CN116376917A - 核酸适体SWL-3G、SWL-3bG在制备治疗膀胱癌药物中的应用 - Google Patents
核酸适体SWL-3G、SWL-3bG在制备治疗膀胱癌药物中的应用 Download PDFInfo
- Publication number
- CN116376917A CN116376917A CN202211305767.6A CN202211305767A CN116376917A CN 116376917 A CN116376917 A CN 116376917A CN 202211305767 A CN202211305767 A CN 202211305767A CN 116376917 A CN116376917 A CN 116376917A
- Authority
- CN
- China
- Prior art keywords
- swl
- aptamer
- bladder cancer
- cells
- gemcitabine
- 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
- 206010005003 Bladder cancer Diseases 0.000 title claims abstract description 43
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 title claims abstract description 41
- 201000005112 urinary bladder cancer Diseases 0.000 title claims abstract description 41
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 4
- 239000003814 drug Substances 0.000 title abstract description 13
- 229940079593 drug Drugs 0.000 title abstract description 11
- 108091008104 nucleic acid aptamers Proteins 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 238000012217 deletion Methods 0.000 claims description 6
- 230000037430 deletion Effects 0.000 claims description 6
- 102000014914 Carrier Proteins Human genes 0.000 claims description 5
- 108091008324 binding proteins Proteins 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims 2
- 239000000126 substance Substances 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 238000007792 addition Methods 0.000 claims 1
- 239000011616 biotin Substances 0.000 claims 1
- 229960002685 biotin Drugs 0.000 claims 1
- 235000020958 biotin Nutrition 0.000 claims 1
- 239000000941 radioactive substance Substances 0.000 claims 1
- 238000006467 substitution reaction Methods 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 229960005277 gemcitabine Drugs 0.000 abstract description 24
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 abstract description 23
- 230000027455 binding Effects 0.000 abstract description 22
- 206010028980 Neoplasm Diseases 0.000 abstract description 13
- 201000011510 cancer Diseases 0.000 abstract description 10
- 101001135344 Homo sapiens Polypyrimidine tract-binding protein 1 Proteins 0.000 abstract description 6
- 102100033073 Polypyrimidine tract-binding protein 1 Human genes 0.000 abstract description 6
- 108020004707 nucleic acids Proteins 0.000 abstract 1
- 102000039446 nucleic acids Human genes 0.000 abstract 1
- 150000007523 nucleic acids Chemical class 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 96
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 28
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 14
- 239000008103 glucose Substances 0.000 description 14
- 229910001629 magnesium chloride Inorganic materials 0.000 description 14
- MJQHZNBUODTQTK-WKGBVCLCSA-N (2s,3r,4s,5r,6r)-2-[[(1s,3s,4s,5s,8r)-3-[(2s,3r,4s,5s,6r)-2-[[(1s,3r,4s,5s,8r)-3,4-dihydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-4-hydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-6-(hydroxymethyl)oxane-3,4,5- Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H]2OC[C@@H]1O[C@@H](O[C@@H]1[C@H]([C@H](O[C@H]3[C@H]4OC[C@@H]3O[C@@H](O)[C@H]4O)O[C@H](CO)[C@@H]1O)O)[C@H]2O MJQHZNBUODTQTK-WKGBVCLCSA-N 0.000 description 12
- 238000011534 incubation Methods 0.000 description 12
- 239000011534 wash buffer Substances 0.000 description 10
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 9
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 9
- 239000002246 antineoplastic agent Substances 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 238000011580 nude mouse model Methods 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 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 6
- 230000008901 benefit Effects 0.000 description 6
- 229940044683 chemotherapy drug Drugs 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- QHPQWRBYOIRBIT-UHFFFAOYSA-N 4-tert-butylphenol Chemical compound CC(C)(C)C1=CC=C(O)C=C1 QHPQWRBYOIRBIT-UHFFFAOYSA-N 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 5
- 239000012148 binding buffer Substances 0.000 description 5
- 210000005068 bladder tissue Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 108010052285 Membrane Proteins Proteins 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 230000037429 base substitution Effects 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 238000001917 fluorescence detection Methods 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108010019372 Heterogeneous-Nuclear Ribonucleoproteins Proteins 0.000 description 3
- 102000006479 Heterogeneous-Nuclear Ribonucleoproteins Human genes 0.000 description 3
- 102000018697 Membrane Proteins Human genes 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000012377 drug delivery Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 208000002109 Argyria Diseases 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 230000001114 myogenic effect Effects 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 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 2
- 230000004083 survival effect Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- 101710159080 Aconitate hydratase A Proteins 0.000 description 1
- 101710159078 Aconitate hydratase B Proteins 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 1
- 101710105008 RNA-binding protein Proteins 0.000 description 1
- 108091008105 X-aptamers Proteins 0.000 description 1
- 238000011226 adjuvant chemotherapy Methods 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 238000009096 combination chemotherapy Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000014 effect on malignancy Effects 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940026778 other chemotherapeutics in atc Drugs 0.000 description 1
- 102000015585 poly-pyrimidine tract binding protein Human genes 0.000 description 1
- 108010063723 poly-pyrimidine tract binding protein Proteins 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
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
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- 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
- C12N2310/16—Aptamers
-
- 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)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Public Health (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种检测人膀胱癌细胞的核酸适体,其核苷酸序列如SEQ ID NO.1至SEQ ID NO.6任一所示,以及两种核酸适体‑药物结合物在制备治疗膀胱癌药物中的应用。本发明获得了膀胱癌特异性结合适体SWL‑3,发现PTBP1蛋白是适体SWL‑3的靶标,其截短链SWL‑3b比SWL‑3具有更优的结合能力。本发明将膀胱癌治疗一线药物吉西他滨与SWL‑3、SWL‑3b结合,构建了两种核酸适体‑药物结合物SWL‑3G、SWL‑3bG,改变了吉西他滨内化到细胞的途径,提高了吉西他滨对于膀胱癌细胞的选择性。本发明为膀胱癌的诊断和治疗提供了一种很有前景的工具,并为有效控制癌症提供了新思路。
