CN105385752A - Detection method of human colon cancer protein marker Spondin-2 and detection kit thereof - Google Patents
Detection method of human colon cancer protein marker Spondin-2 and detection kit thereof Download PDFInfo
- Publication number
- CN105385752A CN105385752A CN201510128247.6A CN201510128247A CN105385752A CN 105385752 A CN105385752 A CN 105385752A CN 201510128247 A CN201510128247 A CN 201510128247A CN 105385752 A CN105385752 A CN 105385752A
- Authority
- CN
- China
- Prior art keywords
- spondin
- colon cancer
- expression
- sample
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of genetic engineering and medical detection, and particularly provides a detection method of a human colon cancer marker Spondin-2. The detection method includes the steps that genome DNA or protein in a sample is separated and extracted; and/or the content of the Spondin-2 in the obtained genome DNA or protein is detected. The invention further provides an application method of the human colon cancer protein marker Spondin-2. The Spondin-2 is used for preparing preparations for diagnosing or predicting or detecting or screening colon cancer and also can be used for preparing preparations for diagnosing or predicting or detecting or screening proliferation and transfer of colon cancer cells, colon cancer clinical staging and colon cancer patient prognosis. The scientific basis is provided for effectively judging the canceration process of the colon cancer. A detection kit is efficient, sensitive and high in specificity. The related detection method is fast, simple, convenient to use and low in cost.
Description
Technical field
The invention belongs to genetically engineered and medical science, relate to the detection method of a kind of secretory protein Spondin-2 as human colon carcinoma mark, detection kit and application method thereof.
Background technology
Colorectal carcinoma is worldwide the third-largest male tumor and second largest female tumor.According to the statistics of World Health Organization GLOBOCAN2012, pre-in 2012 in respect of 74.6 ten thousand male sex and 61.4 ten thousand female patients, overall lethality rate is 50.1% (male sex) and 52.1% (women), shows that prognosis existence is very poor.Tumour occurs not only to affect individual family, also brings serious social influence, increases Public Health Expenditure and financial burden.Therefore, strive for early discovery, early confirmation, early treatment, the curative ratio and prolongation prognosis survival time improving colorectal carcinoma is had very important significance.The onset stage general symptom of colorectal carcinoma is not obvious, is easily left in the basket, once after confirmation, often be in middle and advanced stage and make result for the treatment of not good, more affecting postoperative existence.So a key for the treatment of colorectal carcinoma or other cancers is to find colorectal carcinoma as early as possible.The diagnosis of general colorectal carcinoma, comprise observe bowl evacuation habit change, have blood in stool, stomachache etc., rectum refers to disease, and laboratory examination comprises routine blood test, routine urinalysis and stool routine examination, fecal occult blood testing, image check, B ultrasonic, CT scan, colonoscopy etc.Serum blood inspection be then pass through the most, conventional inspection, conventional mark is carcinomebryonic antigen (CEA), colorectal cancer antigen (CCA), CA19-9, but the clinical effectiveness comparison in difference of these Detection of antigen is large, and the sensitivity detected and specificity have much room for improvement.In this sense, find new while there is highly sensitive, the blood markers thing of high specific is a very urgent task.Utilize such Novel marker, in routine physical examination examination, just can find the existence of early stage colorectal carcinoma as soon as possible.
Secondly, for the outcome of patient, the prediction of Survival after Post operation or chemicotherapy, ordinary method depend on pathological parameter, TNM by stages, the Dukes method such as by stages, but on pathology judges, sometimes there is interference from human factor, be a long felt need for significant difference, highly sensitive, specificity good, easy to operate molecular marker is used as auxiliary judgment means, therefore, find the promising tumor marker of colorectal carcinoma, accurately judge that the clinical stages of adenocarcinoma of lung and prognosis become the urgent task of numerous medical investigators.
Summary of the invention
The object of this invention is to provide the application method of a kind of human colon carcinoma protein markers Spondin-2.
Another object of the present invention is to provide detection method and the detection kit thereof of human colon carcinoma protein markers Spondin-2.
In the present invention, SPON2 and Spondin-2.According to result of study of the present invention, quantitative PCR, enzyme immunoassay and histogenic immunity hybridization prove further, and the expression amount of Spondin-2 in colon cancer tissue is significantly higher than the expression amount in normal colonic tissue.Further, the high expression level of Spondin-2 will shorten colorectal cancer patients postoperative lifetime.Therefore, Spondin-2 can as the target of a kind of new human colon carcinoma mark and selective mechanisms, treatment drugs against colon cancer for effectively judging that colorectal carcinoma canceration process provides scientific basis.The present invention completes on this basis.
The invention provides the detection method of a kind of human colon carcinoma mark Spondin-2, comprise step:
Genomic dna in separation and Extraction sample or albumen;
And/or,
Detect the content of Spondin-2 in the genomic dna of gained or albumen.
Described sample is tissue or the body fluid in vitro people source.
Utilize the genomic dna in the primer amplified sample of Spondin-2, detect the DNA content of Spondin-2.In a preferred embodiment of the invention, utilize the primer amplified sample DNA of sequence as shown in SEQIDNO1 and SEQIDNO2, to determine existence and the content of Spondin-2 in sample DNA.
Utilize the albumen in the antibody of Spondin-2 and sample to react, detect the expressing quantity of Spondin-2.Such as, in a preferred embodiment of the invention, existence and the content of Spondin-2 albumen in westernblot technology determination sample is adopted.
Accordingly, present invention also offers the detection kit of human colon carcinoma mark Spondin-2, it comprises the primer of specific amplification Spondin-2, or for the antibody of Spondin-2 albumen.
Such as, the sequence of the primer of described specific amplification Spondin-2 is as shown in SEQIDNO1 and SEQIDNO2.
Preferably, the standard substance, buffer reagent, specification sheets etc. of Spondin-2 are also comprised in this test kit.
Human colon carcinoma protein markers detection kit of the present invention may be used for preparation diagnosis, prediction, detect or examination human colon carcinoma cancer cells diffusion, colorectal carcinoma drench carry down the sixth of the twelve Earthly Branches move, colorectal cancer clinical by stages with colorectal cancer patients prognosis, etc.
On the other hand, the invention provides the application method of human colon carcinoma mark Spondin-2, i.e. the application of Spondin-2 at preparation diagnosis or the transfer of prediction colorectal carcinoma, colorectal cancer clinical by stages, in the preparation of colorectal cancer patients prognosis.
Such as, the application of described mark Spondin-2 at preparation diagnosis or the transfer of prediction colorectal carcinoma, colorectal cancer clinical by stages, in the test kit of colorectal cancer patients prognosis.The application of described mark Spondin-2 in preparation diagnosis, alleviation or treatment colorectal carcinoma medicament.Spondin-2 can as screening preparation diagnosis, the target of alleviating or treating colorectal carcinoma medicament.
The invention provides one for distinguishing human colon cancer and Ai Pang normal colonic tissue, without nodus lymphoideus transferring rate colon cancer tissue and the plasmalemma protein mark Spondin-2 having nodus lymphoideus transferring rate colon cancer tissue, and utilize this mark to prepare to judge that human colon carcinoma, colorectal carcinoma spread, the preparation of colorectal carcinoma nodus lymphoideus transferring rate, clinical stages, colorectal cancer patients prognosis and prognosis and test kit.The present invention is for effectively judging that human colon carcinoma canceration process provides scientific basis.Detection kit of the present invention is efficient, sensitive, high specificity, and relevant detection method is quick, easy, and cost is lower.
Accompanying drawing explanation
Fig. 1 is the expression of gene expression chip data analysis Spondin-2 gene in colorectal cancer and normal colorectal carcinoma utilized in Oncomine public database.Fig. 1-A is the scatter diagram utilizing the expression of GaedckeColorectal data analysis Spondin-2 in 65 pairs of rectum and rectum cancer tissue, numeral case under loose point or the number of normal sample, p value is analyze with Studentttest (T inspection) significance value obtained, and what asterisk represented this T inspection is paired-samples T-test.Fig. 1-B is the scatter diagram utilizing the expression of GraudensColon data analysis Spondin-2 in 12 routine colons and 48 routine colon cancer tissues.Fig. 1-C is the scatter diagram utilizing the expression of HongColorectal data analysis Spondin-2 in 12 routine colons and 48 routine colon cancer tissues.Fig. 1-D is the scatter diagram utilizing the expression of SkrzypczakColorectal data analysis Spondin-2 in 24 routine colons and 81 routine colon cancer tissues.Fig. 1-E is the scatter diagram utilizing the expression of KiColon data analysis Spondin-2 in 28 routine colons and 82 routine colon cancer tissues.Fig. 1-F is the scatter diagram utilizing the expression of SkrzypczakColorectal2 data analysis Spondin-2 in 20 routine colons, 10 routine adenoma of colon and 10 routine colon cancer tissues.Fig. 1-G is the scatter diagram utilizing the expression of TCGAColorectal data analysis Spondin-2 in 22 routine colorectal carcinomas and 101 routine adenocarcinoma of colon, 60 routine colorectal carcinomas, 22 routine colonic mucus gland cancer and 22 routine adenocarcinom of cecum tissues.Fig. 1-H is the scatter diagram utilizing KaiserColon data analysis Spondin-2 to express in 12 routine colons and 41 routine adenocarcinoma of colon, 13 routine colonic mucus gland cancer, 17 routine adenocarcinoms of cecum, 8 routine second shape rectal adenocarcinomas and 4 routine rectal mucus adenocarcinoma tissue.
Fig. 2 is mRNA and the expression of protein in colorectal cancer cell system and tissue of analyzing Spondin-2.Fig. 2-A is the expression level of methods analyst Spondin-2 gene in 10 pairs of Colorectal Carcinomas with quantitative PCR.P value is analyze with Studentttest (T inspection) significance value obtained.Fig. 2-B is with the expression level of Westernblot methods analyst Spondin-2 albumen at colon carcinoma cell line.Fig. 2-C is that Spondin-2 albumen is added up in the expression of colorectal carcinoma and cancer beside organism, utilizes a commercialization organization chip (containing 90 routine cancer and cancer beside organisms) to carry out the immunohistochemical analysis of Spondin-2.Fig. 2-D is that Spondin-2 albumen is added up in the expression of colorectal carcinoma and cancer beside organism, utilizes another commercialization organization chip (containing 90 routine cancer and cancer beside organisms) to carry out the immunohistochemical analysis of Spondin-2.N representative enters effective number of cases of analysis.SPON2 represents Spondin-2.GAPDH represents dehydephosphatedehydrogenase and phosphoglyceraldehy-de dehydrogenase, as interior mark.Ordinate zou Opticaldensityvalue is optical density value, refers to the dyeing depth value obtained with software scans histochemical stain picture.The size reflection protein expression height of value, value is then expressed greatly high, is worth little, expresses low.
Fig. 3 is the result utilizing commercialization organization chip to carry out Spondin-2 immunohistochemical analysis.Fig. 3-A is the colon showing that Spondin-2 feminine gender is expressed.Fig. 3-B is the Ai Pang colon of Spondin-2 positive expression.Fig. 3-C is the colon cancer tissue that Spondin-2 feminine gender is expressed.Fig. 3-D is the colon cancer tissue of the low positive expression of Spondin-2.Fig. 3-E is the colon cancer tissue of the positives expression of Spondin-2.Fig. 3-F is the colon cancer tissue of the positive expression of Spondin-2.
Fig. 4 is the graph of a relation that the gene of Kaplan-Meier tracing analysis Spondin-2 and protein expression and colorectal carcinoma are survived.Fig. 4-A is the Kaplan-Meier tracing analysis figure of Spondin-2 genetic expression and colon cancer patient overall survival.Gene expression data derives from Oncomine genetic expression public database.Fig. 4-B is the Kaplan-Meier tracing analysis figure of Spondin-2 genetic expression and colon cancer patient disease free survival.Gene expression data derives from Oncomine genetic expression public database.Fig. 4-C is the Kaplan-Meier tracing analysis figure of Spondin-2 protein expression and colon cancer patient overall survival.SPON2 represents Spondin-2.
Fig. 5 is the Spondin-2 protein expression detected by the method for ELISA in colon cancer patient and human normal plasma.Fig. 5-A is the Spondin-2 protein expression in elisa assay colon cancer patient and human normal plasma, according to the amount of flow measurement Spondin-2 in patient and human normal plasma of standard substance, p value is analyze with Studentttest (T inspection) significance value obtained.N represents case load.Fig. 5-B is experimenter's performance characteristic (ROC) tracing analysis that Spondin-2 proteinplasm is expressed.X-coordinate represents false positive or 1-specificity, and ordinate zou represents true positives or sensitivity.Diagonal lines is reference line.AUC represents area under line.SPON2 represents Spondin-2.
Embodiment
Human colon carcinoma protein markers Spondin-2 of the present invention may be used for preparing diagnosis, prediction, detect or the diffusion of examination human colon carcinoma cancer cells, colorectal carcinoma nodus lymphoideus transferring rate, colorectal cancer clinical by stages, the preparation of colorectal cancer patients prognosis.Human colon carcinoma protein markers Spondin-2 may be used for preparing diagnosis, prediction, detecting or the preparation of examination human colon carcinoma; Especially for preparation diagnosis, prediction, detect or the test kit of examination human colon carcinoma.Below in conjunction with preferred embodiment, the present invention is elaborated, and can not the present invention be limited.
Embodiment 1 utilizes bioinformatics method to analyze the expression of Spondin-2 gene in colorectal carcinoma and cancer beside organism
Oncomine public database (https: //www.oncomine.org/resource/login.html) is utilized to analyze the expression of Spondin-2 gene in colorectal carcinoma and cancer beside organism.Analytical procedure: log in above-mentioned Oncomine database, input SPON2 gene name, tumor type selects colorectal carcinoma, data type selects mRNA, variance analysis type selecting cancer and normal assay, then analyze the expression of Spondin-2 gene in each data centralization, whether difference that its expression was downloaded, analyzed separately to each data centralization Spondin-2 gene respectively in cancer and the other expression values of cancer is remarkable.By analysis, prove that Spondin-2 gene presents remarkable high expression level trend compared to cancer beside organism in cancerous tissue, this prove by the analysis of multiple data set, comprise GaedckeColorectal (Fig. 1-A, n=65, p=4.3E-20), GraudensColondataset (figure-1B, n=48, p=0.0006), HongColorectaldataset (figure-1C, n=70, p=2.3E-7), SkrzypczakColorectaldataset (figure-1D, n=81, and KiColondataset (figure-1E, n=82, p=8.4E-9) p=0.0002).Spondin-2 raises and in colorectal carcinoma, expresses rising further in adenoma of colon, proves that Spondin-2 works in colorectal carcinoma generation evolution.And Spondin-2 gene all significantly rises in multiple Colorectal Carcinoma, as adenocarcinoma of colon, colonic mucus gland cancer, adenocarcinom of cecum, second shape rectal adenocarcinoma and rectal mucus gland cancer (figure-1G and figure-1H).
Embodiment 2 quantitative PCR technique analyzes the expression of Spondin-2 gene in colorectal carcinoma and cancer beside organism
Operating process is as follows:
1, colon cancer tissue sample collection.10 pairs of human colon cancers and the other normal colon mucosa tissues of cancer are collected and are placed in liquid nitrogen cryogenics immediately and preserve in operation, and cancer other mucous membrane of colon distance cancerous tissue is more than at least 5 centimetres, and tissue slice confirms through Pathology Doctors ' diagnosis.
2, from colon cancer tissue, RNA sample is extracted
1) the in vitro rear liquid nitrogen freezing rapidly of sample tissue, if organize comparatively large, will be cut into tissue the bulk of soya bean size as far as possible, put into cryopreservation tube, be stored in liquid nitrogen;
2) before extracting RNA, Liquid nitrogen precooler mortar is first used.Take out sample from liquid nitrogen container after, mortar tissue block being put into precooling grinds rapidly, and grinding limit, limit adds liquid nitrogen, and whole process does not all make liquid nitrogen volatilize;
3) after tissue grinder is powdered, when liquid nitrogen is evaporated completely substantially, gets and be dispensed in right amount in 1.5mL centrifuge tube, add 1mLTrizol, room temperature leaves standstill 5min (this sample also can be preserved at-80 DEG C for a long time);
4) add 0.2mL chloroform, cover tightly centrifuge tube pipe lid, with hand thermal agitation 15sec, room temperature places 2-3min;
5) 4 DEG C of centrifugal 15min of 12000g;
6) the careful colourless aqueous phase in upper strata of drawing is in another new 1.5mL centrifuge tube, and every mLTrizol about can draw 0.5mL upper strata aqueous phase;
7) add 0.5mL Virahol, mixing for several times of turning upside down, room temperature leaves standstill 10min;
8) 4 DEG C of centrifugal 10min of 12000g;
9) careful supernatant discarded, at the bottom of pipe, visible white precipitation, adds 75% ethanol 1mL, abundant washing precipitation of turning upside down;
10) 4 DEG C of centrifugal 5min of 7500g;
11) abandon supernatant, be inverted EP pipe and blot residual ethanol on dust-free paper, room temperature places about 15min, makes RNA dry;
12) 35 μ LRNasefreeddH are added
2o dissolves RNA, BioTekEpoch many volumes spectrophotometer and detects 260/280 value ,-80 DEG C of preservations.
3, reverse transcription synthesis cDNA is carried out with RNA
1) reverse transcription operation is carried out according to PrimeScript1stStrandcDNASynthesisKit (TaKaRa) specification sheets.Concrete operation step is as follows:
2) in Microtube, following mixed solution is prepared
OligodTPrimer(50μM)，1μL
dNTPMixture(10mMeach)，1μL
TotalRNA，2μg
RNasefreeddH
2O，upto10μL
3) 65 DEG C of 5min in PCR instrument, rapidly chilling on ice.
4) in above-mentioned Microtube pipe, following inverse transcription reaction liquid is prepared
Reaction solution after above-mentioned sex change, annealing, 10 μ L
5×PrimeScriptBuffer，4μL
RNaseInhibitor(40U/μL)，0.5μL
PrimeScriptRTase(200U/μL)，1μL
RNasefreeddH2O，4.5μL
Total，20μL
5) 30 DEG C of 10min in PCR instrument, 42 DEG C of 45min, 95 DEG C of 5min carry out reverse transcription reaction.
4, real-time quantitative PCR reaction
1) shown in following, PCR reaction solution (operating) is prepared on ice:
Reagent (TaKaRa), usage quantity, final concentration
SYBRPremixExTaqTM(2×)，10μL，1×
PCRForwardPrimer(10μM)，0.4μL，0.2μM
PCRReversePrimer(10μM)，0.4μL，0.2μM
ROXReferenceDyeII(50×)，0.4μL，1×
DNA profiling, 2.0 μ L, 100ng
ddH2O(RNasefreeddH2O)，6.8μL，
Total，20μL。
Quantification PCR primer sequence
spon2-F(AAGAACCAGTACGTCAGTAACGG)(SEQIDNO1)
spon2-R(CACAAACGAGACCAGCGAGT).(SEQIDNO2)
2) RealtimePCR reaction (ABIPRISM7500Real-TimePCRsystem) is carried out
Two-step approach pcr amplification standard program:
Stage1: denaturation, Reps:1,95 DEG C of 30sec
Stage2:PCR reacts, Reps:40,95 DEG C of 5sec, 60 DEG C of 34sec
DissociationStage:Reps:1,95℃15sec,60℃1min,95℃15sec
3) interpretation.Reaction terminates amplification curve and the solubility curve of rear confirmation RealtimePCR, carries out analyzing the relative expression difference of each gene in colon cancer tissue according to two △ Ct method.
5, interpretation of result
We analyze the expression level of Spondin-2 gene in 10 routine normal peoples and 10 routine colorectal cancer patients tissues, result proves that Spondin-2 gene significantly raises (p=0.026) (figure-2A) at colon cancer tissue, illustrates that the result of gene microarray analysis is consistent with the result of experimental verification.
Embodiment 3 Westernblot method detects the expression of Spondin-2 albumen at colon carcinoma cell line
Operating process is as follows:
1, main solution preparation:
1) PBS solution: measure raw work 20 × PBS solution 25mL, ultrapure water is settled to 500mL, room temperature preservation.
2) 50 × Proteininhibitorcocktail (proteinase inhibitor mother liquor): get a Roche Protease inhibitor (completeEDTA-freeProteaseInhibitorcocktailtablets), add 1mL ultrapure water to dissolve, packing,-20 DEG C of preservations, this is 50 × proteinase inhibitor mother liquor.
3) cell pyrolysis liquid: 50mMTris-HCl (pH7.4), 1%SDS, 2mMEDTA, packing ,-20 DEG C of preservations, every milliliter of lysate used time adds 20 μ L50 × Proteininhibitorcocktail again.
4) acrylamide soln: 30% acrylamide, 0.8% methylene diacrylamide;
5) separation gel damping fluid: 1.5mol/LTris-HClpH8.8;
6) concentrated glue damping fluid: 1.0mol/LTris-HClpH6.8;
7)SDS：10％(m/v)；
8) ammonium persulfate AP:10% (m/v);
9) electrophoretic buffer: 25mmol/LTris, 192mmol/L glycine, 0.1% (m/v) SDS, pH8.3;
10) 3 × sds gel sample loading buffer: 150mmol/LTris-HClpH6.8,300mmol/LDTT, 6%SDS, 0.3% tetrabromophenol sulfonphthalein, 30% glycerine ,-20 DEG C of packing are preserved, and general DTT is now with now adding.
11) electricity turns damping fluid: 25mmol/LTris, 192mmol/L glycine.
12) TBST damping fluid: 8.766gNaCl (i.e. 0.15mol/L), 1mLTween20,20mL1MTris-HCl (pH7.4), add ultrapure water constant volume to 1L.
13) confining liquid: take 5g skim-milk and be dissolved in 100mLTBST and be 5% skim-milk confining liquid.
2, the extracting method of culturing cell whole protein:
1) sop up substratum with vacuum pump, add PBS and wash one time, sop up, repeat once;
2) sopped up by PBS, culture dish is put on ice, adds appropriate cell pyrolysis liquid, leaves standstill 3min;
3), after abundant cracking, scrape with clean cell and cell is scraped to culture dish side rapidly, then with rifle, cell debris and lysate are transferred in 1.5mL centrifuge tube;
4) 4 DEG C of centrifugal 15min of 12000g;
5) the supernatant packing after centrifugal is transferred in 1.5mL centrifuge tube ,-20 DEG C of preservations.
3, protein quantification method: BCA determination of protein concentration test kit green skies article No.: P0010
1) get BSA (Bio-rad) protein standard liquid, it is 0.5mg/mL that PBS is diluted to final concentration;
2) according to protein sample quantity, add 1 volume BCA reagent B by 50 volume BCA reagent A, namely 50:1 prepares appropriate BCA working fluid, fully mixes, stable in this BCA working fluid room temperature 24h;
3) reference liquid is added in the standard sample wells of 96 orifice plates by 0,2,4,8,12,16,20 μ L, adds PBS and supply 20 μ L;
4) draw 2 μ L protein samples in the sample well of 96 orifice plates, add PBS and supply 20 μ L;
5) each hole adds 200 μ LBCA working fluids, places 30min in 37 DEG C of incubators;
6) BioTekEpoch many volumes spectrophotometer OD562nm place measures absorbancy.The protein concentration of sample is calculated according to typical curve.
4, SDS-PAGE gel electrophoresis
1) get complete Bio-Rad and join glue sheet glass ddH
2dry in baking oven after O cleans up, be fixed on base with clip;
2) by following formulated 10% separation gel and 5% concentrated glue.During preparative separation glue, upper strata ddH
2o sealing, more than polymerized at room temperature 40min; When preparing the concentrated glue of 5%, by upper strata sealing ddH
2after O exhaustion, add concentrated glue, insert comb, and prevent bubble;
10% separation gel is formulated as follows table:
Concentrated glue is formulated as follows table:
3) after gelling to be concentrated admittedly, gel slab is transferred in Vertial electrophorestic tank, makes gel slab concave surface inside.Add electrophoretic buffer, extract comb;
4) protein sample having surveyed concentration, by volume for 2:1 adds 3 × loadingbuffer, boils 5min in boiling water, the centrifugal 1min of 12000g, loading (applied sample amount is 30 μ g/ swimming lanes), and loading pre-dyed albumen Marker, contrasts as molecular weight simultaneously.
5) electrophoresis: concentrated glue first carries out electrophoresis with 45V voltage, enter after separation gel until bromophenol blue indicator and convert 65V continuation electrophoresis to, to be instructed dose is run about 0.5cm to separation gel and stops electrophoresis.
5, transferring film and Westernblot
1) prepare electricity and turn damping fluid (matching while using), according to gel size, cutting pvdf membrane and filter paper, after methyl alcohol activation pvdf membrane 1min, turns pvdf membrane and filter paper in damping fluid at electricity and soaks more than 15min;
2) sheet glass is pried open, take out SDS-PAGE gel, put into electricity to turn damping fluid and balance, gel and pvdf membrane being fixed on by the order of blackboard-sponge pad-filter paper-gel-pvdf membrane-filter paper-sponge pad-blank wetly turns in electricity turns and press from both sides, electric turn trough is put in the black flour direction of the black flour corresponding groove of clip, fills it up with electricity and turn damping fluid and carry out transferring film (ice bath).The albumen that usual use 110V2h electricity translocated molecule amount is larger, also can turn over night by 30V constant voltage electricity in 4 DEG C of refrigerators;
3) close: transferring film terminates rear taking-up pvdf membrane, and the one side (protein powder) being close to gel put into upward and hatch box, 5% skim-milk room temperature closes 1h;
4) primary antibodie is hatched: anti-Spondin-2 albumen primary antibodie (purchased from Sigma-Aldrich) diluted with confining liquid 1:1000, overnight incubation on 4 DEG C of shaking tables;
5) TBST washs 3 times, each 5-10min;
6) two anti-to hatch: the goat-anti rabbit two anti-confining liquid corresponding with primary antibodie is diluted to proper concn (1:5000-1:8000), room temperature shaker hatches 1h;
7) TBST washs 3 times, each 5-10min;
8) sealed membrane of the suitable size of clip, the super quick nitrite ion of 200-400 μ LThermoSuperSignalWestFemtoMaximumSensitivitySubstrate (by 1:1 matching while using working fluid) is added above, room temperature leaves standstill 1min, uses Las-3000 imaging system to obtain Westernblot result.
6, result: in the clone that Spondin-2 albumen detects in the overwhelming majority, gene expression abundance is higher (comprises Colon cancer cell line HT-29, HCT-116, SW-620, LoVo, Caco-2, Caco-320DM is only had to make an exception), and do not express CCD-18Co (representing Normal Colon cell) is inner, prove Spondin-2 albumen main relevant to colon cancer cell (figure-2B).
Embodiment 4 ImmunohistochemistryMethods Methods analyzes the expression of Spondin-2 albumen in colorectal carcinoma and Carcinoma side normal tissue
Operating process is as follows:
1, organization chip: use 2 sections of commercialization organization chips (Shanghai Xin Chao biotech firm), HCol-Ade180Sur-04 comprises 90 examples, 180 cancers and cancer beside organism, and clinical information comprises sex, age, TNM, tumor size, by stages, classification, follows up a case by regular visits to information etc.; HCol-Ade180CS-01 comprises 90 example, 180 cancers and cancer beside organism, and clinical information is the same, but does not comprise the information of following up a case by regular visits to.The diameter of chip sample point is all 1.5mm, and fixed form is that formalin is fixed.
2, wax is dried
1) organization chip is put into baking oven, temperature is adjusted to 63 degree, dries wax one hour.
2) reagent preparation.10 × PBS damping fluid (formula: 80gNaCl, 2gKCl, 15.35gNa
2hPO
4, 2gKH
2pO
4, be settled to 1000 milliliters with pure water): 10 × PBS damping fluid is diluted to 1 × PBS damping fluid, then in 1 × PBS damping fluid, adds the tween reagent accounting for cumulative volume 0.05%; The pure water of the citric acid solution+900mL of the sodium citrate solution+18mL0.1mol/L of antigen retrieval buffers: 82mL0.1mol/L is placed in pressure kettle;
3, antigen retrieval
1), after slice, thin piece has toasted, take out in baking oven, put into full-automatic dyeing machine, dewax;
2) dewaxing process: dimethylbenzene two cylinder, every cylinder 15 minutes; Dehydrated alcohol two cylinder, every cylinder 7 minutes; 90% alcohol 1 cylinder, 7 minutes; 80% alcohol 1 cylinder, 7 minutes; 70% alcohol 1 cylinder, 7 minutes;
3) from dyeing machinery, slice, thin piece is taken out, by pure water rinsing 3 times, one time 3 minutes.In flushing process, citric acid repair liquid is put and start heating to electric furnace;
4) antigen retrieval: after the boiling of citric acid repair liquid, slice, thin piece is put into pressure kettle, covers high-pressure pot cover, start timing after giving vent to anger, 5 minutes.Time heats to rear termination, is opened by high-pressure pot cover, makes it naturally cool to room temperature;
4, block
Preparation endogenous peroxydase blocker.The H of 38.4mL anhydrous methanol+12mL30% concentration
2o
2+ 9.6mL pure water; Slice, thin piece is put into blocker 10 minutes.
5, primary antibodie is added
1) slice, thin piece is taken out, with PBS wash buffer 3 times, one time 5 minutes;
2) take out Spondin-2 antibody in refrigerator, to put in whizzer 7200 and leave the heart 30 seconds;
3) take out antibody, dilute according to extent of dilution DAKO antibody diluent 1:50;
4) antibody is dripped;
5) wet box is put into refrigerator, 4 DEG C are spent the night.
6, add two to resist
1) from refrigerator, take out wet box, leave standstill and treat that it returns to room temperature in 1 hour;
2) by slice, thin piece PBS wash buffer 3 times, one time 5 minutes;
3) EnVision is dripped
tM+/HRP rabbit working fluid (from DAKO company), 30 minutes;
4) time rinses 3 times to rear PBS, one time 5 minutes.
7, DAB colour developing: take out DAB test kit from refrigerator, prepare by 1mLDAB diluent+1 DAB chromogen; (from DAKO liquid D AB+ substrate system).Slice, thin piece drips the DAB after dilution, develops the color 5 minutes, tap water 15 minutes then.
8, Hematorylin is redyed
1) on slice, thin piece, drip Hematorylin 2 minutes, the time to after submergence 2 seconds in the hydrochloride alcohol of 0.25%, with tap water 2 minutes;
2) slice, thin piece is put into full-automatic dyeing machine to dewater, take out mounting.
3) dehydration: 75% alcohol 1 cylinder, 3 minutes; 85% alcohol 1 cylinder, 3 minutes; 95% alcohol 1 cylinder, 3 minutes; Dehydrated alcohol two cylinder, every cylinder 5 minutes; Dimethylbenzene two cylinder, every cylinder 5 minutes.
9, scan: with chip scanner ScanscopeXT (Aperio company) scanning.
10, the software analysis of chip dyeing
1) open chip scanning file with software AperioImageScopev.12, the region that the figure of each sample point chooses more than 3 exports as single TIFF picture; Some sample points, lack neoplastic epithelial cells, cannot statistical presentation situation, and such point is abandoned.
2) open these TIFF pictures one by one with software I mage-ProPlus, calculate the accumulation optical density value (IOD) of dyeing, this value, divided by the area of statistics dyeing, obtains average accumulated optical density value (MeanIOD).The average accumulated optical density value calculated from multiple regions of same sample is averaged further again, and this value represents the expression amount of each sample Spondin-2 albumen;
3) expression of the Spondin-2 albumen on cancer and cancer side compares by the average accumulated optical density value of each sample;
4) the expression classification of cancer sample.Be convenient to analyze for simplifying classification, the expression of Spondin-2 albumen in cancer adds auxiliary judgment according to MeanIOD value, simplifies further and is divided into positive and negative expression.
11, result
In the analysis of HCol-Ade180Sur-04 organization chip, the other point of effective cancer is 79 examples, effective cancer point is 85 examples, through T inspection, finds that the expression of Spondin-2 albumen in colorectal carcinoma sample is significantly higher than Carcinoma side normal tissue (p=0.025) (figure-2C);
In the analysis of HCol-Ade180CS-01 organization chip, the other point of effective cancer is 82 examples, effective cancer point is 88 examples, through T inspection, finds that the expression of Spondin-2 albumen in colorectal carcinoma sample is significantly higher than Carcinoma side normal tissue (p=2.4E-5);
Figure-3A is shown in by the sample that the other Spondin-2 feminine gender of cancer is expressed, and figure-3B is shown in by the sample of the other Spondin-2 positive expression of cancer, and figure-3C is shown in by the sample that colorectal carcinoma Spondin-2 feminine gender is expressed, and the sample of the basic, normal, high expression of colorectal carcinoma Spondin-2 is shown in Fig. 3 D, E, F.
These results prove that Spondin-2 albumen is that significant difference is expressed in colorectal carcinoma and cancer side.
Embodiment 5Spondin-2 expresses the relation with colon cancer patient clinical parameter
Analyze (obtaining data method the same) with Oncomine gene expression data base, colorectal carcinoma sample is divided into 2 groups according to the median of the expression of Spondin-2 gene, one group is the high expression level group being greater than median, one group is the low expression group being less than median, analyzes the high and low expression of Spondin-2 gene and the relation of clinical parameter by K quadratic method.Software used is PASWStatistics18.
Result: use BitterColon data set, find the expression of Spondin-2 gene and (BittnerColon by stages, χ 2=16.96, p=0.001), T (BittnerColon, χ 2=13.852, p=0.003), M (BittnerColon by stages by stages, χ 2=3.967, p=0.046), Dukes (BittnerColon, χ 2=13.514, p=0.001 by stages; JorissenColorectal3, χ 2=11.337, p=0.01), smoking period (Fisher ' sExactTest, p=0.047) and sex (GaedckeColorectal, χ 2=8.021, p=0.005; TsujiColorectal, χ 2=3.967, p=0.046) there is remarkable relation (table 1).Generally, Spondin-2 genetic expression rise and tumour progression are proportionate, and male patient Spondin-2 gene upregulation is more obvious.
The expression of table 1.Spondin-2 gene and the relation of colon cancer patient Clinical symptoms
Integrate the analytical results from 2 organization chips and immunohistochemical methods, we find expression and the age (χ of Spondin-2 albumen
2=9.65, p=0.0468), M (χ by stages
2=4.245, p=0.039) and transfer (χ
2=4.332, p=0.037) there is remarkable relation, with relation by stages close to remarkable (χ
2=7.446, p=0.059) (table 2).
The relation (immunohistochemical analysis of 2 organization chips) of table 2.Spondin-2 protein expression and colon cancer patient clinicopathologic features.
The relation of embodiment 6Kaplan-Meier methods analyst Spondin-2 gene and albumen and colon cancer patient prognosis
Utilize the gene expression data collection SmithColorectal2 analysis Spondin-2 genetic expression height of Oncomine database and the relation of the survival time of Post operation colon cancer patient, the expression of Spondin-2 gene is divided into and is greater than median (28 example) and is less than median (27 example) two groups.Figure is by Kaplan-Meier tracing analysis method, two groups of data significance Log-rank methods of inspection are checked, result proves one group of patient that Spondin-2 genetic expression is high, its overall lifetime (Overallsurvival) significantly shortens (Log-rank inspection, p=0.011) (figure-4A), mean survival time during low expression is 93.489 months (predictors, standard error 6.604, fiducial interval 80.545-106.433), and mean survival time during high expression level is 42.486 months (predictors, standard error 5.497, fiducial interval 31.712-53.260).
If consider the factor of recurrence, statistics disease free survival rate (Diseasefreesurvival), one group of patient that then same Spondin-2 genetic expression is high, its disease free survival phase also significantly shortens (Log-rank checks, p=0.0263) (figure-4B).Average disease-free survival time during low expression is 82.138 months (predictors, standard error 8.216, fiducial interval 66.035-98.241), and average disease-free survival time during high expression level is 41.507 months (predictors, standard error 5.384, fiducial interval 30.955-52.059).
Organization chip is utilized to carry out immunohistochemical analysis, the relation that research Spondin-2 albumen and colon cancer patient prognosis are survived.The patient of the Spondin-2 protein expression positive, its lifetime significantly shortens (Log-rank checks, p=0.0363) (figure-4C) than the patient of Spondin-2 protein expression feminine gender.Overall survival time when Spondin-2 protein negative is expressed is 68.087 months (predictors, standard error 5.354, fiducial interval 57.592-78.581), and average disease-free survival time during Spondin-2 protein positive expression is 50.800 months (predictors, standard error 4.134, fiducial interval 42.697-58.903)
These the results shows, the high expression level of Spondin-2 gene and albumen shortens the postoperative survival time of colon cancer patient, and the expression of Spondin-2 gene and albumen can as the judge index of colorectal carcinoma prognosis.
Embodiment 7 single argument and Multivariate Cox Regression analysis
Use single argument and Multivariate Cox Regression method, analysis Spondin-2 protein expression and other clinical parameters are on the impact of colon cancer patient survival time further.Univariate analysis result shows, T (the p=0.018 by stages of colon cancer patient, Hazard ratio 2.2,95% fiducial interval 1.144-4.231), N (p<0.001 by stages, Hazard ratio=2.766,95% fiducial interval=1.8-4.249), classification (p=0.038, Hazard ratio=1.832,95% fiducial interval=1.035-3.242) and Spondin-2 protein expression (p=0.042, Hazard ratio=2.229,95% fiducial interval=1.028-4.832) and colorectal carcinoma prognosis significant correlation (table 3);
Multivariate regression analysis shows, Spondin-2 protein expression (p=0.046, Hazard ratio 2.197,95% fiducial interval=1.013-4.768) and N (p<0.001 by stages, Hazard ratio 2.791,95% fiducial interval=1.801-4.326) prognosis of remarkably influenced colon cancer patient, increase prognostic risk.
Table 3.Spondin-2 protein expression and other clinical parameters affect single argument and the multivariate analysis (organization chip and immunohistochemical analysis) of colon cancer patient survival time
Above result shows, Spondin-2 protein expression is independently, affects the factor of colon cancer patient survival time.By detecting the expression of the Spondin-2 albumen in colon cancer patient cancerous tissue, the prognosis survival risk situation of colon cancer patient just can be predicted.
Embodiment 8 detects the expression level of the Spondin-2 albumen in colon cancer patient blood and the ability of discriminating colorectal carninomatosis people and normal people thereof
Operating process:
Spondin-2 enzyme linked immunological test kit (Cloud-CloneCorp Products) is purchased from Shanghai Wu Hao company.
1, each 20 examples of the blood plasma of colon cancer patient and normal people, wherein colon cancer patient is through making a definite diagnosis.
2, main solution preparation:
1) standard substance (dried frozen aquatic products) dilution: every bottle of standard substance add standard dilutions 1mL, builds rear room temperature and places about 10 minutes, repeatedly put upside down simultaneously/rubbing with hydrotropy solution, its concentration is 10,000pg/mL (storage liquid).And then carried out gradient dilution, be diluted to 5,000pg/mL successively, 2,500pg/mL, 1,250pg/mL, 625pg/mL, 312pg/mL, 156pg/mL, 78pg/mL, standard dilutions (0pg/mL) is directly as blank well.
2) serum sample dilution: dilute 10 times, gets 20 μ L serum or blood plasma adds 180 μ LPBS.
3) detect solution A and detect solution B preparation: DetectionA and DetectionB before use hand get rid of several under, to make at the bottom of the liquid deposition of tube wall or bottle cap to pipe.Dilute (as: 10 μ L detect solution A/990 μ L deionized water) with 1:100 with deionized water respectively before use, abundant mixing, total amount preparation (100 μ L/ hole) before dilution needed for precalculated each experiment, answers polygamy 0.1-0.2mL during actual preparation.
4) washings: dense for 20mL washings is diluted to 600mL with 580mL deionized water, carries out 30 times of dilutions.
3, operation steps:
1) application of sample: establish standard orifice, testing sample hole, blank well respectively.If standard orifice 7 hole, add the standard substance of 100 μ L different concns successively.Blank well adds 100 μ L standard dilutions, and remaining hole adds testing sample 100 μ L, and enzyme plate adds overlay film, 37 DEG C of incubations 2 hours.
2) discard liquid, dry, need not wash.
3) every hole adds and detects solution A working fluid 100 μ L (prepared before use), and enzyme plate adds overlay film, 37 DEG C of incubations 1 hour.
4) discard liquid in hole, every hole washings of 350 μ L washs, and soaks 1-2 minute, gets rid of the liquid in enzyme plate, which floor thieving paper of place mat on experiment table, and enzyme plate is firmly clapped several times down, repeats to wash plate 3 times.After last washing, the washings in hole is dried completely.
5) every hole adds and detects solution B working fluid (prepared before use) 100 μ L, adds overlay film, 37 DEG C of incubations 30 minutes.
6) discard liquid in hole, dry, wash plate 5 times, method is with step 4.
7) every hole adds substrate solution 90 μ L, and enzyme plate adds overlay film, and 37 DEG C of lucifuges develop the color 20 minutes
8) every hole adds stop bath 50 μ L, termination reaction.
9) to guarantee at the bottom of enzyme plate, without after bubble-free in water droplet and hole, to use microplate reader in the optical density(OD) (O.D. value) in each hole of 450nm wavelength measurement immediately.
10) calculate: with the concentration of standard substance for ordinate zou, O.D. value is X-coordinate, draws typical curve.O.D. value (sample has done two multiple holes, averages), finds corresponding concentration by typical curve, is multiplied by extension rate (10 times), be the actual concentrations (pg/mL) of sample per sample.
4, result
1) expression level of the Spondin-2 albumen in colon cancer patient and normal human blood
Enzyme-linked immuno-sorbent assay determines the expression level of the Spondin-2 albumen in colon cancer patient and normal human blood, proves that the level of Spondin-2 albumen in colon cancer patient (20 example) blood plasma is significantly higher than normal people's (20 example) (p=3.2E-9) (figure-5A).In colon cancer patient blood plasma, the mean concns of Spondin-2 albumen is 61.8ng/mL, and normal people is then 17.4ng/mL, and patient is far above normal people.
2) the expression level discriminating colorectal carninomatosis people of Spondin-2 albumen and the ability of normal people in blood plasma
According to the result of enzyme-linked immunosorbent assay, receiver operator curve is utilized to analyze, line lower curve area (AUC) value of Spondin-2 albumen is 0.935 (figure-5B, table 4), illustrate that colon cancer patient and normal healthy people can make a distinction as blood plasma marker thing by Spondin-2 albumen well, Spondin-2 albumen can as the blood plasma index of diagnosis of colon cancer.
3) sensitivity of blood plasma Spondin-2 Protein Detection colorectal carcinoma and specificity: according to the result of enzyme-linked immunosorbent assay, sensitivity and the specificity of blood plasma Spondin-2 Protein Detection colorectal carcinoma can be known, when blood plasma Spondin-2 protein concentration is 40.7ng/mL, the sensitivity that colorectal carcinoma detects is 85%, and specificity is 100% (table 5).
The area under a curve (AUC) that table 4. experimenter rational curve is analyzed
A. under nonparametric hypothesis
B. null hypothesis: solid area=0.5
Table 5. receiver operating characteristic curve analyzes the expression level of Spondin-2 in colon cancer patient and human normal plasma
aminimum limit value is that minimum sight control value subtracts 1, and maximum figure value is that maximum sight control value adds 1.Other boundary values all are all the mean value of the sight control value of two vicinities.
SEQUENCELISTING
<110> Fudan University
The detection method of a <120> human colon carcinoma protein markers Spondin-2 and detection kit thereof
<130>20150303
<160>2
<170>PatentInversion3.1
<210>1
<211>23
<212>DNA
<213>Artificial
<400>1
aagaaccagtacgtcagtaacgg23
<210>2
<211>20
<212>DNA
<213>Artificial
<400>2
cacaaacgagaccagcgagt20
Claims (10)
1. a detection method of human colon carcinoma mark Spondin-2, is characterized in that, described method comprises step:
Genomic dna in (a) separation and Extraction sample or albumen;
And/or
B () detects the content of Spondin-2 in the genomic dna of (a) gained or albumen.
2. detection method as claimed in claim 1, it is characterized in that, described sample is tissue or the body fluid in vitro people source.
3. detection method as claimed in claim 1, is characterized in that, utilize the genomic dna in the primer amplified sample of Spondin-2, detects the content of Spondin-2DNA.
4. detection method as claimed in claim 1, is characterized in that, utilizes the antibody of Spondin-2 and the albumen test in sample, the expressing quantity of detection Spondin-2.
5. a detection kit, is characterized in that, it comprises the primer of specific amplification Spondin-2, or for the antibody of Spondin-2 albumen.
6. detection kit as claimed in claim 5, it is characterized in that, the sequence of the primer of described specific amplification Spondin-2 is as shown in SEQIDNO1 and SEQIDNO2.
7. an application method of human colon carcinoma mark Spondin-2, is characterized in that, the application of Spondin-2 at preparation diagnosis or the transfer of prediction colorectal carcinoma, colorectal cancer clinical by stages, in the preparation of colorectal cancer patients prognosis.
8. application method as claimed in claim 7, is characterized in that, the application of described mark Spondin-2 at preparation diagnosis or the transfer of prediction colorectal carcinoma, colorectal cancer clinical by stages, in the test kit of colorectal cancer patients prognosis.
9. application method as claimed in claim 7, is characterized in that, described mark Spondin-2 diagnoses in preparation, alleviate or treat the application in the medicament of colorectal carcinoma.
10. application method as claimed in claim 7, is characterized in that, Spondin-2 is screening preparation diagnosis, alleviate or the target of medicament for the treatment of colorectal carcinoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510128247.6A CN105385752A (en) | 2015-03-23 | 2015-03-23 | Detection method of human colon cancer protein marker Spondin-2 and detection kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510128247.6A CN105385752A (en) | 2015-03-23 | 2015-03-23 | Detection method of human colon cancer protein marker Spondin-2 and detection kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105385752A true CN105385752A (en) | 2016-03-09 |
Family
ID=55418529
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510128247.6A Pending CN105385752A (en) | 2015-03-23 | 2015-03-23 | Detection method of human colon cancer protein marker Spondin-2 and detection kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105385752A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105807062A (en) * | 2014-12-28 | 2016-07-27 | 复旦大学 | Application of human colorectal carcinoma protein Spondin-2 to preparation of colorectal carcinoma diagnosis preparation |
| CN109457033A (en) * | 2018-12-29 | 2019-03-12 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Marker for colon cancer screening |
| CN110872625A (en) * | 2018-08-29 | 2020-03-10 | 深圳大学 | Colorectal cancer marker and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025890A (en) * | 2010-04-06 | 2013-04-03 | 卡里斯生命科学卢森堡控股 | Circulating biomarkers for disease |
| WO2014085826A2 (en) * | 2012-11-30 | 2014-06-05 | Applied Proteomics, Inc. | Method for evaluation of presence of or risk of colon tumors |
-
2015
- 2015-03-23 CN CN201510128247.6A patent/CN105385752A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025890A (en) * | 2010-04-06 | 2013-04-03 | 卡里斯生命科学卢森堡控股 | Circulating biomarkers for disease |
| WO2014085826A2 (en) * | 2012-11-30 | 2014-06-05 | Applied Proteomics, Inc. | Method for evaluation of presence of or risk of colon tumors |
Non-Patent Citations (1)
| Title |
|---|
| F. SCHMID等: "SPON2, a Newly Identified Transcriptional Target Gene of MACC1, is Associated with Colorectal Cancer Metastasis", 《EUROPEAN JOURNAL OF CANCER》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105807062A (en) * | 2014-12-28 | 2016-07-27 | 复旦大学 | Application of human colorectal carcinoma protein Spondin-2 to preparation of colorectal carcinoma diagnosis preparation |
| CN110872625A (en) * | 2018-08-29 | 2020-03-10 | 深圳大学 | Colorectal cancer marker and application thereof |
| CN109457033A (en) * | 2018-12-29 | 2019-03-12 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Marker for colon cancer screening |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Carmicheal et al. | Elevating pancreatic cystic lesion stratification: Current and future pancreatic cancer biomarker (s) | |
| Wu et al. | Diagnostic value of serum PIVKA-II levels for BCLC early hepatocellular carcinoma and correlation with HBV DNA | |
| Peng et al. | Clinical significance of Ki67 and circulating tumor cells with an epithelial-mesenchymal transition phenotype in non-small cell lung cancer | |
| Zhang et al. | Clinical significance of detecting circulating tumor cells in patients with esophageal squamous cell carcinoma by EpCAM‑independent enrichment and immunostaining‑fluorescence in situ hybridization | |
| CN105385752A (en) | Detection method of human colon cancer protein marker Spondin-2 and detection kit thereof | |
| CN111218509A (en) | Novel diagnostic marker PPEF1 for breast cancer and application thereof | |
| Endo et al. | Tissue expression of melanoma-associated antigen A6 and clinical characteristics of gastric cancer | |
| Knoener et al. | Limited value of KAI1/CD82 protein expression as a prognostic marker in human gastric cancer | |
| CN113777311B (en) | ELISA kit for auxiliary diagnosis of esophageal squamous carcinoma | |
| Yu et al. | Application of circulating tumor cells and circulating free DNA from peripheral blood in the prognosis of advanced gastric cancer | |
| Zhou et al. | Annexin A3 as a prognostic biomarker for breast cancer: A retrospective study | |
| Sun et al. | Serum carboxypeptidaseA4 levels predict liver metastasis in colorectal carcinoma | |
| Lu et al. | Detection of circulating stage III–IV gastric cancer tumor cells based on isolation by size of epithelial tumor: using the circulating tumor cell biopsy technology | |
| CN105467127B (en) | The application process and its detection kit of a kind of human colon carcinoma protein markers COL6A3 | |
| CN107266567A (en) | LCRMP4 monoclonal antibodies and preparation method and application | |
| RU2383339C1 (en) | Method of predicting efficiency of neoadjuvant chemotherapy in case of locally advanced ovarian carcinoma | |
| CN108753981A (en) | Application of the quantitative detection of HOXB8 genes in colorectal cancer Index for diagnosis | |
| CN112229997B (en) | Prognostic diagnosis marker Claudin23 for ovarian cancer and application thereof | |
| AL-Bedairy et al. | Molecular Subtypes by Immunohistochemical for Iraqi Women with Breast Cancer | |
| CN105807062A (en) | Application of human colorectal carcinoma protein Spondin-2 to preparation of colorectal carcinoma diagnosis preparation | |
| CN113687076A (en) | Combined detection serum marker for early diagnosis of lung adenocarcinoma and application thereof | |
| CN106399464A (en) | Human colorectal carcinoma molecular marker COL3A1 and application thereof | |
| Zhu et al. | Deficiency of hMLH1 and hMSH2 expression is a poor prognostic factor in Early Gastric Cancer (EGC) | |
| CN112229998A (en) | Prognostic diagnosis marker Claudin22 for ovarian cancer and application thereof | |
| Tarazona et al. | Mir-203A-3P and MMP-2 proteins are highly expressed in CIRCULATING tumor cells from patients with pancreatic carcinoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160309 |