CN104630380B - Carbonic anhydrase IV application in preparation adenocarcinoma of lung diagnostic preparation - Google Patents
Carbonic anhydrase IV application in preparation adenocarcinoma of lung diagnostic preparation Download PDFInfo
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- 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
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
- G01N33/57423—Specifically defined cancers of lung
Abstract
The present invention relates to carbonic anhydrase IV application in preparation adenocarcinoma of lung diagnostic preparation.Invention is by carrying out Meta analysis to adenocarcinoma of lung high flux transcript profile data, obtain difference expression gene, operation analysis instrument carries out functional analysis to difference expression gene, and then filter out candidate gene, and use classical molecular biological method to demonstrate the dependency of carbonic anhydrase IV and adenocarcinoma of lung, carbonic anhydrase IV can be used for preparing adenocarcinoma of lung assisting in diagnosis and treatment preparation, has important clinical value.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to the carbonic anhydrase IV new use in preparation adenocarcinoma of lung diagnostic preparation
On the way.
Background technology
Carbonic anhydrase (carbonic anhydrase, CA) is the class metalloenzyme containing zinc, and its function is catalysis titanium dioxide
The hydration of carbon and the hydrolysis of carbonic acid, have important regulation effect to transport, Water-Electrolyte and the pH balance of carbon dioxide.The most extremely
Having been found that 15 kinds of different types of carbonic anhydrase isozyme less, wherein the activity of carbonic anhydrase III and IV is relatively strong, carbonic acid
Acid anhydride enzyme IV is primarily present in fatty tissue, liver, cardiac muscle and slow skeletal muscle fiber, and himself antibody is the most respectively in systematicness
Being detected in lupus erythematosus, rheumatoid arthritis and diabetes patient, IV type anti-carbonic anhydrase antibody is at kidney disease simultaneously
Important function is served in developing in terms of humoral immunization.
Adenocarcinoma of lung (lung adenocarcinoma), also known as nonsmall-cell lung cancer (NSCLC), is the one of pulmonary carcinoma.Different
In prognosis of squamous cell lung cancer, adenocarcinoma of lung is easier to betide women and nonsmoker.Normal relatively periphery, tumor enlargement in the position of pulmonary
Speed relatively slow (about 120 days doubling times).In early days without sign, it has been late period when being generally diagnosed.Nonsmall-cell lung cancer
Account for the 75%-80% of pulmonary carcinoma sum.In recent years a lot of scholars from cell, molecular level illustrate fall ill to it relevant etc. in terms of
Factor is studied, to making a prediction its genesis mechanism and invasion and attack, recurrence dependency to and guide clinical treatment.Research is sent out
Existing, there is many factors jointly to participate in the pathogenesis of adenocarcinoma of lung, including oncogene mutation and activity expression, the disappearance of antioncogene
With sudden change and somatomedin unconventionality expression, signal disorder etc..Additionally, the pathogenetic research to adenocarcinoma of lung shows that signal turns
Guiding path tumor generation, develop and shift in play an important role.Simultaneously as internal various regulatory mechanism is complicated and changeable,
Influence factor is numerous, although therefore about adenocarcinoma of lung morbidity molecular mechanism research constantly carry out and achieve many new developments, but
Its definite mechanism waits to illustrate, it is likely that relates to multi-path and participates in regulation and control.
The present invention carries out Meta analysis to the adenocarcinoma of lung high flux transcript profile data delivered, it is thus achieved that 1063 differential expressions
Gene, the gene 464 that wherein expression raises, the gene 599 that expression is lowered, and utilize KEGG, GO, OMIM etc.
The analytical tools such as various biological information data bases and MATLAB carry out functional analysis to difference expression gene, and then filter out time
Select gene carbonic anhydrase IV (CA4), and use molecular biology method to confirm that CA4 gene is with non-further
The relation of small cell lung cancer: CA4 and nonsmall-cell lung cancer have good dependency, can be used for preparing nonsmall-cell lung cancer auxiliary
Diagnosis or prognosis preparation, have important clinical value.
Summary of the invention
It is an object of the present invention to provide CA4 new application in preparation adenocarcinoma of lung diagnostic preparation.
For achieving the above object, the present invention carries out Meta analysis to the adenocarcinoma of lung high flux transcript profile data delivered, and obtains
1063 difference expression genes, wherein expression raise gene 464, expression lower gene 599, and profit
By analytical tools such as the various biological information data base such as KEGG, GO, OMIM and MATLAB, difference expression gene is carried out function
Analyze, so filter out affect adenocarcinoma of lung occur develop key gene and important signal path, be additionally based on data mining
The result analyzed, filters out candidate gene CA4 and RAMP3.And then, the present invention passes through fluorescence quantifying PCR method in 5 example adenocarcinomas of lung
The cancerous tissue of diseased patient and 5 example normal structures are that sample verifies that adenocarcinoma of lung Disease-causing gene CA4 and RAMP3 screened is at lung gland
The cancerous tissue of cancer diseased patient and the expression of normal structure, result display CA4 and RAMP3 is in the cancer of adenocarcinoma of lung diseased patient
Tissue is expressed and all declines, but CA4 gene expression declines more notable.
A kind of adenocarcinoma of lung detection kit, described detection kit detection CA4 gene are it is an object of the present invention to provide.Enter
One step, described test kit also includes other detectable.
Further, described detection kit is to detect the PCR kit for fluorescence quantitative of above-mentioned CA4 expression.Preferably
Use primer to for SEQ ID NO.1 and SEQ ID NO.2.
Further, this PCR kit is suitable for all types fluorescence quantitative gene extender that presently, there are on market, spirit
Sensitivity is high, the most quick and precisely, good stability, has a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes: specific primer, internal reference primer, quantitative fluorescent PCR
Reactant liquor.Wherein said specific primer includes that forward primer and downstream primer, forward primer sequence are SEQ ID NO.1,
Downstream primer sequence is SEQ ID NO.2.Described internal reference primer is β-actin internal reference primer, and forward primer sequence is SEQ ID
NO.3, downstream primer sequence is SEQ ID NO.4.
The invention also discloses the using method of a kind of PCR kit for fluorescence quantitative detecting adenocarcinoma of lung, quantitative fluorescent PCR
System: forward primer；Downstream primer；Sample RNA 10pg-100ng；PowerGreen PCR Master Mix
(invitrogen, article No. 4367659), adds RNase-Free Water to 25 μ L.Quantitative fluorescent PCR program: 95 DEG C of 10min are pre-
Degeneration, connects 30-40 circulation: 95 DEG C of 5-30s, 60 DEG C of 30-90s.
Described test kit also comprises RNA extraction agent.PreferablyReagent (invitrogen, article No.
15596-018) carry out sample rna extraction.
The present invention also have detected this test kit susceptiveness, and result shows that this test kit detection range is 106-102copies/μ
L, minimum concentrations is 100copies/ μ l.
A kind of adenocarcinoma of lung detection kit, described detection kit detection CA4 albumen are it is an object of the present invention to provide.Enter
One step, described test kit also includes other detectable.
Further, described detection kit is the ELISA detection kit of detection CA4 albumen.Resisting in described test kit
Body can use commercially available CA4 monoclonal antibody.Further, described test kit includes: the solid phase being coated CA4 monoclonal antibody carries
Body, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction stop buffer etc., preferably
The IgG antibody of HRP labelling, horseradish peroxidase.
Further, described detection kit is the gold-immunochromatographyreagent reagent for assay box of detection CA4 albumen, and described antibody can use
Commercially available CA4 monoclonal antibody.Further, described gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or colloid
Gold percolation.Further, described detection zone (T) specking on gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-CA4 mono-
Clonal antibody, quality control region (C) specking have goat anti-mouse immunoglobulin IgG.
It is an object of the present invention to provide CA4 gene and/or CA4 albumen in the diagnosis or prognosis product of preparation adenocarcinoma of lung
Application.
The present invention is extracted cancerous tissue and the 5 example normal structures of 5 example adenocarcinoma of lung diseased patient the most respectively, to its total serum IgE
Carrying out extract, devise 2 primer SEQ ID NO.1 and SEQ ID NO.2 for expanding CA4 gene, in being used for expanding
2 primer SEQ ID NO.3 and SEQ ID NO.4 of ginseng gene β-actin.It is prepared for the standard DNA containing CA4 gene order
Template, and carried out sensitivity experiments.And then, use the method for qRT-PCR to compare CA4 gene in pulmonary adenocarcinoma and normal group
Expression in knitting.Result shows: qRT-PCR stable amplification result, and wherein CA4 expression in the normal tissue is obvious
Higher than adenocarcinoma of lung cancerous tissue, experiment show bioinformatics the selection result.Further, inventor is in 116 example adenocarcinomas of lung
Detecting CA4 expression conditions in the cancerous tissue of diseased patient and cancer beside organism, result shows 115 example adenocarcinoma of lung diseased patient's
In cancerous tissue, CA4 gene expression is less than the expression in cancer beside organism, detects rate of accuracy reached 99%, i.e. CA4 gene has with adenocarcinoma of lung
There is good dependency, can be used for preparing adenocarcinoma of lung auxiliary diagnosis or prognosis preparation.
Accompanying drawing explanation
Fig. 1 CA4 gene by fluorescence quantitative amplification curve diagram
Fig. 2 CA4 gene by fluorescence quantitative solubility curve figure
Fig. 3 CA4 gene relative expression's spirogram in cancerous tissue and normal structure
Fig. 4 RAMP3 gene by fluorescence quantitative amplification curve diagram
Fig. 5 RAMP3 gene by fluorescence quantitative solubility curve figure
Fig. 6 RAMP3 gene relative expression's spirogram in cancerous tissue and normal structure
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further, be only used for explain the present invention, and it is not intended that to this
The restriction of invention.It will be understood by those skilled in the art that: can in the case of without departing from the principle of the present invention and objective
These embodiments to carry out multiple change, revises, replace and modification, the scope of the present invention is limited by claim and equivalent thereof
Fixed.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer
Part examinations.
Embodiment 1 adenocarcinoma of lung high flux transcript profile data Meta are analyzed
GEO (Gene Expression Omnibus) data base is by NCBI (US National Biotechnology Information center)
Exploitation is safeguarded, GEO data base is as the data base of maximum gene expression data, and this data base is based on chip data, in addition
Also comprise data such as SAGE (serial analysis of gene expression) data of some non-chip types, (ribosome sequence label is even for SARST
Continuous analyze) data, MS (mass spectrum) data, proteome data and a new generation high-flux sequence data (MPSS, extensive parallel survey
Sequence technology) etc..Retrieval uses key word to be (" lung adenocarcinoma " [MeSH Terms] OR " lung
adenocarcinoma"[All Fields])AND"Homo sapiens"[porgn].Limiting research type is
" expression profiling by array " meets the data set of following standard and will include in our research: 1. selected
Data set must be that expression mRNA transcript profile data 2. these data of full-length genome come from adenocarcinoma of lung case group and matched group
Biopsy or the cell of cultivation；3. this research all considers normalized or raw data set；Through screening by 15 sets of data
Collection is included in our research (being shown in Table 1).
The basic condition of table 1 15 set adenocarcinoma of lung full-length genome data set
After initial data being carried out background correction and standardization by transcript profile data analysis software, utilize microarray notable
Property component software (significance analysis of microarray, SAM) to 15 set gene chips results carry out normalizing
Change carries out t-test after processing, screen difference expression gene, sets false positive rate (false discovery during analysis
Rate, FDR) it is≤0.01, filter out 1063 difference expression genes, the gene 464 that wherein expression raises altogether, express
The gene of horizontal down-regulation 599.In order to preferably study the function of difference expression gene, we pass through DAVID to differential expression
Gene carries out the enrichment of GO function and KEGG path is enriched with, and additionally the gene of up-regulated and the gene of downward are carried out by respectively
GO function is enriched with.Finally we select 2 down-regulated gene RAMP3 (eceptor (G protein-coupled) activity
Modifying protein3) and CA4 (carbonic anhydrase IV) as candidate gene carry out classics molecular biosciences
Learn experimental verification.
Embodiment 2 pulmonary adenocarcinoma and normal structure RAMP3 and CA4 expression conditions
One material and method
1, material
Collect 5 example adenocarcinoma of lung cancerous tissues and 5 example normal structures, it is grouped and numbers.
2, method
2.1 pulmonary adenocarcinoma and the extraction of normal structure total serum IgE
UseReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation
Carrying out by product description, concrete operations are as follows:
After collecting sample, frozen mortar tissue being put into pre-cooling after liquid nitrogen, taking-up is ground, sample to be organized
After this is powdered:
1. Trizol, room temperature preservation 5 minutes are added；
2. adding chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5 minutes-10 minutes；
3. 12000rpm high speed centrifugation draws upper strata aqueous phase (inhale 70%) in another new centrifuge tube pipe after 15 minutes, notes
It is not drawn onto the protein substance between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, the most reverse
Mixing, is placed in 10 minutes on ice；
4. 12000rpm carefully discarded supernatant at a high speed after 15 minutes, added 75% in the ratio of I ml/ml Trizol
Paint precipitation (4 DEG C of preservations) washed by DEPC ethanol, washes paint precipitate, vibration mixing, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discard ethanol liquid, ambient temperatare put 5 minutes fully to dry precipitation, add the water dissolution that processed of DEPC and sink
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C.RNA mass is sentenced
Calibration standard: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophoresis pattern has 28S, 18S band clearly；
70 DEG C of water bath heat preservation electrophoresis patterns after 1 hour and the collection of illustrative plates no significant difference before water bath heat preservation.
2.2 reverse transcription synthesis cDNA
UseIII Reverse Transcriptase (invitrogen, article No. 18080-044) enters
Row cDNA reverse transcription, experimental implementation is carried out by product description, and concrete operations are as follows:
Use Reverse Transcriptase kit, with RT Buffer, l μ g total serum IgE is carried out converse record and synthesize cDNA.Use 25 μ l
Reaction system, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into following components in PCR pipe:
5 × RT Buffer 5 μ l, 10mmol/l dNTP 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm mol/l
Oligo dT 2 μ l, 200U/ μ l MMLV 1.25 μ l, template ribonucleic acid 1 μ g, addition aquesterilisa to total system 25 μ l.Hatch 1 for 42 DEG C
Hour, 72 DEG C 10 minutes, of short duration centrifugal.It is standby that-20 DEG C of refrigerators are put in cDNA preservation.
2.3Real-Time PCR
2.3.1 instrument and the method for analysis
With ABI 7500 type quantitative real time PCR Instrument, 2-△ △ CT method is used to carry out the relative quantitative assay of data.2.3.2
Design of primers
Use online primer-design software, synthesized by invitrogen company after design of primers.Concrete primer sequence is as follows:
Table 2 primer sequence
Operating process is as follows:
(1) reaction system: use PowerGreen PCR Master Mix (invitrogen, article No.
4367659) expanding, experimental implementation is carried out by product description.Amplification program is: 95 ° of 10min, (95 DEG C of 15sec, 60 DEG C
60sec) × 45 circulations.
Table 3 RealTime reaction system
| Component | Addition |
| 2×mix | 10μl |
| Forward primer (10uM) | 0.5μl |
| Downstream primer (10uM) | 0.5μl |
| Template | 2μl |
| Add sterile purified water | To 25 μ l |
(2) primer screening
After being mixed by each sample cDNA, carrying out 5 times of gradient dilutions as template, after dilution, sample respectively takes 2 μ l and makees template,
Expand with genes of interest primer and reference gene primer respectively, carry out melt curve analysis analysis at 60-95 DEG C, according to expansion simultaneously
Increasing Efficiency is high and the unimodal principle of solubility curve carries out primer screening.
(3) sample RealTimePCR detection
Take 2 μ l after each sample cDNA 10 times being diluted and make template, enter with genes of interest primer and reference gene primer respectively
Row amplification.Carry out solubility curve analysis at 60-95 DEG C simultaneously.
Two experimental results
CA4 with RAMP3 real-time quantitative PCR amplification curve (seeing Fig. 1 and Fig. 3 respectively) flex point understands, amplification curve entirety is put down
Row is good, shows that the amplification efficiency of each reaction tube is close, and the limit is flat and nothing raises up now, and exponent phase slope is relatively big, explanation
Amplification efficiency is higher；Sample amplified production solubility curve CA4 (see Fig. 2) and RAMP3 (see Fig. 4) are unimodal, illustrate that amplification is produced
Thing only has one, for specific amplification；Relative quantification formula according to qRT-PCR: 2-Δ Ct × 100%, compare CA4 with
RAMP3 gene expression in pulmonary adenocarcinoma and normal structure.Result shows that (being specifically shown in Fig. 5): qRT-PCR expands knot
Fruit is stable, and wherein CA4 Yu RAMP3 expression in cancerous tissue is below normal structure, and CA4 down regulation of gene expression is relatively
RAMP3 gene becomes apparent from, and result above demonstrates high flux transcript profile and expresses confluence analysis CA4 with RAMP3 of data non-
The result of low expression in Small Cell Lung Cancer With Selective tissue.
Embodiment 3 adenocarcinoma of lung cancerous tissue and cancer beside organism's CA4 expression conditions
One material and method
1, material
Non-small cell type pulmonary carcinoma is taken from 2005-2010 years inpatients, takes 134 example adenocarcinoma of lung cancerous tissues and the other group of cancer
Knit, it is numbered respectively.
2, method
Experimental technique in reference example 2.
Two results
In 116 example adenocarcinoma of lung samples, in 115 example samples, CA4 expression in cancerous tissue is less than cancer beside organism, only has
In 1 example sample, CA4 expression in cancerous tissue is the most clearly distinguished with cancer beside organism, and accuracy rate is 99%, shows CA4 base
Because there is good dependency with adenocarcinoma of lung, can be used for preparing adenocarcinoma of lung auxiliary diagnosis or prognosis preparation.
Embodiment 4 one kinds detects the detection kit of CA4 gene in adenocarcinoma of lung
Reagent constituents:
The present invention uses bioinformatics binding molecule biological experiment to verify, filters out adenocarcinoma of lung pathogenic related gene
CA4, and provide the PCR kit for fluorescence quantitative of detection CA4 expression, both facilitate Clinical practice, in turn ensure that result
Concordance, has good potential applicability in clinical practice.
Claims (8)
1. the reagent of detection CA4 gene expression dose or carbonic anhydrase IV application in the diagnosis and treatment preparation of preparation adenocarcinoma of lung.
Application the most according to claim 1, it is characterised in that the diagnosis and treatment preparation of described adenocarcinoma of lung includes using fluorescent quantitation
In PCR method, method for gene chip detection adenocarcinoma of lung, CA4 gene expression dose or immunization method detect carbonic anhydrase in adenocarcinoma of lung
The product of IV.
Application the most according to claim 2, it is characterised in that described detects adenocarcinoma of lung for fluorescence quantifying PCR method
The product of middle CA4 gene expression dose contains the primer of a pair specific amplification CA4 gene；Described gene chip include with
The probe of the nucleic acid array hybridizing of CA4 gene；The product of described immunization method detection adenocarcinoma of lung includes special with carbonic anhydrase IV
Anisogamy antibody.
Application the most according to claim 3, it is characterised in that the primer upstream of the pair of specific amplification CA4 gene
Primer sequence is SEQ ID NO.1, and downstream primer sequence is SEQ ID NO.2.
5. according to the application described in Claims 2 or 3, it is characterised in that carbonic anhydrase in described immunization method detection adenocarcinoma of lung
The product of IV is ELISA detection kit and/gold-immunochromatographyreagent reagent for assay box.
Application the most according to claim 5, it is characterised in that described ELISA detection kit includes: be coated anti-CA4 mono-
The solid phase carrier of clonal antibody, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme is anti-
Answer stop buffer.
Application the most according to claim 5, it is characterised in that described gold-immunochromatographyreagent reagent for assay box uses colloid gold immune layer
Analysis technology or gold colloidal percolation detection target protein.
Application the most according to claim 5, it is characterised in that on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter
Detection zone is sprayed with anti-CA4 monoclonal antibody.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1256713A (en) * | 1997-03-17 | 2000-06-14 | 约安·普斯卡斯 | Rapid method of cancer diagnosis |
| CN102099485A (en) * | 2007-10-23 | 2011-06-15 | 临床基因组学有限公司 | A method of diagnosing neoplasms - II |
| CN104136629A (en) * | 2011-10-24 | 2014-11-05 | 玛特辛格纳治疗股份有限公司 | Carbonic anhydrase ix-related markers and use thereof |
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2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1256713A (en) * | 1997-03-17 | 2000-06-14 | 约安·普斯卡斯 | Rapid method of cancer diagnosis |
| CN102099485A (en) * | 2007-10-23 | 2011-06-15 | 临床基因组学有限公司 | A method of diagnosing neoplasms - II |
| CN104136629A (en) * | 2011-10-24 | 2014-11-05 | 玛特辛格纳治疗股份有限公司 | Carbonic anhydrase ix-related markers and use thereof |
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