell amnion of crosslinking and its preparation method and application | |
| US20160129044A1 (en) | Use of mesothelial cells in tissue bioengineering and artificial tissues | |
| Dhamodaran et al. | One for all: a standardized protocol for ex vivo culture of limbal, conjunctival and oral mucosal epithelial cells into corneal lineage | |
| CN109517784B (en) | Similar corneal epithelial cell, tissue engineered corneal epithelium, preparation and application | |
| Yan et al. | Exploitation of human mesenchymal stromal cell derived matrix towards the structural and functional restoration of the ocular surface | |
| CN107929811A (en) | A kind of tissue engineering comea | |
| Jurkunas et al. | Cultivated autologous limbal epithelial cell transplantation: new frontier in the treatment of limbal stem cell deficiency | |
| US10415014B2 (en) | Method for the ex vivo cultivation of oral mucosal epithelial progenitor cells and oral mucosal epithelial cells | |
| Wu et al. | Design of functional decellularized matrix for conjunctival epithelial stem cell maintenance and ocular surface reconstruction | |
| Aghamollaei et al. | Safety of grafting acellular human corneal lenticule seeded with Wharton's Jelly-Derived Mesenchymal Stem Cells in an experimental animal model | |
| CN115105637B (en) | Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction | |
| CN113425909B (en) | Biological material for repairing corneal injury and preparation method and application thereof | |
| KR102417981B1 (en) | Artificial scaffold for corneal endothelial cell transplantation, manufacturing method thereof, and ultra-thin descemet stripping endothelial graft using the same | |
| CN109224129B (en) | Skin defect repairing material | |
| CN115068504A (en) | Cornea repairing material and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |