CN114432332A - circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 - Google Patents
circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 Download PDFInfo
- Publication number
- CN114432332A CN114432332A CN202210175224.0A CN202210175224A CN114432332A CN 114432332 A CN114432332 A CN 114432332A CN 202210175224 A CN202210175224 A CN 202210175224A CN 114432332 A CN114432332 A CN 114432332A
- Authority
- CN
- China
- Prior art keywords
- circutrn
- heart failure
- vector
- medicine
- recombinant vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000013598 vector Substances 0.000 title claims abstract description 64
- 206010019280 Heart failures Diseases 0.000 title claims abstract description 47
- 239000003814 drug Substances 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title description 6
- 230000002018 overexpression Effects 0.000 claims abstract description 29
- 238000011282 treatment Methods 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 239000013604 expression vector Substances 0.000 claims description 8
- 238000004806 packaging method and process Methods 0.000 claims description 7
- 206010007556 Cardiac failure acute Diseases 0.000 claims description 6
- 206010007558 Cardiac failure chronic Diseases 0.000 claims description 6
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 6
- 230000003612 virological effect Effects 0.000 claims 1
- 206010063837 Reperfusion injury Diseases 0.000 abstract description 40
- 208000012947 ischemia reperfusion injury Diseases 0.000 abstract description 27
- 230000006907 apoptotic process Effects 0.000 abstract description 15
- 208000031225 myocardial ischemia Diseases 0.000 abstract description 15
- 230000002107 myocardial effect Effects 0.000 abstract description 11
- 206010061216 Infarction Diseases 0.000 abstract description 8
- 230000000747 cardiac effect Effects 0.000 abstract description 8
- 238000002474 experimental method Methods 0.000 abstract description 8
- 230000007574 infarction Effects 0.000 abstract description 8
- 238000011084 recovery Methods 0.000 abstract description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 6
- 229910052760 oxygen Inorganic materials 0.000 abstract description 6
- 239000001301 oxygen Substances 0.000 abstract description 6
- 230000009091 contractile dysfunction Effects 0.000 abstract description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 26
- 241000699670 Mus sp. Species 0.000 description 23
- 241000700605 Viruses Species 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 13
- 241000702421 Dependoparvovirus Species 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000001356 surgical procedure Methods 0.000 description 6
- 210000003462 vein Anatomy 0.000 description 6
- 108091028075 Circular RNA Proteins 0.000 description 5
- 210000004413 cardiac myocyte Anatomy 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 210000005003 heart tissue Anatomy 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000004217 heart function Effects 0.000 description 4
- 210000005240 left ventricle Anatomy 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 210000000038 chest Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 210000003414 extremity Anatomy 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- 238000007363 ring formation reaction Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 230000002861 ventricular Effects 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 208000031229 Cardiomyopathies Diseases 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 206010048858 Ischaemic cardiomyopathy Diseases 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000004113 cell culture Methods 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
- 210000004351 coronary vessel Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003601 intercostal effect Effects 0.000 description 2
- 210000000876 intercostal muscle Anatomy 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000010410 reperfusion Effects 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 210000001562 sternum Anatomy 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 210000000779 thoracic wall Anatomy 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 1
- 241000649044 Adeno-associated virus 9 Species 0.000 description 1
- 206010001526 Air embolism Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 230000008828 contractile function Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002951 depilatory effect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000547 effect on apoptosis Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 230000007946 glucose deprivation Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 230000000004 hemodynamic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 1
- 229960004359 iodixanol Drugs 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 230000003680 myocardial damage Effects 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001147 pulmonary artery Anatomy 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- 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
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hospice & Palliative Care (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及生物医学技术领域,特别是涉及circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物。本发明通过实验证明了circUTRN制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对缺血再灌注损伤引起的心力衰竭有很好治疗效果;具体包括circUTRN具有改善心肌缺血再灌注损伤3周所致的心脏收缩功能不全的作用;另外,circUTRN还可以减小急性心肌缺血再灌注损伤的梗死面积;过表达circUTRN能够抑制氧糖剥夺/恢复所诱导的心肌细胞凋亡。
Description
技术领域
本发明涉及生物医学技术领域,特别是涉及circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物。
背景技术
心脏病的危害极大,几乎所有的心血管疾病最终都会导致心力衰竭的发生,心肌梗死、心肌病、血流动力学负荷过重、炎症等任何原因引起的心肌损伤,均可造成心肌结构和功能的变化,最后导致心室泵血和/或充盈功能低下,严重威胁着人类的生命。其中,缺血性心肌病是危害我国国民健康的重大疾病之一,及时进行再灌注是挽救缺血心脏的必需步骤,然而该过程伴随着严重的心肌缺血/再灌注(I/R)损伤,临床仍缺乏有效的干预手段。目前临床上治疗缺血性心肌病的手段主要由溶栓治疗、介入治疗及手术治疗。但是治疗过程多引起各种并发症,例如出血、空气栓塞、穿刺点出血、感染等。
