CN105842444A - Kit for specific detection on endometrial cancer - Google Patents

Kit for specific detection on endometrial cancer Download PDF

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Publication number
CN105842444A
CN105842444A CN201610361917.3A CN201610361917A CN105842444A CN 105842444 A CN105842444 A CN 105842444A CN 201610361917 A CN201610361917 A CN 201610361917A CN 105842444 A CN105842444 A CN 105842444A
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cypa
aptamer
kit
dna
endometrial cancer
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CN201610361917.3A
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李静
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Individual
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Priority to CN201610361917.3A priority Critical patent/CN105842444A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/99—Isomerases (5.)

Abstract

The invention discloses a kit for specific detection on endometrial cancer. The aptamer disclosed by the invention has relatively good affinity with human Cyclophilin A protein. By adoption of the aptamer disclosed by the invention, Cyclophilin A protein in blood can be captured, endometrial cancer can be detected according to variation of the content of the Cyclophilin A protein, and a corresponding kit can be prepared to screen endometrial cancer. The kit disclosed by the invention has the advantages of high sensitivity and low cost.

Description

A kind of test kit of carcinoma of endometrium specific detection
Technical field
The present invention relates to a kind of test kit for detecting carcinoma of endometrium and detection method thereof.
Background technology
Carcinoma of endometrium, as one of big malignant tumor of female genital tract three, accounts for the 20-of female genital tract malignant tumor 30%, occupy female genital tract malignant tumor in some American-European countries the first, serious threat WomanHealth.Along with social development and The prolongation of average human life, endometrial carcinomas sickness rate rises and year by year with rejuvenation trend.If carcinoma of endometrium can be accomplished Early diagnosis, rational therapy, its prognosis is preferable.The main clinical manifestation of carcinoma of endometrium is irregular colporrhagia, with Other tumors such as ovarian cancer are different, and clinical diagnosis relies primarily on diagnostic curettage, but age of sucking or infiltrative type carcinoma of endometrium Patient, its Uterus wall is weak, is likely to result in perforation, damages to patient during dilatation and curettage of uterine.
Cyclophilin A albumen (CYPA) also known as cyclophilin A (Gene Bank No.NP_066953), be positioned at kytoplasm and Karyon, has peptide acyl prolyl cis-trans isomerase activity, participates in protein folding, assembles and transport；At intracellular and ring spore Element A combines, and suppresses T cell activation approach, shows immunosuppressive action, additionally participates in oxidative stress induction and cholesterol generation Apologize for having done sth. wrong journey, and play the function of cytokine.Prior art is reported that to be come in early days by detection human Cyclophilin A expression Diagnosis of endometrial carcinoma.Therefore, detection human Cyclophilin A becomes particularly important.
Aptamer (Aptamer, also known as aptamers, aptamer) is can high-affinity, certain life of combination of high specific Strand widow's nucleic acid molecules (ssDNA or ssRNA) of material.Aptamer is by index concentration Fas lignand system evolution technology (Systemat1c Evolut1on of L1gands by Exponent1al enr1chment, SELEX) is from synthetic What in DNA/RNA library, screening obtained can combine the single stranded DNA/RNA of target molecules by high degree of specificity.Report aptamer Target include metal ion, organic molecule, polypeptide, protein, cell even tissue etc..The molecular recognition merit of aptamer Can be similar with antibody, there is the target identification ability the most higher with antibody molecule.
Summary of the invention
It is an object of the invention to provide aptamer and the test kit thereof of a kind of specific bond CYPA.
The aptamer that the present invention provides, is the single stranded DNA shown in sequence 1-14 of sequence table.
Described aptamer and CYPA albumen have preferable affinity.
Also described aptamer can be modified or transformed, obtain the derivant of described aptamer.
The derivant of described aptamer can be following any one:
A) described aptamer being deleted part or increases the nucleotide of partial complementarity, obtain has with described aptamer There is the derivant of the aptamer of identical function；
B) described aptamer carrying out nucleotide replacement or part is modified, obtain has identical with described aptamer The derivant of the aptamer of function；
C) transforming the skeleton of described aptamer as phosphorothioate backbone, obtain has phase with described aptamer The derivant of the aptamer of congenerous；
D) aptamer transform peptide nucleic acid(PNA) as, obtain has the aptamer of identical function with described aptamer Derivant；
E) after described aptamer being connected upper fluorescence, radioactivity and therapeutic substance, that obtain with described aptamer There is the derivant of the aptamer of identical function.
Described aptamer can be used for the test kit of preparation detection CYPA.
Utilize the aptamer of the present invention, in can capturing in saliva in CYPA, thus for correlator Endometrium Cancer examination.Utilize the aptamer of the present invention, there is highly sensitive, low cost, the easy advantage prepared, easily preserve.The present invention has The highest using value.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment Method, if no special instructions, is conventional method.
The acquisition of embodiment 1CYPA albumen
CYPA gene shown in Genbank:NP_066953 is carried out table by the eukaryotic expression mode that this area is conventional Reach, it is thus achieved that corresponding desired polypeptides albumen.
The screening of embodiment 2 aptamer and preparation
Design two ends comprise about 20 nucleotide, centre includes that the random nucleic acid library of a nucleotide is as follows:
5‘-ATCGTCACCATGGATACATG(N39)TTGCACAATTGGACAGTTAC-3’；N39 represents 39 stochastic kernels Thuja acid.
Being double-stranded DNA by single-stranded DNA banks amplification, product is through 2% agarose gel electrophoresis and cuts glue recovery purification；To return The double-stranded DNA received is template, and in vitro transcription goes out single stranded RNA random library, and transcription product is through PAGE purification.75 μ g RNA library warps The anti-sieve of nitrocellulose filter removes the RNA molecule being combined with film, then with 2ug CYPA albumen, hatches 30min, reactant liquor for 37 DEG C Filter through nitrocellulose filter, wash filter membrane；Then filter membrane is shredded, be placed in elution buffer (6mol/L carbamide, 0.55mol/ L ammonium acetate, l.5mmol/L EDTA, 0.15%SDS) in boil 5min, centrifugal, take supernatant, dehydrated alcohol precipitation RNA, lay equal stress on Newly it is dissolved in 20 μ 1DEPC water；With RNA for template RT-PCR amplifying doulbe-chain DNA, in vitro transcription goes out RNA library for next round Screening；Often in wheel screening process, RT-PCR obtains double-stranded DNA library, goes out RNA aptamer with this double-stranded DNA for template in vitro transcription Storehouse, screening carries out 11 altogether and takes turns.Having obtained 14 aptamers, its sequence is respectively shown in SEQ ID NO:1-14.Particular sequence is such as Shown in lower:
CYPA-1:ATCGTCACCATGGATACATGACTTAACCACGCTCCATTCTATCAATTAACCCGCACTAATTG CACAATTGGACAGTTAC
CYPA-2:ATCGTCACCATGGATACATGATATATTAACCCTCACTAATCTCCTCATCCTACTATAAGTTG CACAATTGGACAGTTAC
CYPA-3:ATCGTCACCATGGATACATGCACTATATTCACGCTAAACCTTTCTTCCTCGCATACCTATTG CACAATTGGACAGTTAC
CYPA-4:ATCGTCACCATGGATACATGCTATTCCCTTTCCGCTACAATCCAATAATCACGCAACACTTG CACAATTGGACAGTTAC
CYPA-5:ATCGTCACCATGGATACATGTTATTCCTGTCTTCACACCGACTTCACATATATCTTCAATTG CACAATTGGACAGTTAC
CYPA-6:ATCGTCACCATGGATACATGCTACCGCTTCCACTCTCCTTATCCATCTTACATTCCCTC TTGCACAATTGGACAGTTAC
CYPA-7:ATCGTCACCATGGATACATGCATACTACAAACCTCCTATAATCTGTTATCAACACTAATTTG CACAATTGGACAGTTAC
CYPA-8:ATCGTCACCATGGATACATGTATTTTCTTCCACTCCCATACTTAGAATAAGAATCCACTTTG CACAATTGGACAGTTAC
CYPA-9:ATCGTCACCATGGATACATGTATGACAATTATTGCCCACTAGATACTTCCACGCAAATCTTG CACAATTGGACAGTTAC
CYPA-10:ATCGTCACCATGGATACATGACTTATCATATCCACCTTATCACATCATCCCCACTTCCT TTGCACAATTGGACAGTTAC
CYPA-11:ATCGTCACCATGGATACATGTCGTTCTCACGAATTCACACACTAACTCCCACACATACATT GCACAATTGGACAGTTAC
CYPA-12:ATCGTCACCATGGATACATGCACTATATCTTACTATCATTTAATCCTTTCACTCCCAACTT GCACAATTGGACAGTTAC
CYPA-13:ATCGTCACCATGGATACATGTCACTCCGCCGCACCACTAATTCACGCTAAACTTATCTTTT GCACAATTGGACAGTTAC
CYPA-14:ATCGTCACCATGGATACATGATTCTCTCCGAACTTCTACATATAAAACCAAGAATTCTTTT GCACAATTGGACAGTTAC
The performance measurement of embodiment 3 protein binding aptamer
Aptamer taking 2.0 μ g respectively, digests lh with calf intestinal alkaline phosphatase (CIP) 37 DEG C, purification reclaims and removes phosphoric acid The RNA changed；By T4 polynucleotide kinase labelling [γ-32P] ATP in dephosphorylized RNA molecule end.10nmol radioactivity The aptamer of labelling with variable concentrations (1-200nM) CYPA37 DEG C respectively hatches 30min, and each group reactant liquor is through celluloid Membrane filtration mistake, washs filter membrane, is dried filter membrane, and liquid scintillation counter measures the exit dose of residual on filter membrane, and same sample is parallel does twice Measure.Calculate the dissociation constant of each aptamer and destination protein.Result is as follows:
Title Dissociation constant Kd (unit nM)
CYPA-1 11.3
CYPA-2 12.5
CYPA-3 13.0
CYPA-4 12.8
CYPA-5 12.7
CYPA-6 11.9
CYPA-7 11.4
CYPA-8 12.4
CYPA-9 12.7
CYPA-10 12.0
CYPA-11 13.1
CYPA-12 13.0
CYPA-13 12.6
CYPA-14 12.8
PBS blank Without binding ability
Aptamer specificity analyses and stability analysis described in embodiment 4
It is respectively adopted human albumin, immune globulin, pg120 albumen, escherichia coli outer membrane protein A, COCH egg In vain, CYPA albumen, carry out specific detection with 14 aptamers, through binding tests find, these aptamers the most not with these Albumen combines, and only keeps higher specificity with CYPA protein binding.
By described aptamer, take 0.2ug, be respectively placed in the serum of room temperature, aqueous solution, place two weeks.Pass through RT- PCR detects, and finds its Stability Analysis of Structures of placement of three weeks, is not degraded.
The diagnosis of aptamer disease described in embodiment 5
Take 8 endometrial carcinomas and the blood of 4 normal persons, use normal saline dilution, it is thus achieved that target sample.
By 14 markd aptamers of coupling respectively with the secretions mixing 30min of 8 patients and 4 normal persons, Separated by biotin, the content of quantitative analysis CYPA therein albumen, found by analysis, in 8 endometrial carcinomas The content of CYPA albumen dramatically increases, and has exceeded the threshold value of regulation.Reach the diagnostic criteria of carcinoma of endometrium.As can be seen here, Its diagnosis effect is preferable.
These are only the preferred embodiments of the present invention, be not limited to the present invention, for those skilled in the art For Yuan, all any modification, equivalent substitution and improvement etc. done within the spirit and principles in the present invention, should be included in this Within the protection domain of invention.
Sequence table
＜ 110 ＞ Li Jing
The test kit of a ＜ 120 ＞ carcinoma of endometrium specific detection
〈160〉14
〈210〉1
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-1
ATCGTCACCATGGATACATGACTTAACCACGCTCCATTCTATCAATTAACCCGCACTAATTGCACAATTGGACAGTTAC
〈210〉2
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-2
ATCGTCACCATGGATACATGATATATTAACCCTCACTAATCTCCTCATCCTACTATAAGTTGCACAATTGGACAGTTAC
〈210〉3
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-3
ATCGTCACCATGGATACATGCACTATATTCACGCTAAACCTTTCTTCCTCGCATACCTATTGCACAATTGGACAGTTAC
〈210〉4
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-4
ATCGTCACCATGGATACATGCTATTCCCTTTCCGCTACAATCCAATAATCACGCAACACTTGCACAATTGGACAGTTAC
〈210〉5
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-5
ATCGTCACCATGGATACATGTTATTCCTGTCTTCACACCGACTTCACATATATCTTCAATTGCACAATTGGACAGTTAC
〈210〉6
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-6
ATCGTCACCATGGATACATGCTACCGCTTCCACTCTCCTTATCCATCTTACATTCCCTCTTGCACAATTGGACAGTTAC
〈210〉7
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-7
ATCGTCACCATGGATACATGCATACTACAAACCTCCTATAATCTGTTATCAACACTAATTTGCACAATTGGACAGTTAC
〈210〉8
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-8
ATCGTCACCATGGATACATGTATTTTCTTCCACTCCCATACTTAGAATAAGAATCCACTTTGCACAATTGGACAGTTAC
〈210〉9
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-9
ATCGTCACCATGGATACATGTATGACAATTATTGCCCACTAGATACTTCCACGCAAATCTTGCACAATTGGACAGTTAC
〈210〉10
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-10
ATCGTCACCATGGATACATGACTTATCATATCCACCTTATCACATCATCCCCACTTCCTTTGCACAATTGGACAGTTAC
〈210〉11
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-11
ATCGTCACCATGGATACATGTCGTTCTCACGAATTCACACACTAACTCCCACACATACATTGCACAATTGGACAGTTAC
〈210〉12
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-12
ATCGTCACCATGGATACATGCACTATATCTTACTATCATTTAATCCTTTCACTCCCAACTTGCACAATTGGACAGTTAC
〈210〉13
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-13
ATCGTCACCATGGATACATGTCACTCCGCCGCACCACTAATTCACGCTAAACTTATCTTTTGCACAATTGGACAGTTAC
〈210〉14
〈211〉 79
〈212〉DNA
＜ 213 ＞ artificial sequence
〈400〉CYPA-14
ATCGTCACCATGGATACATGATTCTCTCCGAACTTCTACATATAAAACCAAGAATTCTTTTGCACAATTGGACAGTTAC

Claims (3)

1., for a test kit for carcinoma of endometrium detection, it contains the nucleic acid that energy specificity is combined with CYPA protein-specific Fit.
2. test kit as claimed in claim 1, it is characterised in that: the sequence of described aptamer is as described in SEQ ID No:6.
3. the method detecting carcinoma of endometrium, it is characterised in that utilize the test kit described in claim 1.
CN201610361917.3A 2015-11-22 2015-11-22 Kit for specific detection on endometrial cancer Pending CN105842444A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610361917.3A CN105842444A (en) 2015-11-22 2015-11-22 Kit for specific detection on endometrial cancer

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CN201510807002.6A CN105223358B (en) 2015-11-22 2015-11-22 A kind of test kit of carcinoma of endometrium specific detection
CN201610361917.3A CN105842444A (en) 2015-11-22 2015-11-22 Kit for specific detection on endometrial cancer

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CN201610361858.XA Pending CN105954516A (en) 2015-11-22 2015-11-22 Kit for detecting specificity of endometrial carcinoma
CN201610370066.9A Pending CN105842445A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial carcinoma
CN201610370068.8A Pending CN105842446A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial carcinoma
CN201610361917.3A Pending CN105842444A (en) 2015-11-22 2015-11-22 Kit for specific detection on endometrial cancer
CN201510807002.6A Active CN105223358B (en) 2015-11-22 2015-11-22 A kind of test kit of carcinoma of endometrium specific detection
CN201610361860.7A Pending CN105866411A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201610369946.4A Pending CN105866412A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201610361916.9A Pending CN105807069A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201610361859.4A Pending CN105807054A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201610361857.5A Pending CN105807068A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer

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CN201610370066.9A Pending CN105842445A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial carcinoma
CN201610370068.8A Pending CN105842446A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial carcinoma

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CN201610369946.4A Pending CN105866412A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201610361916.9A Pending CN105807069A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201610361859.4A Pending CN105807054A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201610361857.5A Pending CN105807068A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer

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CN105886659A (en) * 2016-06-29 2016-08-24 北京泱深生物信息技术有限公司 DSTN gene and expression product thereof as diagnosis and treatment target of endometrial cancer

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US6591144B2 (en) * 2001-10-23 2003-07-08 The Administrators Of The Tulane Educational Fund Steerable catheter and method for locating coronary sinus
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CN101261279A (en) * 2008-04-11 2008-09-10 四川大学 Endometrium cancer diagnose reagent, reagent kit and controlling medicament
US20130078645A1 (en) * 2010-01-29 2013-03-28 Psynova Neurotech Ltd. Biomarkers
CN104109667A (en) * 2014-06-24 2014-10-22 叶尚勉 Nucleic acid fragment and purpose thereof
CN104830867A (en) * 2015-06-07 2015-08-12 杨洋 Aptamer capable of being specifically combined with DKK1 protein in cancer cells

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CN105866412A (en) 2016-08-17
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CN105223358B (en) 2016-09-14
CN105807068A (en) 2016-07-27
CN105223358A (en) 2016-01-06
CN105842445A (en) 2016-08-10
CN105866411A (en) 2016-08-17
CN105842446A (en) 2016-08-10
CN105807054A (en) 2016-07-27

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Application publication date: 20160810