CN108531597A - A kind of detection kit for oral squamous cell carcinomas early diagnosis - Google Patents
A kind of detection kit for oral squamous cell carcinomas early diagnosis Download PDFInfo
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- CN108531597A CN108531597A CN201810416417.4A CN201810416417A CN108531597A CN 108531597 A CN108531597 A CN 108531597A CN 201810416417 A CN201810416417 A CN 201810416417A CN 108531597 A CN108531597 A CN 108531597A
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- 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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The present invention relates to technical field of gene detection, disclose a kind of detection kit for oral squamous cell carcinomas early diagnosis, including 11 kinds of genes:The upstream and downstream primer sequence of the mRNA sequence of MMP1, PLAU, SPP1, KRT15, NUM, IL1RN, HOPX, TGM3, KRT13, MAL, EMP1.Gene or assortment of genes method the invention also discloses a kind of screening for oral squamous cell carcinomas early diagnosis.The present invention be put forward for the first time for oral squamous cell carcinomas early diagnosis detection kit, can it is objective, early, accurately, delicately make a definite diagnosis oral squamous cell carcinomas, to help to improve Patients With Oral Squamous Cell Carcinoma survival rate.
Description
Technical field
The present invention relates to technical field of gene detection more particularly to a kind of detection reagents for oral squamous cell carcinomas early diagnosis
Box.
Background technology
Oral squamous cell carcinomas is most commonly seen one of the malignant tumour of oromaxillo-facial region, and 5 years survival rates of late period oral squamous cell carcinomas are low, and
And face wound caused by radical surgery and swallow, disfluency seriously affects the life quality and mental health of patient.It is early
Phase makes a definite diagnosis and medical measure appropriate is the sole mode for improving Patients With Oral Squamous Cell Carcinoma survival rate.
Tissue pathology still rely on pathologist judgement at present, and different pathological doctor assessment has subjectivity,
It is less reproducible.With the development of accurate medicine, increasingly it is taken seriously with the technology that molecule parting diagnoses.It is presently the most ripe
Molecule parting diagnostic techniques be use instant PCR (Real-time Polymerase Chain
Reaction, wherein Polymerase Chain Reaction hereinafter referred to as PCR) technology detects the expression of certain gene mRNAs,
It is used as biomolecule marker (gene or the assortment of genes), the feature for mainly utilizing it high-throughput both ensure that and detect
Journey it is reliable, also improve the accurate, credible of testing result, and this kind of predicted gene mRNA is in other position malignant tumours of human body
In have been reported that, but had not been reported in oral squamous cell carcinomas.
Gene mRNA is as its existing unique advantage of biomolecule marker, but there is also some technical problems, samples
Amount is bigger, and independent detection crowd is more, and gene mRNA forecasting accuracy is higher, the feature of closer crowd's entirety, but large sample
Collection there are certain difficulty with processing, the reason is that there are certain requirements to entering a group sample, meet the requirements sample not easily collecting, sample
Keep records of heavy workload.
The means of the candidate marker gene mRNA molecule of mainstream screening at present are usually high throughput method primary dcreening operation, then again with being
When PCR verified, method is stablized, technical comparative maturity, but be also susceptible to some false positive results;
Candidate gene mRNA molecules are detected using sonde method, while promoting detection specificity, can also reduce experiment sensitivity, centainly
There can be rate of missed diagnosis in degree, and can also there are some differences between different batches sample and experiment reagent, and influence result.People
Body is a complicated system, and there are certain mistaken diagnosis and rates of missed diagnosis for term single gene mRNA predictions, pass through efficient mathematical modeling
Method to look for the gene mRNA of tumour-specific be also key to the issue, only determine the gene mRNA in tumour source, and right
It, which carries out analysis, preferably to diagnose and predict.
Therefore, to improve objectivity, accuracy and the specificity of oral squamous cell carcinomas early diagnosis, those skilled in the art causes
In finding the biomolecule marker (gene or the assortment of genes) for oral squamous cell carcinomas early diagnosis, develop a kind of can be used for power
The detection kit of oral squamous cell carcinomas early diagnosis.
Invention content
It is early for oral squamous cell carcinomas the technical problem to be solved by the present invention is to determine in view of the drawbacks described above of the prior art
The biomolecule marker (gene or the assortment of genes) of phase diagnosis develops a kind of detection that can be used for oral squamous cell carcinomas early diagnosis
Kit.
To achieve the above object, the present invention provides a kind of detection kit that can be used for oral squamous cell carcinomas early diagnosis, packets
Include 11 kinds of genes:The mRNA sequence of MMP1, PLAU, SPP1, KRT15, NUM, IL1RN, HOPX, TGM3, KRT13, MAL, EMP1
Upstream and downstream primer sequence.
Further, further include the primer of house-keeping gene as internal reference.
Further, the house-keeping gene is β-actin.
Further, further include:Total serum IgE extraction agent, and/or PCR Reverse Transcriptions, and/or PCR quantitative reagents.
Further, the total serum IgE extraction agent includes TRIzol reagents.
Gene or assortment of genes method the present invention also provides a kind of screening for oral squamous cell carcinomas early diagnosis, including with
Lower step:
Step 1: filtering out oral squamous cell carcinomas relevant difference expressing gene, the oral squamous cell carcinomas relevant difference expression base is synthesized
Simultaneously detection kit is made in the primer of the mRNA sequence of cause, and the oral squamous cell carcinomas relevant difference expressing gene includes:MMP1、
PLAU、SPP1、KRT15、NUM、IL1RN、HOPX、TGM3、KRT13、MAL、EMP1;
Step 2: the tumor tissues of acquisition Patients With Oral Squamous Cell Carcinoma, normal structure set up candidate set, made of step 1
Detection kit quantitatively detects the expression of the expressing gene of oral squamous cell carcinomas relevant difference described in candidate set, wherein checkout procedure
The reaction condition of middle gene magnification is consistent and annealing temperature is 60 DEG C;
Step 3: according to the expression of the oral squamous cell carcinomas relevant difference expressing gene, using OPLS-DA modeling methods
It is analyzed with ROC curve, determines the gene of oral squamous cell carcinomas early diagnosis.
Further, following steps are gone back:
Step 4: in addition the tumor tissues of acquisition Patients With Oral Squamous Cell Carcinoma, normal structure set up individual authentication group, step is utilized
Detection kit made of one quantitatively detects the expression of the early diagnosis gene of oral squamous cell carcinomas described in validation group, wherein examining
The reaction condition of gene magnification is consistent in the process and annealing temperature is 60 DEG C;
Step 5: according to the oral squamous cell carcinomas early diagnose gene expression, using OPLS-DA models verification and
ROC curve is analyzed, and Stability and veracity of the oral squamous cell carcinomas early diagnosis gene in oral squamous cell carcinomas early diagnosis is verified.
Further, the number of Patients With Oral Squamous Cell Carcinoma is greater than or equal to 30.
Further, in step 1, the method for screening the oral squamous cell carcinomas relevant difference expressing gene includes:To primary
Property oral squamous cell carcinomas tumor tissues and pairing self normal structure carry out chip gene expression profile detection, sieved by statistical method
Choosing obtains oral squamous cell carcinomas relevant difference expressing gene;And/or from the relevant chip gene expression profile Research Literature of oral squamous cell carcinomas
Middle screening obtains oral squamous cell carcinomas relevant difference expressing gene.
The present invention also provides MMP1, PLAU, SPP1, KRT15, NUM, IL1RN, HOPX, TGM3, KRT13, MAL, EMP1
The assortment of genes is being prepared for the application in the detection kit of oral squamous cell carcinomas early diagnosis.
Compared with prior art:The present invention is the detection kit for oral squamous cell carcinomas early diagnosis being put forward for the first time, energy
It is objective, early, accurately, delicately make a definite diagnosis oral squamous cell carcinomas, to help to improve Patients With Oral Squamous Cell Carcinoma survival rate.
The technique effect of design, specific implementation and the generation of the present invention is described further below with reference to attached drawing, with
It is fully understood from the purpose of the present invention, feature and effect.
Description of the drawings
Fig. 1 is the S-polt figures of 78 oral squamous cell carcinomas relevant difference expressing genes of screening group in present embodiment;
Fig. 2 is the MMP1+PLAU+SPP1+KRT15+NUM+IL1RN+HOPX+TGM3 of screening group in present embodiment
The Heatmap of+KRT13+MAL+EMP1 genes schemes;
Fig. 3 is the normal cancer beside organism of screening group in present embodiment and the OPLS-DA figures of oral squamous cell carcinoma tissues;
Fig. 4 is the MMP1+PLAU+SPP1+KRT15+NUM+IL1RN+HOPX+TGM3 of screening group in present embodiment
The ROC curve of+KRT13+MAL+EMP1 the assortments of genes;
Fig. 5 is the MMP1+PLAU+SPP1+KRT15+NUM+IL1RN+HOPX+TGM3 of validation group in present embodiment
The Heatmap of+KRT13+MAL+EMP1 genes schemes;
Fig. 6 is the normal cancer beside organism of validation group in present embodiment and the PCA-plot figures of oral squamous cell carcinoma tissues;
Fig. 7 is the MMP1+PLAU+SPP1+KRT15+NUM+IL1RN+HOPX+TGM3 of validation group in present embodiment
The ROC curve of+KRT13+MAL+EMP1 the assortments of genes.
Specific implementation mode
The preferred embodiment of the present invention is introduced below with reference to Figure of description, keeps its technology contents more clear and convenient for reason
Solution.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention is not limited only to text
In the embodiment mentioned.
Kit in the present embodiment, including total serum IgE (ribonucleic acid) extraction agent (producer:The winged generation that science and technology of Sai Mo (in
State) Co., Ltd, article No.:15596018), PCR Reverse Transcriptions (producer:Sai Mo flies scientific and technological (China) Co., Ltd of generation that, goods
Number:4374967), PCR quantitative reagents (producer:Sai Mo flies scientific and technological (China) Co., Ltd of generation that, article No.:11736059).Reagent
Box further includes the upstream and downstream primer sequence of following 78 oral squamous cell carcinomas relevant difference expressing gene mRNA sequences, selects β-
Actin house-keeping genes are synthesized as internal reference, all primers by Shanghai life work biology Co., Ltd.
TRIzol reagents (producer is used to collected 200mg people's flesh tissue:Invitrogen companies) carry out total serum IgE
Extracting, the total serum IgE that extracting is obtained utilize the High-Capacity cDNA Reverse with ribonuclease inhibitor
Transcription Kit (translated names:High power capacity cDNA Reverse Transcriptase kits, producer:Sai Mo flies scientific and technological (China) the limited public affairs of generation that
Department, article No.:4374967) reverse transcription is carried out.
Real-time PCR conditions are optimized:It is required that the reaction condition of all gene magnifications is consistent and annealing temperature
It is 60 DEG C.This step is one of committed step of this kit, this step utilizes the consistency of annealing temperature, substantially increases
Specificity in kit detection process.
The sample that reverse transcription is obtained carries out sonde method quantitative PCR reaction, obtains the opposite table of oral squamous cell carcinomas related gene
It is for statistical analysis up to value.
The specific preparation process of kit further includes in the present embodiment:
(1) oral squamous cell carcinomas relevant difference expressing gene is screened
Affymetrix HU95 bases are carried out by the self normal structure to 22 primary oral squamous cell carcinoma tissues and pairing
Because chip of expression spectrum (chip production person be Affymetrix companies of the U.S.) detection and 7 kinds of statistical methods (T-test,
Wilcoxon rank-sum,paired t-test,Significant Analysis of Microaary,Predict
Parameter Value function of Gene Spring software,Mininum Distance to Modal
Ranking, WEighted Punishment on Overlap) screening obtain oral squamous cell carcinomas differential gene expression spectrum, in conjunction with
Clinical tumor sample determines 38 oral squamous cell carcinomas relevant difference expressing genes in genetic transcription and the verification of protein level.
" microarray analysis (microarray analysis) " and " head and neck scale carcinoma (head and neck are used again
Squamous cell carcinoma) " or " oral squamous cell carcinomas (oral squamous cell carcinoma) " based on epigraph exist
Retrieval is reported during having consulted 2000-2018 in public database (Pubmed) on US National Bioinformatics Institute website
With the relevant chip gene expression profile Research Literature of oral squamous cell carcinomas 42.With above-mentioned 38 oral squamous cell carcinomas relevant difference gene phase
It compares and searches new oral squamous cell carcinomas related gene.Searching oral squamous cell carcinomas relevant difference gene specific standards in document is:1. 4
Document above reports that the gene is oral squamous cell carcinomas relevant difference expressing gene, and the gene is raised and lowered in all documents
Gesture is consistent;2. in chip of expression spectrum, the tumour of the gene is more than 2 times and P < relative to the fold differences of normal tissue expression
0.05.It finally screens to 78 oral squamous cell carcinomas relevant difference expressing genes, wherein up-regulation gene 45, down-regulated gene 33.This
A little gene distributions find these gene wide participation items important physiological function tune in various tumour associated signal paths, GO classification
Control, including immune response, inflammatory reaction, growth and development, cycle regulating, cytoskeleton, stress reaction, Apoptosis, cell increasing
Grow regulation and control and cell adhesion etc..
The mRNA sequence that this 78 oral squamous cell carcinomas relevant difference expressing genes are found in ncbi database Genbank, is answered
With the upstream and downstream primer sequence of this 78 mRNA sequences of 5.0 Software for Design of Primer, select β-actin house-keeping genes as in
Reference, all primers are synthesized by Shanghai life work biology Co., Ltd.
(2) gene of oral squamous cell carcinomas early diagnosis is determined
Set up candidate set:Collect 120 Patients With Oral Squamous Cell Carcinomas oral squamous cell carcinoma tissues (N=120) and pairing it is self just
Normal cancer beside organism (N=120), can also be other normal structures.In the reaction condition of gene magnification, consistent and annealing temperature is equal
In the case of 60 DEG C, above-mentioned 78 oral squamous cell carcinomas relevant differences in candidate set are detected by qRT-PCR technologies (real-time and quantification PCR)
The expression of expressing gene, and by partial least squares analysis-discriminant analysis (OPLS-DA) modeling method of quadrature alignment and
Receiver operating curve analyzes the gene spectrotyping that (ROC curve) determines oral squamous cell carcinomas early diagnosis, and specific modeling method is such as
Under:
Model foundation:Xtr=TtrWT+TtroWo T Ytr=UtrCT
Difference expression gene decision criteria:The related coefficient of the one-component of expression and OPLS-DA is more than 0.5 simultaneously
And corresponding p value is less than 0.05 after Bonferroni corrections.
Fig. 1, Fig. 2, Fig. 3 are the OPLS-DA modeling result figures of candidate set.
According to as a result, wherein MMP1, PLAU, SPP1, KRT15, NMU, 1L1RN, HOPX, TGM3, KRT13, MAL, EMP1
United accuracy rate is best, and the area under the curve (AUC) of ROC curve is 0.99361, sees Fig. 4, thus select MMP1, PLAU,
The gene that SPP1, KRT15, NMU, 1L1RN, HOPX, TGM3, KRT13, MAL, EMP1 are early diagnosed as oral squamous cell carcinomas.
(3) gene of verification oral squamous cell carcinomas early diagnosis
Set up validation group:In addition oral squamous cell carcinoma tissues (N=37) and the pairing of 37 Patients With Oral Squamous Cell Carcinomas are independently had collected
Normal cancer beside organism (N=37).Reaction condition in gene magnification is consistent and annealing temperature is 60 DEG C, passes through
QRT-PCR technologies detect the expression of the candidate gene of above-mentioned 11 oral squamous cell carcinomas early diagnosis in screening group.Pass through OPLS-
DA models are verified and ROC curve analysis further assesses this 11 gene molecule expression spectral patterns in oral squamous cell carcinomas early diagnosis
Stability and veracity.
Model is verified
Model risk is predicted:To=Xtest Wo(Wo TWo)-1 Po=XtrTtro(Ttro TTtro)-1
Xtest=Xtest-ToPo TTest=XtestW(WTW)-1
Risk score:Riskscore=Ttest(TT trTtr)-1TT trUtrCT
Risk score>0 is diagnosed as oral squamous cell carcinomas, risk score<0 is diagnosed as non-oral squamous carcinoma
Fig. 5, Fig. 6 are the OPLS-DA verification result figures of validation group.
According to verification result, it is determined that MMP1+PLAU+SPP1+KRT15+NUM+IL1RN+HOPX+TGM3+KRT13+MAL
+ EMP1 combines the Stability and veracity in oral squamous cell carcinomas early diagnosis, and the AUC of ROC curve is 0.9664, sees Fig. 7, therefore
Further determine that MMP1, PLAU, SPP1, KRT15, NMU, 1L1RN, HOPX, TGM3, KRT13, MAL, EMP1 as oral squamous cell carcinomas
Early diagnose gene.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. a kind of detection kit for oral squamous cell carcinomas early diagnosis is characterized in that, including 11 kinds of genes:MMP1、PLAU、
The upstream and downstream primer sequence of the mRNA sequence of SPP1, KRT15, NUM, IL1RN, HOPX, TGM3, KRT13, MAL, EMP1.
2. the detection kit for oral squamous cell carcinomas early diagnosis as described in claim 1, which is characterized in that further include conduct
The primer of the house-keeping gene of internal reference.
3. the detection kit for oral squamous cell carcinomas early diagnosis as claimed in claim 2, which is characterized in that house keeper's base
Because of β-actin.
4. the detection kit as described in any one of claims 1-3 for oral squamous cell carcinomas early diagnosis, which is characterized in that also wrap
It includes:Total serum IgE extraction agent, and/or PCR Reverse Transcriptions, and/or PCR quantitative reagents.
5. the detection kit for oral squamous cell carcinomas early diagnosis as claimed in claim 4, which is characterized in that the total serum IgE
Extraction agent includes TRIzol reagents.
6. a kind of screening is characterized in that, including following step for the gene of oral squamous cell carcinomas early diagnosis or the method for the assortment of genes
Suddenly:
Step 1: filtering out oral squamous cell carcinomas relevant difference expressing gene, the oral squamous cell carcinomas relevant difference expressing gene is synthesized
Simultaneously detection kit is made in the primer of mRNA sequence, and the oral squamous cell carcinomas relevant difference expressing gene includes:MMP1、PLAU、
SPP1、KRT15、NUM、IL1RN、HOPX、TGM3、KRT13、MAL、EMP1;
Step 2: the tumor tissues of acquisition Patients With Oral Squamous Cell Carcinoma, normal structure set up candidate set, detected using made of step 1
Kit quantification detects the expression of oral squamous cell carcinomas relevant difference expressing gene described in candidate set, wherein base in checkout procedure
The reaction condition of gene-amplification is consistent and annealing temperature is 60 DEG C;
Step 3: according to the expression of the oral squamous cell carcinomas relevant difference expressing gene, using OPLS-DA modeling methods and
ROC curve is analyzed, and determines the gene of oral squamous cell carcinomas early diagnosis.
7. screening as claimed in claim 6 is for the gene of oral squamous cell carcinomas early diagnosis or the method for the assortment of genes, feature
It is, it is further comprising the steps of:
Step 4: in addition the tumor tissues of acquisition Patients With Oral Squamous Cell Carcinoma, normal structure set up validation group, made of step 1
Detection kit quantitatively detects the expression of the early diagnosis gene of oral squamous cell carcinomas described in validation group, wherein base in checkout procedure
The reaction condition of gene-amplification is consistent and annealing temperature is 60 DEG C;
Step 5: the expression of gene is early diagnosed according to the oral squamous cell carcinomas, it is bent using the verification of OPLS-DA models and ROC
Line analysis verifies Stability and veracity of the oral squamous cell carcinomas early diagnosis gene in oral squamous cell carcinomas early diagnosis.
8. screening as claimed in claims 6 or 7 is for the gene of oral squamous cell carcinomas early diagnosis or the method for the assortment of genes, special
Sign is that the number of Patients With Oral Squamous Cell Carcinoma is greater than or equal to 30.
9. screening as claimed in claims 6 or 7 is for the gene of oral squamous cell carcinomas early diagnosis or the method for the assortment of genes, special
Sign is, in step 1, the method for screening the oral squamous cell carcinomas relevant difference expressing gene includes:To primary oral squamous cell carcinomas
Tumor tissues and the self normal structure of pairing carry out chip gene expression profile detection, are screened by statistical method and obtain oral cavity
Squamous carcinoma relevant difference expressing gene;And/or it screens and obtains from the relevant chip gene expression profile Research Literature of oral squamous cell carcinomas
Oral squamous cell carcinomas relevant difference expressing gene.
It is prepared by 10.MMP1, PLAU, SPP1, KRT15, NUM, IL1RN, HOPX, TGM3, KRT13, MAL, EMP1 assortment of genes
For the application in the detection kit of oral squamous cell carcinomas early diagnosis.
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CN113804901A (en) * | 2020-06-15 | 2021-12-17 | 南京市口腔医院 | Serum lipid marker for early noninvasive diagnosis of oral squamous cell carcinoma and application thereof |
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CN114672553A (en) * | 2020-12-24 | 2022-06-28 | 中国医学科学院肿瘤医院 | Application of KRT15 in auxiliary diagnosis and targeted therapy of esophageal cancer |
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CN113834927A (en) * | 2020-06-08 | 2021-12-24 | 南京市口腔医院 | Salivary metabolism marker for predicting canceration of oral mucosa precancerous lesion patient and application thereof |
CN113804901A (en) * | 2020-06-15 | 2021-12-17 | 南京市口腔医院 | Serum lipid marker for early noninvasive diagnosis of oral squamous cell carcinoma and application thereof |
CN113804901B (en) * | 2020-06-15 | 2023-06-23 | 南京市口腔医院 | Serum lipid marker for early noninvasive diagnosis of oral squamous carcinoma and application thereof |
CN112201356A (en) * | 2020-10-09 | 2021-01-08 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
CN112201356B (en) * | 2020-10-09 | 2022-02-01 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
CN114672553A (en) * | 2020-12-24 | 2022-06-28 | 中国医学科学院肿瘤医院 | Application of KRT15 in auxiliary diagnosis and targeted therapy of esophageal cancer |
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