环状RNA(circular RNA,circRNA)是一类特殊的非编码RNA分子,与传统的线性RNA(linear RNA,含5’-和3’-末端)不同,circRNA分子呈封闭环状结构,不受核糖核酸外切酶影响,表达更稳定,不易降解。在功能上,circRNA可以作为微小RNA或蛋白质的海绵体,从而实现对下游分子和信号通路的调控;部分circRNA还具有编码潜能,能够翻译成蛋白质或小肽发挥功能。越来越多的证据,circRNA在疾病的发生发展中发挥着重要的调控作用。在心血管领域,已有研究表明,circZNF292在心肌发育过程中促进内皮细胞的增殖;circANRIL在动脉粥样硬化疾病中促进细胞凋亡;circHRCR通过上调心脏保护蛋白ARC从而抑制病理性心肌肥厚和心力衰竭的发生;circFOXO3在心肌病中起到蛋白质海绵的作用,调控心脏衰老等。因此,需要找到一种普遍的缺血再灌注损伤相关的调节基因,并对其进行干预,才能够广泛实现对缺血再灌注损伤引起的心力衰竭的治疗。
发明内容
为了解决上述问题,本发明提供了circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物。本发明利用circUTRN制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对缺血再灌注损伤引起的心力衰竭有很好治疗效果。
为了实现上述目的,本发明提供如下技术方案:
本发明提供了circUTRN在制备治疗心力衰竭药物中的应用,所述circUTRN的核苷酸序列如SEQ ID NO.1所示。
优选的,所述心力衰竭包括急性心力衰竭或慢性心力衰竭。
本发明还提供了一种表达circUTRN的重组载体,所述重组载体包括核苷酸序列如SEQ ID NO.1所示的circUTRN和成环载体。
优选的,所述成环载体包括含有启动子CMV的成环载体。
优选的,所述成环载体包括慢病毒过表达载体或腺病毒过表达载体。
优选的,所述circUTRN序列位于慢病毒过表达载体的EcoRI和NdeI酶切位点之间。
优选的,所述circUTRN序列位于AAV过表达载体的EcoRI和BamHI酶切位点之间。
本发明还提供了一种治疗心力衰竭的药物,所述药物包括:上述重组载体。
优选的,所述药物还包括慢病毒载体系统或腺病毒载体系统。
优选的,所述心力衰竭包括急性心力衰竭或慢性心力衰竭。
有益效果:
本发明提供了circUTRN在制备治疗心力衰竭药物中的应用,所述circUTRN的核苷酸序列如SEQ ID NO.1所示。本发明通过实验证明了circUTRN制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对缺血再灌注损伤引起的心力衰竭有很好治疗效果;具体包括circUTRN具有改善心肌缺血再灌注损伤3周所致的心脏收缩功能不全的作用;另外,circUTRN还可以减小急性心肌缺血再灌注损伤的梗死面积;过表达circUTRN能够抑制氧糖剥夺/恢复所诱导的心肌细胞凋亡。
附图说明
图1为荧光定量PCR检测尾静脉注射腺相关病毒9(AAV9)包装的circUTRN后心肌组织中circUTRN的表达明显上调;
图2为超声心动图检测静脉注射AAV9包装的circUTRN改善心肌缺血再灌注损伤3周所致的心脏收缩功能不全;
图3为静脉注射AAV9包装的circUTRN后降低了急性心肌缺血再灌注损伤引起的心肌梗死严重程度;AAR/LV:Area at risk/Left ventricle weight,危险区/左心室;INF/AAR:Infarct size/Area at risk,梗死区/危险区;
图4为circUTRN能够抵抗OGD/R(氧糖剥夺/再恢复模型)诱导的新生小鼠心肌细胞凋亡(n=6);EV,对照载体;circUTRN,circUTRN过表达载体;TUNEL阳性指示心肌细胞凋亡阳性;图中白色箭头指示心肌细胞凋亡;α-Actinin,α-Actinin阳性表示该细胞是心肌细胞;DAPI,指示细胞核;
其中,Sham,假手术;IRI 3W,IRI后3周;AAV9-EV,AAV9对照病毒;AAV9-OE-circUTRN,circUTRN过表达AAV9;*,P<0.05;**,P<0.01;***,P<0.001。
具体实施方式
本发明提供了circUTRN在制备治疗心力衰竭药物中的应用,所述circUTRN的核苷酸序列如SEQ ID NO.1所示,具体如下:5’-TGGATCTCTTAGAGCTGAATACGACGAATGAAGTTTTCAAGCAGCACAAACTGAACCAAAATGATCAGCTCCTGAGTGTCCCAGACGTCATCAACTGTCTGACCACCACTTACGATGGGCTTGAGCAGCTGCACAAGGACTTGGTCAATGTTCCACTCTGCGTCGATATGTGTCTCAACTGGCTGCTCAACGTATACGACACGGGCCGGACTGGAAAAATTCGGGTACAGAGTCTGAAGATTGGATTGATGTCTCTCTCCAAAGGCCTCTTAGAAGAGAAATACAGATGTCTCTTTAAGGAGGTGGCAGGGCCAACAGAGATGTGTGACCAGCGGCAGCTTGGCCTGCTACTTCACGATGCCATCCAGATCCCTAGGCAGCTGGGGGAAGTAGCAGCCTTTGGGGGCAGTAACATTGAGCCCAGTGTCCGCAGCTGCTTCCAGCAGAATAACAACAAGCCAGAAATCAGTGTGAAGGAGTTTATAGACTGGATGCATTTGGAACCCCAGTCCATGGTGTGGTTGCCGGTTCTGCATCGGGTCGCAGCTGCTGAGACTGCAAAACATCAGGCCAAATGCAACATCTGCAAAGAATGCCCGATTGTTGGGTTCAGATACAGGAGCCTAAAGCATTTTAATTATGATGTCTGCCAGAGTTGCTTCTTTTCTGGAAGAACAGCAAAGGGCCACAAGTTACATTACCCGATGGTAGAATACTGCATACCG-3’。在本发明中,所述circUTRN的circAtlas ID为mmu-Utrn_0055。本发明通过实验证明了circUTRN制备的治疗心力衰竭药物可以治疗或改善心力衰竭,尤其是对缺血再灌注损伤引起的心力衰竭有很好治疗效果;具体包括circUTRN具有改善心肌缺血再灌注损伤3周所致的心脏收缩功能不全的作用;另外,circUTRN还可以减小急性心肌缺血再灌注损伤的梗死面积;过表达circUTRN能够抑制氧糖剥夺/恢复所诱导的心肌细胞凋亡。
在本发明中,所述心力衰竭优选包括急性心力衰竭或慢性心力衰竭,更优选包括缺血再灌注损伤引起的心力衰竭。
