CN117025594A - Oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof - Google Patents
Oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof Download PDFInfo
- Publication number
- CN117025594A CN117025594A CN202310867041.XA CN202310867041A CN117025594A CN 117025594 A CN117025594 A CN 117025594A CN 202310867041 A CN202310867041 A CN 202310867041A CN 117025594 A CN117025594 A CN 117025594A
- Authority
- CN
- China
- Prior art keywords
- awcpdo
- oligodeoxyribonucleic acid
- acid
- oligodeoxyribonucleic
- liver fibrosis
- 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
- 239000002253 acid Substances 0.000 title claims abstract description 36
- 208000019425 cirrhosis of liver Diseases 0.000 title claims abstract description 34
- 239000002773 nucleotide Substances 0.000 claims abstract description 14
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 44
- 206010019668 Hepatic fibrosis Diseases 0.000 claims description 12
- 229940046166 oligodeoxynucleotide Drugs 0.000 claims description 11
- 239000002202 Polyethylene glycol Chemical group 0.000 claims description 9
- 229920001223 polyethylene glycol Chemical group 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 9
- 238000005516 engineering process Methods 0.000 claims description 8
- 230000004048 modification Effects 0.000 claims description 8
- 238000012986 modification Methods 0.000 claims description 8
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- KBDWGFZSICOZSJ-UHFFFAOYSA-N 5-methyl-2,3-dihydro-1H-pyrimidin-4-one Chemical compound N1CNC=C(C1=O)C KBDWGFZSICOZSJ-UHFFFAOYSA-N 0.000 claims description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical group 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 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- JTBBWRKSUYCPFY-UHFFFAOYSA-N 2,3-dihydro-1h-pyrimidin-4-one Chemical compound O=C1NCNC=C1 JTBBWRKSUYCPFY-UHFFFAOYSA-N 0.000 claims description 4
- 108091034117 Oligonucleotide Proteins 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 3
- -1 amino, carboxyl Chemical group 0.000 claims description 3
- 239000011616 biotin Chemical group 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 238000007385 chemical modification Methods 0.000 claims description 3
- 235000012000 cholesterol Nutrition 0.000 claims description 3
- 125000003827 glycol group Chemical group 0.000 claims description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 3
- 208000031873 Animal Disease Models Diseases 0.000 claims description 2
- 238000011558 animal model by disease Methods 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 210000004962 mammalian cell Anatomy 0.000 claims description 2
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 claims description 2
- 238000011321 prophylaxis Methods 0.000 claims 1
- 210000004024 hepatic stellate cell Anatomy 0.000 abstract description 14
- 108010008929 proto-oncogene protein Spi-1 Proteins 0.000 abstract description 13
- 102100027654 Transcription factor PU.1 Human genes 0.000 abstract description 6
- 230000004913 activation Effects 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 28
- 210000004185 liver Anatomy 0.000 description 27
- 239000000843 powder Substances 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 19
- 230000014509 gene expression Effects 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 102000008186 Collagen Human genes 0.000 description 12
- 108010035532 Collagen Proteins 0.000 description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 12
- 229920001436 collagen Polymers 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 108091023040 Transcription factor Proteins 0.000 description 10
- 102000040945 Transcription factor Human genes 0.000 description 10
- 239000000835 fiber Substances 0.000 description 10
- 238000002156 mixing Methods 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 238000010171 animal model Methods 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 8
- 239000007983 Tris buffer Substances 0.000 description 8
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 8
- 210000003734 kidney Anatomy 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 102000007469 Actins Human genes 0.000 description 7
- 108010085238 Actins Proteins 0.000 description 7
- 210000003995 blood forming stem cell Anatomy 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 210000002950 fibroblast Anatomy 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 6
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000027455 binding Effects 0.000 description 6
- 210000002744 extracellular matrix Anatomy 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 description 5
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 210000002216 heart Anatomy 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 230000000284 resting effect Effects 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 230000017074 necrotic cell death Effects 0.000 description 4
- 239000004006 olive oil Substances 0.000 description 4
- 235000008390 olive oil Nutrition 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000012137 tryptone Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 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 3
- 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 3
- 206010016654 Fibrosis Diseases 0.000 description 3
- 206010067125 Liver injury Diseases 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 3
- 229960002327 chloral hydrate Drugs 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000004761 fibrosis Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 3
- 230000002440 hepatic effect Effects 0.000 description 3
- 231100000753 hepatic injury Toxicity 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000000651 myofibroblast Anatomy 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108091060592 XDNA Proteins 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000003176 fibrotic effect Effects 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 210000004969 inflammatory cell Anatomy 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
- 210000001503 joint Anatomy 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000003032 molecular docking Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical group C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 208000007848 Alcoholism Diseases 0.000 description 1
- 101100328883 Arabidopsis thaliana COL1 gene Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 101000823089 Equus caballus Alpha-1-antiproteinase 1 Proteins 0.000 description 1
- 101000651211 Homo sapiens Transcription factor PU.1 Proteins 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 235000002296 Ilex sandwicensis Nutrition 0.000 description 1
- 235000002294 Ilex volkensiana Nutrition 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
- 206010030113 Oedema Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108020004459 Small interfering RNA Proteins 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
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 206010001584 alcohol abuse Diseases 0.000 description 1
- 208000025746 alcohol use disease Diseases 0.000 description 1
- 229960000723 ampicillin Drugs 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
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003510 anti-fibrotic effect Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000007894 caplet Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000009693 chronic damage Effects 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 238000000942 confocal micrograph Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001965 increasing effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002206 pro-fibrotic effect Effects 0.000 description 1
- OCQZXMCGTAWGEQ-UHFFFAOYSA-N prop-2-enamide;n-[(prop-2-enoylamino)methyl]prop-2-enamide Chemical compound NC(=O)C=C.C=CC(=O)NCNC(=O)C=C OCQZXMCGTAWGEQ-UHFFFAOYSA-N 0.000 description 1
- GPTFURBXHJWNHR-UHFFFAOYSA-N protopine Chemical group C1=C2C(=O)CC3=CC=C4OCOC4=C3CN(C)CCC2=CC2=C1OCO2 GPTFURBXHJWNHR-UHFFFAOYSA-N 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000006177 thiolation reaction Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- 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
- A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
-
- 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
- A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/322—2'-R Modification
-
- 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
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
Abstract
The invention discloses an oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof, wherein the nucleotide sequence of the oligodeoxyribonic acid is SEQ ID NO. 1, can specifically enter activated hepatic stellate cells, can competitively bind with a transcription factor PU.1, further acts on a downstream target spot, and inhibits HSC activation and liver fibrosis. The oligodeoxyribonucleic acid provided by the invention can be used for preparing medicines for preventing and treating liver fibrosis.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof.
Background
Liver fibrosis is an inflammatory pathological process and result from excessive deposition of extracellular matrix (Extracellular matrix, ECM) and various chronic injuries in the post-injury repair of liver tissue caused by a variety of factors (e.g., hepatitis virus infection, alcohol abuse, non-alcoholic fatty liver, immune response, drug and chemical poison damage, etc.). ECM is produced by Myofibroblasts (MFB), which are mainly derived from activated hepatic stellate cells (hepatic stellate cell, HSC), and liver injury activates hepatic stellate cells and causes accumulation of extracellular matrix is recognized as a key link in liver fibrosis.
Hepatic stellate cells are located in the perihepatic sinus space and are the major cell types involved in the healing and scarring of liver injury. Hepatic stellate cells are normally in a quiescent state. Upon liver injury, resting hepatic stellate cells are transformed into activated myofibroblasts, resulting in upregulation of α -smooth muscle actin (α -smooth muscle actin, α -SMA) and extracellular matrix deposition. Activation and proliferation of hepatic stellate cells involves a variety of cell signaling pathways, ultimately leading to imbalance in extracellular matrix synthesis and degradation, increased extracellular matrix production, or decreased degradation, are the root causes of liver fibrosis.
PU.1 (Purine-rich box 1) is a member of ETS (E26 transformation-specific) transcription factor family, encoded by SPI1 (human/mouse), which is a major regulator of hematopoietic stem cell proliferation and differentiation, maintaining cell homeostasis, playing an important role in inflammation, cell reprogramming and immunity. Pu.1 inhibits the growth of hepatocytes through TGF- β/Smad signaling pathways, thereby inducing and promoting precursor cell interstitial transformation, causing excessive deposition of ECM, leading to the occurrence of liver fibrosis. After knocking out the pu.1 gene from fibroblasts of human fibrous tissue, collagen release from fibroblasts was reduced, and α -SMA and F-actin (Filamentous actin) expression levels were similar to resting fibroblasts. In contrast, overexpression of PU.1 in resting fibroblasts can induce a transition from resting fibroblasts to a highly activated pro-fibrotic phenotype, an up-regulation of collagen release and expression of alpha-SMA and F-actin. Upregulation of PU.1 in fibrotic disease causes fibrosis-related genes to be induced, fibroblast phenotypes to change, and ECM production to increase. Whereas inactivation of pu.1 is effective to restore the fibrotic phenotype of fibroblasts to a quiescent state and induce regression of tissue fibrosis. In liver fibrosis, pu.1 is highly expressed in activated hepatic stellate cells and not in resting hepatic stellate cells.
The technology of trap oligonucleotides (decoy oligodeoxynucleotide, decoy ODN) is a gene therapy strategy, the principle of which is: based on the core motif of the transcription factor binding site (Transcription Factor binding sites, TFBS), DNA fragments containing TFBS (i.e., decoy ODN) are designed, synthesized, and transfected into cells to competitively capture the transcription factor, prevent the transcription factor from binding to TFBS on the promoter, block transcription of DNA, and regulate expression of downstream target genes. It has the advantages of easy synthesis, high specificity, relatively low synthesis cost, is more stable than siRNA sequences, and is considered as an effective therapeutic tool, and can inhibit the expression of specific genes under various medical conditions. The decoy ODN strategy is an effective method of inhibiting specific gene expression both in vitro and in vivo, and has been studied to confirm its role in cancer and fibrosis. Such as N F- κb decoy ODN, SP1 decoy ODN, etc., but the number and kinds of substances in the decoy ODN technology that can be used for preventing and treating liver fibrosis are small at present, and further development is still required.
Disclosure of Invention
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of cell culture, molecular biology, nucleic acid chemistry, etc., as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
The term "decoy ODN" generally refers to a synthetic DNA fragment designed based on the core motif of the transcription factor binding site, with the ability to competitively capture transcription factors; the term "liver fibrosis" generally refers to a pathophysiological process that refers to the abnormal proliferation of connective tissue within the liver caused by various pathogenic factors.
In order to achieve the above object, the present invention provides an oligodeoxyribonucleic acid, which is an exogenously synthesized nucleotide chain that can specifically enter activated HSCs and inhibit expression of liver fibrosis-promoting genes, inhibiting activation of HSCs and liver fibrosis; the nucleotide sequence of the oligodeoxyribonucleic acid is SEQ ID NO:1, and a nucleotide sequence of 1.
The oligodeoxyribonucleic acid comprises at least one chemical modification: modification of ribose in the oligodeoxyribonucleic acid sequence, including 2' -H quilt F, NH 2 Substitution of OMe; modifying any base in the oligodeoxyribonucleic acid sequence by amino, carboxyl, sulfhydryl, biotin, cholesterol and polyethylene glycol groups; modification of polyethylene glycol (PEG) at the 5 'end and deoxyuracil (dU), deoxythymine (dT) and deoxyhypoxanthine (dL) at the 3' end in the nucleotide sequence of the oligodeoxyribonucleic acid; at least one nucleotide in the nucleotide sequence of the oligodeoxyribonucleic acid is a locked nucleic acid.
The invention also provides an application of the oligodeoxyribonucleic acid in preparing a medicament for preventing and treating liver fibrosis diseases; the prevention and treatment of liver fibrosis disease is based on decoy ODN technology.
The invention also provides a pharmaceutical composition comprising an effective dose of oligodeoxyribonucleotide and a pharmaceutically acceptable carrier or diluent.
Preferably, the effective dose of the oligodeoxyribonucleic acid is 100-400nM.
The invention also provides application of the pharmaceutical composition in preparing medicines for preventing and treating hepatic fibrosis cells and animal disease models.
Preferably, the cell is a mammalian cell.
The invention has the beneficial effects that: the invention screens and obtains an oligodeoxyribonucleic acid sequence, predicts a secondary structure in The UNAFold website, selects a structure with The lowest Gibbs free energy delta G, and generates a tertiary structure of The polydeoxyribonucleic acid in Discovery Studio, which is named Awcpdo. The invention verifies that Awcpdo can target and activate HSC cells in a cell line and an animal model, and verifies that Awcpdo regulates the expression of hepatic fibrosis related genes alpha-SMA, COL1 alpha 2 and TIMP1 in activated HSCs; the observation of Awcpdo in animal model to improve liver fibrosis proves that the oligodeoxyribonucleic acid Awcpdo can be used for preventing and treating liver fibrosis progress, provides a new substance for decoy ODN technology, and provides scientific basis for application and delivery strategy research in liver fibrosis diseases.
Drawings
FIG. 1 is a simulated docking result of the core base sequence of Awcpdo in example 1 and the PU.1 protein, wherein A is a potential base sequence specifically binding to the PU.1 protein, and the letter size indicates the binding potential of each base at the corresponding position; b is a simulated graph of the butt joint of the core base sequence of the Awcpdo and the PU.1 protein, a green structure in the graph is a PU.1 protein DNA binding domain, a double-helix structure is a fragment of the Awcpdo combined with the PU.1 protein, and red dots are water molecules participating in the simulated butt joint.
FIG. 2 shows the results of the optimization of the working concentration of Awcpdo in example 3.1.
FIG. 3 is a fluorescence microscopy image validating that Awcpdo targets activated HSC in example 3.2.
FIG. 4 is a Western Blot chart showing the inhibition of expression of liver fibrosis promoting genes by Awcpdo in example 4, wherein A is a Western Blot gel electrophoresis chart, B is a COL1α2 expression level histogram, C is an α -SMA expression level histogram, and D is a TIMP1 expression level histogram; ns represents no statistical difference, P <0.05, P <0.01, P <0.001, and the values are mean±sd.
FIG. 5 is a serum stability experiment of Awcpdo in example 5. Lanes are samples of DNA Ladder, awcpdo, serum, awcpdo incubated with serum for 0h,0.5h,1h,2h,4h,6h,8h, respectively from left to right.
FIG. 6 is a photograph of a small animal imaged to verify that Awcpdo targets the liver in an animal model as in example 6.1, wherein A is CCL after various times of injection of Awcpdo 4 Living body of molded mouseImaging photograph, B is CCL after injecting Awcpdo for different time 4 Tissue imaging photographs of the model mice, wherein in B, heart, spleen, liver, lung and kidney are sequentially arranged from left to right.
FIG. 7 is a laser scanning confocal microscopy image of Awcpdo targeted activated HSC in an animal model verified in example 6.2, wherein A is an immunofluorescence photograph of co-localization of alpha-SMA with Awcpdo, and B is the presence of Awcpdo in CCL 4 Immunofluorescence photographs of heart, spleen, lung and kidneys in the model mice.
FIG. 8 is a graph showing the effect of Awcpdo on anti-hepatic fibrosis in example 7, wherein A is the liver of a normal mouse and B is CCL 4 Model mouse liver, C is the mouse liver of control group, D is the mouse liver of Awcpdo treatment group.
FIG. 9 is a graph showing the effect of thiomodified Awcpdo on anti-hepatic fibrosis in example 8, wherein A is the liver of a normal mouse and B is CCL 4 Model mouse liver, C is the mouse liver of Awcpdo group, D is the mouse liver of Awcpdo group after being modified by thio.
Detailed Description
The technical solution of the present invention will be further explained below with reference to the accompanying drawings and specific embodiments, and it should be noted that the following embodiments are only preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention shall be defined by the claims. Modifications and substitutions made by those skilled in the art without the inventive effort fall within the scope of the present invention.
The main reagent comprises:
(1) Neonatal bovine serum (NBCS): holly bioengineering materials limited (Hangzhou);
(2) Fetal bovine serum, DMEM medium: gibco corporation (USA);
(3) Trypsin Trypsin, double antibody (penicillin/streptomycin): is of the science and technology limited company of the biological products of the ocean (Tianjin city);
(4) PBS powder (0.01M), ECL chemiluminescent kit, 5 x protein loading buffer: seville biotechnology Co., ltd;
(5) DMSO, EB, β -actin antibodies: sigma Co., USA;
(6) Cell transfection reagent (Touborfect), xhoI, sal I, ecoR I, protein Ladder, protein quantitative analysis kit, DNA Ladder: us Thermo Fisher scientific;
(7) Cell transfection reagent (Neofect): zero-passenger creating intelligent (Beijing) biotechnology limited company;
(8) RIPA lysate: solarbio (beijing);
(9) Awcpdo, scr, fluorescent probe (cy 5, FAM, etc.) labeled Awcpdo/Scr and thio modified Awcpdo synthesis: shanghai Bioengineering Co., ltd;
(10) Plasmid extraction kit: nanjinouzan biotechnology Co., ltd;
(11) Transfer ribonucleic acid (yeast): moeiy biotechnology limited;
(12) YEAST EXTRACT (YEAST EXTRACT), TRYPTONE (TRYPTONE): oxyid LTD (uk);
(13) Competent preparation kit: takara (Takara doctor materials technologies Co., ltd.);
(14) AGAROSE (agarsose), tween20 (Tween 20): bioFrox, germany;
(15) Sodium chloride, tris-base, glycine (Glycine): shanghai Miclin Biochemical technologies Co., ltd;
(16) Ammonium Persulfate (APS) and Sodium Dodecyl Sulfate (SDS): henan Huamei bioengineering Co., ltd;
(17) Skimmed milk powder: inner Mongolian illi Co., ltd;
(18) TEMED (tetramethyl ethylenediamine): life Technologies (U.S.);
(19) PVDF film: millipore (Germany);
(20) 30% acrylamide-methylene bisacrylamide (30% acr-Bis): boschia martensii;
(21) COL 1a 2 antibodies: santa cruz, inc. of America;
(22) alpha-SMA antibody, TIMP1 antibody: abcronal Biotechnology Inc.;
(23) HRP-labeled goat anti-mouse/goat anti-rabbit secondary, 75% alcohol, and 84 antiseptic solution: wuhan flying agent Co., ltd.
