CN104141013B - A kind of diagnosis of colon cancer kit and preparation method thereof - Google Patents
A kind of diagnosis of colon cancer kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses FOXD4L4 gene purposes in the kit preparing diagnosing colon.The present invention utilizes NCBI GEO data retrieval to include satisfactory 5 set colon cancer cases and normal population genome mrna expression chip data in research range, by said chip data are carried out Meta analysis, filtering out FOXD4L4 gene is significant difference gene, compare normal structure, FOXD4L4 gene expresses downward in colon cancer tissue, and uses the above-mentioned conclusion of fluorescence real-time quantitative PCR method validation.Accordingly, the kit that the invention also discloses a kind of diagnosing colon and the method utilizing this kit diagnosing colon.The molecule mechanism that one aspect of the present invention is research colon cancer provides new thinking, on the other hand provides the sensitiveest diagnostic kit for early diagnosis colon cancer.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of diagnosis of colon cancer kit and FOXD4L4 gene at preparation knot
Application in intestinal cancer diagnostic kit.
Background technology
Colon cancer is the common cancer of masculinity and femininity the 3rd, points out in american cancer statistical reports in 2013
2012 annual U.S. colon cancer new cases 103170.And in China, along with life style and the change of eating habit in recent years,
Colon cancer morbidity and death the most quickly rise, and according to statistical result showed in 2011, colon cancer occupied China's Cancer Mortality
3rd, be the 4th malignant tumour causing death, and M & M, the most close to same time world standard, becomes tight
Heavily threaten the public health problem of crowd's life and health.The pathogenesis of colon cancer illustrates the most completely, but Study of Etiology is
Through showing, overwhelming majority colon cancer is that multi-step, a multistage, environmental factor and inherent cause are coefficient multiple
Miscellaneous process.Its development that occurs relates to the change of multiple gene, shows as collaborative accumulation and the phase interaction of polygenes multi-step
With.In recent years, the technology such as genetic chip and high flux screening had the most been applied to carry out more to colon cancer gene expression profile
Research.But the difference of different experiment porch and sample causes analysis result to there is a lot of inconsistency.
Meta analyze can correlative study delivered to same problem report result be collected, statistical integration,
To obtaining more accurate or more result.This is analyzed and can produce a lot of significantly difference expression gene, is avoided that single grinding
Study carefully the incorrectness brought.It is the powerful tool of Recognition Different expressing gene in multiple array experiment are studied that Meta analyzes.
Can recognize that, additionally, analyze with Meta, the differential gene that single research can not identify, and one single chip number can be effectively reduced
According to false positive.
The present invention carries out Meta analysis by the colon cancer transcript profile delivered is expressed data, filters out and affects colon cancer
There is the key gene of development, and use the expression in fluorescence real-time quantitative PCR (QPCR) detection checking clinical sample, the present invention
Excavate the effect in colon cancer of these genes by bioinformatics means, thus the pathogenesis of colon cancer is visited
Study carefully, provide certain Research foundation for its diagnosis and treatment.
Summary of the invention
The present invention carries out Meta analysis and QPCR checking by the colon cancer transcript profile delivered is expressed data, finds
The expression in colon cancer tissue of the FOXD4L4 gene there are differences with expression in the normal tissue.Compared with normal structure,
FOXD4L4 gene expresses downward in colon cancer tissue, by detecting the expression of FOXD4L4 gene transcription level, permissible
Judge whether experimenter suffers from colon cancer.
It is an object of the invention to provide FOXD4L4 gene purposes in preparing diagnosis of colon cancer kit.Described examine
Disconnected kit comprises SYBR Green PCR system, for expanding the primer of FOXD4L4 gene and house-keeping gene
Right.SYBR Green PCR system comprises PCR buffer solution, dNTPs, SYBR Green fluorescent dye.
In technique scheme, the prior art that PCR buffer solution is known to the skilled person, it is preferable that PCR buffers
Liquid comprises: 25mM KCl, 2.5mM MgCl2, 200mM (NH4)2SO4。
Primer is to being that those skilled in the art can design according to the conventional design principle of design of primers.Preferably embodiment party
In case, the forward primer sequence of amplification FOXD4L4 gene is 5 '-GAGGCGAGACAGCAGTTCCTAGAG-3 ', reverse primer sequence
It is classified as 5 '-GATGTACGAGTAGGGGGGCTTTG-3 '；The preferred GAPDH of house-keeping gene, expands the forward primer sequence of this gene
Being 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also comprises M-MLV reverse transcription body
System, this reverse transcription system comprises: T repetition oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptase, RNase press down
Preparation, dNTPs.
Preferably reverse transcription reaction liquid comprises: the MgCl of the KCL of the Tris-HCL of 250mM PH8.3,375mM, 15mM2,
The DTT of 50mM.
RNase inhibitor can be selected for the noncompetitive of RNase inhibitor commonly used in the art, preferably Bacillus coli expression
The recombinant protein enzyme of suppression RNase.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also comprises RNA and extracts reagent,
RNase extracts reagent and comprises Trizol, chloroform, isopropanol, 75% ethanol.
The diagnostic kit of the present invention is stored in-20 DEG C, reduces multigelation as far as possible.
A further object of the present invention is to provide the kit of a kind of diagnosing colon.Described diagnostic kit comprises
SYBR Green PCR system, for expanding the primer pair of FOXD4L4 gene and house-keeping gene.Described SYBR
Green PCR system comprises PCR buffer solution, dNTPs, SYBRGreen fluorescent dye.
In technique scheme, the prior art that PCR buffer solution is known to the skilled person, it is preferable that PCR buffers
Liquid comprises: 25mM KCl, 2.5mM MgCl2, 200mM (NH4)2SO4。
Primer is to being that those skilled in the art can design according to the conventional design principle of design of primers；Preferably embodiment party
In case, the forward primer sequence of amplification FOXD4L4 gene is 5 '-GAGGCGAGACAGCAGTTCCTAGAG-3 ', reverse primer sequence
It is classified as 5 '-GATGTACGAGTAGGGGGGCTTTG-3 '；The preferred GAPDH of house-keeping gene, expands the forward primer sequence of this gene
Being 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also comprises M-MLV reverse transcription body
System, this reverse transcription system comprises: T repetition oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptase, RNase press down
Preparation, dNTPs.
Preferably reverse transcription reaction liquid comprises: the MgCl of the KCL of the Tris-HCL of 250mM PH8.3,375mM, 15mM2,
The DTT of 50mM.
RNase inhibitor can be selected for the noncompetitive of RNase inhibitor commonly used in the art, preferably Bacillus coli expression
The recombinant protein enzyme of suppression RNase.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also comprises RNA and extracts reagent,
RNase extracts reagent and comprises Trizol, chloroform, isopropanol, 75% ethanol.
The method that present invention also offers diagnosing colon, described method includes:
(1) utilize RNA to extract reagent and extract sample total serum IgE；
(2) the RNA reverse transcription that step (1) obtains is become cDNA；
(3) on fluorescence real-time quantitative PCR instrument, FOXD4L4 gene and house-keeping gene are carried out augmentation detection；
(4) determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis；
(5) FOXD4L4 down regulation of gene expression, shows that research object is colorectal cancer patients.
The specific implementation method of step (1) is: collects and frozen after sample after liquid nitrogen, taking-up, tissue is put into precooling
Mortar is ground, after tissue samples is powdered:
1. Trizol, room temperature preservation 5min are added；
2. adding chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5min-10min；
3. draw upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15min in another new centrifuge tube, note not
Protein substance between two-layer aqueous phase to be drawn onto.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, the most reverse mixed
Even, it is placed in 10min on ice；
4. 12000rpm carefully discards supernatant at a high speed after 15min, adds 75% in the ratio of 1ml/ml Trizol
DEPC ethanol washing precipitation (4 DEG C of preservations), washing precipitate, vibration mixing, 12000rpm high speed centrifugation 5min at 4 DEG C；
5. discarding ethanol liquid, ambient temperatare puts 5min fully to dry precipitation, add the water that DEPC processed dissolve heavy
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet specrophotometer, frozen in-70 DEG C.
The specific implementation method of step (2) is: with RT Buffer, l μ g total serum IgE is carried out reverse transcription and synthesizes cDNA.Adopt
By 25 μ l reaction systems, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into following components: DEPC in PCR pipe
Water, 5 × RT Buffer, 10mmol/L dNTP, 0.1mmol/l DTT, 30 μm mol/l Oligo dT, 200U/ μ l M-
MLV RT, template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
The specific implementation method of step (3) is: use 25 μ l reaction systems, and each sample arranges 3 parallel pipes, all expansions
Increase reaction above to ensure the reliability of result.Prepare following reaction system: SYBR Green polymerase chain
Reaction system 12.5 μ l, forward primer (5 μMs/μ l) 1 μ l, reverse primer (5 μMs/μ l) 1 μ l, template cDNA 2.0 μ l, without enzyme water
8.5μl；The forward primer sequence of amplification FOXD4L4 gene is 5 '-GAGGCGAGACAGCAGTTCCTAGAG-3 ', reverse primer
Sequence is 5 '-GATGTACGAGTAGGGGGGCTTTG-3 '；The forward primer sequence of amplification GAPDH gene is 5 '-
TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 ', operations
All carry out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulations.Using SYBR Green as
Fluorescent marker, reacts at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument, true by melt curve analysis analysis and electrophoresis
Determining purpose band, Δ Δ CT method carries out relative quantification.
Advantages of the present invention and having the beneficial effects that: (1) present invention firstly discloses FOXD4L4 gene and colon cancer phase
Closing, FOXD4L4 gene is expected to become the molecular marker of diagnosing colon, and the molecule mechanism for research colon cancer provides new
Thinking.(2) method utilizing the presence or absence of the mode diagnosing colon of detection gene expression is sensitiveer, and beneficially disease is early
The diagnosis of phase.
Accompanying drawing illustrates:
Fig. 1 represents that fluorescence real-time quantitative PCR measures the phase of FOXD4L4 gene mRNA in normal structure and colon cancer tissue
To expression.
Specific embodiment:
Further illustrating the present invention below in conjunction with specific embodiment, embodiments of the invention are only used for explaining the present invention,
It is not intended to limit protection scope of the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The gene that embodiment 1 screening is relevant to colon cancer
1.1NCBI GEO (Gene Expression Omnibus) data retrieval
GEO (Gene Expression Omnibus) database is by NCBI (US National Biotechnology Information center)
Exploitation is safeguarded, GEO database is as the database of maximum gene expression data, and this database is based on chip data, in addition
Also comprise data such as SAGE (serial analysis of gene expression) data of some non-chip types, (ribosomes 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..
A. search key: (" colorectal cancer " [MeSH Terms] OR " colorectal cancer "
[All Fields])AND"Homo sapiens"[porgn]
B. the screening sample strategy in research:
Limiting research type is that " expression profiling by array " meets the data set of following standard and will receive
Enter in our research: 1. selected data collection must be the expression mRNA transcript profile data of full-length genome, and is all RNA-seq number
According to collection；2. these data come from colorectal cancer case group and the biopsy of control group or the cell of cultivation；3. this research is all examined
Consider normalized or raw data set；Finally, 5 set RNA-seq data sets are had to include in our research (table 1).
The basic condition of table 18 set colon cancer full-length genome data set
1.2 colon cancer microarray data Meta analysis results
After initial data being carried out background correction and standardization by transcript profile DAS, utilize microarray notable
Property component software (significance analysis of microarray, SAM) is to 5 sets of data collection standardizations, and it is poor to screen
Different expressing gene, setting false positive rate (false discovery rate, FDR) during analysis is≤0.01, filters out altogether
883 difference expression genes.
Embodiment 2QPCR checking candidate gene and the relation of colon cancer
Based on the result analyzing colon cancer microarray data Meta, according to the size of P value, we select
FOXD4L4 gene (Meta analyzes and shows compared with normal structure, and FOXD4L4 gene expresses downward in colon cancer tissue) is carried out
Checking.Collect 50 colon cancer tissue samples, collect 50 normal structure samples simultaneously, use fluorescence real-time quantitative PCR to carry out
Classical molecular biology experiment checking (qRT-PCR), concrete operation step is as follows:
(1) RNA extracts
After collecting sample, frozen mortar tissue being put into precooling after liquid nitrogen, taking-up is ground, sample to be organized
After this is powdered:
1. Trizol, room temperature preservation 5min are added；
2. adding chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5min-10min；
3. draw upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15min in another new centrifuge tube, note not
Protein substance between two-layer aqueous phase to be drawn onto.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, the most reverse mixed
Even, it is placed in 10min on ice；
4. 12000rpm carefully discards supernatant at a high speed after 15min, adds 75% in the ratio of 1ml/ml Trizol
DEPC ethanol washing precipitation (4 DEG C of preservations), washing precipitate, vibration mixing, 12000rpm high speed centrifugation 5min at 4 DEG C；
5. discarding ethanol liquid, ambient temperatare puts 5min fully to dry precipitation, add the water that DEPC processed dissolve heavy
Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet specrophotometer, frozen in-70 DEG C.
(2) reverse transcription
With RT Buffer, l μ g total serum IgE is carried out reverse transcription and synthesize cDNA.Use 25 μ l reaction systems, each sample
Take 1 μ g total serum IgE as template ribonucleic acid, PCR pipe is separately added into following components: DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.Hatch for 42 DEG C
1h, 72 DEG C of 10min, of short duration centrifugal.
(3) QPCR amplification inspection
Using 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to protect
The reliability of card result.Prepare following reaction system: SYBR Green PCR system 12.5 μ l, forward primer (5
μM/μ l) 1 μ l, reverse primer (5 μMs/μ l) 1 μ l, template cDNA 2.0 μ l, without enzyme water 8.5 μ l；Amplification FOXD4L4 gene is just
It is 5 '-GAGGCGAGACAGCAGTTCCTAGAG-3 ' to primer sequence, reverse primer sequences is 5 '-
GATGTACGAGTAGGGGGGCTTTG-3’；, the forward primer sequence of amplification GAPDH gene is 5 '-
TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 ', operations
All carry out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulations.Using SYBR Green as
Fluorescent marker, reacts at the Light Cycler enterprising performing PCR of fluorescence real-time quantitative PCR instrument, is analyzed by melt curve analysis and electricity
Swimming determines that purpose band, Δ Δ CT method carry out relative quantification, result as it is shown in figure 1, compared with normal structure, FOXD4L4 gene
Expression in colon cancer tissue is lowered, consistent with Meta analysis result.
The preparation of embodiment 3 diagnosis of colon cancer kit
According to the correlation of FOXD4L4 gene Yu colon cancer, can be examined by the expression of detection FOXD4L4 gene
Whether disconnected colon cancer occurs, and the invention provides a kind of examination carrying out diagnosing colon based on detection FOXD4L4 gene expression accordingly
Agent box, the component in this diagnostic kit is as follows: SYBR Green PCR system；Amplification FOXD4L4 gene and
The primer pair of GAPDH gene.The forward primer sequence of amplification FOXD4L4 gene is 5 '-GAGGCGAGACAGCAGTTCCTAGAG-
3 ', reverse primer sequences is 5 '-GATGTACGAGTAGGGGGGCTTTG-3 '；The forward primer sequence of amplification GAPDH is 5 '-
TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.SYBR
Green PCR system comprises PCR buffer solution, dNTPs, SYBR Green fluorescent dye.PCR buffer components
For: 25mM KCL, 2.5mM MgCL2、200mM(NH4)2SO4。
The preparation of embodiment 4 diagnosis of colon cancer kit
According to the correlation of FOXD4L4 gene Yu colon cancer, can be examined by the expression of detection FOXD4L4 gene
Whether disconnected colon cancer occurs, and the invention provides a kind of examination carrying out diagnosing colon based on detection FOXD4L4 gene expression accordingly
Agent box, the component in this diagnostic kit is as follows: SYBR Green PCR system, amplification FOXD4L4 gene and
The primer of GAPDH gene is to, M-MLV reverse transcription system.The forward primer sequence of amplification FOXD4L4 gene is 5 '-
GAGGCGAGACAGCAGTTCCTAGAG-3 ', reverse primer sequences is 5 '-GATGTACGAGTAGGGGGGCTTTG-3 '；Amplification
The forward primer sequence of GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-
GGTGGAATCATATTGGAACA-3’.SYBR Green PCR system comprises PCR buffer solution, dNTPs, SYBR
Green fluorescent dye.PCR buffer composition is: 25mM KCL, 2.5mMMgCL2、200mM(NH4)2SO4.M-MLV reverse transcription body
It is that component is: T repeats oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptase, RNase inhibitor, dNTPs.
Reverse transcription reaction liquid component is: 250mM Tris-HCL (PH8.3), 375mM KCL, 15mM MgCL2, the DTT of 50mM.RNase
Inhibitor is the recombinant protein enzyme of the Noncompetition inhibition RNase of Bacillus coli expression.
The preparation of embodiment 5 diagnosis of colon cancer kit
According to the correlation of FOXD4L4 gene Yu colon cancer, can be examined by the expression of detection FOXD4L4 gene
Whether disconnected colon cancer occurs, and the invention provides a kind of examination carrying out diagnosing colon based on detection FOXD4L4 gene expression accordingly
Agent box, the component in kit is as follows: SYBR Green PCR system, amplification FOXD4L4 gene and GAPDH base
, RNA is extracted reagent by the primer of cause.The forward primer sequence of amplification FOXD4L4 gene is 5 '-
GAGGCGAGACAGCAGTTCCTAGAG-3 ', reverse primer sequences is 5 '-GATGTACGAGTAGGGGGGCTTTG-3 '；Amplification
The forward primer sequence of GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-
GGTGGAATCATATTGGAACA-3’.SYBR Green PCR system comprises PCR buffer solution, dNTPs, SYBR
Green fluorescent dye.PCR buffer composition is: 25mM KCL, 2.5mM MgCL2、200mM(NH4)2SO4.RNA extracts reagent
Comprise Trizol, chloroform, isopropanol, 75% ethanol.
The preparation of embodiment 6 diagnosis of colon cancer kit
According to the correlation of FOXD4L4 gene Yu colon cancer, can be examined by the expression of detection FOXD4L4 gene
Whether disconnected colon cancer occurs, and the invention provides a kind of examination carrying out diagnosing colon based on detection FOXD4L4 gene expression accordingly
Agent box, reagent constituents is as follows: SYBR Green PCR system, amplification FOXD4L4 gene and GAPDH gene
Primer to, M-MLV reverse transcription system, RNA extract reagent.The forward primer sequence of amplification FOXD4L4 gene is 5 '-
GAGGCGAGACAGCAGTTCCTAGAG-3 ', reverse primer sequences is 5 '-GATGTACGAGTAGGGGGGCTTTG-3 '；Amplification
The forward primer sequence of GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-
GGTGGAATCATATTGGAACA-3’.SYBR Green PCR system comprises PCR buffer solution, dNTPs, SYBR
Green fluorescent dye.PCR buffer composition is: 25mM KCL, 2.5mM MgCL2、200mM(NH4)2SO4.M-MLV reverse transcription
System component is: T repeat oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptase, RNase inhibitor,
dNTPs.Reverse transcription reaction liquid component is: the Tris-HCL (PH8.3) of 250mM, the MgCL of KCL, 15mM of 375mM2, 50mM's
DTT.RNase inhibitor is the recombinant protein enzyme of the Noncompetition inhibition RNase of Bacillus coli expression.RNA extracts reagent and comprises
Trizol, chloroform, isopropanol, 75% ethanol.
Claims (5)
1.FOXD4L4 gene purposes in preparing diagnosis of colon cancer kit, it is characterised in that described diagnostic kit comprises
SYBR Green PCR system, for expanding the primer pair of FOXD4L4 gene and house-keeping gene；Described SYBR
Green PCR system comprises: PCR buffer solution, dNTPs, SYBR Green fluorescent dye.
Purposes the most according to claim 1, it is characterised in that the sequence of the primer pair of described amplification FOXD4L4 gene is such as
Under: forward primer sequence is 5 '-GAGGCGAGACAGCAGTTCCTAGAG-3 ', reverse primer sequences is 5 '-
GATGTACGAGTAGGGGGGCTTTG-3’；Described house-keeping gene is GAPDH, and the forward primer sequence of amplification GAPDH gene is
5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
Purposes the most according to claim 1, it is characterised in that described PCR buffer solution comprises: 25mM KCl, 2.5mM
MgCl2、200mM(NH4)2SO4。
4. according to the purposes according to any one of claim 1-3, it is characterised in that described diagnostic kit also comprises M-MLV
Reverse transcription system or RNA extract reagent；Described reverse transcription system comprise T repeat oligonucleotides Oligo dT, reverse transcription reaction liquid,
M-MLV reverse transcriptase, RNase inhibitor, dNTPs；Described RNA extracts reagent and comprises Trizol, chloroform, isopropanol, 75% second
Alcohol.
Purposes the most according to claim 4, it is characterised in that described reverse transcription reaction liquid comprises: 250mM pH8.3's
The MgCl of KCl, 15mM of Tris-HCl, 375mM2, the DTT of 50mM.
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| Title |
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| DNA甲基化在卵巢癌中的研究进展;王文文等;《国际生殖健康/计划生育杂志》;20120731;第31卷(第4期);全文 * |
| Identification of candidate epigenetic biomarker for varian cancer detection;Huang Y W等;《Oncol Rep.》;20091031;第22卷(第4期);853-861 * |
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