本发明还提供了一种表达circUTRN的重组载体,所述重组载体包括核苷酸序列如SEQ ID NO.1所示的circUTRN和成环载体。
在本发明中,所述成环载体优选包括含有启动子CMV的成环载体,进一步优选包括慢病毒过表达载体或腺病毒过表达载体,更优选包括慢病毒过表达载体pLO-ciR或腺病毒过表达载体pK5ssAAV-ciR。
在本发明中,所述circUTRN序列优选位于慢病毒过表达载体的EcoRI和NdeI酶切位点之间。本发明及实施例所述慢病毒过表达载体pLO-ciR优选购自广州吉赛生物科技股份有限公司,货号为GS0103。本发明提供的重组载体可以使目的基因(SEQ ID NO.1所示的序列)在真核细胞内表达为环状RNA circUTRN,从而发挥改善和/或治疗心力衰竭的作用。
在本发明中,所述circUTRN序列优选位于AAV过表达载体的EcoRI和BamHI酶切位点之间。本发明及实施例所述AAV过表达载体pK5ssAAV-ciR优选购自广州吉赛生物科技股份有限公司,货号为GS0109。本发明提供的重组载体可以使目的基因(SEQ ID NO.1所示的序列)在真核细胞内表达为环状RNA circUTRN,从而发挥改善和/或治疗心力衰竭的作用。
本发明还提供了一种治疗心力衰竭的药物,所述药物包括:上述重组载体。在本发明中,当所述重组载体的成环载体为AAV过表达载体时,所述药物的制备方法优选包括腺相关病毒包装;所述腺相关病毒优选包括AAV9病毒。本发明对所述药物的包装没有特殊要求,采用本领域技术人员所熟知的操作方式即可。本发明所述腺相关病毒不参与任何疾病的发生,免疫原性低,基因可持续表达半年以上,其中AAV9病毒对心脏组织存在强的亲和性,可以作为载体将目的基因序列在心脏组织中稳定高效的表达。
在本发明中,当所述重组载体的成环载体为慢病毒过表达载体时,所述药物的制备方法优选包括慢病毒包装。本发明对所述药物的包装没有特殊要求,采用本领域技术人员所熟知的操作方式即可。
在本发明中,所述药物优选还包括药学上可接受的辅料。
在本发明中,所述心力衰竭优选包括急性心力衰竭或慢性心力衰竭,更优选包括缺血再灌注损伤引起的心力衰竭。
为了进一步说明本发明,下面结合实施例对本发明提供的circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
一种表达circUTRN的重组载体,由以下步骤构建得到:
1)将小鼠心脏组织总RNA逆转录后依次进行PCR扩增,双酶切,回收获得circUTRN的cDNA序列(如SEQ ID NO.1所示);所述PCR扩增的上游引物的核苷酸序列如SEQ ID NO.2所示,具体如下:5’-CGGAATTCTGAAATATGCTATCTTACAGTGGATCTCTTAGAGCTGAATACGACG-3’,所述PCR扩增的下游引物的核苷酸序列如SEQ ID NO.3所示,具体如下:
5’-GGAATTCCATATGTCAAGAAAAAATATATTCACCGGTATGCAGTATTCTACCATCGG-3’;
每50μLPCR扩增反应体系中,由以下组分组成:上游引物(10μM)1μL(终浓度为0.2μM)、下游引物(10μM)1μL(终浓度为0.2μM)、2×TransStart FastPfuPCR SuperMix(全式金生物,货号AS221)25μL、cDNA模板(500ng/μL)1μL和无酶水22μL。
PCR反应进程为:95℃预变性2min;95℃变性20s,58℃退火20s,72℃延伸30s,36个循环;4℃,保温。
所述双酶切的体系为:EcoRI-HF(星选酶)1μL、NdeI(星选酶)1μL、10x CutSmart缓冲液5μL、PCR产物/或者载体1μg和无酶水(体积定容到50μL)。
双酶切反应:37℃,4h;
双酶切后的PCR产物采用天根生物超薄DNA产物纯化试剂盒(货号DP203)清洁回收。
2)将成环载体(慢病毒过表达载体pLO-ciR,购自广州吉赛生物科技股份有限公司,货号为GS0103)进行双酶切(方法同步骤1)后,采用天根生物超薄琼脂糖凝胶DNA回收试剂盒(货号DP208)回收大片段载体。
3)将步骤1)回收的片段插入步骤2)回收的成环载体上,得到连接产物。具体反应体系如下:T4 DNA连接酶1μL、10×T4 DNA连接酶缓冲液1μL、双酶切的载体(100ng)2μL、双酶切的基因(100ng)1μL和无酶水5μL,总体积为10μL。
反应条件为:16℃,8h;
4)50μL感受态细胞(感受态细胞TransStbl3 Chemically Competent Cell全式金生物,货号CD521)冰上化开,加入5μL步骤3)制备的连接产物,轻弹混匀,在冰上放置25min;42℃热激45s,然后放置回冰上2min;后加入500μL培养基(无抗LB培养基,品牌为全式金,货号为Cat#CD521),混匀后37℃,180转/min培养1h,复苏细菌。涂布在氨苄抗性的培养平板上过夜培养。过夜培养后挑取单克隆摇菌,送Sanger测序。
实施例2
一种与实施例1相似的表达circUTRN的重组载体,区别在于:
1、将步骤1)中的下游引物替换为核苷酸序列如SEQ ID NO.4所示的下游引物,具体如下:
5’-CGGGATCCAGTTGTTCTTACCGGTATGCAGTATTCTACCATCGG-3’;2、将步骤1)中的NdeI(星选酶)替换为BamHI-HF(星选酶);
3、将步骤2)中的成环载体替换为AAV过表达载体pK5ssAAV-ciR(购自广州吉赛生物科技股份有限公司,货号为GS0109)。
实施例3
一种治疗心力衰竭的药物,所述药物的制备方法如下:
使用293T细胞在合适的细胞密度(80%)时加入包装体系;每皿10cm培养皿加入1mL包装体系:10μgAAV2/9(购自Addgene Plasmid,货号#112865)、10μg pAdDeltaF6质粒(购自Addgene Plasmid,货号#112867)、10μg实施例2构建的重组载体,加DMEM培养基(无FBS无双抗)至910μl,加90μl PEI转染试剂(总体积:1mL);细胞转染60h之后收集上清及其细胞;采用碘克沙醇浓缩超速离心纯化病毒(收集上清后用浓缩柱(Merck UFC905096)4000rpm 4℃,将所有上清离心浓缩至10~15mL即可),得到的病毒悬液为所述药物;所述病毒悬液滴度为1×1013vg/mL。
应用例1
心肌缺血再灌注损伤(IRI)模型建立:IRI模型采用最严重的损伤效果,通过结扎冠状动脉左前降支造成心肌缺血30min后再进行血液复灌。取8~10周龄的野生型雄性小鼠,给小鼠以10μL/g的剂量腹腔注射4%水合氯醛对小鼠进行麻醉,麻醉后使用医用胶带将小鼠腹部朝上固定在恒温毯上,使用脱毛膏去除小鼠后颈部毛发。75%乙醇对脱毛部分进行消毒,在显微镜下,使用小剪刀将小鼠胸骨左缘第四第五肋间位置做一横形切口,切口长约1.2cm,并逐层分离胸壁肌肉直至肋间肌暴露,使用显微镊钝性分离肋间肌肉,暴露心脏,结扎左前降支动脉(左心耳至肺动脉圆锥间,因肉眼不可见,故结扎成功视下方心尖部缺血(变白)情况而定)。缝合肋间肌和胸壁肌肉。30min后再次打开胸腔,将结扎线剪断取出。将小鼠至于恒温毯上保温等待复苏后转移至鼠笼中饲养24小时(IRI急性模型)或21天(IRI 3周,长期重构模型)后处死小鼠,称量小鼠体重、胫骨长度、心脏重量等称重后留样进行后续检测。本实施例中所述假手术与上述IRI手术相似,唯一区别在于,未进行结扎处理。
实验分组分为4个组,具体分组信息:
第1组对照病毒组(实施例3中的AVV2/9)+假手术组,对小鼠心脏进行注射对照病毒,均按照10^11vg/只小鼠,一周后进行假手术,样本数为10只;
第2组AAV9-circUTRN病毒(实施例3制备的药物)+假手术组,对小鼠心脏进行注射AAV9-circUTRN病毒,均按照10^11vg/只小鼠,一周后进行假手术,样本数为10只;
第3组对照病毒(实施例3中的AVV2/9)+IRI手术组,对小鼠心脏进行注射对照病毒,均按照10^11vg/只小鼠,一周后进行上述IRI手术,样本数为14只;
第4组AAV9-circUTRN病毒(实施例3制备的药物)+IRI手术组,对小鼠心脏进行注射AAV9-circUTRN病毒,均按照10^11vg/只小鼠,一周后进行上述IRI手术,样本数为13只。
利用RNAiso Plus提取上述4组小鼠心脏组织中的总RNA,采用荧光定量PCR法检测假手术及IRI手术后三周的circUTRN相对表达量;
具体如下:以18s作为内参引物(具体序列为F1:5’-TCAAGAACGAAAGTCGGAGG-3’,SEQ ID NO.5和R1:5’-GGACATCTAAGGGCATCAC-3’,SEQ ID NO.6);circUTRN的qPCR引物序列为:F2:5’-GGCCACAAGTTACATTACCCG-3’,SEQ ID NO.7和R2:5’-acgttgagcagccagttgag-3’,SEQ ID NO.8,qPCR的结果采用相对定量法进行分析,运用2-ΔΔCt进行计算,如下:ΔCt=靶基因Ct值-内参Ct值,ΔΔCt=各实验组ΔCt值-对照组ΔCt平均值;
具体实时荧光PCR(10μL体系)方法如下:
配制引物工作液:储液(100μM)稀释20倍变成母液(5μM),母液稀释10倍变成工作液(0.5μM);
配制cDNA和SYBR混合液:常用体积比是cDNA:SYBR=1:20(5μL/孔)。
加板:加板顺序为:先加5μL引物工作液,后加5μL cDNA和SYBR的混合液,共10μL体系。
PCR反应程序:BIO-RAD 2STEP
95℃预变性30s;95℃变性15s,60℃退火60s,循环40次;
熔融曲线分析:95℃1s,60℃1s,95℃continuous
(65~95℃0.5℃increments at 2-5sec/step)
Cooling 40℃30s。
具体的,第1组10只小鼠相对RNA含量为0.838568、1.141555、1.018185、1.334223、0.75576、0.835667、0.777007、1.236275、1.249196、1.014663;
第2组10只小鼠相对RNA含量为1.848045、1.522033、1.718322、1.470187、2.940375、2.631709、3.108029、1.712377、1.913216、1.181812;
第3组14只小鼠相对RNA含量为0.454074、0.753145、0.557097、0.503827、0.541863、0.491751、0.560972、0.592957、0.484981、0.473357、0.368823、0.410655、0.373971、0.483303;
第4组13只小鼠相对RNA含量为1.83528、1.575708、2.167452、1.223488、1.516768、2.613531、2.455471、1.53262、0.796088、1.454981、1.642621、2.057653、0.911301。统计结果如图1所示,*,P<0.05,***,P<0.001。
结果表明,circUTRN在急性IRI手术三周后表达下调;尾静脉注射AAV9包装的circUTRN后心脏组织中circUTRN的表达明显上调。
应用例2
采用应用例1相同的分组及处理方式,选取C57BL/6J成年公鼠施行冠状动脉左前降支结扎,结扎30min后松开,在IRI手术3周后,进行心脏超声测量上述四个组心功能相关指标(射血分数(EF)和短轴缩短率(FS))。小动物心脏超声测定小鼠心功能:将小鼠胸部脱毛后,用异氟烷吸入麻醉法将小鼠麻醉,在超声平板上固定好小鼠四肢,并将适量螯合剂涂抹于小鼠腹部心脏处,用探头找到小鼠心脏,待心率稳定在400bpm左右,取胸骨旁左心室长轴及左心室短轴进行检查,测量并计算心功能,获得心功能相关指标:左室射血分数EF和左心室短轴缩短率FS。结果见图2。
具体数据为:第1组10只小鼠FS(%)值为26.57607、28.73468、30.00613、34.14948、32.35228、29.93588、22.36367、26.9502、27.2448、26.21533;
第2组10只小鼠FS(%)值为28.37145、34.25649、31.288、26.87928、31.41054、37.83692、27.23812、22.8164、34.70449、32.0555;
第3组14只小鼠FS(%)值为25.7799、25.72851、17.9281、16.89639、16.79336、18.24466、17.54019、20.96864、16.8033、23.96844、14.63545、24.6609、16.61429、23.86121;
第4组13只小鼠FS(%)值为31.41518、30.00557、30.17961、28.22829、27.84015、34.28185、34.86179、29.70271、28.46201、22.60534、34.172137、27.64563、24.13351。
第1组10只小鼠EF(%)值为52.58755、55.87751、58.14579、64.05795、61.45018、57.95299、45.50572、53.6604、53.85443、52.0395;
第2组10只小鼠EF(%)值为55.51394、64.23814、59.70567、53.01966、60.23159、68.61732、53.49452、46.05206、65.00643、60.71237;
第3组14只小鼠EF(%)值为51.63186、51.77215、37.77709、35.63613、35.49855、38.65654、37.26884、43.38542、35.61092、48.38518、32.21303、49.7293、35.34495、49.15511;
第4组13只小鼠EF(%)值为60.09334、57.32902、58.13966、55.29248、54.55704、64.67837、65.48337、57.59445、56.06144、46.36027、63.965966、54.38887、49.07793。统计结果如图2所示,**,P<0.01。
由图2可知,尾静脉注射circUTRN过表达AAV9能够改善心肌缺血再灌注损伤3周所致的心脏收缩功能不全。
应用例3
TTC染色实验分组分为2个组,每组各10只小鼠,具体为:
第1组:在IRI模型造模1周前开始,采用一次性1mL无菌注射器通过尾静脉注射对照AAV9(实施例3中的AVV2/9),按照10^11vg/只小鼠的浓度进行处理;
第2组:在IRI模型造模1周前开始,采用一次性1mL无菌注射器通过尾静脉注射AAV9-circUTRN(实施例3制备的药物)),按照10^11vg/只小鼠的浓度进行处理。一周后两组采用应用1所述的方法进行IRI手术,24小时之后取样。
TTC(Triphenyltetrazolium Chloride,2,3,5-氯化三苯基四氮唑)染色:将接受IRI手术后的小鼠用4%的水合氯醛腹腔注射麻醉,按照每20g体重200μL麻醉。待小鼠麻倒后,将小鼠腹部向上平躺固定在泡沫板上,剪刀剪开胸部皮肤、肌肉和肋骨,露出心脏。首先,用7-0带线针再次结扎缺血手术时的心脏结扎位点,结扎完成后,用1mL胰岛素注射器吸取1~2mL 1%的Evans Blue,注射入小鼠心脏左心室,观察小鼠鼻尖和四肢,直到鼻尖和四肢变蓝色,注射完成。取出心脏后将心脏置于1.5mL离心管,-20℃冰箱暂存。待心脏冷冻变硬后便可进行切片。将心脏放与切片模具的凹槽里,按凹槽空格依次将刀片经心脏表面由上向下插进空格。待刀片插满后,平行按压刀片,将心脏切成每片1mm的厚度,取下刀片,将心脏切片放进1.5mL离心管。将事先配好的1%的TTC溶液加到离心管中,每管1mL,加好后将离心管置于37℃避光水浴10min。将4%的PFA溶液加到96孔板中,用于固定心脏切片。水浴结束后,将心脏切片从离心管中取出,放入96孔板中固定,每孔一片。固定时间1.5小时。切片固定后1.5小时拍照,防止时间过久颜色流失。96孔板中取出每片心脏称重,记录。使用Image J软件手动圈绘统计心脏切片正面整片面积,正面白色梗死面积,正面红色面积和反面整片面积,反面白色梗死面积,反面红色面积。将统计得到面积数据对应每片切片重量填入表格。
具体实验结果见附图3,具体的,第1组AAR/LV比值为0.546797124、0.498411923、0.573749793、0.478128607、0.502519571、0.474636482、0.466649783、0.501469873、0.504364335、0.445463737;
第2组AAR/LV比值为0.420656141、0.501991857、0.504419175、0.412981739、0.596882045、0.560244519、0.55786186、0.475217199、0.523069062、0.459159742;
第1组INF/AAR比值为0.412583、0.558483、0.544388、0.485926、0.573036、0.541956、0.484268、0.498673、0.548814、0.542946;
第2组INF/AAR比值为0.278354675、0.261470747、0.473479359、0.387369342、0.245335、0.367833474、0.349436683、0.497779168、0.404129982、0.296345906。统计结果如图3所示,**,P<0.01。
结果表明,尾静脉注射circUTRN过表达AAV9能够改善心肌缺血再灌注损伤引起的心肌梗死面积。
应用例4
心肌细胞氧葡萄糖剥夺恢复(OGD/R)模型建立及质粒转染:NMCM用正常心肌细胞培养基培养,待细胞铺展至80%孔面积时换无血清培养基饥饿处理7小时;将6μL2000加入100μL无血清培养基(DMEM)中,预混5分钟(轻轻吹打混匀后静置5分钟),记为A液;将2μg质粒加入100μl无血清培养基(DMEM)中,预混5分钟(轻轻吹打混匀后静置5分钟),记为B液;预混结束将A液与B液混匀,室温静置20分钟后,以100μl/孔的量加入到96孔板中,转染7小时后,弃转染培养基,换成无血清培养基,于37℃,5%CO2的恒温培养箱中继续培养;换液20h之后,将细胞的培养基换成无糖培养基,并将细胞培养板放置在缺氧盒(一个缺氧盒中放置一个缺氧袋,并用无菌填充物尽量排尽缺氧盒中的空气)中孵育8h;从缺氧盒中取出细胞培养板行复氧操作,并将无糖培养基换回正常心肌细胞培养基孵育12h至实验终点。
在新生小鼠心肌细胞中,转染实施例1制备的表达circUTRN的重组载体(0.02mg/L),处理72h后收集细胞,在实验终点前20小时构建OGD/R模型(缺氧缺糖8h,复氧复糖12h)完成功能获得性实验。具体分组为:1)circUTRN对照组(实施例1中的慢病毒过表达载体pLO-ciR),2)circUTRN对照+OGD/R处理组,3)circUTRN过表达组(实施例1制备的表达circUTRN的重组载体),4)circUTRN过表达+OGD/R处理组。实验终点以Tunel与α-actinin/Hoechst免疫荧光共染检测circUTRN对心肌细胞凋亡的保护作用。
由图4可知,OGDR模型中,NMCM凋亡水平与对照组相比显著上升;并且在基础水平过表达circUTRN对凋亡没有影响;而在OGDR刺激下,过表达circUTRN能够保护NMCM缺血再灌注损伤引起的凋亡。具体的,第1)组凋亡率(%)为4.164398、3.660825、3.954054、5.02763、5.036923、6.062882;
第2)组凋亡率(%)为4.35287、5.562385、4.304403、5.45163、3.801478、4.991256;
第3)组凋亡率(%)为12.97532、15.21066、15.17345、17.85742、14.54959、15.53715;
第4)组凋亡率(%)为9.172941、9.148951、8.449327、11.39639、7.821497、10.04349。统计结果如图4所示,**,P<0.01。
综上所述,circUTRN具有改善心肌缺血再灌注损伤3周所致的心脏收缩功能不全的作用;另外,circUTRN还可以减小急性心肌缺血再灌注损伤的梗死面积;过表达circUTRN能够抑制氧糖剥夺/恢复所诱导的心肌细胞凋亡。
虽然本发明已以较佳的实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可以做各种改动和修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 上海大学
<120> circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 725
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
tggatctctt agagctgaat acgacgaatg aagttttcaa gcagcacaaa ctgaaccaaa 60
atgatcagct cctgagtgtc ccagacgtca tcaactgtct gaccaccact tacgatgggc 120
ttgagcagct gcacaaggac ttggtcaatg ttccactctg cgtcgatatg tgtctcaact 180
ggctgctcaa cgtatacgac acgggccgga ctggaaaaat tcgggtacag agtctgaaga 240
ttggattgat gtctctctcc aaaggcctct tagaagagaa atacagatgt ctctttaagg 300
aggtggcagg gccaacagag atgtgtgacc agcggcagct tggcctgcta cttcacgatg 360
ccatccagat ccctaggcag ctgggggaag tagcagcctt tgggggcagt aacattgagc 420
ccagtgtccg cagctgcttc cagcagaata acaacaagcc agaaatcagt gtgaaggagt 480
ttatagactg gatgcatttg gaaccccagt ccatggtgtg gttgccggtt ctgcatcggg 540
tcgcagctgc tgagactgca aaacatcagg ccaaatgcaa catctgcaaa gaatgcccga 600
ttgttgggtt cagatacagg agcctaaagc attttaatta tgatgtctgc cagagttgct 660
tcttttctgg aagaacagca aagggccaca agttacatta cccgatggta gaatactgca 720
taccg 725
<210> 2
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cggaattctg aaatatgcta tcttacagtg gatctcttag agctgaatac gacg 54
<210> 3
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggaattccat atgtcaagaa aaaatatatt caccggtatg cagtattcta ccatcgg 57
<210> 4
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgggatccag ttgttcttac cggtatgcag tattctacca tcgg 44
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tcaagaacga aagtcggagg 20
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggacatctaa gggcatcac 19
<210> 7
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggccacaagt tacattaccc g 21
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
acgttgagca gccagttgag 20
Claims (10)
1.circUTRN在制备治疗心力衰竭药物中的应用,其特征在于,所述circUTRN的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于,所述心力衰竭包括急性心力衰竭或慢性心力衰竭。
3.一种表达circUTRN的重组载体,其特征在于,所述重组载体包括核苷酸序列如SEQID NO.1所示的circUTRN和成环载体。
4.根据权利要求3所述的重组载体,其特征在于,所述成环载体包括含有启动子CMV的成环载体。
5.根据权利要求3或4所述的重组载体,其特征在于,所述成环载体包括慢病毒过表达载体或腺病毒过表达载体。
6.根据权利要求5所述的重组载体,其特征在于,所述circUTRN序列位于慢病毒过表达载体的EcoRI和NdeI酶切位点之间。
7.根据权利要求5所述的重组载体,其特征在于,所述circUTRN序列位于AAV过表达载体的EcoRI和BamHI酶切位点之间。
8.一种治疗心力衰竭的药物,其特征在于,所述药物包括:权利要求3~7任一项所述的重组载体。
9.根据权利要求8所述的药物,其特征在于,所述药物的制备方法包括病毒包装。
10.根据权利要求8或9所述的药物,其特征在于,所述心力衰竭包括急性心力衰竭或慢性心力衰竭。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210175224.0A CN114432332B (zh) | 2022-02-25 | 2022-02-25 | circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 |
US17/837,248 US20230272417A1 (en) | 2022-02-25 | 2022-06-10 | Use of circutrn in preparation of drug for treating heart failure, recombinant vector, and drug for treating heart failure |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210175224.0A CN114432332B (zh) | 2022-02-25 | 2022-02-25 | circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114432332A true CN114432332A (zh) | 2022-05-06 |
CN114432332B CN114432332B (zh) | 2023-06-30 |