Animal experiment related materials:
(1) Sterile syringes (1 mL, 5mL, and 10 mL): hunan oasis Huikang development Co., ltd;
(2) Sterile insulin syringe (U-40): bidi medical instruments (Shanghai) Inc.;
(3) Pathological blade: shanghai Lianhui medical supplies Co;
(4) Microscope cover slip, microscope slide, and adhesive slide: jiangsu Shitai laboratory equipment company;
(5) Light-resistant slide moisture box: huasheng experiment consumables (Taobao shop);
(6) Dehairing instrument: ant nationality flagship (Taobao shop).
Animal experiment related drugs and reagents:
(1) Carbon tetrachloride (CCL) 4 ) Sucrose, chloral hydrate: shanghai Miclin Biochemical technologies Co., ltd;
(2) Edible olive oil: sanchang good office limited (hong Kong);
(3) Paraformaldehyde fixative, 6 XDNA loading buffer, RIPA lysate, 10 XTBE, gelRed, anti-fluorescence quenching caplet, DAPI staining reagent: seville biotechnology Co., ltd;
(4) OCT frozen section embedding agent: jiangsu Meiger Biotech Co., ltd;
(5) Triton X-100, 5% bsa blocking solution, masson/HE staining kit and FITC-labeled secondary antibody: solarbio (beijing);
(6) PBS powder (0.01M): whanbo Oute Biotechnology Co., ltd;
(7) Glycerol, boric acid, glacial acetic acid, methanol and EDTA: national pharmaceutical group chemical reagent company.
The common solution is prepared:
(1) Cell cryopreservation solution: DMEM (-): NBCS: volume ratio of DMSO was 7:2:1, preparing, uniformly mixing, and storing in a dark place at 4 ℃;
(2) PBS solution: pouring 1 package of PBS powder (0.01M) into a measuring cylinder, adding 1L of double distilled water, stirring until the PBS powder is fully dissolved, and fixing the volume to 2L; sterilizing under high pressure, cooling to room temperature, and storing in a refrigerator at 4deg.C;
(3) DMEM (-): high sugar culture solution (without serum and antibiotics) with the specification of 500 mL/bottle;
(4) DMEM (+): to 267mL of DMEM (-) medium, 3mL of penicillin/streptomycin mixed solution (1%) and 30mL of newborn calf serum were added respectively, and mixed uniformly to prepare 300mL of DMEM (+) medium;
(5) Ampicillin solution (Amp): weighing 3g of Amp powder, adding a proper amount of ddH 2 Dissolving O sufficiently, fixing volume to 30mL (100 μg/μl), filtering with 0.22 μm sterile filter, packaging, and storing at-20deg.C;
(6) LB (-) solid Medium: weighing agar powder 3g, yeast extract 1g, tryptone 2g and sodium chloride 2g, adding appropriate amount of ddH 2 After O is fully dissolved, the volume is fixed to 200mL; sterilizing under high pressure, rapidly pouring into a sterile culture dish after the temperature is reduced to about 55 ℃ in an ultra-clean bench, sealing the whole culture dish with a preservative film after the culture dish is cooled and solidified, and inverting the culture dish in a refrigerator at 4 ℃ for later use;
(7) LB (-) liquid Medium: weighing 2g of sodium chloride, 2g of tryptone, 1g of yeast extract and adding a proper amount of ddH 2 After O is fully dissolved, the volume is fixed to 200mL; sterilizing under high pressure, cooling to room temperature, and preserving at 4deg.C;
(8) Amp (+) solid medium: preparing 200mL of LB (-) culture medium solution according to the formula (6), sterilizing under high pressure, cooling to about 55 ℃, adding 200 mu L of Amp with concentration of (100 mu g/. Mu.L), rapidly pouring into a sterile culture dish, sealing the whole culture dish with a preservative film after cooling and solidifying, and inverting the culture dish in a refrigerator with the temperature of 4 ℃ for later use;
(9) Amp (+) liquid medium: 200mL of LB (-) medium solution was prepared according to the formula (7), autoclaved, cooled to about 55℃and then added with 200. Mu.L of Amp at a concentration of (100. Mu.g/. Mu.L),
cooling to room temperature, and preserving at 4deg.C;
(10) 50×tae solution: weigh 24.2g Tris-Base and 37.2g EDTA Na 2 ·2H 2 Adding O powder into a measuring cup, adding 57.1mL of glacial acetic acid, and adding a proper amount of ddH 2 After O is fully dissolved, the volume is fixed to 1L (ph=8.5), and the O is stored at room temperature and diluted to 1×tae when in use;
(11) 10% Sodium Dodecyl Sulfate (SDS): SDS powder (10 g) was weighed and a proper amount of ddH was added 2 Stirring O until the O is fully dissolved, and then keeping the volume to 100mL, and preserving at room temperature for later use;
(12) Agarose gel (1%): weighing 0.4g agarose powder in a conical flask, adding 40mL 1 xTAE into the conical flask, heating the conical flask in a microwave oven until the powder is completely dissolved, shaking the conical flask slightly, adding 1.5 mu L EB when the temperature of the solution is reduced to about 50 ℃, mixing uniformly, immediately pouring into a mold groove, inserting a comb into the hole teeth, and naturally solidifying the solution to obtain the agarose powder;
(13) Weak acid working solution: according to weak acid solution: ddH 2 O=1: 2, the volume ratio is configured and used at present;
(14)20％ CCL 4 : CCL (CCL) 4 : olive oil at 1:4, respectively measuring CCL of the required volume 4 The solution and olive oil solution are stirred in a beaker until being fully and evenly mixed to prepare 20 percent CCL 4 The olive oil mixed solution is preserved in dark at 4 ℃;
(15) Chloral hydrate (10%): 3g of chloral hydrate powder is weighed and a proper amount of autoclaved ddH is added 2 After O is fully dissolved, the volume is fixed to 30mL (100 mg/mL), and the O is preserved in a dark place at 4 ℃;
(16) 70% ethanol solution: ddH sterilized in an amount of 30mL 2 Adding O into 70mL of absolute ethyl alcohol, fully and uniformly mixing, and storing at normal temperature;
(17) 100. Mu.M of Awcpdo and Scr stock solution were prepared: according to the instructions volume, the powder was dissolved in autoclaved ddH 2 O, keeping at-20 ℃ for standby;
(18) DAPI solution (10%): 200 mu L of DAPI solution is added into 2mL of sterile PBS solution, and the mixture is fully and uniformly mixed for use;
(19) Triton (2%): according to PBS: triton=50: 1, respectively measuring the solution with the required volume into a beaker, and fully stirring and uniformly mixing the solution to obtain the finished product;
(20) 1×running Buffer: weighing 75.2g glycine powder, 4g SDS powder, 12.12g Tris Base powder, adding a proper amount of ddH 2 O is stirred, after the dissolution is sufficient, the volume is fixed to 4L, and the mixture is preserved at the temperature of 4 ℃;
(21) 1 XTrans Buffer: 57.6g glycine powder, 4g SDS powder, 12.12g Tris Base powder were weighed and a proper amount of ddH was added 2 O is stirred, after the dissolution is complete, 800mL of methanol is added, the volume is fixed to 4L after the mixture is uniformly mixed, and the mixture is preserved at 4 ℃;
(22) 1 XTBST: weighing 35.2g NaCl,9.68g Tris Base powder, adding appropriate amount of ddH 2 O is stirred, after the solution is fully dissolved, 4mL of 0.05% Tween20 is added into the solution, the mixture is continuously mixed evenly, and then ddH is added 2 O is fixed to 4L, pH is regulated to 7.4, and the mixture is preserved at 4 ℃;
(23) 5% skim milk solution: weighing 2g of skimmed milk powder into a 50mL centrifuge tube, adding 40mL of 1 XTBST solution, and shaking by vortex until the skimmed milk powder is fully dissolved for preparation;
(24) 10% APS: 5g of ammonium persulfate powder was weighed into a beaker, and 50mL of ddH was added thereto 2 Stirring O until it is fully dissolved, packaging into 1.5mL EP tube (1 mL/tube), and storing at 20deg.C in dark place;
(25) 10 XTBE: weighing 216g of Tris-base, 110g of boric acid and 80mL of 0.5M EDTA, adding 1600mL of distilled water for complete dissolution, then, fixing the volume to 2L, adjusting the pH to 8.3, and preserving at normal temperature;
(26) 50% glycerol: taking 100mL of glycerol and 100mL of distilled water, fully and uniformly stirring, and preserving at high pressure and normal temperature;
(27) 2 XBuffer: tris-base 90.84g,10% SDS 20mL was weighed and dissolved in 400mL ddH 2 O, stirring until the mixture is completely dissolved, then, fixing the volume to 1000mL, and adjusting the pH to 8.8;
(28) Tris-HCl (1M): tris-base 48.44g was weighed and dissolved in 200mL ddH 2 O, after stirring to complete dissolution, constant volume to 400mL, ph=6.8.
Plasmid, cell line and animal:
plasmid PCDNA3.1 (+), PCDNA3.1-ALK3, PCDNA3.1-ALK5: tumor microenvironment at university of three gorges and professor Liu Changbai to the focus laboratory for immunotherapy;
HSC-T6 cells (rat hepatic stellate cells): giving by Song Yuhu professor (university of science and technology, china, same-aid Hospital);
specific pathogen free (Specific pathogen free, SPF) grade male C57BL/6 mice: 6-8 weeks old (body weight 20.+ -.2 g), purchased and fed to the university of three gorges laboratory animal center, laboratory animal production license number: SYXK 2022-0012, barrier system, drinking water, ingestion and illumination all meet the Experimental animal quality control Standard in Hubei province.
Example 1 design of Awcpdo
Potential base sequences for specific binding of PU.1 proteins were obtained by database Jaspar (https:// Jaspar. Geneg. Net /), the results are shown in FIG. 1A; the sequence of the obtained oligonucleotide sequence Awcpdo based on the sequence extension is SEQ ID NO. 1. The obtained Awcpdo was subjected to simulated docking with PU.1 protein, and as a result, awcpdo could bind to PU.1 protein as shown in FIG. 1B.
EXAMPLE 2 culture of HSC-T6 cells
(1) Taking out HSC-T6 cell frozen stock solution from-80deg.C refrigerator, rapidly placing into water bath of 37deg.C, rapidly shaking to defrost within 1min,
(2) Transferring the thawed cell suspension into a 15mL centrifuge tube with 3mL of DMEM (+) cell culture solution added in advance, lightly mixing, and centrifuging at 800rpm for 3 min;
(3) After the supernatant was aspirated, 3.5mL of DMEM (+) was added, gently mixed, transferred to a cell culture dish, and 5% CO was added 2 Culturing in an incubator at 37 ℃;
(4) HSC-T6 cells were harvested for use when cells grew on the wall and reached 80% density.
Example 3 validation of Awcpdo-targeted activated HSC in cell lines
3.1 optimization of the working concentration of Awcpdo
(1) The HSC-T6 cell suspension obtained in example 2 was taken at a cell amount of 1X 10 per well 5 Adding the cells into a 24-well plate to be subjected to transfection when the cells are attached and the fusion degree reaches 70-80%;
(2) Discarding old culture medium in the plate, adding PBS for washing twice, adding 400 mu L of DMEM (-) in a 24-pore plate, adding 900 mu L of DMEM (-) in a 6-pore plate, and performing starvation treatment;
(3) Taking sterile EP tubes (500 mu L) corresponding to each hole, adding 100 mu L of DMEM (-) into each tube, adding 1 mu g of plasmid pCDNA3.1-ALK5, gently mixing, and standing for 6min;
(4) The transfection reagent (Turbofect) was added to the EP tube of (3) in a volume ratio of: plasmid = 2:1, gently mixing, and standing for 20min;
(5) All the mixed liquid in the step (4) is dripped into the corresponding hole, and the culture plate is put into an incubator for continuous culture after being gently rocked left and right and up and down; after 6h, cell replacement is carried out, 500 mu LDMEM (+) is added into each hole of DMEM (-), and transfection is carried out for 24h;
(6) Awcpdo was added to each well of (5) to a final concentration of 0nM, 25nM, 50nM, 100nM, 150nM, 200nM, 300nM, 400nM, 500nM, co-cell incubation and after 24h, cell-line cytometry analysis was collected.
As a result, as shown in FIG. 2, the fluorescence intensity of Awcpdo reached the maximum at 300nM and as the concentration continued to increase, the fluorescence intensity in the cells did not increase any more; comprehensive analysis, 300nM is the optimum working concentration of Awcpdo.
3.2, fluorescence microscopy experiments with Awcpdo-targeted activated HSC verify
(1) Transfecting plasmid pCDNA3.1-ALK5 into HSC-T6 cells according to steps (1) - (5) in 3.1, obtaining activated HSC-T6 cells after 24 hours;
(2) FAM-labeled Atcpdo was added to the non-activated HSC-T6 cells (i.e., NC panel in FIG. 3) and activated HSC-T6 cells (i.e., ALK5 panel in FIG. 3) for 24h incubation at a final concentration of 20 nM; qualitative and quantitative analysis of HSC-T6 cells in ALK5 and NC groups was performed using fluorescence microscopy and flow cytometry. As a result, atcpdo can enter activated HSC-T6 cells without entering unactivated HSC-T6 cells, as shown in FIG. 3.
Example 4Awcpdo inhibits liver fibrosis Gene expression
The experimental groupings were as follows:
(1) Mock group: blank control, HSC-T6 cells were untreated;
(2) ALK5 group: transfecting plasmid pCDNA3.1-ALK5 into HSC-T6 cells according to steps (1) - (5) in 3.1, obtaining activated HSC-T6 cells after 24 hours;
(3) Scr group: transfecting plasmid pCDNA3.1-ALK5 into HSC-T6 cells according to steps (1) - (5) in 3.1, obtaining activated HSC-T6 cells after 24 hours; subsequently 300nM of Scr was added per well and incubated for a further 24h;
(4) Awcpdo group: transfecting plasmid pCDNA3.1-ALK5 into HSC-T6 cells according to steps (1) - (5) in 3.1, obtaining activated HSC-T6 cells after 24 hours; subsequently 300nM of Awcpdo was added per well and incubated for an additional 24h;
(5) ALK3 group: transfecting plasmid pCDNA3.1-ALK3 into HSC-T6 cells according to steps (1) - (5) in 3.1, obtaining activated and inhibited HSC-T6 cells after 24 hours;
collecting HSC-T6 cells obtained in the steps (1) - (5), extracting protein, performing Western blot to detect expression conditions of liver fibrosis related genes COL1α2, TIMP1 and alpha-SMA, and performing statistical analysis, wherein beta-actin is used as an internal reference.
As a result, as shown in FIG. 4, COL1α2, TIMP1, and α -SMA were all up-regulated in the ALK 5-treated activated HSC-T6, whereas the protein expression was down-regulated after the respective treatment with Awcpdo, and returned to be consistent with or slightly lower than that of the Mock group, whereas that of the Scr group did not. Taken together, the results demonstrate that Awcpdo is capable of inhibiting expression of genes associated with pro-hepatic fibrosis in activated HSC-T6 cells.
Example 5 serum stability experiments on Awcpdo
(1) Eyeball blood of a male C57BL/6 liver fibrosis model mouse is taken in a sterilized 1.5mL EP tube, centrifuged at room temperature at 3000rpm for 5min, and upper serum is taken in a new EP tube;
(2) Respectively mixing Awcpdo with the concentration of 10 mu M in serum with medium volume, incubating in a water bath kettle at 37 ℃, respectively collecting samples at 0h,0.5h,1h,2h,4h,6h,8h and 10h, and temporarily storing in a refrigerator at-80 ℃ for later use;
(3) Preparing 10% non-denaturing polyacrylamide gel (Native-PAGE), and the formulation of the gel is as follows;
10％ Native-PAGE
(4) After vortex mixing, injecting the solution into two layers of glass plates, after the glue is completely solidified, placing the glue into an electrophoresis tank, filling the inner tank with 0.5 XTBE, adding a proper amount of 0.5 XTBE buffer solution into the outer tank, and pre-electrophoresis for 2 hours;
(5) Taking out the sample from the refrigerator at-80 ℃, thawing on ice, adding 6 XDNA loading with corresponding volume, mixing uniformly, and then instantly centrifuging for about 10 s;
(6) Adding sample, 100V electrophoresis for about 2 hours, and soaking and dyeing glue with GelRed working solution at room temperature for 30min; the position of the band and the extent of dispersion of the band were observed with a gel imaging system.
As a result, the Awcpdo was degraded at 0.5h and the degradation was substantially complete at 4h, as shown in FIG. 5.
Example 6 Small animal imaging and laser scanning confocal microscopy verification of Awcpdo-targeted activated HSC in animal models
6.1 imaging verification of Awcpdo targeting liver in animal model by small animals
(1) A20% CCL injection was subcutaneously administered to mice at 5mL/kg body weight 4 Injecting once every 3 days for 8 weeks to obtain CCL 4 Mice induced liver fibrosis for 8 weeks;
(2) 100 μl (10 μM) of the mice obtained in step (1) were injected via tail vein to form Cy 5-labeled Awcpdo for biological synthesis in Shanghai, and the mice were imaged in vivo and in tissue at 0h,0.5h,1h,2h,3h, and 5h, respectively.
As a result, awcpdo starts to aggregate in the liver at 0.5h and metabolize to the kidney as shown in FIG. 6A; the aggregation was evident in the liver at 1h, and the fluorescence at the kidney metabolism was not as strong as the fluorescence at the liver, and 5h was almost completed from the kidney metabolism. In the same period, hearts, spleens, lungs and kidneys of mice were taken out and photographed, and as a result, as shown in fig. 6B, fluorescence was not seen in the hearts, spleens, lungs, but was accumulated in the liver and gradually metabolized the kidneys with the lapse of time. Taken together, awcpdo has a certain targeting to the liver.
6.2, laser scanning confocal microscopy validation of Awcpdo-targeted activated HSC in animal models
To further verify whether Awcpdo is on CCL 4 The hepatic stellate cells activated by the model-making mice have a targeting effect, and the liver imaged by the tissue is adopted for fixation, dehydration and embedding of paraformaldehyde to prepare frozen sections for immunofluorescence. The activated hepatic stellate cell markers α -SMA were co-localized with Awcpdo, respectively. As shown in fig. 7a, the first column shows DAPI blue fluorescence, the nuclei are displayed, the second column shows Awcpdo red fluorescence, the third column shows α -SMA green fluorescence, and the fourth column shows three combined (merger) images, wherein representative regions of the 0.5h and 3h merger images are shown in column 5 after enlargement, and the combined images are visible: the green fluorescence of α -SMA was largely concentrated around the blood vessel and hepatic blood sinus, and red light of the Cy 5-labeled Awcpdo was found under the high power microscope to be concentrated near the hepatic stellate nucleus around the hepatic blood sinus, and approximately coincident with the green light of α -SMA and appeared yellow, consistent with the expected results. It was demonstrated that Awcpdo has a targeting effect on hepatic stellate cells activated by model-producing mice. The results of heart, spleen, lung and kidney are shown in FIG. 7B, and red fluorescence is not seen. Indicating that Awcpdo has no targeting effect on these organs.
Example 7 measurement of the anti-hepatic fibrosis Effect of Awcpdo
To verify whether Awcpdo has anti-fibrotic effect in mice, CCL was tested 4 Mice induced liver fibrosis for 8 weeks were given 2 times a week by tail vein injection of Awcpdo or Scr (negative control), respectively, with CCL4 modeling (on monday and friday CCL 4), monday, friday and friday Awcpdo or Scr) for 4 weeks, and normal mice, CCL were taken after 4 weeks 4 Model, awcpdo treatment group and Scr group mouse livers were observed and photographed as a whole, and H was performed&E staining and Masson staining, results are shown in FIG. 8, CCL compared to normal mice (A of FIG. 8) 4 The liver surface of the model (B of FIG. 8) was not smooth, had a hard texture, and had pronounced particulate nodules, H&E staining has massive inflammatory cell infiltration accompanied by massive cell necrosis, masson staining shows significant proliferation of collagen fibers, and sink region and bloodThe thickening of collagen fibers around the tube is obvious, the collagen fibers of the hyperplasia among the lobules extend along the inflammatory necrosis area, and a plurality of collagen fiber bundles are crosslinked to divide liver lobules. Scr group mice (C of FIG. 8) and CCL 4 No significant difference was seen from the general view of the liver to pathological examination compared to model mice. While Awcpdo treated mice (D of FIG. 8) and CCL 4 Compared with modeling, the particles on the surface of the liver are obviously reduced, the texture is softer, H&E staining showed reduced inflammatory cell infiltration and cell necrosis, masson staining showed a significant reduction in the proliferated collagen fibers. Taken together, it is demonstrated that Awcpdo has an anti-hepatic fibrosis effect in mice.
Example 8 detection of the anti-hepatic fibrosis Effect of thio-modified Awcpdo
Awcpdo binds to PU.1 through specific base sequence, and various chemical modifications to Awcpdo including but not limited to "modification of ribose in the oligodeoxyribonucleic acid sequence including 2' -H being F, NH 2 Substitution of OMe; modifying any base in the oligodeoxyribonucleic acid sequence by amino, carboxyl, sulfhydryl, biotin, cholesterol and polyethylene glycol groups; modification of polyethylene glycol (PEG) at the 5 'end and deoxyuracil (dU), deoxythymine (dT) and deoxyhypoxanthine (dL) at the 3' end in the nucleotide sequence of the oligodeoxyribonucleic acid; at least one nucleotide in the nucleotide sequence of the oligodeoxyribonucleic acid is a locked nucleic acid ", and can still have the anti-liver fibrosis effect of the original sequence.
To verify the anti-hepatic fibrosis effect of the chemically modified Awcpdo, we performed an Awcpdo thio modification between the deoxyguanosine at position 18 and the deoxyuracil at position 19, followed by animal experiments following the method of example 8, i.e.: to CCL (CCL) 4 Mice induced liver fibrosis for 8 weeks were administered 2 times a week by tail vein injection of either Awcpdo or Awcpdo after thioation, respectively, for a duration of CCL 4 Moulding (in Tuesday and friday CCL) 4 Administration, monday, wednesday and Saturday Awcpdo or Scr), and normal mice were harvested after 4 weeks 4 The model, the Awcpdo group and the mouse liver of the Awcpdo group after being thioated are integrally observed and photographedAnd Masson staining was performed, and the results were shown in FIG. 9, which shows CCL compared with normal mice (A of FIG. 9) 4 The liver surface of the model (B of FIG. 9) is not smooth, the texture is hard, obvious granular nodules are formed, masson staining shows that liver cell edema is obvious, collagen fibers are obviously proliferated, thickening of collagen fibers around a manifold area and blood vessels is obvious, and the proliferated collagen fibers between the leaflets extend along an inflammatory necrosis area, and a plurality of collagen fiber bundles are crosslinked to divide liver leaflets. Awcpdo group mice (C and D of FIG. 9) and CCL after thiolation of Awcpdo group 4 Compared with model mice, the particles on the surface of the liver are obviously reduced, the texture is softer, and the Masson dyeing shows that the collagen fibers which are proliferated are obviously reduced. Taken together, it is demonstrated that the thio-modified Awcpdo has an anti-hepatic fibrosis effect in mice similar to the original sequence.
The object of the present invention is to provide a novel oligodeoxyribonucleic acid, awcpdo, with specific binding and access to activated HSCs, inhibiting HSC activation and liver fibrosis. The present invention detects the optimum working concentration of Awcpdo, and the results show that Awcpdo has the best affinity for HSC-T6 at 300 nM; confirming that Awcpdo can enter activated HSC in a targeting way through means such as a laser scanning confocal microscope, small animal imaging and the like; through Western Blot and other means, it is confirmed that Awcpdo can inhibit liver fibrosis promotion gene expression; in the mouse liver fibrosis model, injection of Awcpdo significantly improved liver fibrosis. Awcpdo is expected to be applied to the prevention and treatment of hepatic fibrosis.
Claims (7)
1. An oligodeoxyribonucleic acid, characterized by: the nucleotide sequence of the oligodeoxyribonucleic acid is SEQ ID NO:1, said oligodeoxyribonucleic acid is capable of specifically entering activated HSC cells.
2. An oligodeoxyribonucleic acid according to claim 1, characterised in that: the oligodeoxyribonucleic acid comprises at least one chemical modification: modification of ribose in the oligodeoxyribonucleic acid sequence, including substitution of 2' -H by F, NH, OMe; modifying any base in the oligodeoxyribonucleic acid sequence by amino, carboxyl, sulfhydryl, biotin, cholesterol and polyethylene glycol groups; modification of polyethylene glycol (PEG) at the 5 'end and deoxyuracil (dU), deoxythymine (dT) and deoxyhypoxanthine (dL) at the 3' end in the nucleotide sequence of the oligodeoxyribonucleic acid; at least one nucleotide in the nucleotide sequence of the oligodeoxyribonucleic acid is a locked nucleic acid.
3. Use of the oligodeoxyribonucleic acid according to claim 1 or 2 for the preparation of a medicament for the prophylaxis and treatment of liver fibrosis diseases; the prevention and treatment of liver fibrosis disease is based on decoy ODN technology.
4. A pharmaceutical composition characterized by: the pharmaceutical composition comprises an effective amount of the oligodeoxyribonucleotide according to claim 1 or 2, and a pharmaceutically acceptable carrier or diluent.
5. The pharmaceutical composition of claim 4, wherein: the effective dose of the oligodeoxyribonucleic acid is 100-400nM.
6. Use of a pharmaceutical composition according to claim 4 or 5 for the preparation of a medicament for the prevention and treatment of hepatic fibrosis cells and animal disease models.
7. The use according to claim 6, characterized in that: the cell is a mammalian cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310867041.XA CN117025594A (en) | 2023-07-14 | 2023-07-14 | Oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310867041.XA CN117025594A (en) | 2023-07-14 | 2023-07-14 | Oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117025594A true CN117025594A (en) | 2023-11-10 |
Family
ID=88601308
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310867041.XA Pending CN117025594A (en) | 2023-07-14 | 2023-07-14 | Oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117025594A (en) |
-
2023
- 2023-07-14 CN CN202310867041.XA patent/CN117025594A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3553077B1 (en) | Peptide for repairing cartilage and treating osteoarthritis | |
| EP3336190A1 (en) | Methods, systems, and compositions for cell-derived/vesicle-based microrna delivery | |
| Zhou et al. | Activated macrophage membrane-coated nanoparticles relieve osteoarthritis-induced synovitis and joint damage | |
| CN101874111A (en) | Utilize the endogenous chylomicron to send to be used to the system of the nucleic acid that suppresses expression of target gene | |
| Carotenuto et al. | Dietary flaxseed mitigates impaired skeletal muscle regeneration: in vivo, in vitro and in silico studies | |
| Huang et al. | Nanoparticle-mediated inhibition of notch signaling promotes mitochondrial biogenesis and reduces subcutaneous adipose tissue expansion in pigs | |
| Zhou et al. | Exosomes derived from dental pulp stem cells accelerate cutaneous wound healing by enhancing angiogenesis via the Cdc42/p38 MAPK pathway | |
| US11622964B2 (en) | Method for destroying cellular mechanical homeostasis and promoting regeneration and repair of tissues and organs, and use thereof | |
| Zhao et al. | Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats | |
| CN107353333A (en) | A kind of bone protectiveness molecule Semaphorine3A and its pharmacy application | |
| Guo et al. | Influence of serum collected from rat perfused with compound Biejiaruangan drug on hepatic stellate cells | |
| CN110684774B (en) | Aptamer specifically binding to DEK protein and application thereof | |
| CN117025594A (en) | Oligodeoxyribonucleic acid Awcpdo for improving liver fibrosis and application thereof | |
| CN116904463A (en) | Oligodeoxyribonucleic acid Atcpdo for improving liver fibrosis and application thereof | |
| CN115896111A (en) | Small interfering RNA for inhibiting ACSL4 expression and application thereof | |
| CN111388499A (en) | Application of miR-31 in preparation of colon-targeted nano-drug for preventing and treating ulcerative colitis | |
| CN110229215A (en) | A kind of medicament slow release polypeptide hydrogel and its preparation method and application | |
| Xie et al. | Effect of a subset of adipose‐derived stem cells isolated with liposome magnetic beads to promote cartilage repair | |
| CN108403665B (en) | Prostate cancer targeted drug delivery carrier modified by EpDT3 aptamer, delivery system, preparation method and application thereof | |
| CN113521080A (en) | Application of CX-5461 in preparation of PHF6 mutant acute myeloid leukemia medicine | |
| CN112516318A (en) | Application of CUL4B in treatment of type II diabetes | |
| Peng et al. | Locally administered lentivirus-mediated siRNA inhibits wear debris-induced inflammation in murine air pouch model | |
| CN101524548A (en) | Nanometer carrier combination and application thereof to preparation of anti-tumor drug | |
| CN111388452A (en) | Adipose tissue targeting peptide P3-chitosan oligomeric lactic acid-polyethylene glycol delivery system and application thereof in nucleic acid drug delivery | |
| WO2023077711A1 (en) | Screening method of medicament for preventing prostatic cancer and application of nitazoxanide in pharmacy |
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 |