CN113694076A - Application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis - Google Patents
Application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis Download PDFInfo
- Publication number
- CN113694076A CN113694076A CN202110919356.5A CN202110919356A CN113694076A CN 113694076 A CN113694076 A CN 113694076A CN 202110919356 A CN202110919356 A CN 202110919356A CN 113694076 A CN113694076 A CN 113694076A
- Authority
- CN
- China
- Prior art keywords
- mir
- mirna
- osteoarthritis
- mesenchymal stem
- human mesenchymal
- 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
- 239000002679 microRNA Substances 0.000 title claims abstract description 38
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 36
- 241000282414 Homo sapiens Species 0.000 title claims abstract description 28
- 108091070501 miRNA Proteins 0.000 title claims abstract description 16
- 239000003814 drug Substances 0.000 title claims abstract description 11
- 206010003246 arthritis Diseases 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title description 8
- 201000008482 osteoarthritis Diseases 0.000 claims abstract description 43
- 238000011282 treatment Methods 0.000 claims abstract description 22
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 239000000090 biomarker Substances 0.000 claims description 2
- 238000012377 drug delivery Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 108091091751 miR-17 stem-loop Proteins 0.000 abstract description 55
- 108091044046 miR-17-1 stem-loop Proteins 0.000 abstract description 50
- 108091065423 miR-17-3 stem-loop Proteins 0.000 abstract description 50
- 210000001519 tissue Anatomy 0.000 abstract description 23
- 210000004027 cell Anatomy 0.000 abstract description 22
- 238000000034 method Methods 0.000 abstract description 9
- 102000055008 Matrilin Proteins Human genes 0.000 abstract description 4
- 108010072582 Matrilin Proteins Proteins 0.000 abstract description 4
- 230000001684 chronic effect Effects 0.000 abstract description 3
- 210000000845 cartilage Anatomy 0.000 description 31
- 108700011259 MicroRNAs Proteins 0.000 description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 18
- 210000000629 knee joint Anatomy 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 210000001188 articular cartilage Anatomy 0.000 description 13
- 238000001356 surgical procedure Methods 0.000 description 13
- 239000012188 paraffin wax Substances 0.000 description 12
- 108091007167 extracellular matrix enzymes Proteins 0.000 description 10
- 102000036444 extracellular matrix enzymes Human genes 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 210000000988 bone and bone Anatomy 0.000 description 9
- 230000018044 dehydration Effects 0.000 description 9
- 238000006297 dehydration reaction Methods 0.000 description 9
- 230000006866 deterioration Effects 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 8
- 108020005345 3' Untranslated Regions Proteins 0.000 description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 7
- 101001120822 Homo sapiens Putative microRNA 17 host gene protein Proteins 0.000 description 7
- 102100026055 Putative microRNA 17 host gene protein Human genes 0.000 description 7
- 229960001484 edetic acid Drugs 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 108091005663 ADAMTS5 Proteins 0.000 description 5
- 102000051389 ADAMTS5 Human genes 0.000 description 5
- 102100027995 Collagenase 3 Human genes 0.000 description 5
- 101000577887 Homo sapiens Collagenase 3 Proteins 0.000 description 5
- 101000990915 Homo sapiens Stromelysin-1 Proteins 0.000 description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 5
- 210000001612 chondrocyte Anatomy 0.000 description 5
- 210000004748 cultured cell Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 108091069239 miR-17-2 stem-loop Proteins 0.000 description 5
- 230000008439 repair process Effects 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 4
- 108060001084 Luciferase Proteins 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 4
- 230000008355 cartilage degradation Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 230000004064 dysfunction Effects 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 210000003127 knee Anatomy 0.000 description 4
- 210000003041 ligament Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 101001124991 Homo sapiens Nitric oxide synthase, inducible Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 208000037273 Pathologic Processes Diseases 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000009054 pathological process Effects 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 239000001993 wax Substances 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- 208000006820 Arthralgia Diseases 0.000 description 2
- 238000000116 DAPI staining Methods 0.000 description 2
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 2
- 206010015548 Euthanasia Diseases 0.000 description 2
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 description 2
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- 108010021487 Nitric Oxide Synthase Proteins 0.000 description 2
- 102000008299 Nitric Oxide Synthase Human genes 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 2
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- QOSATHPSBFQAML-UHFFFAOYSA-N hydrogen peroxide;hydrate Chemical compound O.OO QOSATHPSBFQAML-UHFFFAOYSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229960003299 ketamine Drugs 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000003349 osteoarthritic effect Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 229960001603 tamoxifen Drugs 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 210000003954 umbilical cord Anatomy 0.000 description 2
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 2
- 229960001600 xylazine Drugs 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 108010041390 Collagen Type II Proteins 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102100037241 Endoglin Human genes 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100038595 Estrogen receptor Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101000958041 Homo sapiens Musculin Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 206010072970 Meniscus injury Diseases 0.000 description 1
- 108091030146 MiRBase Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 206010057178 Osteoarthropathies Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000037099 Prosthesis Failure Diseases 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 208000026137 Soft tissue injury Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000000981 basic dye Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- DERZBLKQOCDDDZ-JLHYYAGUSA-N cinnarizine Chemical compound C1CN(C(C=2C=CC=CC=2)C=2C=CC=CC=2)CCN1C\C=C\C1=CC=CC=C1 DERZBLKQOCDDDZ-JLHYYAGUSA-N 0.000 description 1
- 229960000876 cinnarizine Drugs 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010864 dual luciferase reporter gene assay Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 210000004394 hip joint Anatomy 0.000 description 1
- 102000046949 human MSC Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 210000005067 joint tissue Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000002278 reconstructive surgery Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 210000004353 tibial menisci Anatomy 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physical Education & Sports Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Developmental Biology & Embryology (AREA)
- Rheumatology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Pathology (AREA)
- Hematology (AREA)
Abstract
The invention provides an application of miRNA in preparing a medicament for treating arthritis, wherein the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1. Also provides a human mesenchymal stem cell expressing the miRNA. Also provides the application of the human mesenchymal stem cells in preparing the medicament for treating arthritis. The invention identifies miR-17-5p capable of repairing cartilage matrix and tissue related to osteoarthritis in a wide-range targeted manner, identifies osteoarthritis treatment capacity of miR-17-5p in a mouse DMM model, identifies the relation between the osteoarthritis severity and miR-17-5p expression level in a clinical sample, simultaneously prepares human mesenchymal stem cells capable of autonomously and continuously expressing miR-17-5p, establishes a miR-17-5p delivery treatment system taking cells as a carrier, and can be used as a treatment method of chronic osteoarthritis.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to a biological small molecule and a cell, in particular to a microRNA (miRNA) for effectively delaying osteoarthritis deterioration and a human mesenchymal stem cell for autonomously and continuously expressing the miRNA.
Background
Osteoarthritis, also known as degenerative osteoarthropathy, is a chronic disease mainly involving damage of articular cartilage and affecting the whole joint tissues, and is clinically manifested as chronic disability caused by joint pain and related dysfunction. Because damaged articular cartilage has no self-repair capability, no obvious and effective method for preventing further deterioration of osteoarthritis exists in the current medicine, and most patients with advanced diseases can only select artificial joint replacement to improve the quality of life.
The structural integrity of articular cartilage is one of the important markers of cartilage health, primarily determined by the extracellular matrix of chondrocytes. Due to the stimulation of external biomechanics and biochemistry, a plurality of matrix degrading enzymes are locally generated, so that the cartilage matrix is gradually degraded, the structural integrity of the cartilage is damaged, an internal calcified layer is continuously abraded, and finally, the generation and progressive deterioration of osteoarthritis are caused.
Mesenchymal Stem Cells (MSCs) have wide sources, can self-renew and differentiate in multiple directions, have strong inflammation regulating capacity and homing capacity, migrate to tissue injury parts in a targeted manner, and secrete factors, exosomes and the like to regulate microenvironment; or the tissue cells are differentiated into specific types of tissue cells to carry out cell replacement, so that the repair of the damaged part is promoted, and the tissue cells have a wide application prospect in the treatment of various clinical diseases. However, MSCs show great heterogeneity due to differences in their donor sources, tissue sources, isolation methods and culture systems, and thus proper in vitro pretreatment-induced culture can modulate and improve the disease-specific therapeutic capabilities of MSCs. Among them, the MSCs derived from fat, skin and cord blood become ideal donors and tissue sources for MSCs culture due to easy acquisition, low tumor formation rate, high cost performance, little harm to patients and no ethical issues.
mirnas are small endogenous RNAs of about 20-24 nucleotides in length, and have a variety of important regulatory roles in vivo. The presently discovered mirnas can delay part of the pathological process of osteoarthritis through various mechanisms including regulation of target mRNA stability, endogenous inhibitory RNA pathway, etc., but are not satisfactory in terms of effective repair of chondrocytes and their matrix. Researches find that the skeletal dysfunction caused by miR-17-92 cluster gene dysfunction is accompanied with the shortening of bone units and the fusion of bone joints, and suggest that the miR-17-92 cluster gene can be a potential joint disease target. Meanwhile, the discovered miRNAs can only generate unidirectional targeting effect on specific single matrix degrading enzymes, and the discovery and identification of the miRNA which can widely target osteoarthritis-related matrix degrading enzymes are helpful for developing molecular therapeutic measures for effectively repairing osteoarthritis lesions.
At present, gradient treatment measures are clinically adopted for treating osteoarthritis, and combination treatment of staged physics, medicines and joint cavity repair surgery is adopted, and the measures can only slightly delay the development of osteoarthritis; when the disease worsens to an advanced stage, joint reconstructive surgery treatments, including expensive and complex joint replacement procedures, are required to relieve joint pain and restore joint mobility. With the development of joint replacement, more than 90% of cases have no advanced stage for more than 10 years after operation, but infection around the prosthesis at the early stage after operation and prosthesis loosening at the long stage after operation both need to be repaired again after operation, thus extremely reducing the life quality of patients. Therefore, a reversible therapeutic drug molecule or combined cell therapeutic measure with simple administration route, small wound and low operation difficulty is urgently needed to inhibit the progress of osteoarthritis.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a microRNA (miRNA) and a human mesenchymal stem cell for effectively delaying osteoarthritis deterioration, and the miRNA and the human mesenchymal stem cell for effectively delaying osteoarthritis deterioration aim at solving the technical problem that the osteoarthritis treatment effect in the prior art is poor.