Family
ID=81374552
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210175224.0A Active CN114432332B (zh) | 2022-02-25 | 2022-02-25 | circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 |
Country Status (2)
Country | Link |
---|---|
US (1) | US20230272417A1 (zh) |
CN (1) | CN114432332B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115216473A (zh) * | 2021-06-21 | 2022-10-21 | 上海大学 | 一种工程化环状RNA circmiR-29b及其在制备治疗肌萎缩的药物中的应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106222173A (zh) * | 2016-08-12 | 2016-12-14 | 青岛大学 | circRNA MNCR核苷酸、含有该核苷酸的药物组合物及其用途 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7863017B2 (en) * | 2006-12-01 | 2011-01-04 | Wisconsin Alumni Research Foundation | TAT-utrophin as a protein therapy for dystrophinopathies |
CN109706172B (zh) * | 2019-01-03 | 2022-10-11 | 广州吉赛生物科技股份有限公司 | 一种基于Alu元件的精准型环形RNA表达框架和载体及其应用 |
-
2022
- 2022-02-25 CN CN202210175224.0A patent/CN114432332B/zh active Active
- 2022-06-10 US US17/837,248 patent/US20230272417A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106222173A (zh) * | 2016-08-12 | 2016-12-14 | 青岛大学 | circRNA MNCR核苷酸、含有该核苷酸的药物组合物及其用途 |
Non-Patent Citations (1)
Title |
---|
刘国銮等: "circRNA _000203 与心力衰竭患者心功能及预后的关系研究", 《国际检验医学杂志》 * |
Also Published As
Publication number | Publication date |
---|---|
CN114432332B (zh) | 2023-06-30 |
US20230272417A1 (en) | 2023-08-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112472690B (zh) | 一种制备增强CNPase活性的化合物或生物药物的方法用于治疗心脏疾病 | |
CN107385033B (zh) | piRNA-5938及其反义核酸在诊断、治疗缺血性心脏疾病中的应用 | |
CN114432332B (zh) | circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 | |
CN113234811A (zh) | CircRNA000338的应用及其药物 | |
CN108187029B (zh) | 白细胞免疫球蛋白样受体亚家族b成员4在制备预防、缓解和/或治疗心肌肥厚药物的应用 | |
CN105194660A (zh) | 泛素特异蛋白酶18(usp18)在治疗心肌肥厚中的功能及应用 | |
CN111705061B (zh) | 和心脏疾病相关的piRNA-P1与piRNA-P1反义核苷酸、应用和药物 | |
CN112294835B (zh) | LncRNA-266在制备诱导棕色脂肪细胞分化药物中的应用 | |
CN111808186B (zh) | 一种人源性分泌型fndc5蛋白及其制备方法和用途 | |
CN115068632A (zh) | Ago2在制备治疗心衰的药物方面的用途及其蛋白、基因、转化体、药物与制备方法 | |
CN107625781B (zh) | miRNA抑制子在制备防治心肌梗死药物中的应用 | |
CN116790612B (zh) | 一种促进心肌再生修复的过氧化物酶体生发蛋白3及其应用 | |
CN105194673A (zh) | 生长抑制特异性蛋白6(gas6)在治疗心肌肥厚中的功能及应用 | |
CN114588264B (zh) | 敲低或抑制egr3的试剂在制备心肌缺血再灌注损伤药物中的应用 | |
CN113559266B (zh) | Ckip-1 3` UTR在预防和/或治疗心力衰竭疾病药物中的应用 | |
CN114517209B (zh) | 环状RNA circTTC3过表达腺相关病毒载体、腺相关病毒及其应用 | |
CN114507677B (zh) | Ndufs1基因在治疗心梗后心衰中的应用和相关产品 | |
CN114432453B (zh) | 敲低或抑制clec4d的试剂在制备心肌缺血再灌注损伤药物中的应用 | |
CN110433170B (zh) | 心脏抽吸液miR-28-5p在心脏疾病中的应用 | |
CN118286434A (zh) | Ythdf3抑制剂在制备防治病理性心肌肥厚和心室重构的药物中的应用 | |
CN106512009B (zh) | Ph同源域家族a成员3(phlda3)在治疗心肌肥厚中的应用 | |
CN117721194A (zh) | Ap2a1基因在制备高血压防治产品中的用途 | |
CN117100838A (zh) | Dnajc9在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 | |
CN117530953A (zh) | circRcor3在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物 | |
CN117180435A (zh) | Frizzled 2作为靶点在治疗压力超负荷性心肌损伤中的作用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |