CN116650528A - Application of immune energized MSC-EVS in preparation of medicament for treating retina diseases - Google Patents
Application of immune energized MSC-EVS in preparation of medicament for treating retina diseases Download PDFInfo
- Publication number
- CN116650528A CN116650528A CN202310616975.6A CN202310616975A CN116650528A CN 116650528 A CN116650528 A CN 116650528A CN 202310616975 A CN202310616975 A CN 202310616975A CN 116650528 A CN116650528 A CN 116650528A
- Authority
- CN
- China
- Prior art keywords
- msc
- evs
- immune
- energized
- retina
- 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
- 210000001525 retina Anatomy 0.000 title claims abstract description 39
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 20
- 239000003814 drug Substances 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title claims description 4
- 201000010099 disease Diseases 0.000 title abstract description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 7
- 208000017442 Retinal disease Diseases 0.000 claims description 24
- 102000004127 Cytokines Human genes 0.000 claims description 23
- 108090000695 Cytokines Proteins 0.000 claims description 23
- 206010038923 Retinopathy Diseases 0.000 claims description 19
- 108010074108 interleukin-21 Proteins 0.000 claims description 17
- 230000028993 immune response Effects 0.000 claims description 16
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 11
- 230000003712 anti-aging effect Effects 0.000 claims description 8
- 239000008194 pharmaceutical composition Substances 0.000 claims description 8
- 239000000049 pigment Substances 0.000 claims description 7
- 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 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 230000002434 immunopotentiative effect Effects 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 241000699670 Mus sp. Species 0.000 abstract description 27
- 238000000034 method Methods 0.000 abstract description 12
- 230000014509 gene expression Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 9
- 201000007737 Retinal degeneration Diseases 0.000 abstract description 7
- 230000032683 aging Effects 0.000 abstract description 7
- 230000004258 retinal degeneration Effects 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 5
- 210000001578 tight junction Anatomy 0.000 abstract description 5
- 230000004913 activation Effects 0.000 abstract description 4
- 238000003125 immunofluorescent labeling Methods 0.000 abstract description 4
- 230000037361 pathway Effects 0.000 abstract description 4
- 230000005779 cell damage Effects 0.000 abstract description 3
- 208000037887 cell injury Diseases 0.000 abstract description 3
- 210000003986 cell retinal photoreceptor Anatomy 0.000 abstract description 3
- 230000002757 inflammatory effect Effects 0.000 abstract description 3
- 210000000608 photoreceptor cell Anatomy 0.000 abstract description 3
- 238000004393 prognosis Methods 0.000 abstract description 3
- 238000011084 recovery Methods 0.000 abstract description 3
- 230000004382 visual function Effects 0.000 abstract description 3
- 101150099493 STAT3 gene Proteins 0.000 abstract description 2
- 229940124644 immune regulator Drugs 0.000 abstract description 2
- 238000007910 systemic administration Methods 0.000 abstract description 2
- 102000000591 Tight Junction Proteins Human genes 0.000 abstract 1
- 108010002321 Tight Junction Proteins Proteins 0.000 abstract 1
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 35
- 241000872931 Myoporum sandwicense Species 0.000 description 28
- 230000002207 retinal effect Effects 0.000 description 13
- 102100030704 Interleukin-21 Human genes 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- -1 IL-23 Proteins 0.000 description 10
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 10
- 102100040247 Tumor necrosis factor Human genes 0.000 description 9
- 238000012014 optical coherence tomography Methods 0.000 description 9
- 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 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 238000000465 moulding Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 210000003462 vein Anatomy 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 206010064930 age-related macular degeneration Diseases 0.000 description 5
- 210000005252 bulbus oculi Anatomy 0.000 description 5
- 230000004300 dark adaptation Effects 0.000 description 5
- 208000002780 macular degeneration Diseases 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 4
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 4
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 230000004438 eyesight Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000009758 senescence Effects 0.000 description 4
- 206010002091 Anaesthesia Diseases 0.000 description 3
- 235000008474 Cardamine pratensis Nutrition 0.000 description 3
- 240000000606 Cardamine pratensis Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000037005 anaesthesia Effects 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 210000004087 cornea Anatomy 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 210000003583 retinal pigment epithelium Anatomy 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 208000006550 Mydriasis Diseases 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 2
- 229960002327 chloral hydrate Drugs 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 208000030533 eye disease Diseases 0.000 description 2
- 239000003889 eye drop Substances 0.000 description 2
- 229940012356 eye drops Drugs 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 108091008695 photoreceptors Proteins 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000004243 retinal function Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 229960004394 topiramate Drugs 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical class [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 102000036541 Heterogeneous Nuclear Ribonucleoprotein D0 Human genes 0.000 description 1
- 108091021225 Heterogeneous Nuclear Ribonucleoprotein D0 Proteins 0.000 description 1
- 101001128431 Homo sapiens Myeloid-derived growth factor Proteins 0.000 description 1
- 101001077660 Homo sapiens Serine protease inhibitor Kazal-type 1 Proteins 0.000 description 1
- 101000997835 Homo sapiens Tyrosine-protein kinase JAK1 Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102000042838 JAK family Human genes 0.000 description 1
- 108091082332 JAK family Proteins 0.000 description 1
- 108010008148 Member 4 Subfamily A ATP Binding Cassette Transporter Proteins 0.000 description 1
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 1
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 206010057430 Retinal injury Diseases 0.000 description 1
- 102100033617 Retinal-specific phospholipid-transporting ATPase ABCA4 Human genes 0.000 description 1
- 102100025144 Serine protease inhibitor Kazal-type 1 Human genes 0.000 description 1
- 208000027073 Stargardt disease Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 108020000411 Toll-like receptor Proteins 0.000 description 1
- 102000002689 Toll-like receptor Human genes 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102100033438 Tyrosine-protein kinase JAK1 Human genes 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000000546 chi-square test Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000007831 electrophysiology Effects 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- UKZQEOHHLOYJLY-UHFFFAOYSA-M ethyl eosin Chemical compound [K+].CCOC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 UKZQEOHHLOYJLY-UHFFFAOYSA-M 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 102000056374 human MYDGF Human genes 0.000 description 1
- 102000057041 human TNF Human genes 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000007108 local immune response Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 230000026269 optomotor response Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000011697 sodium iodate Substances 0.000 description 1
- 229940032753 sodium iodate Drugs 0.000 description 1
- 235000015281 sodium iodate Nutrition 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Hematology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Ophthalmology & Optometry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to application of immune energized MSC-EVS in preparing a medicament for treating retina diseases. The invention proves that the immune energized MSC-EVs can obviously reduce the RPE and photoreceptor cell damage of mice with retinal degeneration, protect visual function and delay retinal degeneration, and provides a new way for injecting the immune energized MSC-EVs into the vitreous cavity, which has better effect than the systemic administration mode. Immunofluorescent staining demonstrated that immunoenergized MSC-EVs promoted tight junction recovery of RPE cells in mice with model of retinal degeneration. The immune energized MSC-EVs stem prognosis can inhibit the activation of Stat3 pathway, inhibit the expression of related inflammatory factors, delay the aging of RPE cells and block the process of retinal photoreceptor cell injury. The invention provides experimental basis and theoretical basis for the immune-energized MSC-EVs to treat retina diseases and the potential clinical application of the immune-energized MSC-EVs as an immune regulator, and provides wide application prospect for the immune-energized MSC-EVs to treat retina diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to application of immune energized MSC-EVS in preparation of a medicament for treating retina diseases.
Background
Retinal diseases are refractory blinding eye diseases which seriously damage the visual function of human beings, become increasingly prominent main blinding eye diseases in China, and comprise age-related macular degeneration (AMD), retinitis Pigmentosa (RP), stargardt diseases and the like, and an effective treatment method is not yet clinically available. Dysfunction and loss of retinal pigment epithelial cells (RPE) are common pathological features common to such diseases. In the past, the RPE cells are considered to participate in the pathophysiological process of retina through the actions of nutrition, barrier, phagocytosis and the like, but in the disease process, the RPE cells are aged, and can express immune related receptors such as TLRs, fc-gamma and the like, mediate local immune response and influence cell survival and repair. The search for controlling the aging of RPE cells and promoting the repair of the RPE cells is an important research direction for treating retinal diseases.
The immunomodulatory capacity of mesenchymal stem cells is not constitutive, but is conferred and determined by the type and intensity of inflammation. Under the action of pro-inflammatory factors, the immune regulatory function of mesenchymal stem cells may be enhanced, a process called immune-energization (Primed MSCs). Studies have reported that pretreatment of MSC with IFN-Y and TNF-A may promote MSC immunosuppression viSub>A the JAK1/JAK 2-dependent pathway. Han et al found that IL-17 enhances the immunosuppressive function of MSCs by down regulating AUF1 expression in mesenchymal stem cells, thereby increasing the stability of various immune molecules IL-2, GM-CSF, TNF α, iNOS, and IL-6 mRNA. In addition, IL-1β -energized mesenchymal stem cell Exosomes can inhibit sw982 cell IL-1β and TNF- α mediated inflammatory responses in osteoarthritis.
Interleukin-21, 23, 27 (IL-21, IL-23, IL-27) and TNF- α are a class of pro-inflammatory cytokines that are normally tightly regulated and expressed at low levels, except during infection, trauma or other stress, but are elevated as the immune system ages. These cytokines activate intracellular Stat3 signaling pathway activation, exacerbating the local inflammatory response. However, there is no report on how the extracellular vesicles of mesenchymal stem cells after the immune energization of interleukin-21, 23, 27 (IL-21, IL-23, IL-27) or TNF-alpha can inhibit the immune regulation of retinal cells, whether the retinopathy can be inhibited.
Disclosure of Invention
The invention aims to solve the problems existing in the prior art of retinal disease treatment, can effectively improve the aging state of retinal pigment epithelium and delay the death process of retinal photoreceptor cells by using immune energized MSC-EVS as an active ingredient, has anti-inflammatory and neuroprotective effects, can reduce retinal damage and improve retinal function, and provides a practical theoretical basis for clinical treatment of retinal disease.
In order to solve the technical problems, the invention is realized by the following technical scheme.
In a first aspect, the invention provides the use of mesenchymal stem cell extracellular vesicles (MSC-EVS) which are immunopotentiated by an immune response cytokine in the manufacture of a medicament for the treatment of retinopathy and/or for improving physiological indicators associated with retinopathy.
Preferably, the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF-alpha.
Preferably, the physiological index related to retinopathy is selected from one or more of the group consisting of the number of RPE pigment disorder clusters deposited under retina, the thickness of the outer nuclear layer of retina, the fold condition of the outer nuclear layer of retina, and the level of anti-aging substance expressed by the RPE cells of retina.
Preferably, the retinal RPE cells express one or more anti-aging agents selected from Areg, sfrp2, csf2, spink 3.
Preferably, the mesenchymal stem cell extracellular vesicles which are energized by the immune response cytokine are prepared by the following method: adding one or more immune response cytokines into MSC cells, taking supernatant after intervention treatment, centrifuging and enriching.
Preferably, the MSC cells are selected from one or more of human-derived MSC cells, murine-derived MSC cells.
Preferably, the time of the intervention is 24 hours.
In a second aspect, the invention provides the use of an immune response modulating cytokine in the manufacture of a product for the immunopotentiation of mesenchymal stem extracellular vesicles.
Preferably, the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF-alpha.
In a third aspect, the invention provides a pharmaceutical composition for treating retinopathy and/or improving physiological indicators associated with retinopathy, comprising mesenchymal stem cell extracellular vesicles which are immunopotentiated by an immune response cytokine and a solvent.
Preferably, the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF-alpha.
Preferably, the physiological index related to retinopathy is selected from one or more of the group consisting of the number of RPE pigment disorder clusters deposited under retina, the thickness of the outer nuclear layer of retina, the fold condition of the outer nuclear layer of retina, and the level of anti-aging substance expressed by the RPE cells of retina.
Preferably, the retinal RPE cells express one or more anti-aging agents selected from Areg, sfrp2, csf2, spink 3.
Preferably, the solvent is selected from physiological saline.
Preferably, the concentration of the immunoresponsive cytokine immunopotentiating mesenchymal stem cell extracellular vesicles is 1 μg/μl.
In a fourth aspect, the invention provides a pharmaceutical formulation for the treatment of retinopathy and/or for improving physiological indicators associated with retinopathy, comprising mesenchymal stem extracellular vesicles immunopotentiated by immunocompetent cytokines and pharmaceutically acceptable excipients.
Preferably, the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF-alpha.
Preferably, the physiological index related to retinopathy is selected from one or more of the group consisting of the number of RPE pigment disorder clusters deposited under retina, the thickness of the outer nuclear layer of retina, the fold condition of the outer nuclear layer of retina, and the level of anti-aging substance expressed by the RPE cells of retina.
Preferably, the retinal RPE cells express one or more anti-aging agents selected from Areg, sfrp2, csf2, spink 3.
Preferably, the concentration of the immunoresponsive cytokine immunopotentiating mesenchymal stem cell extracellular vesicles is 1 μg/μl.
Preferably, the dosage form of the pharmaceutical preparation is selected from one or more of solution, sol, emulsion and suspension; most preferably, the dosage form of the pharmaceutical formulation is selected from solutions.
Preferably, the pharmaceutically acceptable auxiliary materials are selected from one or more of solvents, solubilizers, surfactants, bacteriostats, antioxidants, chelating agents and stabilizers.
Compared with the prior art, the invention has the following beneficial effects:
(1) Through a great deal of researches, the invention fully proves the relevant therapeutic effect of the immune-energized MSC-EVS on the retina diseases, and proposes that the immune-energized MSC-EVS has better therapeutic effect than the traditional MSC-EVS. Wherein, immune energized MSC-EVS regulates the aging state of retina RPE cells mainly by inhibiting STAT3 mediated inflammatory pathway activation, so that the amplitude of the Electroretinogram (ERG) waveform is improved, the RPE pigment disorder mass deposited under the retina is reduced, and the vision is improved. The immune energized MSC-EVS has the functions of anti-aging and neuroprotection, increases the thickness of the outer nuclear layer of retina, reduces the damage to retina, improves the function of retina, and opens up a new treatment idea for clinically treating retina diseases.
(2) The invention carries out intensive research on the related mechanism of the immune-energized MSC-EVS in treating retinal diseases, and confirms that the immune-energized MSC-EVS can inhibit STAT3 pathway activation at the downstream of retina RPE cells, reduce the aging level of the retina RPE cells, inhibit retinal inflammatory reaction and provide practical theoretical basis for the subsequent treatment and drug development of retinal related diseases.
Drawings
FIG. 1 is a graph showing the results of a wave change under different light intensity stimuli.
FIG. 2 is a graph showing the results of b wave change under different light intensity stimuli
Figure 3 is a graph showing the effect of immune-energized MSC-EVS on visual conditions.
Fig. 4 is a graph showing the effect of immune-energized MSC-EVS on ONL thickness.
Fig. 5 is a graph showing the effect of immune-energized MSC-EVS on membrane thickness.
Fig. 6 is a graph showing the effect of immune-energized MSC-EVS on retinal structural changes.
FIG. 7 is a schematic representation of the results of immunoenergized MSC-EVS on RPE-scleral complex patch immunofluorescence detection of the status of RPE tight junctions and their expression of STAT3 factor.
FIG. 8 is a graph showing the effect of immune-energized MSC-EVS on the expression of senescence-associated factors at the mRNA level in the RPE-scleral complex.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention more clear and clear, the present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Unless otherwise specified, all of the reagents used in the present invention are commercially available. The experimental methods used in the present invention, such as animal experiments, immunohistochemistry, and the like, are all conventional methods and techniques in the art. For the development of animal experiments, the experimental protocols were approved by the central animal experimental ethics committee of the department of ophthalmic center of the university of Zhongshan and followed the guidelines.
Representative results of selection from the biological experimental replicates are presented in the context figures, and data are presented as mean±sd and mean±sem as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 22.0 software. And comparing the average value difference of two or more groups by adopting conventional medical statistical methods such as t-test, chi-square test, analysis of variance, rank sum test and the like. p < 0.05 was considered a significant difference.
EXAMPLE 1 construction of disease model
Sodium iodate (NaIO) 3 ) Is a stable oxidant, and can increase the conversion of glycine in melanocyte into glyoxylic acid with cytotoxicity compound by means of specific chemical reaction with melanin, thus NaIO 3 RPE cells are selectively damaged. NaIO (NaIO) 3 Induced retinochoroidal lesions exhibit a course of disease and pathological changes similar to those of non-exudative age-related macular lesions (AMD), a classical drug-induced animal model for modeling non-exudative AMD. In non-exudative AMD patients, RPE dysfunction leads to death of the outer nuclear layer photoreceptor cells; similarly, naIO 3 Selective action on RPE cells causes their death, with secondary damage to photoreceptors. Thus the invention adopts NaIO 3 And constructing a related disease model. The method comprises the following specific steps:
(1) Human umbilical cord MSCs or murine bone marrow MSCs are cultured in vitro DMEM/F12+10% FBS+1% P/S medium, after the cell fusion degree is 70-80%, the medium is replaced by DMEM/F12+10% exosome-free FBS+1% P/S, 20ng/mL recombinant human IL-23 cytokine (Preprotective), 25ng/mL recombinant murine IL-21 cytokine (R & D Systems), 10ng/mL recombinant human IL-27 cytokine (Preprotective), or 10ng/mL recombinant human TNF-alpha cytokine (R & D Systems) is added, after 24 hours, the supernatant is collected, the enriched extracellular vesicles are separated and concentrated on the centrifugal tube wall by a differential centrifugation method, and the concentration of 1 mu g/mu L is prepared by using physiological saline, so that an immune-energized MSC-EVS solution is obtained for standby.
(2) C57BL/6J mice (purchased from Jiangsu Jiugang Biotech Co., ltd.) at 8-12 weeks were randomly divided into four groups, which were designated as groups 1-4, 10 each.
(3) Group 1 (blank) was injected with physiological saline at the tail vein, while 1 μl of physiological saline was injected into the vitreous cavity; group 2 (model group) NaIO in tail vein 3 1 mu L of physiological saline is injected into the vitreous cavity; group 3 (treatment group) NaIO was injected into the tail vein 3 1. Mu.L of the immunopotentiated MSC-EVS solution (IL-21 or IL-23 or IL-27 or TNF-. Alpha.) prepared in step (1) was injected into the vitreous cavity. Group 4 (treatment group) NaIO was injected into the tail vein 3 The vitreous cavity was injected with 1. Mu.L of non-immunopotentiated MSC-EVS solution. Tail vein injection of 20mg/kg 0.02% NaIO in group 2-4 3 (injection volume about 0.1-0.2mL, based on mouse body weight), group 1 tail vein was injected with an equal volume of saline.
(4) The same day of tail vein molding is given once to the vitreous cavity for drug injection, and the materials are taken on the 7 th day after molding, so that the treatment effect is evaluated.
Example 2 Electroretinogram (ERG) evaluation of retinal function in mice following immune-energized MSC-EVS treatment
The whole experiment process is carried out under the condition of weak red light illumination. The method comprises the following specific steps:
(1) In example 1NaIO 3 Electroretinogram (ERG) measurements were performed on each of the above groups of mice on day 7 of molding using a electroretinogram (Celeris-Diagnosys system). Mice were dark adapted for 12 hours prior to examination. 4.3% chloral hydrate (10 mL/kg) was injected intraperitoneally for anesthesia, and the compound topiramate eye drops mydriasis.
(2) Mice were fixed on a laboratory plate and electrodes were mounted. A few methylcellulose is dripped on the cornea, a recording electrode (a ring cornea electrode made of 0.2mm copper wire) is clung to the corner consolidated edge, a reference electrode (a needle electrode) is placed under the skin of the same side cheek of the mouse, and a grounding electrode is placed under the skin of the tail of the mouse.
(3) The relevant examination starts after a dark adaptation again of about 5 minutes. Dark adaptation ERG (Scotopic ERG) detection and recording was performed using an international clinical visual electrophysiology standardization protocol. Separately record0.03cd.s·m -2 ，0.1cd.s·m -2 ，0，3cd.s·m -2 ，1.0cd.s·m -2 ，3.0cd.s·m -2 And 10.0 cd.s.m -2 The ERG waveform of the mice under illumination intensity was measured for a standard flash stimulus of 30Hz at the time of strobe response, and the first three response waves were discarded. The application software analyzes and counts the waveforms.
The detection results are shown in FIGS. 1-2. Wherein FIG. 1 shows the change of a wave under different light intensity stimuli; fig. 2 shows a change in b wave. In the figure, the normal saline group is blank group 1, naIO 3 The +normal saline group is the intervention model control group of the mice with retinal degeneration, naIO 3 The + (IL-21/IL-23/IL-27/TNF-alpha) primed-MSC-EVs group is the immune energized MSC-EVS intravitreal administration treatment group of mice with retinal degeneration, naIO 3 The +MSC-EVs group is the traditional MSC-EVs administration treatment group.
The result shows that after dark adaptation for 12 hours, under different illumination intensities, the a-wave and b-wave amplitudes of ERG are counted, and NaIO can be observed 3 The amplitude of the a wave and the b wave of the +normal saline mice is obviously reduced compared with that of the normal saline control group; after IL-21, IL-23, IL-27 or TNF-alpha immune energized MSC-EVs intravitreal injection treatment, a wave and a b wave of ERG are found to be one compared with NaIO under dark adaptation and two different stimulus intensities 3 The statistically significant waveform amplitude increases in the +saline group or the conventional MSC-EVs group, and there is no statistical difference between the four treatment groups, IL-21 or IL-23 or IL-27 or TNF-alpha, suggesting that the immune-energized MSC-EVs are more effective than the conventional MSC-EVs. The immune-energized MSC-EVs of the four factors can improve the retinal nerve function by intravitreal injection, and other functional experiments are carried out by taking the immune-energized MSC-EVs of IL-23 as a representative.
Example 3OMR (Optomotor Response) assessment of mice vision following immune-energized MSC-EVS treatment
(1) In example 1NaIO 3 On day 7 of molding, fundus imaging examinations were performed on each group of mice using a Optokinetic response detection system. The mice remained awake.
(2) After dark adaptation, mice were tested freely on a platform in the center of a box consisting of four identical sized LED screens showing a rotating vertical black and white stripe. The stimulus light source was a sinusoidal grating with a stimulus rotated at 12 degrees/second, and ten spatial frequencies, including 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, and 0.6 were tested in steps.
(3) The movements of the mice were recorded using an infrared camera placed over the chamber to calculate the number of head tracking movements during each spatial frequency.
The detection results are shown in FIG. 3. The results showed that NaIO 3 The visual acuity of the sick mice was significantly reduced, indicating that the more severe the retinopathy, the immune-energized (IL-23) mice had a lower treatment group (primed-MSC-EVs) for MSC-EVs than saline-interfered NaIO 3 Vision of the sick mice is obviously improved, and compared with the traditional MSC-EVs treatment group (NaIO) 3 +msc-EVs) vision is better, indicating a reduction in the degree of retinopathy, p < 0.01, p < 0.001.
Example 4 evaluation of retinal thickness in immunocompetent MSC-EVS treated mice by Optical Coherence Tomography (OCT)
(1) In example 1NaIO 3 On day 7 of molding, OCT measurements were performed on each group of mice using an electroretinogram (SPECTRALIS-OCT, heidelberg, germany). 4.3% chloral hydrate (10 ml/kg) is injected into abdominal cavity before examination for anesthesia, and the compound topiramate eye drops mydriasis;
(2) After anesthesia, the mouse head is fixed on the proper position of the OCT jaw frame, and the bulbar conjunctiva is clamped by forceps to rotate the eyeball position, so that the OCT detection light source is aligned to the laser spot area. The laser spot is scanned by a three-dimensional scanning method set in a computer, and a point 3mm away from the disk nipple is used as a circle center, and 1.5mm is used as a radius for scanning. The scan range covers the four directions of the retina, lateral, longitudinal, nasal, temporal, to calculate the average thickness of the retina.
The detection results are shown in FIGS. 4-5. Wherein FIG. 4 is a photograph showing the Optical Coherence Tomography (OCT) of mouse eyes after MSC-EVS treatment with immune-energized (IL-23), and the dotted line shows the Outer Nuclear Layer (ONL) where photoreceptor cells are located, naIO 3 After molding, photoreceptors were lost and ONL was thickThe ONL thickness is increased after the vitreous cavity is injected with the immune-energized MSC-EVS treatment, and the ONL thickness is obviously increased after the immune-energized MSC-EVS treatment is applied to the whole body. Fig. 5 is a statistical plot of retinal thickness, showing that the structure of the diseased retina is significantly recovered after MSC-EVS treatment by intravitreal injection of immune-energized (IL-23), p < 0.01.
EXAMPLE 5 Paraffin section H & E staining to observe changes in retinal structure in immunocompetent MSC-EVS treated mice
(1) In example 1NaIO 3 On day 7 of molding, each group of mice was sacrificed for cervical dislocation, eyeballs were obtained, and 10% formalin was used for fixation for 18-24 hours.
(2) Dehydrating in gradient ethanol for 12 hours; soaking in xylene for 2 hours, and soaking in paraffin at 65 ℃ for 3-4 hours to carry out permeabilization and paraffin soaking.
(3) The tissue is packed by an automatic embedding machine, and a slicing machine is used for taking 3-4 mu m slices and attaching the slices to a glass slide.
(4) Xylene dewaxing was used, and 100%, 95%, 75% gradient alcohol washing was performed to hydration.
(5) Staining was performed by hematoxylin dip for 10-15 minutes, and washing with running water.
(6) Differentiation is carried out for 1-2 seconds by using 1% hydrochloric acid alcohol, and saturated ammonia water is blued for a plurality of seconds, and the mixture is washed for 15-30 minutes by running water.
(7) Dip-dyeing with 1% alcohol-soluble eosin for 5-15 seconds; conventional dehydration, transparency and sealing.
The detection results are shown in FIG. 6. The results showed that NaIO 3 Fold appears in the retinal ONL layer structure of sick mice, the subretinal RPE pigment arrangement is disordered, the aggregate accumulation is obviously increased, and the therapeutic group of MSC-EVS (NaIO) of vitreous cavity injection immune energized (IL-23) 3 +primed-MSC-EVs) compared to NaIO 3 The ONL layer fold of the +normal saline pathological group is reduced, and the RPE pigment lump is obviously reduced; more traditional MSC-EVs treatment group (NaIO) 3 +MSC-EVs) ONL layer cell density was higher.
Example 6 immunofluorescence staining for detection of the status of RPE tight junctions and STAT3 expression
The RPE-sclera complex is spread and subjected to immunofluorescence staining, and the specific steps are as follows:
(1) In example 1NaIO 3 On the 7 th day of molding, each group of mice was sacrificed for cervical dislocation to obtain eyeballs.
(2) The eyeball was fixed in 4% PFA in an amount of 20 times or more by volume for 60min.
(3) The eyeball is fixed by toothed forceps under an dissecting microscope, the optic nerve is firstly removed, connective tissue and muscle of the bulbar wall are removed, the cornea is sheared off, and tissues such as iris, crystal, retina and the like are removed, so that the complete RPE-sclera complex is obtained.
(4) The RPE-scleral complex was blocked at room temperature for 1 hour or overnight at 4℃with 0.5% Triton X-100/PBS+5% BSA.
(5) Freshly prepared immunofluorescent primary antibody (ZO-1, p-STAT 3): 5% BSA at 1: 100-scale dilution, RPE-scleral complex was placed in antibody, incubated overnight at 4 ℃, during which the EP tube was gently shaken every few hours to fully contact the antibody.
(6) Taking out the RPE-sclera complex, incubating the secondary antibody, and keeping out of light at room temperature for 1-2 hours; PBST was washed 6 times for 10 minutes each.
(7) Transferring the RPE-sclera complex onto a glass slide, removing impurities under an anatomic microscope by using toothless forceps, radially cutting a 4-6 knife with a video disc as a center, dripping a small amount of anti-fluorescence quenching sealing tablet sealing sheet upwards on the RPE layer, and observing and photographing under a fluorescence microscope or a confocal microscope.
The detection results are shown in FIG. 7. The results showed that NaIO 3 After dry prognosis, p-STAT3 is mainly found in NaIO 3 RPE cell surface expression increased after intervention, whereas p-STAT3 expression decreased after intravitreal intervention with primed MSC-EVs. The RPE cells of the normal mice are in a honeycomb hexagonal structure to form a complete tight connection barrier, and NaIO 3 The tight junctions of RPE cells of the lesion group are destroyed, and the tight junctions are restored after the treatment of the primed MSC-EVs (IL-23), which indicates that the primed MSC-EVs can act on the RPE cells through the p-STAT3 and promote the restoration of the barrier function thereof.
EXAMPLE 7 RPE-scleral Complex qPCR detection of mRNA level Gene expression changes
(1) Extraction of mouse RPE-scleral complex RNA: a set of 2 RPE-scleral complexes was placed in 1mL Trizol, vigorously shaken, and the tissues were minced.
(2) 200-300 mu L of chloroform is added, and the mixture is vigorously shaken and kept stand for 10-15 minutes at room temperature.
(3) Centrifuge at 4℃for 30-40 min at 13.2 ten thousand revolutions and carefully transfer 400-500. Mu.L of the supernatant clear supernatant to a new EP tube.
(4) Adding equal volume of isopropanol, shaking vigorously, standing at-20deg.C for more than 2 hr, centrifuging, and discarding supernatant.
(5) 1mL of 70% RNase-free ethanol was added, the pellet was sprung, the ethanol was aspirated after centrifugation again, and the pellet was allowed to air dry in a fume hood.
(6) Add 20-30. Mu.L of DEPC water (depending on the size of the pellet), place on ice and check A260/280 and concentration. Using PrimeScript TM RT reagent Kit cDNA reverse transcription of cDNA is carried out by a reverse transcription kit; then, according to the instructions, a real-time quantitative PCR was performed using SYBERGREEN kit, GAPDH as an internal control, with 2 -ΔΔCt Analysis of the results was performed in which PCR primers are shown in Table 1 below.
TABLE 1 qPCR primers
The detection results are shown in FIG. 8. Areg, sfrp2, csf2 and Spink3 reflect aging. The results show that, in NaIO 3 In the model group of intervention, the expression of senescence-associated genes is increased, and NaIO in the model group is obtained after the treatment of the MSC-EVS with immune-energized (IL-23) 3 The expression of inflammatory factors is reduced, which shows that the immune energized MSC-EVS can effectively improve the aging state of the retinal pigment epithelium and promote the functional recovery of retina.
From the above, experiments such as ERG, OCT, H and E prove that the immune energized MSC-EVS can obviously reduce RPE cell damage of mice with retinal degeneration, protect visual function, delay retinal thinning and achieve better intravitreal injection effect than systemic administration. Immunofluorescence staining proves that the immune energized MSC-EVS can act on the p-STAT3 transcription factor of the retinal pigment epithelial cells, regulate and control the tight connection of the RPE cells of the mice in the retinal degeneration model to obtain recovery. The immune-energized MSC-EVS dry prognosis can reduce the expression level of the RPE scleral complex senescence-associated factors, thereby improving the senescence stress state of the retinal pigment epithelium and delaying the progression of retinal photoreceptor cell death. The invention provides experimental basis and theoretical basis for the immune-energized MSC-EVS to treat retina diseases and the potential clinical application of the immune-energized MSC-EVS as an immune regulator, and the immune-energized MSC-EVS has wide application prospect in preparing a pharmaceutical preparation for treating retina degenerative diseases.
The above detailed description describes the analysis method according to the present invention. It should be noted that the above description is only intended to help those skilled in the art to better understand the method and idea of the present invention, and is not intended to limit the related content. Those skilled in the art may make appropriate adjustments or modifications to the present invention without departing from the principle of the present invention, and such adjustments and modifications should also fall within the scope of the present invention.
Claims (10)
1. Use of mesenchymal stem cell extracellular vesicles immunopotentiated by immunocompetent cytokines in the manufacture of a medicament for treating retinopathy and/or improving physiological indicators related to retinopathy.
2. The use according to claim 1, wherein the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF- α.
3. The use according to claim 1, wherein the physiological index associated with retinopathy is selected from one or more of the group consisting of the number of RPE pigment disorder clusters deposited under the retina, the thickness of the outer nuclear layer of the retina, the condition of the outer nuclear layer folds of the retina, and the level of anti-aging substance expressed by RPE cells of the retina.
4. Use of an immune response cytokine in the preparation of a product for the immunopotentiation of mesenchymal stem extracellular vesicles.
5. The use according to claim 4, wherein the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF- α.
6. A pharmaceutical composition for treating retinopathy and/or improving physiological indicators associated with retinopathy, comprising mesenchymal stem extracellular vesicles that are immunopotentiated by an immune response cytokine and a solvent.
7. The pharmaceutical composition of claim 6, wherein the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF- α.
8. The pharmaceutical composition of claim 6, wherein the solvent is selected from the group consisting of physiological saline.
9. A pharmaceutical formulation for treating retinopathy and/or improving physiological criteria associated with retinopathy, comprising mesenchymal stem extracellular vesicles which are immunopotentiated by immune response cytokines and pharmaceutically acceptable excipients.
10. The pharmaceutical formulation of claim 9, wherein the immune response cytokine is selected from one or more of IL-21, IL-23, IL-27, TNF- α.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310616975.6A CN116650528A (en) | 2023-05-29 | 2023-05-29 | Application of immune energized MSC-EVS in preparation of medicament for treating retina diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310616975.6A CN116650528A (en) | 2023-05-29 | 2023-05-29 | Application of immune energized MSC-EVS in preparation of medicament for treating retina diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116650528A true CN116650528A (en) | 2023-08-29 |
Family
ID=87713138
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310616975.6A Pending CN116650528A (en) | 2023-05-29 | 2023-05-29 | Application of immune energized MSC-EVS in preparation of medicament for treating retina diseases |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116650528A (en) |
-
2023
- 2023-05-29 CN CN202310616975.6A patent/CN116650528A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7032616B2 (en) | Use of gold clusters or substances containing gold clusters in the manufacture of drugs for the prevention and / or treatment of glaucoma | |
| KR20130099926A (en) | Compounds for the treatment/prevention of ocular inflammatory diseases | |
| He et al. | Topical administration of a suppressor of cytokine signaling-1 (SOCS1) mimetic peptide inhibits ocular inflammation and mitigates ocular pathology during mouse uveitis | |
| Barsegian et al. | Corneal neuropathy: an underrated manifestation of diabetes mellitus | |
| Ramos et al. | Safety evaluation of ocular drugs | |
| Wang et al. | αB-Crystallin alleviates endotoxin-induced retinal inflammation and inhibits microglial activation and autophagy | |
| Liu et al. | A novel rat model of ocular hypertension by a single intracameral injection of cross‐linked hyaluronic acid hydrogel (Healaflow®) | |
| Paiva et al. | Licarin A as a novel drug for inflammatory eye diseases | |
| CN113559122A (en) | Eye drops containing exosome and preparation method and application thereof | |
| Engelhardt et al. | Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells | |
| CN110621320A (en) | Treatment of glaucoma | |
| CN116650528A (en) | Application of immune energized MSC-EVS in preparation of medicament for treating retina diseases | |
| CN115531416A (en) | Application of astrocyte exosome and melatonin pretreatment in optic nerve injury treatment | |
| Ahmed | A textbook of ophthalmology | |
| CN116478239A (en) | Active peptide for resisting xerophthalmia and application thereof | |
| Santos-Anderson et al. | A histopathologic study of retinitis pigmentosa | |
| US20180200339A1 (en) | Methods of reducing corneal endothelial cell loss | |
| RU2766083C1 (en) | Biologically active substance in the form of a bioregulatory composition, exhibiting a supportive and restorative effect on tissues and organs, and pharmacological composition | |
| Peresypkina | A comparative evaluation of the efficacy of dimethylaminoethanol derivative 7–16, C7070 and picamilon in correction of experimental hypertensive neuroretinopathy. Research Results in Pharmacology 4 (3): 1–8 | |
| CN107998384A (en) | Alpha1-antitrypsin is applied to the medicine for preparing treatment nervus retrogression illness in eye | |
| Korbmacher et al. | Feasibility of intravitreal injections and ophthalmic safety assessment in marmoset (Callithrix jacchus) monkeys | |
| Ramos et al. | Ocular immunopathology | |
| CN114246938A (en) | Use of IL-4 in the manufacture of a medicament for the treatment of retinal degenerative diseases | |
| Prause et al. | The eye | |
| RU2161335C2 (en) | Method for modeling proliferative vitroretinopathy |
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 |