The invention provides application of miRNA (miR-17-5p) in preparation of a medicament for treating arthritis, wherein the nucleotide sequence of the miRNA is caaagugcuuacagugcagguag (shown as SEQ ID NO. 1).
The invention also provides a human mesenchymal stem cell for expressing miRNA, wherein the nucleotide sequence of the miRNA is caaagugcuuacagugcagguag (shown as SEQ ID NO. 1).
The invention also provides application of the human Mesenchymal Stem Cells (MSCs) in preparing a medicament for treating arthritis.
The invention also provides application of the miRNA in preparing a biomarker for diagnosing osteoarthritis, wherein the nucleotide sequence of the miRNA is caaagugcuuacagugcagguag (shown as SEQ ID NO. 1).
Specifically, the human mesenchymal stem cells are derived from human fat, skin or umbilical cord.
The invention also provides a drug delivery system, which takes the mesenchymal stem cells as a vector, wherein the vector autonomously and continuously expresses miRNA, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.
The invention provides a miRNA for effectively relieving osteoarthritis progression, and the connection between the level and osteoarthritis, the action and the treatment mechanism are demonstrated through an osteoarthritis (DMM) mouse model of medial meniscus injury; and has identified a link between the extent of cartilage tissue pathology in osteoarthritis patients and the levels of miRNA expression provided.
The miRNA provided by the invention and the human mesenchymal stem cell capable of effectively expressing and secreting the miRNA can be used for the following purposes, such as: can be used for treating osteoarthritis of different stages.
The principle that the miR-17-5p delays osteoarthritis deterioration and repairs the cartilage extracellular matrix of the bone joint comprises the following steps: the method has the advantages that four matrix degrading enzymes capable of destroying the articular cartilage extracellular matrix are subjected to wide targeted inhibition, namely matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5 and nitric oxide synthase NOS2, and targeted inhibition is performed through recognition sites of 3' UTRs of the four matrix degrading enzymes (shown in figure 1); and maintaining the balance of anabolism and catabolism in the extracellular matrix of chondrocytes, and maintaining the structural integrity of articular cartilage.
The invention discloses a MSCs delivery system capable of autonomously and continuously expressing miR-17-5p, which is realized according to the following principle: intravenous or in situ infusion of MSCs can provide some disease relief; the Agomir is double-stranded small RNA which is modified by cholesterol at the 3' end, two thio frameworks at the 5' end and four full-strand methoxy groups at the 3' end according to a mature miRNA antisense chain, has higher affinity with a cell membrane, has expression time of up to one week after being transfected into cells, and has biosafety.
Compared with the prior art, the invention has remarkable technical progress. The invention identifies miR-17-5p capable of repairing cartilage matrix and tissue related to osteoarticular in a wide-target mode, identifies osteoarthritis treatment capacity of miR-17-5p in a mouse DMM model, identifies the relation between the osteoarthritis severity and miR-17-5p expression level in a clinical sample, simultaneously prepares human MSCs capable of autonomously and continuously expressing miR-17-5p, establishes a miR-17-5p delivery treatment system with cells as a carrier, can be used as a treatment method of chronic osteoarthritis, and has important clinical significance.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 shows that miR-17-5p targets the inhibition of four matrix degrading enzymes through the 3' UTR region.
FIG. 2 shows that the mouse DMM model is successfully constructed, and the expression level of miR-17-5p is closely related to the deterioration degree of cartilage degradation and osteoarthritis.
FIG. 3 shows that both DMM surgery and miR-17-92 cluster gene knockout can lead to increased osteoarthritis score, and the score is reduced after Agomir-17 injection, which indicates that miR-17-5p can inhibit osteoarthritis pathological process.
FIG. 4 shows that the expression level of miR-17-5p in normal human articular cartilage is far higher than that of osteoarthritis patients, about 2 times in early stage and 4 times in middle and late stages, and the similarity of the expression pattern and mechanism of miR-17-5p in human cartilage and mouse cartilage is suggested.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text. It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
1. Test object
The C57BL/6 mouse is purchased from Shanghai Weitonglihua laboratory animal technology Limited company, miR-17-92fl/flMouse and Col2CreERTMice were purchased from Jackson laboratories and housed in the university of congruence laboratory animal center SPF grade environment. The internal operation training of the experimental animal center of college university has been performed in the experiment involving the operation, surgery and euthanization of the animals.
2. Experimental methods
The following cell experiment procedures were all completed in GMP laboratories in compliance with clinical standards; animal experiments were all done in an SPF environment.
2.1 miR-17-5p can be widely targeted to articular cartilage related matrix degrading enzymes
2.1.1 MiRanda database (http:// www.microrna.org /) provides the prediction information about miRNA target genes of human, Drosophila and zebrafish genomes and the expression profiles of miRNA in different tissues, and TargetScan (http:// www.targetscan.org /) predicts target genes by searching for mRNA sites matching each miRNA region; articular cartilage related matrix degrading enzymes of mRNA 3' UTR containing miR-17-5p recognition sites, including matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5, nitric oxide synthase NOS2, are predicted by miRanda and TargetScan, and the binding sites are shown in figure 1A, and specific site mutation (mutation) is carried out according to the binding sites; the wt 3'UTR sequence and the mutant 3' UTR are synthesized by biological engineering Limited company and loaded into a fluorescent reporter gene vector psiCHECK-2, and the vector is purchased from Promega company and can monitor the change of the expression of a target gene fused with a reporter gene (Renilla luciferase);
caaagugcuuacagugcagguag (shown as SEQ ID NO. 1) provided by the invention can be prepared from miRBase (A), (B), (C) and C)http://www.mirbase.org/) And (6) database query acquisition.
2.1.2 luciferase reporter experiments were performed on HEK293T cells (purchased from ATCC, CRL-11268), HEK293T cells were seeded in 96-well plates and cultured, the transfection reagent Lipofectamine 2000 was purchased from Invitrogen, transfection procedures were performed according to the instructions, 10pmol of miR-17-5p and 10ng of 3' UTR-psiCHECK-2 were transfected per well, cells were collected after 48 hours of transfection, and luciferase activity was assayed according to the instructions of the Dual luciferase reporter assay kit (purchased from Promega). As shown in FIG. 1B, miR-17-5p can significantly reduce the luciferase activity of wt 3'UTR, but cannot reduce the luciferase activity of mutant 3' UTR, and similar level changes are observed in matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5, and nitric oxide synthase NOS 2. Therefore, the miR-17-5p has wide matrix degrading enzyme targeting property, and simultaneously targets and regulates matrix metallopeptidase MMP3 and MMP13, chondroprotein ADAMTS5 and nitric oxide synthase NOS 2.
2.2 in the constructed mouse DMM model, the expression level of miR-17-5p is related to the development degree of osteoarthritis
2.2.1 mice of 10 weeks of age were anesthetized with ketamine and xylazine and subjected to a surgical procedure that transects the medial meniscal ligament of the left knee, destroying the cartilage of the left knee and inducing osteoarthritis associated with meniscal instability. The mice in the control group were only subjected to sham surgery treatment without transecting the medial meniscal ligament of the left knee joint;
2.2.2 two groups of mice are euthanized at 0, 4, 8 and 12 weeks after the operation, the left knee joint is obtained and stained with safranin O-fast green and Hematoxylin Eosin (HE), and the expression of microRNA-17 is detected by Fluorescence In Situ Hybridization (FISH);
the method comprises the following specific steps:
2.2.2.1 tissue fixation and Paraffin embedding
After collecting the left knee joint of the mouse, fixing the left knee joint for 48 hours by paraformaldehyde with the mass-volume ratio concentration (g/ml) of 4 percent, then decalcifying the left knee joint for 10 days by an Ethylene Diamine Tetraacetic Acid (EDTA) solution with the mass-volume ratio concentration (g/ml) of 10 percent, and placing the trimmed tissue and the corresponding label in a dehydration box;
putting the dehydration box into a dehydrator, and dehydrating with gradient alcohol (volume percentage concentration) for 90 minutes, 85 minutes, 90 minutes, 60 minutes and 60 minutes respectively by using 50 percent alcohol and 70 percent alcohol;
and (3) transparency: 5-10 minutes of alcohol benzene, 5-10 minutes of xylene I and 5-10 minutes of xylene II;
paraffin penetration: benzene wax 90 minutes, paraffin I60 minutes, paraffin II 60 minutes, and paraffin III 60 minutes;
embedding: embedding the left knee joint tissue permeated with the paraffin in an embedding machine, taking out the wax block from the embedding box after the paraffin is solidified, and finishing the wax block;
slicing: slicing the cartilage tissue on a paraffin slicer, slicing the cartilage tissue on a sagittal plane or a coronal plane with the thickness of 6 mu m, and storing the cartilage tissue at room temperature for later use after baking;
2.2.2.2 safranin O-fast Green staining
Basophilic cartilage tissue is combined with basic dye safranin O to be red, while acidophilic bone is combined with acid dye fast green to form green or blue, when cartilage is damaged, glycoprotein in the cartilage can be released, so that matrix components are unevenly distributed, and accordingly, the safranin O is slightly or not stained.
Dewaxing the paraffin section to distilled water conventionally, preparing Weigart dye liquor according to the conventional freshness, and dyeing for 3-5 minutes;
differentiation was carried out for 15 seconds by a hydrochloric acid-ethanol solution, followed by light washing with distilled water for 1 minute;
dyeing with a fast green dye for 5 minutes, and then washing with distilled water for 1 minute;
dip dyeing with safranin o (safranin o) dye for 2 minutes followed by light washing with distilled water for 1 minute;
the sections were washed gently with acetic acid solution for 1 minute to further remove residual dye, and then washed gently with distilled water for 1 minute;
dehydrating the mixture by ethanol with the volume percentage concentration of 95% and absolute ethanol in sequence, then performing xylene transparency, and sealing the mixture by using optical resin for subsequent observation and evaluation;
2.2.2.3 HE staining
Dewaxing the paraffin sections to distilled water conventionally, and preparing hematoxylin and eosin ethanol dyes according to the conventional method;
staining with hematoxylin for 10-30 min, and slightly washing with tap water to turn blue;
carrying out differentiation for about 15 seconds by using a hydrochloric acid-ethanol solution until the slices become light red, and then slightly washing by using tap water flow to recover the color to blue;
and (3) dehydration I: sequentially dehydrating the mixture by using 50 percent ethanol by volume, 70 percent ethanol by volume and 80 percent ethanol by volume for 3 to 5 minutes respectively;
re-dyeing for 1-3 minutes by using an eosin ethanol dye with the mass-volume ratio concentration (g/ml) of 0.5 percent;
and (II) dehydration: dehydrating the mixture by ethanol with the volume percentage concentration of 95% and absolute ethanol for 3-5 minutes respectively, carrying out dimethylbenzene transparentization twice after air drying, and sealing the mixture by using optical resin for subsequent observation and evaluation;
2.2.2.4 miR-17-5p-FISH detection
FISH of miR-17-5p is performed according to the international standard protocol. Digoxigenin (DIG) -labeled miR-17-5p oligonucleotide probes were purchased from Qiagen for labeling miR-17-5p molecules in tissue sections; DAPI staining was used to visualize the nuclei.
In brief, after collecting the left knee joint of the mouse, fixing the left knee joint by using 4% paraformaldehyde with mass-volume ratio concentration (g/ml), decalcifying the left knee joint by using 10% EDTA solution with mass-volume ratio concentration (g/ml), immersing the left knee joint in sucrose solution for gradient dehydration, embedding and freezing the left knee joint in a compound (OCT) with the optimal cutting temperature, and slicing the left knee joint in a thickness of 10 mu m; sections were permeabilized with proteinase K for 5 minutes at 37 ℃ and treated with 3% hydrogen peroxide water by volume concentration to block endogenous peroxidase activity; slides bearing frozen sections of cartilage tissue were then incubated overnight with the relevant probe (1 nM); finally, carrying out color development by using digoxin-peroxidase resistance (DIG-POD resistance) and Cy 3-tyramide (dissolved in hydrogen peroxide water with the volume percentage concentration of 0.001%), carrying out DAPI staining for cell nucleus color development, and further observing the statistical result;
2.2.3 introduction of the OARSI score of the International Association for osteoarthritis, scored by two observers to assess the extent of cartilage destruction, with higher scores evidencing greater severity of destruction;
2.2.4 As shown in FIG. 2A, with safranin O-fast green staining, the DMM mouse model is increased with time, and the distribution of cartilage tissues deeply stained on the surface of the bone joint is disordered and even faulted, which shows that the integrity of the cartilage tissues in the bone joint is progressively destroyed in the model; as shown in fig. 2A, loss of tissue integrity and continuity of the articular cavity surface was also observed by HE staining, indicating that the degree of articular cartilage degradation in mice progressively worsened after DMM surgery; FISH detection shows that the miR-17-5p expression quantity is remarkably reduced along with progressive worsening of the mouse articular cartilage degradation degree; as shown in fig. 2B, the OARSI score of mice in the operative group was about 2-fold that of the control group at week 4 after the operation, and stabilized at weeks 8 and 12, which was about 5-fold that of the control group; the number of miR-17-5p positive cells on the articular surface of mice in the operation group is about one half of that in the control group at the 4 th week after the operation, and the cells tend to be stable at the 8 th and 12 th weeks and are about one quarter of that in the control group. The legend and the experimental results prove that the mouse DMM model is successfully constructed, and the expression level of miR-17-5p is closely related to the deterioration degree of cartilage degradation and osteoarthritis.
2.3 miR-17-5p can inhibit osteoarthritis pathological process
2.3.1 miR-17-5p analogue can effectively repair bone joint soft tissue injury and dysfunction in mouse DMM model
2.3.1.1 mice of 10 weeks of age were anesthetized with ketamine and xylazine and subjected to surgery to transect the medial meniscal ligament of the left knee, destroying the cartilage of the left knee and inducing osteoarthritis associated with meniscal instability. The mice in the control group were only subjected to sham surgery treatment without transecting the medial meniscal ligament of the left knee joint;
2.3.1.2 Agomir-17 (1.5 nmol) was dissolved in 8. mu.l of phosphate buffer and injected into the mouse joint cavity 1 time a week for 4 times starting 4 weeks after DMM surgery; injecting Agomir-NC into mice in a control group;
2.3.1.3 at 8 weeks after DMM surgery, the left knee joints of three groups of mice (sham surgery group, surgery and Agomir-NC injection group, surgery and Agomir-17 injection group) were collected after euthanasia, fixed for 48 hours after 4% paraformaldehyde in mass-to-volume concentration (g/ml), then decalcified for 10 days with 10% Ethylene Diamine Tetraacetic Acid (EDTA) solution in mass-to-volume concentration (g/ml), the trimmed tissues and corresponding labels were placed in a dehydration box, embedded with paraffin after dehydration, further sliced and dewaxed to distilled water, and the structural integrity of articular cavity cartilage tissues was examined by safranin O-fast green staining, the operation was the same as 2.2.2.1-2 in example 1;
2.3.1.4 As shown in figure 3A, in the mouse DMM model, after 4 weeks of joint cavity injection treatment of miR-17-5p analogue Agomir-17, the articular cartilage layer structure is complete, while the cartilage tissue in the Agomir-NC treatment group is unevenly distributed and discontinuous, and the integrity is destroyed; accordingly, the OARSI score for the Agomir-17 treatment group was significantly lower than for the Agomir-NC treatment group, tending towards the sham group;
2.3.2 knocking out miR 17-92 cluster gene leads to pathological changes similar to osteoarthritis in joint cavity, and miR-17-5p analogue can effectively reverse corresponding pathological changes
2.3.2.1 miR-17～92fl/flMouse and Col2CreERTMouse hybridization to obtain Col2CreERT；miR-17～92fl/+Mice, and in turn Col2CreERT；miR-17～92fl/+Mice and miR-17-92fl/flMouse is hybridized to obtain Col2CreERT；miR-17～92fl/flIn mice (miR-17-92 cKO mice), miR-17-92 cluster genes in chondrocytes and other cells which usually express endogenous type II collagen genes in miR-17-92 cKO mice can be inductively knocked out;
2.3.2.2 activator tamoxifen of estrogen receptor ER is purchased from Sigma company, 7-week-old miR-17-92 cKO mice receive tamoxifen with a dose of 100 mug/g through intraperitoneal injection, the dose is once a day for 6 days, and miR-17-5p is inductively knocked out;
2.3.2.3 Agomir-17 (1.5 nmol) was dissolved in 8. mu.l of phosphate buffer, and 1 Agomir-17 dilution was injected into the mouse joint cavity 4 times a week from 4 weeks after induction knockout; the control group mice are injected with Agomir-NC, and the blank control group is miR-17-92fl/flA mouse;
2.3.2.4 at 8 weeks after induction knockout, collecting the left knee joints of three groups of mice (blank control group, induction and injection of Agomir-NC group, induction and injection of Agomir-17 group) after euthanasia, fixing for 48 hours after 4% paraformaldehyde with mass-volume ratio concentration (g/ml), decalcifying for 10 days with 10% Ethylene Diamine Tetraacetic Acid (EDTA) solution with mass-volume ratio concentration (g/ml), placing the trimmed tissues and corresponding labels in a dehydration box, embedding paraffin after dehydration, further slicing and dewaxing into distilled water, and checking the structural integrity of articular cavity cartilage tissues by safranin O-fast green staining, which is the same as 2.2.2.1-2 in example 1;
2.3.2.5 As shown in figure 3B, after miR-17-5p is knocked out, joint cavity injection treatment of miR-17-5p analogue Agomir-17 is carried out for 4 weeks, the structure of the articular cartilage layer is complete, and cartilage tissues in an Agomir-NC treatment group are unevenly and discontinuously distributed and the integrity is damaged; correspondingly, the OARSI score of the Agomir-17 treated group was significantly lower than that of the Agomir-NC treated group, tending toward the blank control group;
in conclusion, the expression level of miR-17-5p is highly related to the pathological deterioration degree of the bone joint in a mouse DMM model, and miR-17-5p can widely target 4 matrix degrading enzymes related to the bone joint cartilage matrix, including matrix metallopeptidase MMP3 and MMP13, cartilage proteoglycan ADAMTS5 and nitric oxide synthase NOS2, and maintain the structural integrity and the function of the joint cartilage tissue.
Example 2
1. Test object
Non-arthritic normal knee joint or hip joint cartilage tissue was surgically obtained from 6 osteosarcoma or trauma patients (1 male and 5 female, median age 53 years), and osteoarthritic cartilage specimens were obtained from 18 osteoarthritic patients (4 male and 14 female, median age 66 years) within 2 hours after joint reconstruction, and patients were graded and diagnosed according to the osteoarthritis diagnostic criteria of the american college of rheumatology. The collection of waste human tissue was approved by the research ethics committee of the affiliated hospital of xu medical university, and all patients participating in the study provided written informed consent.
2. Experimental methods
2.1 extraction of total RNA from human cartilage tissue and specific reverse transcription
The collected human cartilage Tissue was disrupted to homogenate by Tissue lyser-48L (purchased from kyoto new biomedical limited, shanghai), total RNA extraction was performed according to RNeasy fibers Tissue Mini Kit (purchased from Qiagen), then 0.5 μ g of total RNA was used for reverse transcription to synthesize cDNA library, and the reaction system was configured according to the reverse transcription Kit instructions of Takara in combination with the designed miR-17-5p specific stem-loop primers, substantially as follows:
| components | Volume of |
| 5×PrimeScript buffer | 4μl |
| PrimeScript RT Enzyme Mix I | 1μl |
| Stem loop primer | 1μl |
| Total RNA | 0.5μg |
| RNase Free dH2O | Up to 20μl |
Wherein the stem-loop Primer sequences are as follows (designed using software Primer Premier 5.0):
5'-gtcgtatccagtgcagggtccgaggtattcgcactggatacgacctacct-3', respectively; (as shown in SEQ ID NO. 2)
After fully mixing, centrifuging for a short time, placing on a PCR instrument, and running a program, wherein the reverse transcription program comprises the following steps:
15 minutes at 37 ℃; 5 seconds at 85 ℃; infinity at 4 ℃;
the reverse transcribed cDNA products were immediately used for Quantitative RT-PCR (qRT-PCR) detection or stored at-20 ℃;
2.2 detection of miR-17-5p relative expression level by human cartilage tissue qRT-PCR
A reaction system with a total volume of 20. mu.l was prepared according to the SYBR Green mix qRT-PCR kit from TaKaRa, all primers were diluted to 10. mu.M, and the cDNA library synthesized in example 2 at 2.1 was diluted at a ratio of 1:10, as follows:
| components | Volume of addition |
| cDNA | 1μl |
| 2×SYBR Green qPCR Super Mix | 10μl |
| Upstream primer (10. mu.M) | 0.5μl |
| Upstream primer (10. mu.M) | 0.5μl |
| ROX | 0.4μl |
| RNase Free dH2O | 7.6μl |
Wherein the upstream and downstream Primer sequences are respectively (designed by software Primer Premier 5.0):
an upstream primer: 5'-cgcaaagtgcttacagtgc-3', respectively; (as shown in SEQ ID NO. 3)
A downstream primer: 5'-agtgcagggtccgaggtatt-3' are provided. (as shown in SEQ ID NO. 4)
After loading, centrifugation was carried out at 2000rpm for 1 minute, and the program was run in an ABI 7500 Fast System (Applied Biosystems) apparatus:
10 minutes at 95 ℃ (15 seconds at 95 ℃, 30 seconds at 56 ℃, 30 seconds at 72 ℃) multiplied by 40 cycles, and a dissolution curve is added to analyze the specificity of the primer;
2.3 data analysis
After the amplification of the qRT-PCR instrument is finished, observing a dissolution curve and copying a CT value, and according to a fluorescent quantitative PCR relative quantitative formula 2-△△CtSample fold expression calculation was performed, and the results were analyzed using SPSS software for Student's t-test, which is considered to be when P<0.05 has statistical significance.
2.4 As shown in figure 4, the expression level of miR-17-5p in normal human articular cartilage is far higher than that of osteoarthritis patients, about 2 times of the early stage and 4 times of the middle and late stages, which suggests that the expression patterns of miR-17-5p in human cartilage and mouse cartilage are similar. These findings indicate that miR-17-5p can be used for clinical osteoarthritis treatment.
Example 3
The following cellular experimental procedures were all performed in a GMP laboratory in compliance with clinical standards.
1. The safety of the single miR-17-5p micromolecule injection or miR-17-5p analogue Agomir-17 injection in the joint cavity is difficult to control, and the side effect is difficult to evaluate; clinical trials on the treatment of the MSCs are carried out in various diseases, and the safety, reliability and effectiveness are evaluated to a certain extent; agomir-17 is pretreated by MSCs to obtain miR-17-5p autonomous and continuous expression capacity, and miR-17-5p combined cell therapy is established by taking the MSCs as a carrier, so that the method is a safer and more effective potential strategy for clinically treating osteoarthritis.
2. Preparation of MSCs (messenger ribonucleic acids) and preparation of MSCs for autonomously and continuously expressing miR-17
2.1 preparation and quality control of MSCs
2.1.1 human umbilical cord tissue samples are placed in physiological saline buffer containing antibiotics, transported to GMP cells on ice, washed with physiological saline buffer containing antibiotics, transferred to a clean petri dish, stripped to remove vascular tissue, the Fahrenheit gel fraction is taken, washed and cut into about 1mm3Adding collagenase with the mass-volume ratio concentration (g/ml) of 0.2% into the tissue blocks, transferring the tissue blocks into a 15ml centrifuge tube, and putting the centrifuge tube into a water bath kettle at 37 ℃ for digestion for 1 hour, wherein similarly, the tissue sources such as human fat, skin and the like are also suitable;
2.1.2 preparing an alpha-MEM complete medium containing 5% by volume of KSR (human serum replacement), 0.032% by volume of sodium heparin, terminating the digestion, followed by filtering out the undigested tissue mass with a 70 μm cell sieve and centrifuging for 5 minutes at 1200rpm, the centrifuge setting parameter;
2.1.3 washing with complete Medium 2 times, after counting according to 5X 106Cell density per ml plated at 37 ℃ in 5% CO2Culturing in a humid incubator; removing non-adherent cells when the cells grow to reach the confluence degree of about 80%, and carrying out passage according to a ratio of 1:3 after digestion;
2.1.4 performing mycoplasma pollution detection, and determining that the cultured cells are not polluted by mycoplasma;
2.1.5 the cultured cells can grow adherent to the wall through visual observation; the cultured cells have the capacity of differentiating into fat cells, chondroblasts and osteoblasts through a three-line differentiation test; the cultured cells expressed positive cell surface markers CD44, CD73, CD90 and CD105 on the surface and did not express negative cell surface markers CD45, CD19, CD11b, CD34 and HLA-DR by flow identification of the cells. The three points prove that the cultured cells are MSCs;
2.2 preparation of MSCs containing miR-17-5p expression modules
2.2.1 synthetic Agomir-17 from RiboBio (RiboBio) science and technology, Inc., Guangzhou;
2.2.2 MSCs cultured in step 2.1 of example 3 were cultured to a density of 70% and the KSR halved medium was replaced 1 hour before transfection;
2.2.3 will
HD (purchased from Promega corporation) reagent and Agomir-17 solution were equilibrated to room temperature;
2.2.41.5 nmol of Agomir-17 was dissolved in 50. mu.l of RNase-free ultrapure water and stored at a concentration of 30. mu.M, and 1. mu.l was diluted in 49. mu.l of Opti-MEM medium at the time of transfection to a final concentration of 600 nM; get
HD 0.1μl/cm2Mixing with the Agomir-17 diluent, incubating at room temperature for 5-15min, and adding into cell culture system;
2.2.5, continuing to culture for 6-16h after transfection, and replacing the culture medium with a complete culture medium to obtain the successfully prepared MSCs containing the miR-17-5p expression components.
Sequence listing
<110> cinnarizine
<120> application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
caaagugcuu acagugcagg uag 23
<210> 2
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacctacct 50
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cgcaaagtgc ttacagtgc 19
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Claims (5)
1. An application of miRNA in preparing a medicine for treating arthritis, wherein the nucleotide sequence of miRNA is shown in SEQ ID NO. 1.
2. A human mesenchymal stem cell expressing miRNA, wherein the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.
3. Use of the human mesenchymal stem cells of claim 2 in the manufacture of a medicament for the treatment of arthritis.
4. The application of miRNA in preparing biomarker for diagnosing osteoarthritis is disclosed, wherein the nucleotide sequence of miRNA is shown in SEQ ID NO. 1.
5. A drug delivery system is characterized in that mesenchymal stem cells are used as a vector, miRNA is expressed in the vector, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110919356.5A CN113694076A (en) | 2021-08-11 | 2021-08-11 | Application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110919356.5A CN113694076A (en) | 2021-08-11 | 2021-08-11 | Application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113694076A true CN113694076A (en) | 2021-11-26 |
Family
ID=78652254
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110919356.5A Pending CN113694076A (en) | 2021-08-11 | 2021-08-11 | Application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113694076A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114984225A (en) * | 2022-06-22 | 2022-09-02 | 中山大学附属第八医院（深圳福田） | Application of hsa _ circ _0005773 in preventing and treating mesenchymal stem cell aging and osteoarthritis |
-
2021
- 2021-08-11 CN CN202110919356.5A patent/CN113694076A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| JIE JIA; ET AL.: ""MiR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in non-traumatic osteonecrosis"", 《EXPERIMENTAL & MOLECULAR MEDICINE》 * |
| YONG LI;ET AL.: ""miR-17-5p and miR-19b-3p prevent osteoarthritis progression by targeting EZH2"", 《EXPERIMENTAL AND THERAPEUTIC MEDICINE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114984225A (en) * | 2022-06-22 | 2022-09-02 | 中山大学附属第八医院（深圳福田） | Application of hsa _ circ _0005773 in preventing and treating mesenchymal stem cell aging and osteoarthritis |
| CN114984225B (en) * | 2022-06-22 | 2024-02-02 | 中山大学附属第八医院（深圳福田） | Application of hsa_circ_0005773 in prevention and treatment of mesenchymal stem cell aging and osteoarthritis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cherubini et al. | FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition | |
| CN102421917B (en) | Means and methods for counteracting, preventing and/or determining heart failure, or a risk of heart failure | |
| DE102007052114B4 (en) | Method for modulating the function, growth or differentiation of a cell | |
| Zhong et al. | MicroRNA-337 is associated with chondrogenesis through regulating TGFBR2 expression | |
| JP6157852B2 (en) | Use of microRNA to treat diseases associated with ciliary dysfunction in multicilia epithelial cells | |
| WO2009023525A2 (en) | Methods of modulating mesenchymal stem cell differentiation | |
| Xia et al. | Altered function in cartilage derived mesenchymal stem cell leads to OA-related cartilage erosion | |
| US20230355652A1 (en) | Fetal stromal cell derived exosomes for tissue repair | |
| Wang et al. | Small RNA sequencing reveals a novel tsRNA‐06018 playing an important role during adipogenic differentiation of hMSCs | |
| Crobu et al. | Differentiation of single cell derived human mesenchymal stem cells into cells with a neuronal phenotype: RNA and microRNA expression profile | |
| Huang et al. | MiR-26b regulates cartilage differentiation of bone marrow mesenchymal stem cells in rats through the Wnt/β-catenin signaling pathway. | |
| CN113694076A (en) | Application of miRNA and human mesenchymal stem cells in preparation of medicine for treating arthritis | |
| Greene et al. | miRNA transcriptome and myofiber characteristics of lamb skeletal muscle during hypertrophic growth1 | |
| WO2008053909A1 (en) | Agent for controlling growth and/or differentiation of mesenchymal stem cell | |
| CN106350519A (en) | MicroRNA for adjusting rapid growth of cornua cervi pantotrichum cartilage and application thereof | |
| CN104862316A (en) | MiRNA-2400 with biological function of significantly promoting bovine preadipocyte proliferation | |
| WO2015088162A1 (en) | Composition for promoting differentiation from stem cell to chondrocyte | |
| EP3704228B1 (en) | Skeletal muscle differentiation of mesodermal ipsc derived progenitors | |
| JP2022545376A (en) | Method for in vitro production of hyaline cartilage tissue | |
| CN111560437A (en) | Biomarkers for predicting oral squamous carcinoma and their use in therapy | |
| Zhao et al. | MiR-181a regulates the chondrogenic differentiation in pig peripheral blood mesenchymal stem cells | |
| CN111944751B (en) | Abca4 gene related to stem cell proliferation and application thereof | |
| CN108300763A (en) | A method of screening miR-101-3P target genes | |
| Bardell | microRNA: target interactions in tendinopathy | |
| He | A Novel Role for microRNA-214 in Inflammatory Smooth Muscle Cell Differentiation from Adventitia Stem/progenitor Cells and Arterial Remodelling |
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: 20211126 |
|
| RJ01 | Rejection of invention patent application after publication |