Description
技术领域
本发明涉及一种核酸适体及其检测、治疗应用,特别是涉及一种可用于人膀胱癌细胞及临床样本组织检测的核酸适体及基于该核酸适体与化疗药物偶联用于膀胱癌的检测与治疗。
背景技术
膀胱癌指的是发生在膀胱黏膜上的恶性肿瘤,是泌尿系统发病率最高的肿瘤,其发病率在所有肿瘤中排第12位,根据美国癌症协会调查数据显示,2020年全球膀胱癌新发病例有573278例,死亡病例有212536例,其中,在亚洲地区,男性的发病率约是女性的四倍。虽然 70%-85% 的患者最初被诊断为非肌层浸润性膀胱癌 (NMIBC),但这些患者中的大多数复发和进展为肌层浸润性膀胱癌(MIBC)。此外,癌症会发生远处转移,转移患者的5年生存率极低。对于这些患者,主要的临床治疗是手术联合化疗。化疗仍然是延长膀胱癌患者生存时间的唯一选择。常用的化疗药物有丝裂霉素C、表柔比星、吉西他滨等。然而,这些化疗药物亟待解决的问题是靶向性差。因此,迫切需要一种具有更高敏感性和特异性的分子探针用于膀胱癌的诊断和靶向治疗。
核酸适体(nucleic acid aptamer) 是与靶分子特异性结合的单链寡核苷酸,靶标范围广,包括小分子、离子或蛋白质等。核酸适体与某些靶蛋白结合后可以内化到细胞中,并已被研究作为药物载体。与抗体不同,核酸适体具有许多优点,例如易于合成、价格低廉、稳定性好、易于修饰、高亲和力和低免疫原性。所有这些优点使核酸适体有希望成为分子探针和靶向部分。在过去的几年中,基于核酸适体-药物偶联物 (ApDC) 的靶向给药已被广泛用于提高化疗药物的安全性和有效性。
吉西他滨作为一种广泛使用的化学治疗药物,本质上是一种胞嘧啶核衍生物,对各种恶性肿瘤具有广泛的抗肿瘤活性。由于与其他化疗药物相比毒性相对较低,吉西他滨联合顺铂已成为晚期膀胱癌辅助化疗的一线方案。然而,由于吉西他滨的生物半衰期短、生物利用度低、代谢失活快和靶标特异性差,其临床获益有限。已经探索了各种药物递送系统,包括纳米颗粒、脂质体、气溶胶、肽和核酸适体,以克服吉西他滨的这些缺点。在递送系统中,基于核酸适体-药物偶联物(ApDC)的靶向药物递送已成为提高化疗药物疗效和安全性的潜在策略。
多嘧啶束结合蛋白1(PTBP1;也称为PTB或hnRNP1)是一种RNA结合蛋白,属于异质核核糖核蛋白(hnRNPs)家族,参与调控mRNA剪接、翻译、稳定性和定位等细胞内功能。生物学过程。研究表明,PTBP1在多种肿瘤组织或细胞中高表达,促进肿瘤细胞生长、侵袭和转移。虽然它对恶性肿瘤的影响似乎与细胞类型有关,但它仍然在肿瘤的恶性特征中发挥着至关重要的作用,可能是癌症治疗的靶点。
发明内容
本发明人建立了一个新的文库——Swan Library 2.0,该文库均由本发明人课题组前期对不同癌症组织筛选所得到的众多序列已知但功能不明确的ssDNA组成。SwanLibrary 2.0文库目前包含近50条ssDNA,且数量正在不断增加。与普通的筛选文库相比的优点,我们的Swan Library 2.0文库的序列较短,均来自X-Aptamer微球库中的序列,该文库序列前期经过生物信息学的结构分析预测和化学修饰,在提高筛选效率及核酸适体的功能性方面具有一定的优势。
我们将人膀胱癌细胞系5637、T24和其对照细胞人胚胎膀胱组织来源细胞CCC-HB-2分别与Swan Library中的ssDNA进行结合验证,进行数十次的结合验证实验后,最终得到一条与人膀胱癌细胞系5637、T24结合且不与人胚胎膀胱组织来源细胞CCC-HB-2结合的ssDNA,并将其命名为SWL-3。同时,我们对SWL-3进行截短分析,发现SWL-3b也能够保持原有序列SWL-3的结合能力。
SWL-3全序列如下:
5’-TTTTTAACACGACTCGAGTTCGTCTCCCGAACCACACGCGTGTGGGCCCATG -3’(SEQ ID NO.1)
SWL-3b的核苷酸序列如下:
5’-TTTTTAACACGACTCGAGTTCGAACCACACGCGTGTGGGCCCATG-3’(SEQ ID NO .3)
通过对SWL-3的质谱结果分析以及验证发现,SWL-3的靶标为多嘧啶束结合蛋白1(PTBP1)。通过Westernblot实验发现,PTBP1在膀胱癌细胞T24和5637中高表达,而在786-O以及HEK293细胞中低表达。
通过将化疗药物吉西他滨分别与特异性核酸适体SWL-3、SWL-3b进行碱基替换,设计并合成了一种基于核酸适体功能化小分子药物,即核酸适体-吉西他滨结合物SWL-3G和SWL-3bG。其中,吉西他滨的碱基替换方式为:从5’端数,将SWL-3的第22至28处的所有C碱基替换为吉西他滨;从5’端数,将SWL-3b的第15至26处所有C碱基替换为吉西他滨。SWL-3G、SWL-3bG具有与原链相同的识别能力,并且通过流式竞争实验发现,SWL-3G、SWL-3bG的结合靶标仍为PTBP1。通过细胞毒性实验发现,SWL-3G、SWL-3bG具有很好的靶向性,选择性的杀伤T24细胞并降低了药物的毒副性作用。因此,这些结果可能为利用SWL-3G、SWL-3bG开发新的膀胱癌靶向治疗药物提供思路及策略。因而,本发明的目的首先是提供一种高度特异性、稳定性、可用于人膀胱癌检测的核酸适体及其制备检测试剂的应用方法,并且提供一种能靶向运输抗癌药物到细胞内的载药系统。
与现有技术相比,本发明的优点在于:本发明通过Swan Library 2.0文库得到的核酸适体亲和力与特异性高;无免疫原性;能够体外化学合成,分子量小,可以对不同部位进行修饰和取代,且序列稳定、易于保存、便于标记等优势。采用本发明的核酸适体的靶标明确,为多嘧啶束结合蛋白1(PTBP1),可以有针对性地进行设计与改造。将本发明通过筛选到的核酸适体与化疗药物吉西他滨进行碱基替换,可以提高吉西他滨的靶向性,降低其对正常细胞的杀伤作用。
附图说明
图1为swan library中的ssDNA与人膀胱癌细胞系T24的结合情况。
图2为流式检测SWL-3分别与膀胱癌细胞5637、膀胱癌细胞T24、对照细胞人胚胎膀胱组织来源细胞CCC-HB-2的结合情况。
图3为共聚焦检测SWL-3与膀胱癌细胞5637、膀胱癌细胞T24、对照细胞人胚胎膀胱组织来源细胞CCC-HB-2的结合情况。
图4 SWL-3应用于膀胱癌裸鼠模型活体成像。
图5为SWL-3的靶标鉴定,其中,A为下拉蛋白的银染分析结果图,B为下拉蛋白的Westernblot分析结果图。
图6为SWL-3序列经过一系列的删减后与膀胱癌细胞T24、膀胱癌细胞5637、对照细胞HEK293的结合情况。
图7为SWL-3G、SWL-3bG分别与人膀胱癌细胞系T24、5637细胞、人胚胎肾细胞HEK293的结合情况分析。
图8为SWL-3G、SWL-3bG与SWL-3的流式竞争结合分析,其中,A为用浓度梯度的无标记的SWL-3与FAM标记的SWL-3G以及FAM标记的SWL-3bG的流式竞争结果示意图,B为用浓度梯度的无标记的Library与FAM标记的SWL-3G以及FAM标记的SWL-3bG的流式竞争结果示意图。
图9为吉西他滨对人膀胱癌细胞系T24、人胚胎肾细胞HEK293的毒性分析,其中,A为浓度梯度的吉西他滨对T24细胞的杀伤效果,B为浓度梯度的吉西他滨对HEK293细胞的杀伤效果。
图10为SWL-3G、SWL-3bG分别对人膀胱癌细胞系T24、人胚胎肾细胞HEK293的毒性分析。
具体实施方式
以下的实施例便于更好的理解本发明,但并不限定于本发明。以下实施例中的实验方法如无特殊说明,均为常规方法。下属实施例中所用的实验材料如无特殊说明,均来自常规生化试剂商店购买所得到的。
细胞来源:本实验所用到的细胞系人膀胱癌细胞5637、人膀胱移形细胞癌细胞T24、正常的人胚胎膀胱组织来源细胞CCC-HB-2、正常的人胚胎肾细胞HEK293均来源于中科院上海细胞库。
实施例1:确定与T24细胞系结合能力最强的序列
首先,用0.2%EDTA将贴壁状态的T24细胞从培养皿上消化下来,分别将细胞收集到离心管中,并用洗涤缓冲液(PBS,含0.45 % 的葡萄糖,5 mM氯化镁)离心洗涤几次;其次,将终浓度为250nM的Swan Library 2.0中的所有序列分别加入到结合缓冲液(D-PBS,含0.45%的葡萄糖,5 mM 氯化镁,100mg/L tRNA,1g/L BSA)重悬的T24细胞中;然后放置于4℃摇床孵育 60min;孵育完成之后并用洗涤缓冲液(PBS,含0.45 % 的葡萄糖,5 mM氯化镁)离心洗涤两次,并通过流式细胞仪进行荧光检测,结果如图1所示,位移最大的被认为与T24细胞结合能力最强的序列,我们将其命名为SWL-3。
实施例2 :SWL-3分别特异性识别5637细胞和T24细胞
首先,用0.2%EDTA分别将贴壁状态的5637细胞、T24细胞和CCC-HB-2细胞从培养皿上消化下来,分别将细胞收集到离心管中,并用洗涤缓冲液(PBS,含 0.45 % 的葡萄糖,5mM氯化镁)离心洗涤几次;分别向5637细胞、T24细胞和CCC-HB-2细胞中加入等量的结合缓冲液(D-PBS,含0.45%的葡萄糖,5 mM 氯化镁,100mg/L tRNA,1g/L BSA),并分别投入终浓度为250nMSWL-3与对照序列;然后放置于4℃摇床孵育60min;孵育完成之后并用洗涤缓冲液(PBS,含0.45 % 的葡萄糖,5 mM氯化镁)离心洗涤两次,并通过流式细胞仪进行荧光检测。图2结果显示SWL-3分别与5637细胞、T24细胞的结合具有高亲和力且特异性,而几乎不与CCC-HB-2细胞结合。
其次,对SWL-3、对照文库链进行预处理。用灭菌水将其稀释到终浓度为 400nM,然后在95℃金属浴中变性5min,冰上复性10min,然后置于常温。同时分别对5637细胞、T24细胞、CCC-HB-2细胞进行预处理。分别用DPBS清洗5637细胞、T24细胞、CCC-HB-2细胞 2-3次。最后分别在5637细胞、T24细胞、CCC-HB-2细胞中加入预处理后的候选序列和结合缓冲液(Binding Buffer:DPBS,0 .45%的葡萄糖,5 mM 氯化镁,100mg/L tRNA,1g/L BSA)。然后将处理好的细胞样品放置于4℃摇床孵育1h。孵育完成之后再用洗涤缓冲液(PBS,含0 .45 %的葡萄糖,5 mM氯化镁)清洗3次,并通过激光共聚焦进行荧光检测,结果见图3。
实施例3:3-吲哚菁类染料(Cy5)标记的SWL-3应用于膀胱癌裸鼠模型活体成像
购买四周龄的裸鼠,喂养几天以适应新环境后,往每只裸鼠皮下植入5×107个T24细胞,待2周后裸鼠长成瘤。将Cy5标记的SWL-3、文库序列25μmol/L分别通过尾静脉注射到不同植瘤裸鼠中,用小动物活体成像仪进行拍照观察。结果如图4所示,Cy5标记的SWL-3在裸鼠肿瘤部位有富集,文库序列没有富集。
实施例4:核酸适体SWL-3靶标为多嘧啶束结合蛋白1(PTBP1)
(1)将T24细胞接种在10cm培养皿中,培养24h,约为30个皿左右。吸弃旧培养基,DPBS清洗两次。细胞中加入EDTA室温消化,后将EDTA吸弃,用DPBS将细胞吹打下来,收集在15ml离心管中。用洗涤缓冲液(PBS,含0.45 % 的葡萄糖,5 mM氯化镁)离心洗涤,弃上清。向15mL离心管中加入膜蛋白抽提液,离心,14000g,5min,收集的上清即为细胞全膜蛋白溶液。
取三个1.5mL EP管,分别加入100μl的琼脂糖凝胶珠,离心5500rpm,5min,分别标记为空白组琼脂糖凝胶珠、文库组琼脂糖凝胶珠、SWL-3组琼脂糖凝胶珠。向每个EP管中加入1mL的5%的BSA,震荡混匀,在4℃摇床封闭1h。封闭完成后,用洗涤缓冲液(PBS,含0.45 %的葡萄糖,5 mM氯化镁)洗涤5次,5500rpm,4℃,5 min,置于冰上待用。向收集的蛋白样品中加入3mL的DNA封闭液,震荡匀后在4℃,孵育1h,封闭完之后,取出适量留作全膜蛋白样品组。将封闭之后的全膜蛋白按照空白琼脂糖凝胶珠样本组、文库链、文库组琼脂糖凝胶珠、SWL-3、SWL-3组琼脂糖凝胶珠的顺序于4℃摇床孵育1h。孵育完成后,用洗涤缓冲液(PBS,含0.45 % 的葡萄糖,5 mM氯化镁)对空白组琼脂糖凝胶珠、文库组琼脂糖凝胶珠、SWL-3组琼脂糖凝胶珠分别洗涤5次,5500rpm,4℃,5 min。之后加入与琼脂糖凝胶珠等体积的2×loading buffer,100℃变性10min,置于冰上10min,5500rpm,4℃,5 min后,进行银染和Westernblot的分析。结果如图5所示,SWL-3下拉蛋白泳道在60KDA条带处出现了一条明显的差异条带,而文库组琼脂糖凝胶珠和空白组琼脂糖凝胶珠均未出现,用PTBP1抗体进行Westernblot分析发现,在57KDa处,只有SWL-3下拉蛋白泳道出现了特异性的条带。因此证明,PTBP1是SWL-3的靶标。
实施例5:SWL-3序列全序列的删减方法
为了探究哪一段序列分别是5637细胞、T24细胞结合关键区域,我们通过如下的删减方法进行序列的优化:
SWL-3全序列为:
5’-TTTTTAACACGACTCGAGTTCGTCTCCCGAACCACACGCGTGTGGGCCCATG -3’(SEQ ID NO.1)
SWL-3a:(左边删减8个碱基,右边删除7个碱基)
5’-ACGACTCGAGTTCGTCTCCCGAACCACACGCGTGTGG-3’(SEQ ID NO .2)
SWL-3b:(中间删减7个碱基)
5’-TTTTTAACACGACTCGAGTTCGAACCACACGCGTGTGGGCCCATG-3’(SEQ ID NO .3)
SWL-3c:(中间删减11个碱基)
5’-TTTTTAACACGACTCGAGTTCGTCTCCCGAACCGGCCCATG-3’ (SEQ ID NO .4)
SWL-3d:(左边删减13个碱基)
5’-TCGAGTTCGTCTCCCGAACCACACGCGTGTGGGCCCATG-3’(SEQ ID NO .5)
SWL-3e:(右边删减11个碱基)
5’-TTTTTAACACGACTCGAGTTCGTCTCCCGAACCACACGCGT-3’(SEQ ID NO .6)
我们将上述全序列及一系列删减后的序列分别与5637细胞、T24细胞孵育,进行流式细胞仪检测并计算出每条链相对结合情况。如图6所示删减后SWL-3b的结合能力比全序列增加,SWL-3a结合能力最差,推测关键结合位点是删减部分。基于此设计吉西他滨碱基替换方案。
实施例6:SWL-3G、SWL-3bG与T24、5637、HEK293细胞的结合情况分析
首先,用0.2%EDTA分别将贴壁的T24细胞、5637细胞和HEK293细胞从培养皿上消化下来,将细胞收集到离心管中,并用洗涤缓冲液(PBS,含0.45 % 的葡萄糖,5 mM氯化镁)离心洗涤几次;其次,将终浓度为400nM的SWL-3G、SWL-3bG序列分别加入到结合缓冲液(DPBS,含0.45%的葡萄糖,5 mM 氯化镁,100mg/L tRNA,1g/L BSA)重悬的T24细胞、HEK293细胞中,于4℃摇床孵育 60min;用洗涤缓冲液(PBS,含0.45% 的葡萄糖,5 mM氯化镁)洗涤两次,并通过流式细胞仪进行荧光检测,结果如图7所示,SWL-3G、SWL-3bG分别与T24细胞、5637细胞具有结合特异性,而不能结合PTBP1低表达HEK293细胞。
实施例7:SWL-3G和SWL-3bG具有与母体适体 SWL-3 相同的结合位点
为了鉴定两条载药序列SWL-3G、SWL-3bG与全长序列SWL-3的结合位点是否相同,我们进行了位点竞争实验。用0.2%EDTA将贴壁的T24细胞消化下来,与梯度浓度(0.1、0.5、2.5、5μmol/L)的未标记荧光的SWL-3和文库孵育后,再向其中分别加入FAM标记的终浓度为400nM的SWL-3G、SWL-3bG继续孵育,孵育完成后洗涤,流式检测。实验结果如图8所示,SWL-3G、SWL-3bG的结合能力随着SWL-3浓度的增加而逐渐消失,相比之下,随着文库浓度的增加不会影响SWL-3G、SWL-3bG的结合,这表明两条载药序列SWL-3G、SWL-3bG与全长序列SWL-3结合的是同一位点。
实施例8:吉西他滨基本不具有选择性杀伤功能
分别取生长良好的人膀胱癌T24细胞悬液和HEK293细胞悬液,接种于96孔板,每孔100μL,细胞接种数量控制在1万/孔。接种后置于37℃、5%CO2饱和湿度条件下过夜培养。待细胞贴壁完全后再分别加入浓度50、100、200、400、800nmol/L的吉西他滨,每组设5个平行孔,空白对照组以完全培养基代替吉西他滨。根据实验要求继续培养。10h后弃掉上清,用DPBS 洗涤细胞(注意不能有气泡),后加入新鲜培养基培养62h。弃旧培基,用DPBS清洗后,每孔加入cck8孵育,然后用酶标仪测定OD值(结果见图9)。结果显示吉西他滨对T24细胞的IC50=12.00nM,而图B结果显示HEK293细胞的IC50=15.67nM。说明GEM对两种细胞基本不具有选择性。
实施例9:SWL-3G、SWL-3bG的细胞毒性分析
取生长良好的人膀胱癌T24细胞悬液和HEK293细胞悬液,分别接种于96孔板,每孔100μL,细胞接种数量控制在1×104/孔。接种后置于37℃、5%CO2饱和湿度条件下过夜培养。待细胞贴壁完全后,再分别加入浓度25、50、100、200、400nmol/L的SWL-3G或SWL-3bG,每组设5个平行孔,空白对照组以完全培养基代替SWL-3G、SWL-3bG。根据实验要求继续培养10h后弃掉上清,用DPBS 洗涤细胞,加入新鲜培养基培养62h。弃旧培基并用DPBS清洗,每孔加入cck8试剂孵育,然后用酶标仪测定OD值。结果如图10显示,核酸适体SWL-3G、SWL-3bG均可实现对T24细胞的杀伤,而对HEK293细胞的毒性很小,差异具有显著性。因此证明了SWL-3G、SWL-3bG具有很好的靶向性,选择性的杀伤细胞并降低了药物的毒副性作用。
Claims (7)
1.一种检测人膀胱癌细胞的核酸适体,其特征在于,其核苷酸序列如SEQ ID NO .1至SEQ ID NO .6任一所示。
2.根据权利要求1所述的核酸适体,其特征在于,其核苷酸序列如SEQ ID NO .1或SEQID NO .3所示。
3.根据权利要求1或2所述的检测人膀胱癌细胞的核酸适体,其特征在于,对核苷酸序列进行替换、添加、缺失或修饰而对将核酸适体进行修饰和改造。
4.根据权利要求3所述的检测人膀胱癌细胞的核酸适体,其特征在于,所述修饰是指在核酸适体连接上荧光物质、放射性物质、治疗性物质、生物素、或者酶标记。
5.根据权利要求4所述的核酸适体,其特征在于,所述的修饰是用3-吲哚菁类染料进行修饰。
6.权利要求1-5任一项所述的检测人膀胱癌细胞的核酸适体在制备检测或治疗人膀胱癌细胞的制剂的用途。
7.根据权利要求6所述的用途,其特征在于,所述核酸适体与多嘧啶束结合蛋白1的相互作用以达到治疗膀胱癌的目的。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211305767.6A CN116376917A (zh) | 2022-10-24 | 2022-10-24 | 核酸适体SWL-3G、SWL-3bG在制备治疗膀胱癌药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211305767.6A CN116376917A (zh) | 2022-10-24 | 2022-10-24 | 核酸适体SWL-3G、SWL-3bG在制备治疗膀胱癌药物中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116376917A true CN116376917A (zh) | 2023-07-04 |
