CN116589555B - 一种细胞穿梭肽及其应用 - Google Patents
一种细胞穿梭肽及其应用 Download PDFInfo
- Publication number
- CN116589555B CN116589555B CN202310350766.1A CN202310350766A CN116589555B CN 116589555 B CN116589555 B CN 116589555B CN 202310350766 A CN202310350766 A CN 202310350766A CN 116589555 B CN116589555 B CN 116589555B
- Authority
- CN
- China
- Prior art keywords
- kla
- cells
- cell
- tumor
- ryk
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 106
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 71
- 229920001184 polypeptide Polymers 0.000 claims abstract description 55
- 230000004927 fusion Effects 0.000 claims abstract description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 15
- 239000002246 antineoplastic agent Substances 0.000 claims description 12
- 229940041181 antineoplastic drug Drugs 0.000 claims description 10
- 230000000861 pro-apoptotic effect Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 130
- 210000004881 tumor cell Anatomy 0.000 abstract description 30
- 230000006907 apoptotic process Effects 0.000 abstract description 27
- 230000000259 anti-tumor effect Effects 0.000 abstract description 15
- 101001026262 Homo sapiens Gasdermin-D Proteins 0.000 abstract description 13
- 229940079593 drug Drugs 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 13
- 206010028980 Neoplasm Diseases 0.000 abstract description 10
- 230000005847 immunogenicity Effects 0.000 abstract description 8
- 231100000053 low toxicity Toxicity 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 108020004459 Small interfering RNA Proteins 0.000 abstract description 6
- 239000002105 nanoparticle Substances 0.000 abstract description 6
- 108020004707 nucleic acids Proteins 0.000 abstract description 6
- 102000039446 nucleic acids Human genes 0.000 abstract description 6
- 150000007523 nucleic acids Chemical class 0.000 abstract description 6
- 231100000419 toxicity Toxicity 0.000 abstract description 6
- 102100037388 Gasdermin-D Human genes 0.000 abstract description 5
- 230000001988 toxicity Effects 0.000 abstract description 5
- 230000001093 anti-cancer Effects 0.000 abstract description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 13
- 201000005202 lung cancer Diseases 0.000 description 13
- 208000020816 lung neoplasm Diseases 0.000 description 13
- 201000007270 liver cancer Diseases 0.000 description 12
- 208000014018 liver neoplasm Diseases 0.000 description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 11
- 238000000684 flow cytometry Methods 0.000 description 11
- 239000013641 positive control Substances 0.000 description 11
- 231100000135 cytotoxicity Toxicity 0.000 description 9
- 230000003013 cytotoxicity Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 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 9
- 238000000034 method Methods 0.000 description 9
- 102000056265 human GSDMD Human genes 0.000 description 8
- 238000003766 bioinformatics method Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 230000030570 cellular localization Effects 0.000 description 6
- 230000008878 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000032258 transport Effects 0.000 description 6
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 5
- 101000911513 Homo sapiens Uncharacterized protein FAM215A Proteins 0.000 description 5
- 102100026728 Uncharacterized protein FAM215A Human genes 0.000 description 5
- 238000004624 confocal microscopy Methods 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 238000000799 fluorescence microscopy Methods 0.000 description 5
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 5
- 230000002194 synthesizing effect Effects 0.000 description 5
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 4
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000004700 cellular uptake Effects 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 210000001700 mitochondrial membrane Anatomy 0.000 description 4
- 230000000149 penetrating effect Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 102000018832 Cytochromes Human genes 0.000 description 2
- 108010052832 Cytochromes Proteins 0.000 description 2
- 108010040476 FITC-annexin A5 Proteins 0.000 description 2
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 230000006882 induction of apoptosis Effects 0.000 description 2
- 230000007762 localization of cell Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000003952 Caspase 3 Human genes 0.000 description 1
- 108090000397 Caspase 3 Proteins 0.000 description 1
- 102000004041 Caspase 7 Human genes 0.000 description 1
- 108090000567 Caspase 7 Proteins 0.000 description 1
- 102000004039 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 1
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940034982 antineoplastic agent Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 231100001141 mammalian cytotoxicity Toxicity 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 231100000324 minimal toxicity Toxicity 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- 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)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Nanotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种细胞穿梭肽及其应用。所述细胞穿梭肽的氨基酸序列如SEQ ID NO.1所示。本发明还公开了一种融合多肽,由所述的细胞穿梭肽与促凋亡多肽KLA偶联。本发明涉及的RKPSSSWFWKPRYK来源于人源GSDMD蛋白质氨基酸序列,因此具有低免疫原性的特点,并且本身具有低毒性,并能将抗癌多肽KLA高效的运送到肿瘤细胞中从而起到抗肿瘤的效果。还可以利用所述细胞穿梭肽将蛋白质、药物、核酸、siRNAs、纳米粒子等运送至肿瘤细胞中从而增强抗肿瘤效果,也可以与抗肿瘤药物偶联,起到降低抗肿瘤药物的使用剂量和毒性,或发挥逆转肿瘤耐药的作用。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种细胞穿梭肽及其应用。
背景技术
肿瘤是危害人类健康的严重疾病之一,目前恶性肿瘤的治疗效果仍不理想,尤其多数恶性肿瘤晚期往往已经失去了手术治疗的时机,只能通过放疗及化疗进行保守治疗,但放疗及化疗副作用大,例如化疗中抗肿瘤化学药物治疗所需的用药剂量较大,毒副作用也大,严重影响了肿瘤患者的化疗效果。肿瘤治疗从基础研究到药物开发的一个难以克服的障碍来源于细胞膜通透性低。因此,改善化疗药物跨膜转位能力将能明显降低抗肿瘤药物的有效剂量,从而减少不良反应。
目前,治疗药物分子的传递不良和生物利用度低是抗肿瘤药物开发过程中的两大主要障碍,尽管电穿孔、显微注射、脂质体转染和病毒载体等传递系统已被广泛应用,但它们仍然存在效率低、细胞毒性大、生物利用度低及不能用于体内研究等缺点。细胞穿梭肽(Cell penetrating peptides,CPPs)能够以无毒的方式将治疗和诊断分子送入细胞内,作为一种有前景的非病毒药物和诊断剂递送工具,受到了相当大的关注。在大多数情况下,CPPs是一类长度为5~30个氨基酸残基组成的小肽,可通过内吞和/或巨噬细胞吞噬作用进行膜转运,而不会对膜造成明显的损伤。CPPs的这种独特能力可以用来改善细胞对各种生物活性分子的吸收,它已成功地应用于体外和体内治疗分子(如多肽、蛋白质、药物、核酸、siRNAs、纳米粒子)的输送。由于CPPs在药物输送方面的巨大应用,鉴定新型高效的CPPs已成为当前研究的热点。虽然以CPPs为基础的药物传递为最安全和最有用的治疗应用提供了巨大的机会,但每个CPP-缀合物的细胞毒性和转运效率取决于多种因素,如CPP氨基酸序列、CPP浓度、孵育时间、温度、细胞类型和药物类型等。
但是目前发现的CPPs由于其细胞毒性大、转运效率低和免疫原性强,其应用仍然局限于狭窄的临床应用。因此,还需进一步寻找低免疫原性、低毒性高转运效率的CPPs,以提高药物如抗肿瘤药物的向细胞内输送的能力。
发明内容
针对现有技术中的缺陷,本发明提出了一种细胞穿梭肽及其应用。本发明涉及的细胞穿梭肽CPPs(多肽序列是RKPSSSWFWKPRYK,简称RYK),是来源于人源GSDMD蛋白质氨基酸序列中的高效低毒的新型CPPs。将RYK融合凋亡诱导肽KLA(具有破坏线粒体膜、诱导癌细胞凋亡的能力,但其穿透真核细胞的能力较差),发现新型RYK能够向肿瘤细胞内输送KLA及提高其融合多肽KLA的抗肿瘤活性。
本发明提供一种细胞穿梭肽,所述细胞穿梭肽的氨基酸序列如SEQ ID NO.1(RKPSSSWFWKPRYK)所示。
本发明还提供一种融合多肽,由所述的细胞穿梭肽与促凋亡多肽KLA偶联,所述融合多肽的氨基酸序列如SEQ ID NO.2(RKPSSSWFWKPRYKGGKLAKLAKKLAKLAK)所示。两条多肽合成时中间用GG连接,GG是把两条多肽连接在一起的连接臂。
本发明还提供一种多肽融合药物,由所述的细胞穿梭肽与抗肿瘤药物偶联。
本发明还提供一种多肽融合蛋白质,由所述的细胞穿梭肽与蛋白质偶联。
本发明还提供一种多肽融合核酸,由所述的细胞穿梭肽与核酸偶联。
本发明还提供一种多肽融合siRNA,由所述的细胞穿梭肽与siRNAs偶联。
本发明还提供一种多肽融合纳米颗粒,由所述的细胞穿梭肽与纳米颗粒偶联。
本发明还提供所述的细胞穿梭肽或所述的融合多肽在制备抗肿瘤药物中的应用。
本发明还提供所述的多肽融合药物或所述的多肽融合蛋白质或所述的多肽融合核酸或所述的多肽融合siRNA或所述的多肽融合纳米颗粒在制备抗肿瘤药物中的应用。
综上,与现有技术相比,本发明达到了以下技术效果:
(1)来源于人源GSDMD蛋白质氨基酸序列,具有高效低毒性和低免疫原性。
(2)利用生物信息学方法预测人源GSDMD蛋白质氨基酸序列中可能存在的CPPs及其特征。
(3)通过流式细胞仪分析不同细胞的摄取能力、荧光显微镜和共聚焦显微镜确认RKPSSSWFWKPRYK具有能进入肿瘤细胞的潜能,而且还能进入肿瘤细胞的细胞核,并且穿梭进入细胞的能力强于公认的阳性对照R8。
(4)结果表明RYK本身对肿瘤细胞是低毒性的,通过将RKPSSSWFWKPRYK与促凋亡多肽KLA偶联,一系列实验结果表明RKPSSSWFWKPRYK具有携带大分子物质KLA进入细胞的能力,且能够引起凋亡相关蛋白的增多从而引起肿瘤细胞的凋亡具有抗肿瘤的作用。说明RKPSSSWFWKPRYK可以将KLA携带进入肿瘤细胞,并不改变KLA的促凋亡作用。
(5)RKPSSSWFWKPRYK融合促凋亡肽KLA后能高效的将KLA运送到肿瘤细胞中并具有明显的抗肿瘤作用。可以将RKPSSSWFWKPRYK-KLA融合肽开发成新的抗癌多肽。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明的技术流程示意图。
图2为本发明实施例1中CellPPD(http://crdd.osdd.net/raghava/cellppd/submission.php)网站预测细胞穿梭肽。
图3为本发明实施例1中根据SVM score对预测到的细胞穿梭肽的序列进行排序,得分越高,是细胞穿梭肽的可能性越大。
图4为本发明实施例2中不同多肽细胞摄取能力的对比。
图5为本发明实施例3中荧光显微镜检测多肽RYK在A549和HpeG2两种细胞系中的定位。
图6为本发明实施例4中共聚焦显微镜检测多肽RYK在A549和HpeG2两种细胞系中的定位。
图7为本发明实施例5中荧光显微镜显示多肽RYK可以将KLA带入A549和HpeG2两种细胞。
图8为本发明实施例5中流式细胞仪分析FITC-KLA-RYK的细胞摄取能力。
图9为本发明实施例5中CCK8法检测多肽对肿瘤细胞的细胞毒性。
图10为本发明实施例5中光学显微镜观察RYK-KLA引起细胞凋亡。
图11为本发明实施例5中流式细胞术仪检测RYK-KLA引起细胞凋亡,具有浓度依赖性。
图12为本发明实施例5中Western blot法检测凋亡相关蛋白的变化。
图13为本发明RYK-KLA引起肿瘤细胞凋亡的机制图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
本发明技术方案如下,如图1流程图所示:
1.生物信息学方法预测GSDMD蛋白质中可能存在的CPPs及其特征。
(1)用两种不同的数据库(Cell PPD web server和C2Pred web server)分别预测GSDMD蛋白质组中可能存在的CPPs,并根据得分由高到低排序。
(2)使用CPPred-RF web server和MLCPP web server评估上一步确定的CPPs的摄取效率。
(3)多肽可以在体内可引起免疫反应,导致机体的过敏反应。因此,使用IEDB webserve评估CPPs的免疫原性。
(4)使用Toxin Pred web server、Aller Top web server和Allergen FP webserver评估CPPs的毒性和致敏性。
(5)使用I-TASSER web server预测CPPs的结构模型,利用NetSurfP web server预测CPPs的二级结构。
2.体外实验检测预测的CPPs的细胞定位和毒性、并定量分析其穿透效率。
(1)Fmoc法合成FITC标记的CPPs(RKPSSSWFWKPRYK,RKPSSSWFWKPR,RKPSSSWFWKPRYKC和R8),由安徽国平药业有限公司合成。
(2)流式细胞仪分析不同细胞穿梭肽的细胞摄取能力。
(3)荧光显微镜筛选能够进入细胞的细胞穿梭肽。
(4)共聚焦显微镜检测CPPs的细胞定位。
3.优选低毒高效的RYK耦联凋亡诱导肽KLA,评估RYK-KLA融合肽的细胞摄取能力、定位及其对肿瘤细胞凋亡的影响。
(1)利用Fmoc法合成FITC标记的多肽系列(FITC-KLA,FITC-R8-KLA,FITC-RYK-KLA),FITC-R8-KLA序列作为阳性对照,由安徽国平药业有限公司合成。CCK8法检测CPPs的细胞毒性。
(2)荧光显微镜和共聚焦显微镜检测FITC-KLA-CPP的细胞摄取、细胞定位。
(3)流式细胞仪分析不同FITC-KLA-CPP的细胞摄取能力。
(4)CCK8法检测KLA-CPP的细胞毒性。
(5)光学显微镜观察RYK-KLA对细胞形态的影响。
(6)流式细胞术仪测定细胞凋亡。
(7)Western blot法检测凋亡相关蛋白的变化。
实施例1生物信息学分析
利用一系列的生物信息学方法在人源GSDMD蛋白质氨基酸序列中筛选新颖有效的细胞穿梭肽,GSDMD蛋白质在NCBI中的参考序列为NP_079012.3。所用到的生物信息学分析网站如表1所示。
表1生物信息学方法预测和分析人源GSDMD蛋白质氨基酸序列中的细胞穿梭肽
人源GSDMD蛋白质氨基酸序列来源的细胞穿梭肽的筛选及特征分析
使用CellPPD网站在人源GSDMD蛋白质氨基酸序列中筛选细胞穿梭肽,只预测了含有10个、15个和20个氨基酸的序列,界面如图2所示。
根据SVM得分进行排序,结果如图3所示,选择得分比较高的前16个多肽进一步分析。其中RKPSSSWFWKPRYK,RKPSSSWFWKPR,RKPSSSWFWKPRYKC,同时被CellPPD和C2Pred以高分预测为细胞穿梭肽。同时利用CPPred-RF和MLCPP两个网站分析16个多肽被细胞摄入的效率,结果如表2~表5所示。
表2预测细胞穿梭肽的穿膜效率
表3分析细胞穿梭肽的特征
表4评估细胞穿梭肽的免疫原性、毒性、抗原性、半衰期和溶血性
表5分析细胞穿梭肽的膜结合能力
综合以上生物信息学的分析结果,选择RKPSSSWFWKPRYK,RKPSSSWFWKPR,RKPSSSWFWKPRYKC三条多肽作为候选的细胞穿梭肽,利用Fmoc法合成FITC标记的多肽系列,并合成FITC标记的R8序列作为阳性对照,由安徽国平药业有限公司合成。以肺癌细胞系A549和肝癌细胞系HepG2作为模型,通过流式细胞仪分析不同细胞的摄取能力、荧光显微镜和共聚焦显微镜确认在细胞中的定位等实验来验证每条多肽的细胞穿膜能力及效率。
实施例2流式细胞仪分析不同细胞穿梭肽的细胞摄取能力
(1)将肺癌A549和肝癌HpeG2两种细胞接种到共聚焦培养皿中(1×104/孔)中,培养24h后,用无血清DMEM洗涤细胞2次,将FITC-CPP(10μM、20μM)在无血清DMEM中37℃孵育1h。
(2)用预冷PBS冲洗3次,然后用胰蛋白酶消化分离,悬浮于400μlPBS中。每个样本的细胞内荧光强度用流式细胞仪(美国美国BD公司)测定。激发波长和检测波长分别为488和525nm。
(3)结果如图4所示,RKPSSSWFWKPRYK,RKPSSSWFWKPR,RKPSSSWFWKPRYKC三条多肽组和A549细胞亲本细胞组相比,都有不同程度的荧光摄入,说明三条多肽都能进入A549细胞中,进入细胞的量因多肽序列的不同而有所不同。和阳性对照组(R8)相比,RKPSSSWFWKPRYK(简称RYK)荧光摄取量多于R8组,说明RYK多肽进入细胞的能力强于阳性对照组。因此选择多肽RKPSSSWFWKPRYK作为进一步探讨的细胞穿梭肽。
实施例3荧光显微镜筛选能够进入细胞的细胞穿梭肽
(1)将肺癌A549和肝癌HpeG2两种细胞接种到24孔板(2×105/孔)中,培养24h后,用无血清DMEM洗涤细胞2次,将FITC-CPP(5μM、10μM、20μM、40μM和80μM)在无血清DMEM中37℃孵育1h。
(2)用PBS洗涤细胞超过三次以上,使用荧光显微镜观察细胞对FITC-CPP的摄取及其细胞定位,并拍照记录。
(3)结果如图5所示,RYK和R8都能进入肺癌A549和肝癌HpeG2两种细胞,都定位于胞浆和胞核。
实施例4共聚焦显微镜检测细胞穿梭肽RYK的细胞定位
(1)将肺癌A549和肝癌HpeG2两种细胞接种到共聚焦培养皿中(1×104/孔)中,培养24h后,用无血清DMEM洗涤细胞2次,将FITC-CPP(5μM、10μM、20μM、40μM和80μM)在无血清DMEM中37℃孵育1h。
(2)用PBS洗涤细胞超过三次以上,使用共聚焦显微镜观察细胞对FITC-CPP的摄取及其细胞定位,并拍照记录。
(3)结果如图6所示,30min时10μM的RYK可以进入A549和HpeG2两种细胞系中细胞核,而10μM的R8 3h时才能进入细胞,说明RYK进入细胞的效能要强于阳性对照R8,并且还能进入细胞核。
以上结果说明RKPSSSWFWKPRYK具有能进入肿瘤细胞的潜能,而且还能进入肿瘤细胞的细胞核,并且穿梭进入细胞的能力强于公认的阳性对照R8。下面通过实验来探究RKPSSSWFWKPRYK是否具有携带大分子物质进入细胞的能力。
实施例5本发明的细胞穿梭肽(CPPs)能够提高抗癌多肽KLA抗肿瘤的作用
凋亡抑制和无限增殖是肿瘤细胞的主要特征。因此,诱导肿瘤细胞凋亡是治疗肿瘤的主要目标之一。线粒体是细胞凋亡和细胞死亡的调控中心,在抗肿瘤药物的研究中,线粒体已成为诱导肿瘤细胞凋亡的药物作用靶点之一。作为一条经典的双亲性抗癌多肽,KLA([KLAKLAK]2)最开始是作为一条具有较低哺乳动物细胞毒性α-螺旋抗菌肽被设计出来的。它可以结合并破坏带负电荷的细菌膜;然而,它通常不能破坏真核细胞质膜,因此对真核细胞几乎没有细胞毒性。另一方面,KLA通过形成α螺旋破坏真核细胞线粒体膜的完整性触发线粒体膜破裂,导致细胞色素C释放并诱导细胞凋亡。由于进入真核细胞的效率很低,KLA本身具有很低的肿瘤细胞毒性。故目前的研究集中在提高KLA的细胞吸收效率上,将KLA连接CPPs极大地提高了KLA的抗肿瘤能力。因此,寻找低免疫原性、低毒性高转运效率的CPPs,也是目前抗肿瘤药物研究的热点之一。本实施例将KLA连接RYK,探索RYK是否能将KLA运送至肿瘤细胞中从而引起肿瘤细胞的凋亡进而发挥KLA的抗肿瘤特性。
利用Fmoc法合成FITC标记的多肽系列(FITC-KLA,FITC-R8-KLA,FITC-RYK-KLA),FITC-R8-KLA序列作为阳性对照,由安徽国平药业有限公司合成。以肺癌细胞系A549和肝癌细胞系HepG2作为模型,以此来验证RYK携带KLA进入细胞能力及效率。
(一)荧光显微镜和共聚焦显微镜检测FITC-KLA-CPP的细胞摄取、细胞定位。
(1)将肺癌A549和肝癌HpeG2两种细胞接种到24孔板(2×105/孔)中,培养24h后,用无血清DMEM洗涤细胞2次,分别加入10μM的FITC-KLA-R8和FITC-KLA-RYK在无血清DMEM中37℃孵育1h。
(2)用预冷PBS洗涤细胞超过三次以上,分别使用荧光显微镜和共聚焦显微镜观察细胞对FITC-KLA-CPP的摄取及其细胞定位,并拍照记录。
(3)结果如图7所示,KLA本身几乎不能进入A549和HpeG2两种细胞,而KLA和RYK偶联后可以进入两种细胞中,并且进入的量要多于KLA和R8偶联。此结果说明RYK可以携带KLA进入细胞中,就有细胞穿梭肽的功能。
(二)流式细胞仪分析FITC-KLA-RYK的细胞摄取能力。
(1)将肺癌A549和肝癌HpeG2两种细胞接种到6孔板(2×105/孔)中,培养24h后,用无血清DMEM洗涤细胞2次,分别加入10μM的FITC-KLA-R8和FITC-KLA-RYK在无血清DMEM中37℃孵育1h。用预冷PBS冲洗3次,然后用胰蛋白酶消化分离,悬浮于400μl PBS中。每个样本的细胞内荧光强度用流式细胞仪(美国美国BD公司)测定。
(2)结果如图8所示,与亲本细胞(A549和HpeG2两种细胞,control)相比,KLA进入细胞的量很少,而RYK-KLA进入细胞的量很多,且多于R8-KLA进入细胞的量。说明RYK携带KLA进入细胞的效率要大于阳性对照R8携带KLA的能力。
(三)CCK8法检测KLA-CPP的细胞毒性。
(1)将肺癌A549和肝癌HpeG2两种细胞分别接种到96孔板中(3×103/孔)中,细胞贴壁后,分别用不同浓度的RYK、R8、KLA、RYK-KLA和R8-KLA(10μM、20μM、20μM和40μM)处理24h。5%CO2,37℃培养细胞24h(培养时间根据细胞种类的不同和每孔内细胞数量的多少而异)。
(2)如果起始的培养液体积为200μl,则需加入20μl CCK-8溶液,其它情况以此类推。
(3)在细胞培养箱内继续孵育24h后,在450nm测定吸光度。
(3)结果如图9所示,RYK、R8、KLA三种多肽对A549和HpeG2两种肿瘤细胞几乎没有毒性作用,且RYK的毒性最低。RYK-KLA和R8-KLA两种多肽都能明显抑制A549和HpeG2两种肿瘤细胞的增殖活性,而且RYK-KLA的抑制能力要强于R8-KLA(图9和表6)。
表6R8-KLA和RYK-KLA在A549和HpeG2培养24小时的IC50值
(四)光学显微镜观察RYK-KLA对细胞形态的影响。
(1)将肺癌A549细胞接种到6孔板中(2×105/孔)中,细胞贴壁后,分别用20μM的RYK、R8、KLA、RYK-KLA和R8-KLA处理细胞,5%CO2,37℃培养细胞24h。
(2)用预冷PBS冲洗3次,光学显微镜观察并拍照。
(3)结果如图10所示,RYK、R8、KLA三种多肽对A549和HpeG2两种肿瘤细胞的形态几乎没有影响,但是RYK-KLA和R8-KLA两种多肽都能明显引起两种肿瘤细胞形态发生凋亡的改变,说明RYK和R8能够将KLA携带进入细胞内,从而发挥KLA诱导细胞发生凋亡。
(五)流式细胞术测定细胞凋亡。
(1)肺癌A549和肝癌HpeG2两种细胞经不同浓度的RYK-KLA(10μM、20μM和40μM)处理24h后,用Annexin v-FITC和PI染色法检测细胞的凋亡情况。用预冷PBS冲洗3次,细胞重新悬浮在含Annexin v-FITC和5μlPI的500μl结合液中,在室温条件下避光孵育30min,然后用流式细胞仪测定细胞凋亡。
(2)结果如图11所示,10μM的RYK-KLA可以引起A549和HpeG2两种细胞发生凋亡,并且会随着RYK-KLA浓度的增加凋亡的细胞数也增多。
(六)Western blot法检测凋亡相关蛋白的变化
(1)将肺癌A549和肝癌HpeG2两种细胞接种到10cm培养皿中培养24h后,用无血清DMEM洗涤细胞2次,经不同浓度的RYK-KLA(10μM、20μM和40μM)处理24h后,用预冷PBS冲洗3次,用蛋白裂解液提取总蛋白并定量。SDS聚丙烯酰胺凝胶电泳分离,电泳转移到PVDF膜上。用抗PARP、Cytochrome C和Caspase-3抗体进行标准Western blot分析。用辣根过氧化物酶联免疫二抗探针检测膜。利用增强化学发光(ECL)和紫外产物成像系统对检测到的蛋白质进行显像。
(2)结果如图12所示,随着RYK-KLA浓度的增加,可以引起A549和HpeG2两种细胞中的凋亡相关活性蛋白Cytochrome C、cleaved-PARP、Procaspase-9和Procaspase-7逐渐增多,从而导致细胞发生凋亡,可能的机制如图13所示。
综合以上实施例,本发明的技术路线如下:
(a)利用生物信息学方法预测人源GSDMD蛋白质氨基酸序列中可能存在的CPPs及其特征。
(b)综合生物信息学的分析结果,选择RKPSSSWFWKPRYK,RKPSSSWFWKPR,RKPSSSWFWKPRYKC三条多肽作为候选的细胞穿梭肽,利用Fmoc法合成FITC标记的多肽系列,并合成FITC标记的R8序列作为阳性对照,由安徽国平药业有限公司合成。以肺癌细胞系A549和肝癌细胞系HepG2作为模型,通过流式细胞仪分析不同细胞的摄取能力、荧光显微镜和共聚焦显微镜确认在细胞中的定位等实验来验证每条多肽的细胞穿膜能力及效率。
(c)利用凋亡诱导肽KLA具有破坏线粒体膜、诱导癌细胞凋亡的能力,但其穿透真核细胞的能力较差的特点,将RKPSSSWFWKPRYK偶联至KLA,探索RKPSSSWFWKPRYK是否能将KLA运送至肿瘤细胞中从而引起肿瘤细胞的凋亡进而发挥KLA的抗肿瘤特性。
综合以上结果,能够得出以下结论:
(1)来源于人源GSDMD蛋白质氨基酸序列中的RKPSSSWFWKPRYK具有进入肿瘤细胞的潜能,而且还能进入肿瘤细胞的细胞核,并且进入细胞的能力强于公认的阳性对照R8。
(2)通过将RKPSSSWFWKPRYK与促凋亡多肽KLA偶联,一系列实验结果表明RKPSSSWFWKPRYK具有携带大分子物质进入细胞的能力,它是一种新颖的细胞穿梭肽,并且具有高效低毒的特点。
(3)RYK-KLA进入肿瘤细胞后能够引起凋亡相关蛋白的增多从而引起肿瘤细胞的凋亡具有抗肿瘤的作用,并不改变KLA的促凋亡作用,RYK-KLA可以开发成抗肿瘤多肽,为肿瘤的治疗提高新的方法。
本发明中涉及到的细胞穿梭肽(多肽序列:RKPSSSWFWKPRYK)主要用于将不能穿过细胞膜进入细胞内的生物大分子运送至细胞内,而且它具有高效性、低毒性和低免疫原性的特点,可以利用它将蛋白质、药物、核酸、siRNAs、纳米粒子等运送至肿瘤细胞中从而增强抗肿瘤效果。也可以与抗肿瘤药物偶联,起到降低抗肿瘤药物的使用剂量和毒性,或发挥逆转肿瘤耐药的作用。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.一种细胞穿梭肽,其特征在于,所述细胞穿梭肽的氨基酸序列如SEQ ID NO.1所示。
2.一种融合多肽,其特征在于,由权利要求1所述的细胞穿梭肽与促凋亡多肽KLA偶联,所述融合多肽的氨基酸序列如SEQ ID NO.2所示。
3.权利要求2所述的融合多肽在制备抗肿瘤药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310350766.1A CN116589555B (zh) | 2023-03-31 | 2023-03-31 | 一种细胞穿梭肽及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310350766.1A CN116589555B (zh) | 2023-03-31 | 2023-03-31 | 一种细胞穿梭肽及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116589555A CN116589555A (zh) | 2023-08-15 |
CN116589555B true CN116589555B (zh) | 2024-02-06 |
Family
ID=87603376
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310350766.1A Active CN116589555B (zh) | 2023-03-31 | 2023-03-31 | 一种细胞穿梭肽及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116589555B (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105112383A (zh) * | 2015-08-25 | 2015-12-02 | 三峡大学 | 细胞膜穿透肽hPP5及其用途 |
CN105175526A (zh) * | 2015-08-25 | 2015-12-23 | 三峡大学 | 细胞膜穿透肽hPP8及其用途 |
CN107827953A (zh) * | 2017-11-08 | 2018-03-23 | 哈尔滨商业大学 | 一种靶向穿膜肽、具有其的药物以及应用 |
CN112533640A (zh) * | 2018-05-04 | 2021-03-19 | 双亥生命科学株式会社 | 利用基于蜂毒肽的细胞凋亡诱导肽的m2型肿瘤相关巨噬细胞的靶向 |
CN113773394A (zh) * | 2020-06-10 | 2021-12-10 | 山东大学 | 一种融合肽及在制备抗肿瘤制剂中的应用 |
-
2023
- 2023-03-31 CN CN202310350766.1A patent/CN116589555B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105112383A (zh) * | 2015-08-25 | 2015-12-02 | 三峡大学 | 细胞膜穿透肽hPP5及其用途 |
CN105175526A (zh) * | 2015-08-25 | 2015-12-23 | 三峡大学 | 细胞膜穿透肽hPP8及其用途 |
CN107827953A (zh) * | 2017-11-08 | 2018-03-23 | 哈尔滨商业大学 | 一种靶向穿膜肽、具有其的药物以及应用 |
CN112533640A (zh) * | 2018-05-04 | 2021-03-19 | 双亥生命科学株式会社 | 利用基于蜂毒肽的细胞凋亡诱导肽的m2型肿瘤相关巨噬细胞的靶向 |
CN113773394A (zh) * | 2020-06-10 | 2021-12-10 | 山东大学 | 一种融合肽及在制备抗肿瘤制剂中的应用 |
Also Published As
Publication number | Publication date |
---|---|
CN116589555A (zh) | 2023-08-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2018000657A1 (zh) | 一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒 | |
Meller et al. | TH17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26 | |
Krull et al. | Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket | |
Eike et al. | The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells | |
US9499604B2 (en) | Trail mutant membrane-penetrating peptide-alike and methods of preparation thereof | |
Guo et al. | Proinflammatory macrophage-derived microvesicles exhibit tumor tropism dependent on CCL2/CCR2 signaling axis and promote drug delivery via SNARE-mediated membrane fusion | |
CN114053427B (zh) | 一种多肽靶向药物及其制备方法和应用 | |
CN103304637B (zh) | 细胞穿膜肽hPP3及其用途 | |
CN105112383A (zh) | 细胞膜穿透肽hPP5及其用途 | |
CN111905105B (zh) | 一种用于癌症靶向治疗的蛋白类纳米药物及其制备方法 | |
CN113956342B (zh) | 一种肿瘤新生抗原多肽及其应用 | |
Nan et al. | A comprehensive assessment of the biocompatibility of Magnetospirillum gryphiswaldense MSR-1 bacterial magnetosomes in vitro and in vivo | |
CN116589555B (zh) | 一种细胞穿梭肽及其应用 | |
Christiansen et al. | The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage | |
Shen et al. | Benzyl stapled modification and anticancer activity of antimicrobial peptide A4K14-Citropin 1.1 | |
EP3266796B1 (en) | Trail membrane-penetrating peptide-like mutant mur5, preparation method therefor, and application thereof | |
WO2023093303A1 (zh) | 靶向p53的多肽及其在制备用于治疗癌症的药物中的应用 | |
CN110669145B (zh) | 三聚体trail融合蛋白基因修饰间充质干细胞的方法及其用途 | |
Xia et al. | Novel Peptide CM 7 Targeted c-met with antitumor activity | |
WO2012169911A1 (en) | Peptides, constructs and uses therefor | |
Huo et al. | Potential Resistance to Antineoplastic Aminated Fullerenes Mediated by M2-Like Monocyte-Derived Exosomes | |
US6232443B1 (en) | Renal nuclear matrix proteins, polynucleotide sequences encoding them, and their use | |
CN118271465A (zh) | Trx-SPRR2A蛋白及其在制备抗肿瘤药物中的应用 | |
CN112239487A (zh) | 一种靶向抗凋亡蛋白bfl-1的稳定多肽类蛋白共价抑制剂 | |
Salas-Huenuleo et al. | A simple method to label vesicles for visualization and in vivo tracking |
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 |