CN105866412A - Kit for specific detection of endometrial cancer - Google Patents

Kit for specific detection of endometrial cancer Download PDF

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CN105866412A
CN105866412A CN201610369946.4A CN201610369946A CN105866412A CN 105866412 A CN105866412 A CN 105866412A CN 201610369946 A CN201610369946 A CN 201610369946A CN 105866412 A CN105866412 A CN 105866412A
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cypa
aptamer
kit
dna
endometrial cancer
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李静
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57442Specifically defined cancers of the uterus and endometrial
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115Aptamers, 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/16Aptamers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/99Isomerases (5.)

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Abstract

The invention discloses a kit for specific detection of endometrial cancer. An aptamer provided by the invention has preferable affinity with human Cyclophilin A protein. The aptamer provided by the invention can be utilized to capture Cyclophilin A protein from blood; the endometrial cancer can be detected through the change of the content of the Cyclophilin A protein; the aptamer is prepared into a corresponding kit which is used for screening the endometrial cancer. The utilization of the kit provided by the invention has the advantages of high sensitivity and low cost.

Description

A kind of kit of carcinoma of endometrium specific detection
Technical field
The present invention relates to a kind of kit for detecting carcinoma of endometrium and detection method thereof.
Background technology
Carcinoma of endometrium, as one of big malignant tumour of female genital tract three, accounts for the 20-of female genital tract malignant tumour 30%, occupy female genital tract malignant tumour in some American-European countries the first, serious threat WomanHealth.Along with social development and The prolongation of average human life, the endometrial carcinomas incidence of disease 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 tumours such as oophoroma are different, and clinical diagnosis relies primarily on diagnostic curettage, but lactation or infiltrative type carcinoma of endometrium Patient, its uterine wall is weak, is likely to result in perforation, damages to patient during dilatation and curettage.
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 cell factor.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 Prof. Du Yucang 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 recognition capability the most higher with antibody molecule.
Summary of the invention
It is an object of the invention to provide aptamer and the 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 derivative of described aptamer.
The derivative of described aptamer can be following any one:
A) described aptamer being deleted part or increases the nucleotides of partial complementarity, obtain has with described aptamer There is the derivative of the aptamer of identical function;
B) described aptamer carrying out nucleotides replacement or part is modified, obtain has identical with described aptamer The derivative of the aptamer of function;
C) transforming the skeleton of described aptamer as phosphorothioate backbone, obtain has phase with described aptamer The derivative of the aptamer of congenerous;
D) aptamer transform peptide nucleic acid as, obtain has the aptamer of identical function with described aptamer Derivative;
E) after described aptamer being connected upper fluorescence, radioactivity and therapeutic substance, that obtain with described aptamer There is the derivative of the aptamer of identical function.
Described aptamer can be used for the 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 nucleotides, centre includes that the random nucleic acid library of a nucleotides 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 purifying;To return The double-stranded DNA received is template, and in-vitro transcription goes out single stranded RNA random library, and transcription product purifies through PAGE.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 urea, 0.55mol/ L ammonium acetate, l.5mmol/L EDTA, 0.15%SDS) in boil 5min, centrifugal, take supernatant, absolute ethyl alcohol precipitation RNA, lay equal stress on Newly it is dissolved in 20 μ 1 DEPC water;With RNA for template RT-PCR amplifying doulbe-chain DNA, in-vitro transcription goes out RNA library for next Wheel screening;Often in wheel screening process, RT-PCR obtains double-stranded DNA library, goes out RNA with this double-stranded DNA for template in-vitro transcription adaptive Word bank, 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 As follows:
CYPA-1:ATCGTCACCATGGATACATGACTTAACCACGCTCCATTCTATCAATTAACCCGCACTAATTG CACAATTGGACAGTTAC
CYPA-2:ATCGTCACCATGGATACATGATATATTAACCCTCACTAATCTCCTCATCCTACTATAAGTTG CACAATTGGACAGTTAC
CYPA-3:ATCGTCACCATGGATACATGCACTATATTCACGCTAAACCTTTCTTCCTCGCATACCTATTG CACAATTGGACAGTTAC
CYPA-4:ATCGTCACCATGGATACATGCTATTCCCTTTCCGCTACAATCCAATAATCACGCAACACTTG CACAATTGGACAGTTAC
CYPA-5:ATCGTCACCATGGATACATGTTATTCCTGTCTTCACACCGACTTCACATATATCTTCAATTG CACAATTGGACAGTTAC
CYPA-6:ATCGTCACCATGGATACATGCTACCGCTTCCACTCTCCTTATCCATCTTACATTCCCTCTTG CACAATTGGACAGTTAC
CYPA-7:ATCGTCACCATGGATACATGCATACTACAAACCTCCTATAATCTGTTATCAACACTAATTTG CACAATTGGACAGTTAC
CYPA-8:ATCGTCACCATGGATACATGTATTTTCTTCCACTCCCATACTTAGAATAAGAATCCACTTTG CACAATTGGACAGTTAC
CYPA-9:ATCGTCACCATGGATACATGTATGACAATTATTGCCCACTAGATACTTCCACGCAAATCTTG CACAATTGGACAGTTAC
CYPA-10:ATCGTCACCATGGATACATGACTTATCATATCCACCTTATCACATCATCCCCACTTCCTTT GCACAATTGGACAGTTAC
CYPA-11:ATCGTCACCATGGATACATGTCGTTCTCACGAATTCACACACTAACTCCCACACATACATT GCACAATTGGACAGTTAC
CYPA-12:ATCGTCACCATGGATACATGCACTATATCTTACTATCATTTAATCCTTTCACTCCCAACTT GCACAATTGGACAGTTAC
CYPA-13:ATCGTCACCATGGATACATGTCACTCCGCCGCACCACTAATTCACGCTAAACTTATCTTTT GCACAATTGGACAGTTAC
CYPA-14:ATCGTCACCATGGATACATGATTCTCTCCGAACTTCTACATATAAAACCAAGAATTCTTTT GCACAATTGGACAGTTAC
The performance measurement of embodiment 3 protein combination aptamer
Aptamer is taken respectively 2.0 μ g, digests lh with calf intestinal alkaline phosphatase (CIP) 37 DEG C, purify recovery and remove phosphoric acid The RNA changed;By T4 polynucleotide kinase mark [γ-32P] ATP in dephosphorylized RNA molecule end.10nmol radioactivity The aptamer of mark 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
Described in embodiment 4, aptamer is specifically analyzed and stability analysis
It is respectively adopted human serum albumin, immune serum globulin (ISG), 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 specific with CYPA protein combination.
By described aptamer, take 0.2ug, be respectively placed in the serum of normal temperature, the 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 secretion 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 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 kit for carcinoma of endometrium detection, it contains the nucleic acid that can be specifically combined with CYPA protein-specific Fit.
2. kit as claimed in claim 1, it is characterised in that: the sequence of described aptamer is as described in SEQ ID No:2.
3. the method detecting carcinoma of endometrium, it is characterised in that utilize the kit described in claim 1.
CN201610369946.4A 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer Pending CN105866412A (en)

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CN201610369946.4A CN105866412A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
CN201510807002.6A CN105223358B (en) 2015-11-22 2015-11-22 A kind of test kit of carcinoma of endometrium specific detection

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CN201510807002.6A Active CN105223358B (en) 2015-11-22 2015-11-22 A kind of test kit of carcinoma of endometrium specific detection
CN201610361858.XA Pending CN105954516A (en) 2015-11-22 2015-11-22 Kit for detecting specificity of endometrial carcinoma
CN201610361917.3A Pending CN105842444A (en) 2015-11-22 2015-11-22 Kit for specific detection on 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
CN201610361860.7A Pending CN105866411A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
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
CN201610361916.9A Pending CN105807069A (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

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CN201610361858.XA Pending CN105954516A (en) 2015-11-22 2015-11-22 Kit for detecting specificity of endometrial carcinoma
CN201610361917.3A Pending CN105842444A (en) 2015-11-22 2015-11-22 Kit for specific detection on 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
CN201610361860.7A Pending CN105866411A (en) 2015-11-22 2015-11-22 Kit for specific detection of endometrial cancer
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
CN201610361916.9A Pending CN105807069A (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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CN104109667A (en) * 2014-06-24 2014-10-22 叶尚勉 Nucleic acid fragment and purpose thereof
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US6451609B1 (en) * 1996-05-30 2002-09-17 The Cleveland Clinic Foundation Method of detecting gynecological carcinomas
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CN108165603A (en) * 2013-01-25 2018-06-15 艾克斯赛尔生物科学公司 For the method for selective enrichment target cell, composition, kit and system

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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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CN105223358A (en) 2016-01-06
CN105866411A (en) 2016-08-17
CN105842445A (en) 2016-08-10
CN105223358B (en) 2016-09-14
CN105842444A (en) 2016-08-10
CN105954516A (en) 2016-09-21

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Application publication date: 20160817