Family
ID=86966136
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211305767.6A Pending CN116376917A (zh) | 2022-10-24 | 2022-10-24 | 核酸适体SWL-3G、SWL-3bG在制备治疗膀胱癌药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116376917A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114574495A (zh) * | 2020-12-01 | 2022-06-03 | 上海交通大学医学院附属仁济医院 | 核苷类衍生物改性的核酸适体r50 |
-
2022
- 2022-10-24 CN CN202211305767.6A patent/CN116376917A/zh active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114574495A (zh) * | 2020-12-01 | 2022-06-03 | 上海交通大学医学院附属仁济医院 | 核苷类衍生物改性的核酸适体r50 |
CN114574495B (zh) * | 2020-12-01 | 2024-04-09 | 上海交通大学医学院附属仁济医院 | 核苷类衍生物改性的核酸适体r50 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107469088B (zh) | 一种基于dna折纸术的精确识别靶向纳米载体的构建方法及其应用 | |
US20160215279A1 (en) | Nanozymes, methods of making nanozymes, and methods of using nanozymes | |
CN106164293A (zh) | 适配子构建体 | |
CN107868786B (zh) | 多药耐药结肠癌细胞的单链dna适配体 | |
CN116376917A (zh) | 核酸适体SWL-3G、SWL-3bG在制备治疗膀胱癌药物中的应用 | |
CN108866052A (zh) | Ar-v7可变剪切序列在抗去势抵抗性肿瘤中的应用 | |
CN111218443B (zh) | 合成核酸药物偶联物的方法 | |
CN113350366B (zh) | 核酸适体的应用 | |
CN107190052A (zh) | Loc101928926基因的用途 | |
CN103290018B (zh) | 一种与人表皮生长因子受体iii型突变体特异性结合的核酸适配子及其应用 | |
KR102154683B1 (ko) | 글리피칸-3 특이적 변형 압타머 및 이의 용도 | |
CN106282191B (zh) | 特异性靶向骨肉瘤细胞的适配子及其制备方法和应用 | |
JP2014217311A (ja) | がん細胞に結合するdnaアプタマー | |
CN110317878A (zh) | 一种用于膀胱癌诊治监测的长链非编码rna及其应用 | |
CN108239646A (zh) | 结合肝癌细胞的核酸适配体及其应用和应用其的检测方法 | |
US20180016582A1 (en) | Dna aptamer binding to non-small cell lung cancer cells (h1975) | |
US20020103349A1 (en) | Drug-oligonucleotides chimeric molecules | |
CN106729756A (zh) | 生物标志物作为靶标在肺腺癌诊治中的应用 | |
CN104212800A (zh) | 一种与人表皮生长因子受体iii型突变体特异性结合的核酸适配子及其应用 | |
CN102876681A (zh) | 一种用于靶向药物载体的核酸适体 | |
CN107326028B (zh) | 可与cd123结合的核酸适配体及其靶向载药系统的构建与应用 | |
CN108866066A (zh) | 一种检测人纤维肉瘤细胞的核酸适体及其在制备检测制剂中的应用 | |
CN111394461B (zh) | 宫颈鳞癌的分子靶标及其在诊断和治疗中的应用 | |
CN108114283A (zh) | 生物标志物muc21在骨肉瘤诊断和治疗中的应用 | |
CN113528530B (zh) | 一种与猪鼻支原体特异性结合的核酸适体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |