CN113769105A - 修饰miR-155-5p/SOCS1轴的外泌体、制备方法及其应用 - Google Patents
修饰miR-155-5p/SOCS1轴的外泌体、制备方法及其应用 Download PDFInfo
- Publication number
- CN113769105A CN113769105A CN202110688347.XA CN202110688347A CN113769105A CN 113769105 A CN113769105 A CN 113769105A CN 202110688347 A CN202110688347 A CN 202110688347A CN 113769105 A CN113769105 A CN 113769105A
- Authority
- CN
- China
- Prior art keywords
- socs1
- exosome
- mir
- bmsc
- exo
- 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 123
- 101150045565 Socs1 gene Proteins 0.000 title claims abstract description 84
- 102000058018 Suppressor of Cytokine Signaling 1 Human genes 0.000 title claims abstract description 84
- 108700027336 Suppressor of Cytokine Signaling 1 Proteins 0.000 title claims abstract description 84
- 108091083308 miR-155 stem-loop Proteins 0.000 title claims abstract description 78
- 108091091301 miR-155-1 stem-loop Proteins 0.000 title claims abstract description 78
- 108091041686 miR-155-2 stem-loop Proteins 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 210000000274 microglia Anatomy 0.000 claims abstract description 49
- 230000002207 retinal effect Effects 0.000 claims abstract description 35
- 230000014509 gene expression Effects 0.000 claims abstract description 34
- 239000003112 inhibitor Substances 0.000 claims abstract description 25
- 206010061218 Inflammation Diseases 0.000 claims abstract description 24
- 230000004054 inflammatory process Effects 0.000 claims abstract description 22
- 239000003814 drug Substances 0.000 claims abstract description 18
- 206010023332 keratitis Diseases 0.000 claims abstract description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 12
- 201000010099 disease Diseases 0.000 claims abstract description 10
- 230000037361 pathway Effects 0.000 claims abstract description 10
- 230000002757 inflammatory effect Effects 0.000 claims abstract description 6
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 33
- 230000002401 inhibitory effect Effects 0.000 claims description 25
- 206010029113 Neovascularisation Diseases 0.000 claims description 23
- 230000005764 inhibitory process Effects 0.000 claims description 15
- 230000010287 polarization Effects 0.000 claims description 15
- 210000001185 bone marrow Anatomy 0.000 claims description 12
- 210000002540 macrophage Anatomy 0.000 claims description 8
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 claims description 7
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 claims description 7
- 230000002018 overexpression Effects 0.000 claims description 6
- 208000030533 eye disease Diseases 0.000 claims description 5
- 208000022873 Ocular disease Diseases 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 3
- 102000003777 Interleukin-1 beta Human genes 0.000 claims description 3
- 108090000193 Interleukin-1 beta Proteins 0.000 claims description 3
- 102000003945 NF-kappa B Human genes 0.000 claims description 3
- 108010057466 NF-kappa B Proteins 0.000 claims description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 3
- 108090001005 Interleukin-6 Proteins 0.000 claims description 2
- 241000700605 Viruses Species 0.000 claims description 2
- 230000002222 downregulating effect Effects 0.000 claims description 2
- 210000003954 umbilical cord Anatomy 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 36
- 210000001525 retina Anatomy 0.000 abstract description 20
- 230000033115 angiogenesis Effects 0.000 abstract description 16
- 230000004913 activation Effects 0.000 abstract description 11
- 229940079593 drug Drugs 0.000 abstract description 7
- 230000000903 blocking effect Effects 0.000 abstract description 6
- 210000004087 cornea Anatomy 0.000 abstract description 4
- 230000019491 signal transduction Effects 0.000 abstract description 4
- 238000011144 upstream manufacturing Methods 0.000 abstract description 3
- 230000001419 dependent effect Effects 0.000 abstract 1
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 description 42
- 210000004027 cell Anatomy 0.000 description 29
- 241000699666 Mus <mouse, genus> Species 0.000 description 28
- 108090000623 proteins and genes Proteins 0.000 description 26
- 239000000243 solution Substances 0.000 description 26
- 241000699670 Mus sp. Species 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 25
- TZSMWSKOPZEMAJ-UHFFFAOYSA-N bis[(2-methoxyphenyl)methyl] carbonate Chemical class COC1=CC=CC=C1COC(=O)OCC1=CC=CC=C1OC TZSMWSKOPZEMAJ-UHFFFAOYSA-N 0.000 description 23
- 241001465754 Metazoa Species 0.000 description 16
- 238000000034 method Methods 0.000 description 16
- 210000005252 bulbus oculi Anatomy 0.000 description 15
- 206010055665 Corneal neovascularisation Diseases 0.000 description 13
- 208000007135 Retinal Neovascularization Diseases 0.000 description 13
- 201000000159 corneal neovascularization Diseases 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 13
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 11
- 238000001727 in vivo Methods 0.000 description 11
- 210000001642 activated microglia Anatomy 0.000 description 10
- 230000033228 biological regulation Effects 0.000 description 10
- 238000007619 statistical method Methods 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 9
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 9
- 101710088580 Stromal cell-derived factor 1 Proteins 0.000 description 9
- 238000003125 immunofluorescent labeling Methods 0.000 description 9
- 108091070501 miRNA Proteins 0.000 description 9
- 239000002679 microRNA Substances 0.000 description 9
- 239000011259 mixed solution Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- FFTVPQUHLQBXQZ-KVUCHLLUSA-N (4s,4as,5ar,12ar)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1C2=C(N(C)C)C=CC(O)=C2C(O)=C2[C@@H]1C[C@H]1[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]1(O)C2=O FFTVPQUHLQBXQZ-KVUCHLLUSA-N 0.000 description 7
- 230000005012 migration Effects 0.000 description 7
- 238000013508 migration Methods 0.000 description 7
- 229960004023 minocycline Drugs 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 description 6
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 description 6
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 6
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 6
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 6
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 6
- 210000001508 eye Anatomy 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 206010067484 Adverse reaction Diseases 0.000 description 5
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 5
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 5
- 230000006838 adverse reaction Effects 0.000 description 5
- 239000003513 alkali Substances 0.000 description 5
- 230000005754 cellular signaling Effects 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 108010082117 matrigel Proteins 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 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 4
- 102100025222 CD63 antigen Human genes 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 4
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 4
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 4
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 4
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 4
- 108010002352 Interleukin-1 Proteins 0.000 description 4
- 102000000589 Interleukin-1 Human genes 0.000 description 4
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 210000002889 endothelial cell Anatomy 0.000 description 4
- 239000003862 glucocorticoid Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 210000003128 head Anatomy 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 230000002980 postoperative effect Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229940037128 systemic glucocorticoids Drugs 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 229920000742 Cotton Polymers 0.000 description 3
- 206010012689 Diabetic retinopathy Diseases 0.000 description 3
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 210000000795 conjunctiva Anatomy 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 239000003889 eye drop Substances 0.000 description 3
- 229940012356 eye drops Drugs 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 229920006008 lipopolysaccharide Polymers 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000006724 microglial activation Effects 0.000 description 3
- 230000002025 microglial effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 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 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 210000003606 umbilical vein Anatomy 0.000 description 3
- 230000002792 vascular Effects 0.000 description 3
- 210000003556 vascular endothelial cell Anatomy 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 description 2
- 208000003098 Ganglion Cysts Diseases 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 102000005877 Peptide Initiation Factors Human genes 0.000 description 2
- 108010044843 Peptide Initiation Factors Proteins 0.000 description 2
- 208000017442 Retinal disease Diseases 0.000 description 2
- 206010055666 Retinal neovascularisation Diseases 0.000 description 2
- 206010038910 Retinitis Diseases 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 208000005400 Synovial Cyst Diseases 0.000 description 2
- CVUDMNSZAIZFAE-UHFFFAOYSA-N Val-Arg-Pro Natural products NC(N)=NCCCC(NC(=O)C(N)C(C)C)C(=O)N1CCCC1C(O)=O CVUDMNSZAIZFAE-UHFFFAOYSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 206010064930 age-related macular degeneration Diseases 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000002491 angiogenic effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 210000005056 cell body Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 2
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000007726 management method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000001023 pro-angiogenic effect Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 238000006748 scratching Methods 0.000 description 2
- 230000002393 scratching effect Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 210000004127 vitreous body Anatomy 0.000 description 2
- 238000009736 wetting Methods 0.000 description 2
- PCDUALPXEOKZPE-DXCABUDRSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoic acid Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O PCDUALPXEOKZPE-DXCABUDRSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- 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 1
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 1
- VBRDBGCROKWTPV-XHNCKOQMSA-N Ala-Glu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N VBRDBGCROKWTPV-XHNCKOQMSA-N 0.000 description 1
- GRPHQEMIFDPKOE-HGNGGELXSA-N Ala-His-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O GRPHQEMIFDPKOE-HGNGGELXSA-N 0.000 description 1
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 1
- NLYYHIKRBRMAJV-AEJSXWLSSA-N Ala-Val-Pro Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N NLYYHIKRBRMAJV-AEJSXWLSSA-N 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- XPSGESXVBSQZPL-SRVKXCTJSA-N Arg-Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XPSGESXVBSQZPL-SRVKXCTJSA-N 0.000 description 1
- GIVWETPOBCRTND-DCAQKATOSA-N Arg-Gln-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O GIVWETPOBCRTND-DCAQKATOSA-N 0.000 description 1
- RKRSYHCNPFGMTA-CIUDSAMLSA-N Arg-Glu-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O RKRSYHCNPFGMTA-CIUDSAMLSA-N 0.000 description 1
- GNYUVVJYGJFKHN-RVMXOQNASA-N Arg-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N GNYUVVJYGJFKHN-RVMXOQNASA-N 0.000 description 1
- LVMUGODRNHFGRA-AVGNSLFASA-N Arg-Leu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O LVMUGODRNHFGRA-AVGNSLFASA-N 0.000 description 1
- OISWSORSLQOGFV-AVGNSLFASA-N Arg-Met-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCCN=C(N)N OISWSORSLQOGFV-AVGNSLFASA-N 0.000 description 1
- FIQKRDXFTANIEJ-ULQDDVLXSA-N Arg-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FIQKRDXFTANIEJ-ULQDDVLXSA-N 0.000 description 1
- IGFJVXOATGZTHD-UHFFFAOYSA-N Arg-Phe-His Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccccc1)C(=O)NC(Cc2c[nH]cn2)C(=O)O IGFJVXOATGZTHD-UHFFFAOYSA-N 0.000 description 1
- WKPXXXUSUHAXDE-SRVKXCTJSA-N Arg-Pro-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O WKPXXXUSUHAXDE-SRVKXCTJSA-N 0.000 description 1
- ISVACHFCVRKIDG-SRVKXCTJSA-N Arg-Val-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O ISVACHFCVRKIDG-SRVKXCTJSA-N 0.000 description 1
- ZBYLEBZCVKLPCY-FXQIFTODSA-N Asp-Ser-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ZBYLEBZCVKLPCY-FXQIFTODSA-N 0.000 description 1
- NWAHPBGBDIFUFD-KKUMJFAQSA-N Asp-Tyr-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O NWAHPBGBDIFUFD-KKUMJFAQSA-N 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 230000005653 Brownian motion process Effects 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000018380 Chemical injury Diseases 0.000 description 1
- 108010008951 Chemokine CXCL12 Proteins 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 208000028006 Corneal injury Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010015943 Eye inflammation Diseases 0.000 description 1
- HHWQMFIGMMOVFK-WDSKDSINSA-N Gln-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O HHWQMFIGMMOVFK-WDSKDSINSA-N 0.000 description 1
- DLOHWQXXGMEZDW-CIUDSAMLSA-N Gln-Arg-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O DLOHWQXXGMEZDW-CIUDSAMLSA-N 0.000 description 1
- LZMQSTPFYJLVJB-GUBZILKMSA-N Glu-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N LZMQSTPFYJLVJB-GUBZILKMSA-N 0.000 description 1
- NNQDRRUXFJYCCJ-NHCYSSNCSA-N Glu-Pro-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O NNQDRRUXFJYCCJ-NHCYSSNCSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 1
- PMNHJLASAAWELO-FOHZUACHSA-N Gly-Asp-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PMNHJLASAAWELO-FOHZUACHSA-N 0.000 description 1
- LLWQVJNHMYBLLK-CDMKHQONSA-N Gly-Thr-Phe Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LLWQVJNHMYBLLK-CDMKHQONSA-N 0.000 description 1
- AYUOWUNWZGTNKB-ULQDDVLXSA-N His-Phe-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AYUOWUNWZGTNKB-ULQDDVLXSA-N 0.000 description 1
- BCSGDNGNHKBRRJ-ULQDDVLXSA-N His-Tyr-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N BCSGDNGNHKBRRJ-ULQDDVLXSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- YJRSIJZUIUANHO-NAKRPEOUSA-N Ile-Val-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)O)N YJRSIJZUIUANHO-NAKRPEOUSA-N 0.000 description 1
- 230000004163 JAK-STAT signaling pathway Effects 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- GRZSCTXVCDUIPO-SRVKXCTJSA-N Leu-Arg-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O GRZSCTXVCDUIPO-SRVKXCTJSA-N 0.000 description 1
- WGNOPSQMIQERPK-GARJFASQSA-N Leu-Asn-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N WGNOPSQMIQERPK-GARJFASQSA-N 0.000 description 1
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 1
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 1
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 1
- AIRUUHAOKGVJAD-JYJNAYRXSA-N Leu-Phe-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIRUUHAOKGVJAD-JYJNAYRXSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- FBNPMTNBFFAMMH-AVGNSLFASA-N Leu-Val-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-AVGNSLFASA-N 0.000 description 1
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 1
- 206010072138 Limbal stem cell deficiency Diseases 0.000 description 1
- URGPVYGVWLIRGT-DCAQKATOSA-N Lys-Met-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O URGPVYGVWLIRGT-DCAQKATOSA-N 0.000 description 1
- 101100202610 Mus musculus Cxcl12 gene Proteins 0.000 description 1
- 101000617124 Mus musculus Stromal cell-derived factor 1 Proteins 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 208000007920 Neurogenic Inflammation Diseases 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 208000023715 Ocular surface disease Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- UEXCHCYDPAIVDE-SRVKXCTJSA-N Phe-Asp-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 UEXCHCYDPAIVDE-SRVKXCTJSA-N 0.000 description 1
- JKJSIYKSGIDHPM-WBAXXEDZSA-N Phe-Phe-Ala Chemical compound C[C@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](N)Cc1ccccc1)C(O)=O JKJSIYKSGIDHPM-WBAXXEDZSA-N 0.000 description 1
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 description 1
- TUYWCHPXKQTISF-LPEHRKFASA-N Pro-Cys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N2CCC[C@@H]2C(=O)O TUYWCHPXKQTISF-LPEHRKFASA-N 0.000 description 1
- GURGCNUWVSDYTP-SRVKXCTJSA-N Pro-Leu-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GURGCNUWVSDYTP-SRVKXCTJSA-N 0.000 description 1
- FKYKZHOKDOPHSA-DCAQKATOSA-N Pro-Leu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FKYKZHOKDOPHSA-DCAQKATOSA-N 0.000 description 1
- MLKVIVZCFYRTIR-KKUMJFAQSA-N Pro-Phe-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O MLKVIVZCFYRTIR-KKUMJFAQSA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- YUSRGTQIPCJNHQ-CIUDSAMLSA-N Ser-Arg-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O YUSRGTQIPCJNHQ-CIUDSAMLSA-N 0.000 description 1
- UFKPDBLKLOBMRH-XHNCKOQMSA-N Ser-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)C(=O)O UFKPDBLKLOBMRH-XHNCKOQMSA-N 0.000 description 1
- KDGARKCAKHBEDB-NKWVEPMBSA-N Ser-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CO)N)C(=O)O KDGARKCAKHBEDB-NKWVEPMBSA-N 0.000 description 1
- CAOYHZOWXFFAIR-CIUDSAMLSA-N Ser-His-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O CAOYHZOWXFFAIR-CIUDSAMLSA-N 0.000 description 1
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 1
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 1
- WRQLCVIALDUQEQ-UNQGMJICSA-N Thr-Phe-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WRQLCVIALDUQEQ-UNQGMJICSA-N 0.000 description 1
- NQQMWWVVGIXUOX-SVSWQMSJSA-N Thr-Ser-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NQQMWWVVGIXUOX-SVSWQMSJSA-N 0.000 description 1
- JLRGJRBPOGGCBT-UHFFFAOYSA-N Tolbutamide Chemical compound CCCCNC(=O)NS(=O)(=O)C1=CC=C(C)C=C1 JLRGJRBPOGGCBT-UHFFFAOYSA-N 0.000 description 1
- NUQZCPSZHGIYTA-HKUYNNGSSA-N Tyr-Trp-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N NUQZCPSZHGIYTA-HKUYNNGSSA-N 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- PTFPUAXGIKTVNN-ONGXEEELSA-N Val-His-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)NCC(=O)O)N PTFPUAXGIKTVNN-ONGXEEELSA-N 0.000 description 1
- ZTKGDWOUYRRAOQ-ULQDDVLXSA-N Val-His-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N ZTKGDWOUYRRAOQ-ULQDDVLXSA-N 0.000 description 1
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 1
- 239000009466 Valverde Substances 0.000 description 1
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 description 1
- 206010047571 Visual impairment Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 108010087049 alanyl-alanyl-prolyl-valine Proteins 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010059459 arginyl-threonyl-phenylalanine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000005537 brownian motion Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000009956 central mechanism Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 201000005667 central retinal vein occlusion Diseases 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 208000021921 corneal disease Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 208000037888 epithelial cell injury Diseases 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000000893 fibroproliferative effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 210000004713 immature microglia Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000001232 limbus corneae Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000002690 local anesthesia Methods 0.000 description 1
- 230000035168 lymphangiogenesis Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108010016686 methionyl-alanyl-serine Proteins 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000003959 neuroinflammation Effects 0.000 description 1
- 230000009223 neuronal apoptosis Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 201000007914 proliferative diabetic retinopathy Diseases 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000004263 retinal angiogenesis Effects 0.000 description 1
- 230000004283 retinal dysfunction Effects 0.000 description 1
- 208000004644 retinal vein occlusion Diseases 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- GKCBAIGFKIBETG-UHFFFAOYSA-N tetracaine Chemical compound CCCCNC1=CC=C(C(=O)OCCN(C)C)C=C1 GKCBAIGFKIBETG-UHFFFAOYSA-N 0.000 description 1
- 229960002372 tetracaine Drugs 0.000 description 1
- 229940072172 tetracycline antibiotic Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Images
Classifications
-
- 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
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Rheumatology (AREA)
- Pain & Pain Management (AREA)
- Botany (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Ophthalmology & Optometry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明属于生物医药领域,具体涉及修饰miR‑155‑5p/SOCS1轴的外泌体制备方法及其在制备治疗视网膜及角膜炎症‑新生血管疾病药物中的应用。该修饰miR‑155‑5p/SOCS1轴的外泌体内包裹有miR‑155‑5p抑制物和高水平SOCS1,通过增强SOCS1表达以及阻断SOCS1的上游miR‑155‑5p的表达,从而强效抑制NFκB通路,在视网膜中可显著抑制小胶质细胞M1型活化并阻断其促进新生血管作用,在角膜中也能够阻断NFκB依赖性信号通路抑制炎症因子的表达而减轻炎症及新生血管的表现。
Description
技术领域
本发明属于细胞生物学、分子生物学及生物医药领域,具体涉及修饰 miR-155-5p/SOCS1轴的外泌体制备方法及其在制备治疗眼部疾病药物中的应用。
背景技术
眼部新生血管的形成是导致多种致盲性眼部疾病的原因,治疗的关键在于抑制新生血管的生长。目前临床上尚没有治疗眼部新生血管真正的有效药物和方法。
眼部新生血管包括视网膜新生血管和角膜新生血管,视网膜新生血管属于异常血管增生,其主要发生在糖尿病视网膜疾病、视网膜中央静脉阻塞、年龄相关性黄斑变性等,是临床病例中致盲的主要原因之一。迄今为止,视网膜新生血管发生的分子机制并不完全清楚,因此深入揭示其发病机制,对视网膜新生血管的早期干预具有重要意义。
小胶质细胞是中枢神经系统特化的巨噬细胞,也是视网膜的第三类胶质细胞。视网膜小胶质细胞的活化被认为是多种眼科疾病的重要驱动力,其对于触发和维持神经炎症起着关键作用。目前,越来越多的研究认为活化的小胶质细胞促进了视网膜新生血管病变。Ma等(Ma Wenxin,Zhao Lian,Fontainhas Aurora M et al.Microglia in the mouseretina alter the structure and function of retinal pigmented epithelialcells:a potential cellular interaction relevant to AMD.[J].PLoS ONE,2009,4:e7945.)通过视网膜色素上皮细胞与小胶质细胞体外共培养实验以及在体视网膜下抑制小胶质细胞实验,证实了小胶质细胞在视网膜色素上皮细胞损伤下发生激活并促进新生血管生成。Ana等(Arroba Ana I,Valverde M,Modulation of microglia in theretina:new insights into diabetic retinopathy.[J].Acta Diabetol,2017,54:527-533.)研究显示,在增生性糖尿病视网膜病变中,小胶质细胞出现异常活化进而促进新生血管产生。Madhvi等 (Menon Madhvi,Mohammadi Shahin,Davila-Velderrain Jose etal.Single-cell transcriptomic atlas of the human retina identifies cell typesassociated with age-related macular degeneration.[J].Nat Commun,2019,10:4902.)通过单细胞测序及生物信息学技术也证实了小胶质细胞的激活是造成视网膜新生血管病变的关键因素。此外,已经存在的视网膜炎症也能够波及静息小胶质细胞进而促进视网膜新生血管的生成。因此,抑制视网膜小胶质的活化对于减缓视网膜炎症进展、抑制视网膜新生血管具有重要意义。
目前治疗视网膜新生血管的主流方式是在玻璃体腔注射下调血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)或其受体的药物和抑制二者结合后发挥生物学效应的药物。同时,也有许多研究已经证实米诺环素可以早期干预视网膜疾病过程,通过阻碍视网膜小胶质细胞活化进而干预新生血管。
然而,上述两种主流治疗方式存在如下问题:(1)抗VEGF治疗存在不良反应,且无法取代病因治疗;已有较多病例报道抗VEGF治疗会诱发肾脏损害、高血压等全身性不良反应,而且会加重纤维膜增生性视网膜病变等眼部疾病。更重要的是,单纯阻断VEGF往往属于病理进程晚期的干预,更早期的病理因素持续存在,也导致抗VEGF治疗后期可能需要加大剂量和频次。(2)米诺环素是一种广谱抗菌的四环素类抗生素,它能与tRNA结合,达到抑菌的效果,许多研究证实米诺环素通过抑制活化小胶质细胞向M1型即促炎表型极化,因此能够阻碍视网膜小胶质细胞活化进而干预新生血管。但米诺环素副作用较多,许多研究团队证实米诺环素可诱导神经元凋亡等加重视网膜功能紊乱,不良反应较多。另外,米诺环素作为抗生素无法长期使用,否则可能由于破坏正常菌群平衡而引起严重的机会致病性疾病。可见,米诺环素不是一种适宜的临床用药。
角膜新生血管是常见的严重眼表和角膜病变不良后果,如果不及时处理,可能会导致明显的视力障碍。
炎症是引起角膜新生血管的核心机制,包括化学损伤、感染、免疫紊乱、角膜缘干细胞缺乏和缺氧等。炎性细胞(尤其是巨噬细胞和中性粒细胞)和血管生长因子在炎症诱导的膜新生血管之间存在相互作用。VEGF-A介导的巨噬细胞募集可以启动“免疫放大级联”反应,从而促进角膜血管生成和淋巴管生成。在角膜损伤过程中募集的炎性细胞,尤其是巨噬细胞,产生促进血管内皮细胞增殖和迁移的促血管生成因子和蛋白水解酶。前炎性细胞因子和趋化因子是人类血管生成的强有力介质,通常在角膜炎症中过表达。
在大多数病理条件下,炎症是角膜新生血管的关键因素。浸润受损角膜的炎性细胞是强有力的血管生成介质的主要来源,如IL-1、TNF-α和VEGF。此外炎症反应产物,如前列腺素也具有促血管生成特性。糖皮质激素是强有力的炎症抑制剂,已用于角膜新生血管的抗炎和抗血管生成。撇开副作用,糖皮质激素是临床上用于控制炎症和角膜新生血管最有效的药物之一。但糖皮质激素的不良反应较容易出现,需要密切监测长期使用糖皮质激素的患者从而进行潜在不良反应的早期诊断和管理。因此临床上需要更加安全、有效的治疗。
发明内容
本发明的目的在于提供一种治疗视网膜及角膜炎症-新生血管疾病的药物:修饰miR-155-5p/SOCS1轴的外泌体,通过靶向抑制视网膜炎症及角膜炎症而减弱新生血管产生的始动因素,达到抑制眼部新生血管的目的;将上述修饰 miR-155-5p/SOCS1轴的外泌体在制备治疗视网膜炎症、角膜炎症及新生血管疾病药物中的应用作为本发明的另一个目的,具有治疗效果好且安全性高的优势。
基于上述目的,本发明采用的技术方案如下:
第一方面,本发明提供了一种修饰miR-155-5p/SOCS1轴的外泌体,该外泌体内包裹有miR-155-5p抑制物和细胞因子信号转导抑制分子(以下简称: SOCS1)。
外泌体是直径在30~150nm的双层脂质囊泡,具有非免疫性和易于透过细胞膜的特性,因此,外泌体作为药物递送载体具有其独特的天然优势。
miRNA能对多个靶点进行不同程度的调控,且这些靶基因往往存在于相关联的分子网络中,因此对miRNA的干预会比对特定功能蛋白直接改造具有更大的优势。同时miRNA不会完全抑制靶蛋白的表达,相对于基因敲除等方法, miRNA对细胞的毒性更小、安全性更高。申请者前期通过体外实验发现 miR-155-5p的水平与眼部炎症、新生血管正相关。视网膜实验方面,原代视网膜小胶质细胞外源性转染miR-155-5p的模拟物后可观察到细胞出现明显的活化表现,同时视网膜小胶质细胞M1型相关分子如iNOS和IL1β明显上调。此外,体内实验也证实了拮抗miR-155-5p可显著下调视网膜及角膜炎症和新生血管相关分子的表达。因此,通过外源性改造令外泌体携带miR-155-5p的小分子抑制物,通过对miR-155-5p进行人工干预,使其具有眼部疾病治疗价值。
SOCS1全称为细胞因子信号转导抑制分子1,SOCS1自上个世纪发现以来,已被证实负向调控JAK-STAT通路及NFκB通路,具有抑制炎症的作用。申请者前期体内、体外实验也证实了SOCS1对NFκB通路发挥抑制作用,并且发现 SOCS1是miR-155-5p调控视网膜炎症-新生血管的关键靶点。然而现有技术中 SOCS1与视网膜炎症及新生血管的关系未见明确研究,更未公开SOCS1作为 miR-155-5p调控视网膜及角膜炎症-新生血管的关键靶点。
基于申请者前期研究,本发明外泌体携带高水平的SOCS1和miR-155-5p 的小分子抑制物,利用miR-155-5p抑制物拮抗上游miRNA抑制性调控,利用 SOCS1提供更多靶蛋白靶点,使得miRNA能够对多个靶点进行不同程度的调控,从而具有强效抑制NFκB通路的作用,显著抑制视网膜及角膜炎症,阻断其促进新生血管作用,能够用于早期眼部炎症及新生血管疾病的治疗,具有治疗效果好、安全性高的优点。
另外,本申请者前期研究发现,视网膜小胶质细胞活化早于VEGF表达上调,单独抗VEGF治疗只是针对血管新生诱导的终末环节,无法取代病因学治疗。此外,小胶质细胞活化介导的神经炎症还会导致其他病理性改变,如诱导神经节细胞凋亡等,本申请提供的含有SOCS1和miR-155-5p的小分子抑制物的外泌体能够抑制视网膜小胶质细胞激活,阻断其促进新生血管的作用,从而能够病原性地治疗视网膜炎症及新生血管疾病。
进一步地,miR-155-5p抑制物的碱基序列如SEQ ID NO.1所示;SOCS1的氨基酸序列如SEQ ID NO.2所示。
进一步地,外泌体源于间充质干细胞。
间充质干细胞是一类具有自我更新和多向分化能力的多能干细胞,其组织修复和再生作用在临床中得到广泛的应用。本发明采用来源于间充质干细胞的外泌体,是间充质干细胞和外泌体两者优势的承载者,能够通过人工修饰改变其内容物和生物学特性,从而避免了细胞移植的风险,另外,将来源于间充质干细胞的外泌体作为药物的递送载体,具有可控性高、免疫原性低、安全性高的优势,还具有易于获取、储存和管理的优点。
进一步地,间充质干细胞为骨髓间充质干细胞、脐带间充质干细胞、脂肪间充质干细胞中的任一种。
第二方面,本发明提供了上述修饰miR-155-5p/SOCS1轴外泌体的制备方法,包括如下步骤:
(1)由携带SOCS1过表达载体的病毒感染间充质干细胞,制得过表达SOCS1的间充质干细胞;
(2)由过表达SOCS1的间充质干细胞细胞培养液分离得到外泌体;外泌体内包裹过表达的SOCS1;
(3)将miR-155-5p抑制物转染由步骤(2)得到的外泌体,制得修饰 miR-155-5p/SOCS1轴的外泌体。
本发明首先获取过表达SOCS1的外泌体,再经外源性改造另其携带 miR-155-5p的小分子抑制物,能够通过拮抗上游miRNA抑制性调控、外源性增加靶蛋白的量,共同抑制NFκB通路,从而用于视网膜及角膜炎症-新生血管的早期预防与治疗。
进一步地,步骤(2)由SOCS1过表达的间充质干细胞经分离得到外泌体的具体过程为:将SOCS1过表达的间充质干细胞在含有正常DMEM培养基上培养至密度为90%;经PBS缓冲液清洗后,加入去除外泌体的DMEM培养基,于37℃下培养48h得到细胞培养基,将细胞培养基超离得到的沉淀即为外泌体。
进一步地,步骤(3)将miR-155-5p抑制物转染由步骤(2)得到的外泌体的具体过程为:将miR-155-5p抑制物与外泌体混合得到混合液,向混合液中加入氯化钙溶液至混合液中氯化钙的浓度为0.1mol/L,用PBS缓冲液补充混合液体积至300μL;将混合液进行冷热交变处理后,向混合液中加入RNA酶,于37℃下酶解30min后,加入PBS缓冲液,再经超离得到的沉淀即为修饰 miR-155-5p/SOCS1轴的外泌体。
第三方面,上述修饰miR-155-5P/SOCS1轴的外泌体在制备治疗眼部疾病药物中的应用。
进一步地,上述眼部疾病为视网膜炎症或角膜炎症或新生血管疾病。
进一步地,本发明修饰miR-155-5p/SOCS1轴的外泌体通过抑制NFκB通路,逆转小胶质细胞的M1型极化,下调小胶质细胞M1型巨噬细胞相关分子iNOS 和IL-1β的表达,达到治疗视网膜炎症的目的。
进一步地,本发明修饰miR-155-5p/SOCS1轴的外泌体通过抑制抑制NFκB 通路,下调炎症因子TNF-α、IL-6的表达,达到治疗角膜炎症的目的。
进一步地,本发明修饰miR-155-5p/SOCS1轴的外泌体通过靶向抑制视网膜炎症、角膜炎症达到抑制眼部新生血管的目的。
本发明通过对视网膜及角膜炎症-新生血管疾病的发病机制进行研究,利用修饰miR-155-5p/SOCS1轴的外泌体抑制NFκB通路,抑制血管新生相关蛋白 HIF-1α、CXCR4、VEGFA和SDF-1的表达,减弱视网膜及角膜新生血管产生的始动因素,达到抑制视网膜及角膜新生血管的目的,能够对新生血管进行早期干预。
与现有技术相比,本发明的有益效果如下:
(1)本发明提供的修饰miR-155-5p/SOCS1轴的外泌体内包裹有 miR-155-5p抑制物和细胞因子信号转导抑制分子SOCS1,从而强效抑制NFκB 通路,显著抑制炎症,阻断其促进新生血管作用,能够病原性地治疗视网膜及角膜炎症-新生血管疾病。此外,本发明修饰miR-155-5p/SOCS1轴的外泌体的灵活性更强,且具有降低视网膜小胶质细胞活化后伴随的其他病理损害如神经节细胞凋亡等的效果。
(2)本发明将修饰miR-155-5p/SOCS1轴的外泌体作为视网膜及角膜炎症- 新生血管疾病的治疗药物,能够对miRNA及其靶点进行干预,借助间充质干细胞外泌体作递送系统,大大提高治疗效果和安全性。
附图说明
图1为改良BMSC源外泌体(N-BMSC-EXO)制备和特征鉴定图;
图2为改良BMSC源外泌体(N-BMSC-EXO)在体外逆转小胶质M1极化形态及功能图;
图3为改良BMSC源外泌体(N-BMSC-EXO)在体内逆转小胶质细胞M1 极化形态、功能图;
图4为改良BMSC源外泌体(N-BMSC-EXO)在体外抑制活化小胶质细胞介导的促人脉静脉血管内皮细胞(HUVECs)增殖、迁移、成管和血管新生相关蛋白表达图;
图5为改良BMSC源外泌体(N-BMSC-EXO)在体内抑制活化小胶质细胞介导的视网膜新生血管和视网膜血管新生相关蛋白表达图;
图6为改良BMSC源外泌体(N-BMSC-EXO)在碱烧伤模型中抑制小鼠角膜炎症因子表达图;
图7为改良BMSC源外泌体(N-BMSC-EXO)在碱烧伤模型中抑制小鼠角膜新生血管图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合附图和具体实施例对本发明作进一步说明。以下实施例所涉及各原料如无特别说明,均为市售通用产品。
实施例1修饰miR-155-5p/SOCS1轴的外泌体、制备方法及鉴定
一、修饰miR-155-5p/SOCS1轴的外泌体
修饰miR-155-5p/SOCS1轴的外泌体内包裹有miR-155-5p抑制物和SOCS1,其中,外泌体源于骨髓间充质干细胞。其中,修饰miR-155-5p/SOCS1轴的外泌体亦称为改良BMSC源外泌体(以下简写为:N-BMSC-EXO)。
二、修饰miR-155-5p/SOCS1轴的外泌体的制备方法
上述修饰miR-155-5p/SOCS1轴的外泌体的制备方法包括如下步骤:
(1)小鼠骨髓间充质干细胞的培养
收集5~7周龄小鼠骨髓细胞悬液(低糖DMEM培养基冲出骨髓液)于15ml EP管中,在1500rpm/min的转速下,离心5min弃上清,用含10%FBS的DMEM 培养基重悬细胞,在5%CO2、21%O2、37℃条件下,以1×106的细胞数均匀铺在T25培养瓶中,24h后半量换液,洗去未贴壁的细胞,以后每48h全量换液,一般7天细胞可生长至融合状态,制得大量小鼠骨髓间充质干细胞。
所有动物实验程序均严格遵循中山大学中山眼科中心《关于动物应用于伦理学规定》,伦理编号为SYXK(YUE)2017-093。所有动物饲养、实验操作及处死原则均遵循视觉与眼科学研究学会(ARVO)《关于动物实验处理规定相关要求》进行。
(2)过表达SOCS1的骨髓间充质干细胞的培养
利用三质粒共转系统在293FT细胞中转入重组pLJM1-CMV-m-Socs1-Puro,psPAX2,pMD2.G生产并纯化慢病毒。在5%CO2、21%O2、37℃条件下,慢病毒处理骨髓间充质干细胞24h后换液,用PBS清洗3遍后添加含嘌呤霉素的 DMEM培养基筛选48-72h,存活的细胞即为过表达SOCS1的骨髓间充质干细胞,其中,SOCS1的氨基酸序列如SEQ ID NO.2 (MVARNQVAADNAISPAAEPRRRSEPSSSSSSSSPAAPVRPRPCPAVPAPAPGD THFRTFRSHSDYRRITRTSALLDACGFYWGPLSVHGAHERLRAEPVGTFLVRD SRQRNCFFALSVKMASGPTSIRVHFQAGRFHLDGSRETFDCLFELLEHYVAAP RRMLGAPLRQRRVRPLQELCRQRIVAAVGRENLARIPLNPVLRDYLSSFPFQI
)所示。
(3)含有过表达SOCS1的外泌体的分离及鉴定
将过表达SOCS1的小鼠骨髓间充质干细胞(BMSC)在含正常DMEM培养基的T75培养瓶中培养至密度为90%;用PBS清洗3遍后添加20ml含10%去除外泌体FBS的DMEM培养基,并在21%O2、5%CO2、37℃条件下培养。48h 后收获细胞培养基,以300×g离心10min,2000×g离心10min,10000×g、4℃离心30min,收集上清液并经0.22μm滤网过滤除去残留细胞,然后将过滤后的上清液以100000×g、4℃超离70min(BecKman Coulter,California,USA),收集沉淀物,并用PBS重悬沉淀物后再以100000×g、4℃超离70min,收集沉淀,即为含有SOCS1过表达载体的外泌体,并将沉淀重悬于100μL PBS中,得到含有过表达SOCS1的外泌体悬浮液。
(4)改良BMSC源外泌体(N-BMSC-EXO)的制备
miR-155-5p抑制物转染过表达SOCS1的外泌体,制得修饰 miR-155-5p/SOCS1轴的外泌体,即改良BMSC源外泌体(N-BMSC-EXO),具体过程如下:
miR-155-5p抑制剂(miR-155-5p inhibitor)购自锐博生物(中国广州), miR-155-5p inhibitor的序列如SEQ ID NO.1 (UUAAUGCUAAUUGUGAUAGGGGU)所示。取200pmolmiR-155-5p inhibitor 与20μg PBS重悬后的外泌体悬浮液混合形成混合液,向混合液中添加无菌氯化钙溶液使混合液中氯化钙的终浓度为0.1mol/L,并用PBS将终体积补至300μL,随后,于冰上静置30min后,在42℃热激60s,继续在冰上静置5min,向体系中添加1.5μgRNA酶,37℃水浴30min,再向体系中添加20mL PBS后,于 100000×g、4℃超离70min,收集沉淀即得修饰miR-155-5p/SOCS1轴的外泌体,也即改良BMSC源外泌体(N-BMSC-EXO),随后将沉淀重悬于100μL PBS中备用。
三、改良BMSC源外泌体的鉴定
对上述制得的改良BMSC源外泌体(N-BMSC-EXO)进行鉴定,同时,以直接由骨髓间充质干细胞分离的外泌体转染miR-155-5p inhibitor,制得的外泌体 (BMSC-EXO)作为对照,即BMSC-EXO内包裹有miR-155-5p inhibitor,而未包裹SOCS1。
将上述两种不同的外泌体N-BMSC-EXO和BMSC-EXO进行形态、大小以及免疫学鉴定,具体方法及鉴定结果如下:
1)外泌体的形态、大小的鉴定
利用电子显微镜和NanoSight系统对N-BMSC-EXO和BMSC-EXO外泌体的形态、大小进行鉴定,具体过程为:
将N-BMSC-EXO和BMSC-EXO外泌体分别悬浮于3%戊二醛溶液,随后将其分别滴入碳涂覆的铜网格中,5min后用蒸馏水反复洗涤后用4%乙酸铀酰染色10min,再用1%甲基纤维素溶液处理5min,待网格干燥后使用TEM 1011 透射电子显微镜在80KV(JEOL-1200EX)下成像,使用配备有快速视频捕获和粒子追踪软件的Nanosight LM10系统(Nanosight Ltd,Navato,CA)分析外泌体的大小,通过测量布朗运动的速率来计算纳米粒子浓度和粒度分布。
结果如图1中的图1B和图1C所示,其中,图1B给出了N-BMSC-EXO和 BMSC-EXO这两种外泌体的形态电子显微镜图片,两者均呈双凹“茶托样”圆形囊泡;图1C为经NanoSight分析得出N-BMSC-EXO和BMSC-EXO外泌体的直径分布及浓度图;可以看出,N-BMSC-EXO的直径为126.4±23.5;BMSC-EXO 的直径为122.2±20.4。
2)外泌体内SOCS1以及外泌体内标记物CD63、HSP70和TSG101的Western 蛋白印迹分析
将N-BMSC-EXO和BMSC-EXO这两种外泌体分别进行蛋白提取得到蛋白提取物,将蛋白提取物在裂解缓冲液中匀浆,并以12000rpm/min的转速离心 15min,利用二辛可宁酸(BCA)法测定蛋白质浓度。免疫印迹后,将蛋白质转移到PVDF膜上,并与特异性抗体一起孵育。其中特异性抗体包括一抗和荧光素缀合的二抗,孵育后通过显影仪(ChemiDoc XRS+,BIO-RAD)检测蛋白浓度。其中一抗包括:SOCS1(Cell Signaling Technology,3950)、TSG101(Abcam, ab125011)、CD63(Abcam,ab134045)、HSP70(Cell Signaling Technology,4872)。荧光素缀合的二抗为:抗兔Alexa Fluor 555(Cell Signaling Technology,4413),抗鼠Alexa Fluor 488(Cell Signaling Technology,4408)。
对N-BMSC-EXO和BMSC-EXO这两种外泌体的Western蛋白印迹分析结果如图1中的图1A所示,可以看出,相对于BMSC-EXO,N-BMSC-EXO外泌体中SOCS1的水平显著升高;对上述两种外泌体中的标记物CD63、HSP70和TSG101的Western蛋白印迹分析结果如图1D所示,外泌体蛋白CD63、HSP70 和TSG101在N-BMSC-EXO和BMSC-EXO这两种外泌体中均由表达。
上述结果证明了改良BMSC源外泌体具有外泌体性质,并进一步表明,骨髓间充质干细胞转染SOCS1质粒后超离获取的外泌体,再经转染miR-155-5p inhibitor的系列操作依然能收集到正常外泌体且不会破坏其结构。
实施例2修饰miR-155-5p/SOCS1轴的外泌体在体外对小胶质细胞M1极化逆转效果
一、小鼠视网膜小胶质细胞的培养纯化
取SPF级生后1-3天C57乳鼠的视网膜2片,胰酶消化后种至T75培养瓶,用10ml含20%胎牛血清的DMEM/F12培养液培养,每3天加3ml培养液。14 天后培养瓶封口,在37℃、200rpm/min的摇床上震荡90min,上清液离心后种至由0.01%的L-多聚赖氨酸包被的孔板上。30min后换液,利用差速贴壁纯化原代小胶质细胞。
所有动物实验程序均严格遵循中山大学中山眼科中心《关于动物应用于伦理学规定》,伦理编号为SYXK(YUE)2017-093。所有动物饲养、实验操作及处死原则均遵循视觉与眼科学研究学会(ARVO)《关于动物实验处理规定相关要求》进行。
二、修饰miR-155-5p/SOCS1轴的外泌体对小胶质细胞M1极化的抑制效果试验
为了明确改良BMSC源外泌体(N-BMSC-EXO)对小胶质细胞M1极化的抑制作用,首先利用100ng/mL的脂多糖(LPS)刺激原代视网膜小胶质细胞 M1极化,然后分别用PBS,BMSC-EXO和N-BMSC-EXO刺激24小时,并通过如下实验对小胶质细胞的功能改变进行研究。
用外泌体特异性的染料PKH26标记BMSC-EXO和N-BMSC-EXO,将其加入小胶质细胞培养体系中,于24h进行共聚焦显微镜观察,结果如图2A所示,图2A为原代小胶质细胞吞噬PKH26标记的BMSC-EXO和N-BMSC-EXO的共聚焦显微镜代表性图像,比例尺为10μm,图2A显示PKH26标记的BMSC-EXO 和N-BMSC-EXO均可被小胶质细胞成功吞噬。
通过光镜和小胶质细胞特征性标记物Iba-1免疫荧光染色观察,结果如图2B 和图2C所示,图2B为原代小胶质细胞分别经PBS、BMSC-EXO、N-BMSC-EXO 处理后光学显微镜观察的细胞形态图以及由小胶质细胞特征性标记物Iba-1免疫荧光染色的代表性图,比例尺为10μm,可以看到M1极化的小胶质细胞胞体明显变大,细胞突起变短呈毛刺状,而BMSC-EXO与N-BMSC-EXO处理均可显著抑制M1极化的形态,小胞体的圆形或纺锤形静息细胞明显增多;图2C为小胶质细胞M1极化率的统计分析图,可以看出相对于PBS和BMSC-EXO, N-BMSC-EXO抑制小胶质细胞M1极化的效果更加显著。
利用实施例1中Western蛋白印迹分析方法,对NFκB蛋白(磷酸化/总蛋白)以及M1型巨噬细胞相关分子如iNOS和IL-1β的表达进行检测,结果如图 2D所示,与BMSC-EXO相比,N-BMSC-EXO能够显著下调iNOS和IL-1β的表达水平。
上述结论表明,N-BMSC-EXO在体外条件下已经具备逆转小胶质细胞M1 极化的作用,且效果比普通BMSC-EXO更加显著。
实施例3miR-155-5p/SOCS1轴修饰的外泌体在体内对小胶质细胞M1极化逆转效果
为了进一步验证改良BMSC源外泌体(N-BMSC-EXO)在体内是否会抑制小胶质细胞M1极化,首先提前2天利用在小鼠玻璃体腔注射活化小胶质细胞来源外泌体(1μg),造成广泛的小胶质细胞活化,随后每7天玻璃体腔注射PBS, BMSC-EXO(1μg)或N-BMSC-EXO(1μg),第3周评估小胶质细胞形态、位置和功能的改变。其中,在小鼠玻璃体腔内注射PBS的小鼠记为PBS组;在小鼠玻璃体腔内注射BMSC-EXO的小鼠记为BMSC-EXO组;在小鼠玻璃体腔内注射N-BMSC-EXO的小鼠记为N-BMSC-EXO组。
其中,在小鼠玻璃体腔注射的具体方法如下:
在解剖显微镜下固定好小鼠头位,充分暴露眼球,于角膜缘后1mm处,避开血管组织,利用破囊针头做巩膜穿刺。使用33G Hamilton显微注射器以约45 度角从穿刺口避开晶状体斜穿入玻璃体腔内。保持针头不移动的情况下缓慢推注注射器,使药物缓慢释放,推注完成后停留30秒后缓慢拔出针头,立即用消毒棉签封闭针孔,覆盖结膜。术后羟甲基纤维素凝胶涂眼,以确保在小鼠苏醒前眼球表面的湿润。待到小鼠完全复苏后放回鼠笼。在手术中及复苏过程中使用电热毯保温,以确保实验动物的顺利苏醒、避免术后患病和死亡。
所有动物实验程序均严格遵循中山大学中山眼科中心《关于动物应用于伦理学规定》,伦理编号为SYXK(YUE)2017-093。所有动物饲养、实验操作及处死原则均遵循视觉与眼科学研究学会(ARVO)《关于动物实验处理规定相关要求》进行。
随后将上述三组实验小鼠处死,固定取眼球作冰冻切片,对切片进行免疫荧光染色,具体过程如下:
修剪新鲜眼球组织表面肌肉、筋膜等组织,4%新鲜配制的PFA固定眼球, 4℃过夜。次日10%蔗糖溶液脱水2小时后,以30%蔗糖溶液过夜脱水。将过夜脱水后的眼球组织浸入OCT包埋胶,待OCT完全浸入眼球组织后,将包埋模具放入-80℃冰箱,待组织完全冰冻后行组织切片。使用冰冻切片机,将眼球组织切成10μm厚度的组织切片,做好标记,储存于-80℃冰箱备用。
切片洗脱OCT后,用5%BSA封闭1小时,使用以下一抗:Iba-1(Abcam, ab178847),SDF-1(Abcam,ab25117),CXCR4(Abcam,ab124824),PECAM-1(Santa Cruz,sc-376764),Ki67(Cell Signaling Technology,9129)。与第二抗体和DAPI 分别避光孵育后使用防淬灭封片剂封片。
检测结果如图3所示,其中图3A为PBS、BMSC-EXO、N-BMSC-EXO三种不同处理的小鼠眼球冰冻切片Iba-1免疫荧光染色的代表性图像、活化细胞数、迁移细胞数统计分析图,比例尺为100μm;由图3A可以看出,相对于PBS组和BMSC-EXO组,N-BMSC-EXO处理组能够显著减少内、外核层小胶质细胞的数量以及向丛状层迁移的活化细胞数量。
图3B为PBS、BMSC-EXO、N-BMSC-EXO三种不同处理的小鼠视网膜总蛋白p-NFκB、NFκB、iNOS和IL-1β表达水平的Western印迹图及统计分析图;由图3B可以看出,Western印迹检测NFκB蛋白(磷酸化/总蛋白)以及M1型巨噬细胞相关分子iNOS、IL-1β的结果与体外试验结果一致,N-BMSC-EXO处理显著逆转了小胶质细胞的M1型极化相关表型。
综上,N-BMSC-EXO在体内和体外都能够有效抑制视网膜小胶质细胞M1 极化,表明N-BMSC-EXO(即miR-155-5p/SOCS1外泌体)能够用于治疗视网膜炎症。
实施例4修饰miR-155-5p/SOCS1轴的外泌体在体外抑制视网膜新生血管的效果
现有研究发现,活化的小胶质细胞可以促进新生血管产生。为了验证改良BMSC源外泌体(N-BMSC-EXO)对活化小胶质细胞促新生血管的抑制效果,分别从如下四个方面进行验证。
首先收集经LPS诱导活化并经PBS,BMSC-EXO或N-BMSC-EXO处理48h 的小胶质细胞培养上清,与ECM培养液按照1:1的比例混合后,分别处理人脐静脉血管内皮细胞(HUVECs),得到PBS、BMSC-EXO、N-BMSC-EXO三种不同处理的HUVECs,进行下述效果试验。
(1)改良BMSC源外泌体对人脐静脉血管内皮细胞(HUVECs)增殖的抑制效果试验
将PBS、BMSC-EXO、N-BMSC-EXO三种不同处理的HUVECs以2×103 cells/well培养于96孔板内,培养24小时后用不同混合培养液处理24小时,弃培养液,向每孔内加入100μLCCK-8试剂(10μL CCK8溶液+90μL无血清细胞培养液),孵育2~4小时,期间观察培养液颜色变化情况,并用酶标仪(Bio-Tek, USA)测定在450nm波长处的吸光值。每次实验设立3个重复孔,实验重复3次。
利用上述方法对CCK-8细胞活性测定,结果如图4A所示,图4A为不同混合培养液处理HUVECs 24小时后CCK-8检测细胞活性结果,可以看出,活化小胶质细胞的培养液能够显著促进HUVECs的增殖活性,BMSC-EXO处理活化小胶质细胞对HUVECs增殖活性的影响不显著,但N-BMSC-EXO处理后活化小胶质细胞对HUVECs的增殖促进作用明显被削弱。
(2)改良BMSC源外泌体对HUVECs迁移的抑制效果试验
将PBS、BMSC-EXO、N-BMSC-EXO三种不同处理的HUVECs接种至24 孔板,待长至90%融合时,更换培养基为无血清ECM继续培养24小时,待融合接近100%时将无菌直尺放在24孔板表面,用20μL微量加样枪头垂直于24 孔板不同孔中以相同力度、同一枪头均匀划痕。划痕后用PBS冲洗3遍去除脱落悬浮的细胞,此时,在光学显微镜下拍照记录为0小时,拍照后,根据分组给予不同的混合培养基进行相应的干预,继续培养12小时、24小时后分别拍照记录各孔细胞向划痕区域迁移的情况,位置需与0小时相同。该实验至少重复3 次。最后利用ImageJ软件测量各孔不同时间点划痕裸区的面积,并计算迁移率。迁移率=(原始划痕裸区面积-各时间点划痕裸区面积)/原始划痕裸区面积×100%。
划痕实验结果如图4B所示,图4B为不同混合培养液处理HUVECs 0h、12h 和24h划痕实验光学显微镜下代表性图像及统计分析图,比例尺100μm,可以看出BMSC-EXO和N-BMSC-EXO均能抑制HUVECs的迁移,且N-BMSC-EXO 的抑制效果更加显著。
(3)改良BMSC源外泌体对成管分支节点数和成管总长度的抑制效果试验
将Matrigel基质胶置于4℃冰箱过夜,使Matrigel基质胶缓慢融化。实验过程中,Matrigel基质胶始终保持在冰上操作。96孔板中用预冷的枪头在每孔中加入60μLMatrigel基质胶,置于5%CO2、21%O2、37℃条件下培养2小时,使Matrigel基质胶凝固备用。
消化人脐静脉血管内皮细胞,调整HUVECs的密度为2×104cells/mL,每组均给予培养液和细胞悬液共200μL,接种于96孔板中,5%CO2、21%O2、37℃条件下培养6小时。取出96孔板,倒置显微镜下观察,随机选择5个不同视野拍照,对节点分支数和成管总长度进行计数。
成管试验结果如图4C所示,图4C为不同混合培养液处理HUVECs成管实验光学显微镜下代表性图像及分支节点数、成管总长度统计分析图,比例尺 100μm;可以看出,相比于BMSC-EXO组,N-BMSC-EXO组中成管分子节点和成管总长度均最低,表明,N-BMSC-EXO能够显著降低成管分支节点数和成管总长度。
(4)改良BMSC源外泌体对血管新生相关蛋白的表达抑制效果
利用Western蛋白印迹法和酶联免疫吸附法,对血管新生相关蛋白如 HIF-1α、CXCR4、VEGFA和SDF-1的表达进行检测,其中,Western蛋白印迹法参照实施例1中所述方法,酶联免疫吸附法如下:
采用Mouse SDF-1/CXCL12(Stromal Cell Derived Factor 1)ELISA Kit(Elabscience,E-EL-M3046)检测SDF-1的含量。首先在对应板孔中加入100μL标准品工作液或样本,37℃孵育90分钟。弃掉板内液体后,立即加入100μL生物素化抗体工作液,37℃孵育60分钟。弃掉板内液体,洗板3次。每孔加入100μL HRP酶结合物工作液,37℃孵育30分钟,弃掉板内液体,洗板5次。每孔加入 90μL底物溶液,37℃孵育15分钟左右。每孔加入90μL底物溶液,37℃孵育15 分钟。立即在450nm波长下读数,处理数据。
对血管新生相关蛋白如HIF-1α、CXCR4、VEGFA和SDF-1的表达进行检测的结果如图4D和图4E所示,其中,图4D为不同混合培养液处理HUVECs 后SDF-1表达水平的酶联免疫吸附结果;图4E为不同混合培养液处理HUVECs 后HIF-1α、CXCR4、VEGFA表达水平的Western印迹图及统计分析图,结果发现N-BMSC-EXO显著下调了上述蛋白的表达。
综合上述结果分析,证实了N-BMSC-EXO可以在体外有效抑制小胶质细胞的促血管细胞增殖、迁移及成管能力,阻碍血管新生相关蛋白的表达上调。
实施例5修饰miR-155-5p/SOCS1轴的外泌体在体内抑制视网膜新生血管的效果
为了在体内进一步明确改良BMSC源外泌体(N-BMSC-EXO)对活化小胶质细胞促新生血管作用能否抑制,首先每3天利用活化小胶质细胞来源外泌体 (1μg)进行小鼠玻璃体腔注射,持续6周,建立视网膜新生血管小鼠模型,随后将建立视网膜新生血管小鼠模型的小鼠分为三组,每组小鼠每7天玻璃体腔分别注射PBS、BMSC-EXO(1μg)、N-BMSC-EXO(1μg),持续3周后将各组实验小鼠处死,固定取眼球作冰冻切片,进行PECAM-1/Ki67双染色,结果如图5A所示,图5A为视网膜新生血管小鼠分别接受PBS、BMSC-EXO(1μg)、 N-BMSC-EXO(1μg)处理后,眼球冰冻切片PECAM-1/Ki67免疫荧光染色的代表性图像及统计分析图,比例尺100μm,结果显示,PBS处理组有较多突破视网膜内界膜且具有增殖活性的内皮细胞,BMSC-EXO和N-BMSC-EXO均能抑制新生内皮细胞的产生,且N-BMSC-EXO效果更加显著。
图5B为视网膜新生血管小鼠分别接受PBS、BMSC-EXO(1μg)、 N-BMSC-EXO(1μg)处理后,眼球冰冻切片SDF-1免疫荧光染色的代表性图像,比例尺100μm;图5C为视网膜新生血管小鼠分别接受PBS、BMSC-EXO (1μg)、N-BMSC-EXO(1μg)处理后,眼球冰冻切片CXCR4免疫荧光染色的代表性图像,比例尺100μm;图5B和图5C的免疫荧光结果显示,PBS处理组中SDF-1和CXCR4呈现强特异性,表达较高,而BMSC-EXO和N-BMSC-EXO 可减弱上述蛋白的表达。
利用Western蛋白印迹和酶联免疫吸附法,对各组视网膜总蛋白进行检测,检测结果如图5D和图5E所示,其中,图5D为不同处理小鼠视网膜总蛋白SDF-1 表达水平的酶联免疫吸附结果;图5E为不同处理小鼠视网膜总蛋白HIF-1α、 CXCR4、VEGFA表达水平的Western印迹图及统计分析图;可以看出,与PBS 组和BMSC-EXO组相比,N-BMSC-EXO引起血管新生相关蛋白HIF-1α、CXCR4、VEGFA下调更加显著。
实施例6修饰miR-155-5p/SOCS1轴的外泌体在碱烧伤模型中抑制小鼠角膜炎症因子表达效果
为了在体内探究改良BMSC源外泌体(N-BMSC-EXO)对角膜炎症的影响,首先建立小鼠角膜碱烧伤模型,具体过程如下:
采用10%水合氯醛(5mg/kg)腹腔注射麻醉,1%丁卡因滴眼液局部麻醉,棉签拭去过多水分。用镊子夹住直径为0.2mm的单层滤纸片,浸于1M氢氧化钠溶液中,使其达饱和状态,吸水纸吸除多余液体,将滤纸片置于C57/BL小鼠角膜中央10s,弃掉滤纸,立即用20mL的PBS冲洗烧伤区及结膜囊1min。此后每天给予0.5%氯霉素滴眼液滴眼,2次/d。
小鼠角膜碱烧伤模型建立完成后,分为三组,自伤后当日起每组小鼠分别给予结膜下注射PBS、BMSC-EXO(1μg)、N-BMSC-EXO(1μg),每日1次,持续2周后将各组实验小鼠处死,固定取眼球作冰冻切片,进行TNF-α染色,结果如图6A和图6B所示。图6A和图6B为视网膜新生血管小鼠分别接受PBS、 BMSC-EXO(1μg)、N-BMSC-EXO(1μg)处理后,眼球冰冻切片TNF-α免疫荧光染色的代表性图像及统计分析图,比例尺50μm,结果显示,PBS处理组有较多炎症因子TNF-α阳性的细胞,BMSC-EXO和N-BMSC-EXO均能抑制TNF-α阳性细胞的产生,且N-BMSC-EXO效果更加显著。
其中,在小鼠结膜下注射的具体方法如下:
在解剖显微镜下固定好小鼠头位,充分暴露眼球,于角膜缘后1mm处,避开血管组织,使用33G Hamilton显微注射器以约20度角斜穿入球结膜囊中。保持针头不移动的情况下缓慢推注注射器,使药物缓慢释放,推注完成后停留30 秒后缓慢拔出针头,立即用消毒棉签封闭针孔。术后羟甲基纤维素凝胶涂眼,以确保在小鼠苏醒前眼球表面的湿润。待到小鼠完全复苏后放回鼠笼。在手术中及复苏过程中使用电热毯保温,以确保实验动物的顺利苏醒、避免术后患病和死亡。
所有动物实验程序均严格遵循中山大学中山眼科中心《关于动物应用于伦理学规定》,伦理编号为SYXK(YUE)2017-093。所有动物饲养、实验操作及处死原则均遵循视觉与眼科学研究学会(ARVO)《关于动物实验处理规定相关要求》进行。
利用qRT-PCR,对各组视网膜IL-6mRNA水平进行检测,检测结果如图6C 所示。可以看出,与PBS组和BMSC-EXO组相比,N-BMSC-EXO引起IL-6mRNA 下调更加显著。
实施例7修饰miR-155-5p/SOCS1轴的外泌体在碱烧伤模型中抑制小鼠角膜新生血管效果
为了在体内进一步明确改良BMSC源外泌体(N-BMSC-EXO)能否抑制碱烧伤继发的角膜新生血管,在小鼠角膜碱烧伤模型建立完成后分为三组,自伤后当日起每组小鼠分别给予结膜下注射PBS、BMSC-EXO(1μg)、N-BMSC-EXO (1μg),每日1次,持续2周后将各组实验小鼠处死,固定取眼球作角膜铺片,进行CD31染色,结果如图7A和图7B所示。图7A和图7B为角膜碱烧伤小鼠分别接受PBS、BMSC-EXO(1μg)、N-BMSC-EXO(1μg)处理后,角膜铺片 CD31免疫荧光染色的代表性图像(含局部放大图)及统计分析图,全角膜铺片图像比例尺400μm,结果显示,PBS处理组有大量迂曲细小的角膜新生血管, BMSC-EXO和N-BMSC-EXO均能抑制角膜新生血管的产生,且N-BMSC-EXO 效果更加显著。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 中山大学中山眼科中心
<120> 修饰miR-155-5p/SOCS1轴的外泌体、制备方法及其在制备治疗眼部疾病药物中的应用
<130> 0220061
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> RNA
<213> miR-155-5p
<400> 1
uuaaugcuaa uugugauagg ggu 23
<210> 2
<211> 212
<212> PRT
<213> SOCS1
<400> 2
Met Val Ala Arg Asn Gln Val Ala Ala Asp Asn Ala Ile Ser Pro Ala
1 5 10 15
Ala Glu Pro Arg Arg Arg Ser Glu Pro Ser Ser Ser Ser Ser Ser Ser
20 25 30
Ser Pro Ala Ala Pro Val Arg Pro Arg Pro Cys Pro Ala Val Pro Ala
35 40 45
Pro Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ser Asp
50 55 60
Tyr Arg Arg Ile Thr Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe
65 70 75 80
Tyr Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ala
85 90 95
Glu Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys
100 105 110
Phe Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg
115 120 125
Val His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser Arg Glu Thr
130 135 140
Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg
145 150 155 160
Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln
165 170 175
Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val Gly Arg Glu Asn Leu
180 185 190
Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe
195 200 205
Pro Phe Gln Ile
210
Claims (10)
1.一种修饰miR-155-5p/SOCS1轴的外泌体,其特征在于,所述外泌体内包裹有miR-155-5p抑制物和SOCS1。
2.根据权利要求1所述修饰miR-155-5p/SOCS1轴的外泌体,其特征在于,所述miR-155-5p抑制物的碱基序列如SEQ ID NO.1所示;所述SOCS1的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求2所述修饰miR-155-5p/SOCS1轴的外泌体,其特征在于,所述外泌体源于间充质干细胞。
4.根据权利要求3所述修饰miR-155-5p/SOCS1轴的外泌体,其特征在于,所述间充质干细胞为骨髓间充质干细胞、脐带间充质干细胞、脂肪间充质干细胞中的任一种。
5.权利要求1~4任一所述修饰miR-155-5p/SOCS1轴的外泌体的制备方法,其特征在于,包括如下步骤:
(1)由携带SOCS1过表达载体的病毒感染间充质干细胞,制得过表达SOCS1的间充质干细胞;
(2)由过表达SOCS1的间充质干细胞细胞培养液分离得到外泌体;所述外泌体内包裹过表达的SOCS1;
(3)将miR-155-5p抑制物转染由步骤(2)得到的外泌体,制得修饰miR-155-5p/SOCS1轴的外泌体。
6.权利要求1~4任一所述修饰miR-155-5p/SOCS1轴的外泌体在制备治疗眼部疾病药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述眼部疾病为视网膜炎症或角膜炎症或新生血管疾病。
8.根据权利要求7所述的应用,其特征在于,所述修饰miR-155-5p/SOCS1轴的外泌体通过抑制NFκB通路,逆转小胶质细胞的M1型极化,下调小胶质细胞M1型巨噬细胞相关分子iNOS和IL-1β的表达,达到治疗视网膜炎症的目的。
9.根据权利要求7所述的应用,其特征在于,所述修饰miR-155-5p/SOCS1轴的外泌体通过抑制NFκB通路,下调炎症因子TNF-α、IL-6的表达,达到治疗角膜炎症的目的。
10.根据权利要求8或9所述的应用,其特征在于,所述修饰miR-155-5p/SOCS1轴的外泌体通过靶向抑制视网膜炎症、角膜炎症达到抑制眼部新生血管的目的。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110688347.XA CN113769105A (zh) | 2021-06-21 | 2021-06-21 | 修饰miR-155-5p/SOCS1轴的外泌体、制备方法及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110688347.XA CN113769105A (zh) | 2021-06-21 | 2021-06-21 | 修饰miR-155-5p/SOCS1轴的外泌体、制备方法及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113769105A true CN113769105A (zh) | 2021-12-10 |
Family
ID=78835956
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110688347.XA Pending CN113769105A (zh) | 2021-06-21 | 2021-06-21 | 修饰miR-155-5p/SOCS1轴的外泌体、制备方法及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113769105A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103898050A (zh) * | 2014-03-31 | 2014-07-02 | 中国人民解放军军事医学科学院基础医学研究所 | 高效分泌一氧化氮的重组间充质干细胞及其制备方法与应用 |
US20190249250A1 (en) * | 2015-11-20 | 2019-08-15 | Braindtech S.R.L. | Microglia Microvesicles Contained MicroRNA-Based Methods For The Diagnosis, Prognosis And Treatment Monitoring Of Neurological, Neurodegenerative And Inflammation-Based Diseases |
CN111454900A (zh) * | 2020-05-08 | 2020-07-28 | 李立 | 骨髓间充质干细胞外泌体的制备方法 |
-
2021
- 2021-06-21 CN CN202110688347.XA patent/CN113769105A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103898050A (zh) * | 2014-03-31 | 2014-07-02 | 中国人民解放军军事医学科学院基础医学研究所 | 高效分泌一氧化氮的重组间充质干细胞及其制备方法与应用 |
US20190249250A1 (en) * | 2015-11-20 | 2019-08-15 | Braindtech S.R.L. | Microglia Microvesicles Contained MicroRNA-Based Methods For The Diagnosis, Prognosis And Treatment Monitoring Of Neurological, Neurodegenerative And Inflammation-Based Diseases |
CN111454900A (zh) * | 2020-05-08 | 2020-07-28 | 李立 | 骨髓间充质干细胞外泌体的制备方法 |
Non-Patent Citations (3)
Title |
---|
XIAOCHENG ZHOU等: ""Melanoma cell-secreted exosomal miR-155-5p induce proangiogenic switch of cancer-associated fibroblasts via SOCS1/JAK2/STAT3 signaling pathway"" * |
唐乖;龙鼎新;: "has-miR-155-5p靶基因预测及其生物信息学分析" * |
孙园园;郭大东;毕宏生;: "MicroRNA在葡萄膜炎发病过程中的调控作用研究进展" * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wu et al. | Galectin‐1 promotes choroidal neovascularization and subretinal fibrosis mediated via epithelialmesenchymal transition | |
US11801268B2 (en) | Methods of treating ocular inflammation and chemical injuries of the eye with extracellular vesicles | |
JP4361734B2 (ja) | 血管形成の調節に有用なトリプトファニル−tRNAシンテターゼ由来ポリペプチド | |
CN107109410A (zh) | 通道调节剂 | |
NO333837B1 (no) | Anvendelse av en sammensetning som omfatter et trunkert plasminprotein for fremstilling av et medikament for behandling av en lidelse i et øye samt sammensetning og sett. | |
Das et al. | Vimentin knockdown decreases corneal opacity | |
Koh et al. | Subretinal human umbilical tissue-derived cell transplantation preserves retinal synaptic connectivity and attenuates Müller glial reactivity | |
CN112870228B (zh) | 一种多功能微环境保护外泌体水凝胶及其制备方法和应用 | |
Mohammadi et al. | Pharmacology Research and textbook | |
WO2018171411A1 (zh) | 一种融合蛋白及其制备方法和其在制备治疗眼科疾病、抗炎、抗肿瘤药物中的应用 | |
KR20230093400A (ko) | Ccn5를 유효성분으로 포함하는 망막질환 예방 또는 치료용 약학 조성물 | |
CN113769105A (zh) | 修饰miR-155-5p/SOCS1轴的外泌体、制备方法及其应用 | |
JP6440107B2 (ja) | 細胞シートの製造方法、組成物、細胞培養補助剤、及び細胞培養方法 | |
CN114099664B (zh) | 一种基于Treg细胞外泌体的靶向协同药物体系及其制备方法 | |
JP5851692B2 (ja) | 網膜神経節ニューロン変性を予防又は治療するためのビコイドファミリーのホメオタンパク質の使用 | |
Hong et al. | Substance-P blocks degeneration of retina by stimulating migration and proliferation of retinal pigmented epithelial cells | |
CN106075448A (zh) | Angpt2抑制剂在制备用于治疗血管瘤的药物中的应用 | |
KR101916801B1 (ko) | 3차원 생체 모사 시스템을 이용한 맥락막 혈관신생을 수반하는 질환의 치료제 스크리닝 방법 | |
CA3122229A1 (en) | Composition for and method of facilitating corneal tissue repair | |
CN117138048B (zh) | Dock6在制备防治眼部新生血管性疾病药物中的应用 | |
CN106890313A (zh) | 用于治疗病理性近视的药物 | |
Sennlaub et al. | Christophe Roubeix | |
CN113930428B (zh) | 一种用于治疗CNV的miRNA及其制备方法和应用 | |
WO2024119424A1 (zh) | Ptxf在抑制c-rel基因表达方面的应用 | |
De Vivero et al. | Mesenchymal Stromal Cells from Human WhartonLs Jelly Modulate the Intraocular Immune Response in a Glucocorticoid Hypertension Model: An Exploratory Analysis |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211210 |
|
RJ01 | Rejection of invention patent application after publication |