CN105807062A - Application of human colorectal carcinoma protein Spondin-2 to preparation of colorectal carcinoma diagnosis preparation - Google Patents
Application of human colorectal carcinoma protein Spondin-2 to preparation of colorectal carcinoma diagnosis preparation Download PDFInfo
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Abstract
The invention belongs to the fields of molecular biology and medical science, and relates to a new application of a human colorectal carcinoma protein Spondin-2 to preparation of a colorectal carcinoma diagnosis preparation; the invention also provides a corresponding kit and an application thereof. Concretely, the invention provides the application method of the human colorectal carcinoma protein marker Spondin-2 to preparation of the preparation for diagnosis, prediction, detection or screening of colorectal carcinoma; and the application method of the human colorectal carcinoma protein marker Spondin-2 to preparation of the preparation for diagnosis, prediction, detection or screening of colorectal carcinoma cell propagation, cancerometastasis, colorectal carcinoma clinical stages, and prognosis of colorectal carcinoma patients. The preparation provides a scientific basis for effectively judging canceration process of colorectal carcinoma.
Description
Technical field
The invention belongs to molecular biology and medical domain, relate to the new application of a kind of human colon carcinoma Protein S pondin-2, namely its application in preparing diagnosis of colon cancer preparation, present invention also offers corresponding test kit and application thereof.
Background technology
Colon cancer is worldwide the third-largest male tumor and second largest female tumor.Statistics according to World Health Organization (WHO) GLOBOCAN2012, pre-in respect of 74.6 ten thousand male and 61.4 ten thousand female patients in 2012, overall fatality rate is 50.1% (male) and 52.1% (women), it was shown that prognosis existence is very poor.There is not only impact individual family in tumor, also brings serious social influence, increases Public Health Expenditure and financial burden.Therefore, strive for early discovery, early confirmation, early treatment, the cure rate and prolongation prognosis life span improving colon cancer is had very important significance.The onset stage general symptom of colon cancer is inconspicuous, it is easy to be left in the basket, once after confirmation, often be in middle and advanced stage and make therapeutic effect not good, more affect postoperative existence.So, treatment colon cancer or of other cancers it is critical only that and finds colon cancer as early as possible.The diagnosis of general colon cancer, including observe bowl evacuation habit change, have blood in stool, stomachache etc., rectum refers to that disease, lab testing include 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 carcinoembryonic antigen (CEA), colorectal cancer antigen (CCA), CA19-9, but the clinical effectiveness comparison in difference of these Detection of antigen is big, and the sensitivity of detection and specificity have much room for improvement.In this sense, it has been found that have high sensitivity while new, the blood markers thing of high specific is a very urgent task.Utilize such Novel marker, it is possible in routine physical examination examination, find the existence of early stage colon cancer as soon as possible.
Secondly, for the prediction of the outcome of patient, Survival after Post operation or chemicotherapy, conventional 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, finding the promising tumor marker of colon cancer, the accurate clinical stages judging adenocarcinoma of lung and prognosis become the urgent task of numerous medical investigators.
Summary of the invention
It is an object of the invention to provide a kind of human colon carcinoma protein markers and application thereof.
On the one hand, the invention provides the new opplication of human colon carcinoma Protein S pondin-2, i.e. human colon carcinoma Protein S pondin-2 application in preparing diagnosis of colon cancer preparation.
Described diagnosis of colon cancer preparation be diagnosis or prediction colon cancer transfer, colorectal cancer clinical by stages, the preparation of colorectal cancer patients prognosis.
Described diagnosis of colon cancer preparation be diagnosis or prediction colon cancer transfer, colorectal cancer clinical by stages, the test kit of colorectal cancer patients prognosis.
Described diagnosis of colon cancer preparation be the propagation of examination colon cancer cell, transfer, colorectal cancer clinical by stages or the preparation of colon cancer patient prognosis.
Wherein, human colon carcinoma Protein S pondin-2 is the diagnosis marker of human colon carcinoma.
On the other hand, the invention provides a kind of test kit detecting colon cancer, containing the reagent detecting human colon carcinoma Protein S pondin-2 expression in described test kit, for instance the reagent of detection albumen or nucleic acid content.Utilize this test kit detection human colon carcinoma Protein S pondin-2 expression, further for the propagation of colon cancer cell, transfer, colorectal cancer clinical by stages, colon cancer patient prognosis or colon cancer canceration process evidence is provided.
Possibly together with human colon carcinoma Protein S pondin-2 standard sample in described test kit.
Described test kit can contain the primer of detection human colon carcinoma Protein S pondin-2.Preferably, the sequence of described primer is such as shown in SEQIDNO1 and SEQIDNO2.
In the present invention, utilize Oncomine public database (https://www.oncomine.org/resource/login.html) analyze the expression in colon cancer and cancer beside organism of the Spondin-2 gene；Result proves that Spondin-2 gene presents significantly high expression trend compared to cancer beside organism in cancerous tissue, and especially Spondin-2 gene all significantly rises in multiple Colorectal Carcinoma.
In the present invention, analyzing the expression in colon cancer and cancer beside organism of the Spondin-2 gene with quantitative PCR technique, result proves that Spondin-2 gene significantly raises (p=0.026) (as shown in figure-2A) at colon cancer tissue.
In the present invention, the expression at colon carcinoma cell line of the Spondin-2 albumen is detected by Westernblot method, result display Spondin-2 albumen gene expression abundance in colon carcinoma cell line is higher, and do not express in Normal Colon cell, it was shown that Spondin-2 albumen main relevant to colon cancer cell (as shown in figure-2B).
In the present invention, analyzing the expression in colon cancer and Carcinoma side normal tissue of the Spondin-2 albumen with ImmunohistochemistryMethods Methods, result proves that Spondin-2 albumen is that significant difference is expressed by colon cancer and cancer.
In the present invention, carrying out Spondin-2 further and express the relation research with colon cancer patient clinical parameter, result display Spondin-2 gene expression rise is proportionate with tumour progression, and male patient Spondin-2 gene upregulation becomes apparent from.
The invention provides one for distinguishing human colon cancer and Ai Pang normal lung tissue, without lymphatic metastasis colon cancer tissue and the plasmalemma protein mark Spondin-2 having lymphatic metastasis colon cancer tissue, the preparation of this mark is utilized to judge that human colon carcinoma, colon cancer spread, the preparation of colon cancer lymphatic metastasis, clinical stages, colorectal cancer patients prognosis and prognosis.The present invention is for effectively judging that human colon carcinoma canceration process provides scientific basis.
Accompanying drawing explanation
Fig. 1 Spondin-2 gene expresses notable rise in data base's colon cancer sample chip.Utilize the expression in colorectal cancer and normal colorectal carcinoma of the gene expression chip data analysis Spondin-2 gene in Oncomine public database.Fig. 1-A is the scatterplot utilizing GaedckeColorectal data analysis Spondin-2 expression in 65 pairs of rectum and rectum cancer tissue, numeral under scatterplot represents case or the number of normal sample, p value is analyze, with Studentttest (T inspection), the significance value obtained, and what asterisk represented this T inspection is paired-samples T-test.Fig. 1-B is the scatterplot utilizing GraudensColon data analysis Spondin-2 expression in 12 example colons and 48 example colon cancer tissues.Fig. 1-C is the scatterplot utilizing HongColorectal data analysis Spondin-2 expression in 12 example colons and 48 example colon cancer tissues.Fig. 1-D is the scatterplot utilizing SkrzypczakColorectal data analysis Spondin-2 expression in 24 example colons and 81 example colon cancer tissues.Fig. 1-E is the scatterplot utilizing KiColon data analysis Spondin-2 expression in 28 example colons and 82 example colon cancer tissues.Fig. 1-F is the scatterplot utilizing SkrzypczakColorectal2 data analysis Spondin-2 expression in 20 example colons, 10 example adenoma of colon and 10 example colon cancer tissues.Fig. 1-G is the scatterplot utilizing TCGAColorectal data analysis Spondin-2 expression in 22 example colorectal carcinomas and 101 example adenocarcinoma of colon, 60 example colon cancer, 22 example colonic mucus adenocarcinoma and 22 example adenocarcinom of cecum tissues.Fig. 1-H utilizes the KaiserColon data analysis Spondin-2 scatterplot expressed in 12 example colons and 41 example adenocarcinoma of colon, 13 example colonic mucus adenocarcinoma, 17 example adenocarcinoms of cecum, 8 example second shape rectal adenocarcinomas and 4 example rectal mucus adenocarcinoma tissue.
Fig. 2 is mRNA and the protein expression in colorectal cancer cell system and tissue of analyzing Spondin-2.Fig. 2-A is the methods analyst Spondin-2 gene using quantitative PCR expression in 10 pairs of Colorectal Carcinomas.P value is analyze, with Studentttest (T inspection), the significance value obtained.Fig. 2-B uses Westernblot methods analyst Spondin-2 albumen at the expression of colon carcinoma cell line.Fig. 2-C is that Spondin-2 albumen is added up in the expression of colon cancer and cancer beside organism, utilizes a commercialization organization chip (containing 90 example 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 colon cancer and cancer beside organism, utilizes another commercialization organization chip (containing 90 example cancer and cancer beside organisms) to carry out the immunohistochemical analysis of Spondin-2.N represents and enters the effective number of cases analyzed.SPON2 represents Spondin-2.GAPDH represents dehydephosphatedehydrogenase and glyceraldehyde phosphate dehydrogenase, as interior mark.Vertical coordinate 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 then expresses height greatly, is worth little, expresses low.
Fig. 3 is the result utilizing commercialization organization chip to carry out Spondin-2 immunohistochemical analysis.Fig. 3-A shows the colon 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 gene of Kaplan-Meier tracing analysis Spondin-2 and the graph of a relation of protein expression and colon cancer existence.Fig. 4-A is the Kaplan-Meier tracing analysis figure of Spondin-2 gene expression and colon cancer patient overall survival.Gene expression data derives from Oncomine gene expression public database.Fig. 4-B is the Kaplan-Meier tracing analysis figure of Spondin-2 gene expression and colon cancer patient disease free survival.Gene expression data derives from Oncomine gene 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 in the method detection colon cancer patient with ELISA and human normal plasma.Fig. 5-A is the Spondin-2 protein expression in elisa assay colon cancer patient and human normal plasma, Spondin-2 amount in patient and human normal plasma is determined in measurement according to standard substance, and p value is analyze, with Studentttest (T inspection), the significance value obtained.N represents case load.Fig. 5-B is experimenter's operating characteristic (ROC) tracing analysis that Spondin-2 proteinplasm is expressed.Abscissa represents false positive or 1-specificity, and vertical coordinate represents true positives or sensitivity.Diagonal is reference line.AUC represents area under line.SPON2 represents Spondin-2.
Detailed description of the invention
The invention provides a kind of human colon carcinoma protein markers Spondin-2 for preparing the preparation of diagnosis, prediction, detection or examination human colon carcinoma；Test kit especially for preparation diagnosis, prediction, detection or examination human colon carcinoma.The human colon carcinoma protein markers Spondin-2 of the present invention can be also used for preparation diagnosis, prediction, detection or the diffusion of examination human colon carcinoma cancerous cell, colon cancer lymphatic metastasis, colorectal cancer clinical by stages, the preparation of colorectal cancer patients prognosis；Drench especially for preparation diagnosis, prediction, detection or the diffusion of examination human colon carcinoma cancerous cell, colon cancer carry down the sixth of the twelve Earthly Branches shifting, colorectal cancer clinical by stages, the test kit of colorectal cancer patients prognosis.
Below in conjunction with embodiment, the present invention is elaborated, without limiting the present invention.
Embodiment 1
Utilize Oncomine public database (https://www.oncomine.org/resource/login.html) analyze the expression in colon cancer and cancer beside organism of the Spondin-2 gene.Analysis method: log in above-mentioned Oncomine data base, input SPON2 gene name, tumor type selects colon cancer, data type selects mRNA, variation analysis type selecting cancer and normal assay, then analyzing Spondin-2 gene expression in each data set, whether the difference downloading, analyzing separately its expression respectively in the other expression values of cancer and cancer by Spondin-2 gene in each data set is notable.nullBy analysis，Prove that Spondin-2 gene presents significantly high expression trend compared to cancer beside organism in cancerous tissue，This is proved by the analysis of multiple data sets，Including 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 p=0.0002),N=82,P=8.4E-9).Spondin-2 raises in adenoma of colon and expresses further up in colon cancer, it was demonstrated that Spondin-2 works in colon cancer generation evolution.And Spondin-2 gene all significantly rises in multiple Colorectal Carcinoma, such as adenocarcinoma of colon, colonic mucus adenocarcinoma, adenocarcinom of cecum, second shape rectal adenocarcinoma and rectal mucus adenocarcinoma (figure-1G and figure-1H).
Embodiment 2
The expression in colon cancer and cancer beside organism of the Spondin-2 gene is analyzed with quantitative PCR technique.
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 immediately placed on liquid nitrogen cryogenics in operation and preserve, and the other mucous membrane of colon distance cancerous tissue of cancer 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) sample tissue rear liquid nitrogen freezing rapidly in vitro, if tissue is relatively big, will be cut into tissue the bulk of Semen Glycines size as far as possible, put in cryopreservation tube, be stored in liquid nitrogen；
2) before extracting RNA, Liquid nitrogen precooler mortar is first used.After taking out sample from liquid nitrogen container, piece of tissue being put in the mortar of pre-cooling and grind rapidly, 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, taking and be dispensed in 1.5mL centrifuge tube in right amount, add 1mLTrizol, room temperature stands 5min (this sample can also-80 DEG C long-term preservations)；
4) adding 0.2mL chloroform, cover tightly centrifuge tube pipe lid, be shaken vigorously by hand for 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 to another new 1.5mL centrifuge tube, and every mLTrizol about can draw 0.5mL upper strata aqueous phase；
7) adding 0.5mL isopropanol, turn upside down and mix for several times, room temperature stands 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, turns upside down and fully wash precipitation；
10) 4 DEG C of centrifugal 5min of 7500g；
11) abandoning supernatant, be inverted EP pipe and blot residual ethanol on dust-free paper, room temperature places about 15min, makes RNA dry；
12) add 35 μ LRNasefreedH2O and dissolve RNA, BioTekEpoch many volumes spectrophotometer detection 260/280 value ,-80 DEG C of preservations.
3, reverse transcription synthesis cDNA is carried out with RNA
1) reverse transcription operation carries out according to PrimeScript1stStrandcDNASynthesisKit (TaKaRa) description.Concrete operation step is as follows:
2) in Microtube, following mixed liquor is prepared.
| Oligo dT Primer(50μM) | 1μL |
| dNTP Mixture(10mM each) | 1μL |
| Total RNA | 2μg |
| RNase free dH2O | up to 10μL |
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.
| Reactant liquor after above-mentioned degeneration, annealing | 10μL |
| 5×PrimeScript Buffer | 4μL |
| RNase Inhibitor(40U/μL) | 0.5μL |
| PrimeScript RTase(200U/μL) | 1μL |
| RNase free dH2O | 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) preparation PCR reactant liquor shown according to the form below (on ice operation):
| Reagent (TaKaRa) | Make consumption | Final concentration |
| SYBR Premix Ex TaqTM(2×) | 10μL | 1× |
| PCR Forward Primer(10μM) | 0.4μL | 0.2μM |
| PCR Reverse Primer(10μM) | 0.4μL | 0.2μM |
| ROX Reference DyeII(50×) | 0.4μL | 1× |
| DNA profiling | 2.0μL | 100ng |
| dH2O(RNase free dH2O) | 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 method pcr amplification standardization 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 confirms amplification curve and the solubility curve of RealtimePCR after terminating, be analyzed each gene relative expression's difference in colon cancer tissue according to double; two △ Ct methods.
5, interpretation of result
We analyze Spondin-2 gene expression in 10 example normal persons and 10 example 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
The expression at colon carcinoma cell line of the Spondin-2 albumen is detected by Westernblot method.
Operating process is as follows:
1, main solution preparation:
1) PBS solution: measuring raw work 20 × PBS solution 25mL, ultra-pure water is settled to 500mL, room temperature preservation.
2) 50 × Proteininhibitorcocktail (protease inhibitor mother solution): take a Roche Protease inhibitor (completeEDTA-freeProteaseInhibitorcocktailtablets), add 1mL ultra-pure water to dissolve, subpackage,-20 DEG C of preservations, this is 50 × protease inhibitor mother solution.
3) cell pyrolysis liquid: 50mMTris-HCl (pH7.4), 1%SDS, 2mMEDTA, subpackage ,-20 DEG C of preservations, every milliliter of lysate used time adds 20 μ L50 × Proteininhibitorcocktail again.
4) acrylamide solution: 30% acrylamide, 0.8% methylene diacrylamide；
5) separation gel buffer: 1.5mol/LTris-HClpH8.8；
6) concentration glue buffer: 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% bromophenol blue, 30% glycerol ,-20 DEG C of subpackages preserve, and general DTT is now with now adding.
11) electricity turns buffer: 25mmol/LTris, 192mmol/L glycine.
12) TBST buffer: 8.766gNaCl (i.e. 0.15mol/L), 1mLTween20,20mL1MTris-HCl (pH7.4), add ultra-pure water constant volume to 1L.
13) confining liquid: weigh 5g defatted milk powder and be dissolved in 100mLTBST and be 5% defatted milk powder confining liquid.
2, the extracting method of cell whole protein is cultivated:
1) sop up culture medium with vacuum pump, add PBS and wash one time, sop up, be repeated once；
2) being sopped up by PBS, culture dish is put on ice, adds appropriate cell pyrolysis liquid, stands 3min；
3), after fully 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 subpackage 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) taking BSA (Bio-rad) protein standard liquid, PBS is diluted to final concentration of 0.5mg/mL；
2) according to protein sample quantity, adding 1 volume BCA reagent B by 50 volume BCA reagent A, namely 50:1 prepares appropriate BCA working solution, fully mixes, stable in this BCA working solution room temperature 24h；
3) titer 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 solutions, places 30min in 37 DEG C of incubators；
6) BioTekEpoch many volumes spectrophotometer OD562nm place measures absorbance.The protein concentration of sample is calculated according to standard curve.
4, PAGE gel electrophoresis
1) take complete Bio-Rad and join drying in baking oven after glue glass plate ddH2O cleans up, be fixed on base with clip；
2) 10% separation gel and 5% concentration glue are prepared by formula as below.During preparative separation glue, upper strata ddH2O sealing, more than polymerized at room temperature 40min；When preparing the concentration glue of 5%, after sealing ddH2O in upper strata is exhausted, add concentration glue, insert comb, and prevent bubble；
10% separation gel is formulated as follows table:
Concentration 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 adds 3 × loadingbuffer for 2:1 by volume, boils the centrifugal 1min of 5min, 12000g, loading (applied sample amount is 30 μ g/ swimming lanes), simultaneously loading pre-dyed albumen Marker, compare as molecular weight in boiling water.
5) electrophoresis: concentration glue first carries out electrophoresis with 45V voltage, enters until bromophenol blue indicator and converts 65V continuation electrophoresis after separation gel to, and to be instructed dose is run to separation gel about 0.5cm and stop electrophoresis.
5, transferring film and Westernblot
1) prepare electricity and turn buffer (matching while using), according to gel size, cutting pvdf membrane and filter paper, after methanol activation pvdf membrane 1min, pvdf membrane and filter paper are turned immersion more than 15min in buffer at electricity；
2) glass plate is pried open, take out PAGE gel, put into electricity and turn balance in buffer, by the order of blackboard-foam-rubber cushion-filter paper-gel-pvdf membrane-filter paper-foam-rubber cushion-blank, gel and pvdf membrane are fixed on the wet electricity that turns and turn in folder, the black flour direction of the black flour corresponding groove of clip is put in electricity turn trough, fill it up with electricity and turn buffer and carry out transferring film (ice bath).Generally use the albumen that 110V2h electricity translocated molecule amount is bigger, it is possible to 30V constant voltage electricity turns over night in 4 DEG C of refrigerators；
3) close: transferring film takes out pvdf membrane after terminating, and the one side (protein powder) being close to gel is put into upward and hatches in box, and 5% defatted milk powder 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-hatch: the goat-anti rabbit two anti-() corresponding with primary antibodie is diluted to debita spissitudo (1:5000-1:8000) with confining liquid, room temperature shaker is hatched 1h；
7) TBST washs 3 times, each 5-10min；
8) sealed membrane of the suitable size of clip, add the super quick nitrite ion of 200-400 μ LThermoSuperSignalWestFemtoMaximumSensitivitySubstrate (by 1:1 matching while using working solution) herein above, room temperature stands 1min, uses Las-3000 imaging system to obtain Westernblot result.
6, result: gene expression abundance is higher in the cell lines that detect of the overwhelming majority (includes Colon cancer cell line HT-29, HCT-116, SW-620, LoVo, Caco-2 for Spondin-2 albumen, only Caco-320DM exception), and do not express CCD-18Co (representing Normal Colon cell) is inner, it was demonstrated that Spondin-2 albumen main relevant to colon cancer cell (figure-2B).
Embodiment 4
The expression in colon cancer and Carcinoma side normal tissue of the Spondin-2 albumen is analyzed with ImmunohistochemistryMethods Methods.
Operating process is as follows:
1, organization chip: use 2 sections of commercialization organization chips (Shanghai Xin Chao biotech firm), HCol-Ade180Sur-04 includes 90 examples, 180 cancers and cancer beside organism, clinical information include sex, the age, TNM, tumor size, by stages, classification, follow up a case by regular visits to information etc.；HCol-Ade180CS-01 includes 180 cancers of 90 example and cancer beside organism, and clinical information ibid, but does not include 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) putting in baking oven by organization chip, temperature is adjusted to 63 degree, dries wax one hour.
2) reagent preparation.10 × PBS (formula: 80gNaCl, 2gKCl, 15.35gNa2HPO4,2gKH2PO4, it is settled to 1000 milliliters with pure water): 10*PBS buffer is diluted to 1 × PBS, then in 1 × PBS, adds the tween reagent accounting for cumulative volume 0.05%；Antigen retrieval buffers: the pure water of the citric acid solution+900 milliliters of+18 milliliters of 0.1mol/L of sodium citrate solution of 82 milliliters of 0.1mol/L is placed in pressure cooker；
3, antigen retrieval
1), after slice, thin piece has toasted, take out in baking oven, put in full-automatic dyeing machine, dewax；
2) dewaxing process: dimethylbenzene two cylinder, every cylinder 15 minutes；Dehydrated alcohol two cylinder, every cylinder 7 minutes；90% ethanol 1 cylinder, 7 minutes；80% ethanol 1 cylinder, 7 minutes；70% ethanol 1 cylinder, 7 minutes；
3) from staining machine, slice, thin piece is taken out, by pure water rinsing 3 times, one time 3 minutes.Citric acid repair liquid is put by flushing process and begins to warm up to electric furnace；
4) antigen retrieval: after the boiling of citric acid repair liquid, put into by slice, thin piece in pressure cooker, cover high-pressure pot cover, starts timing after giving vent to anger, 5 minutes.Time terminates heating to rear, is opened by high-pressure pot cover so that it is naturally cool to room temperature；
4, block
Preparation endogenous peroxydase blocker.38.4ml the H2O2+9.6ml pure water of absolute methanol+12ml30% concentration；Slice, thin piece is put in blocker 10 minutes.
5, primary antibodie is added
1) take out slice, thin piece, rinse 3 times with PBS, one time 5 minutes；
2) refrigerator takes out Spondin-2 antibody, put in centrifuge 7200 and leave the heart 30 seconds；
3) take out antibody, dilute according to dilution factor DAKO antibody diluent 1:50；
4) dropping antibody；
5) wet box being put into refrigerator, 4 DEG C overnight.
6, add two to resist
1) from refrigerator, take out wet box, stand and treat that it returns to room temperature in 1 hour；
2) slice, thin piece PBS is rinsed 3 times, one time 5 minutes；
3) dropping EnVisionTM+/HRP rabbit working solution (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+1 DAB chromogen of 1mlDAB diluent；(from DAKO liquid D AB+ substrate system).Slice, thin piece drips the DAB after dilution, develops the color 5 minutes, tap water 15 minutes after then.
8, haematoxylin is redyed
1) on slice, thin piece, drip haematoxylin 2 minutes, the time to after in the hydrochloride alcohol of 0.25% submergence 2 seconds, with tap water 2 minutes；
2) slice, thin piece is put into full-automatic dyeing machine and carries out dehydration, take out mounting.
3) dehydration: 75% ethanol 1 cylinder, 3 minutes；85% ethanol 1 cylinder, 3 minutes；95% ethanol 1 cylinder, 3 minutes；Dehydrated alcohol two cylinder, every cylinder 5 minutes；Dimethylbenzene two cylinder, every cylinder 5 minutes.
9, scanning: with chip scanner ScanscopeXT (Aperio company) scanning.
10, the software analysis of chip dyeing
1) opening chip scanning file with software AperioImageScopev.12, the region choosing more than 3 on the figure of each sample point exports as single TIFF picture；Some sample points, lack neoplastic epithelial cells, it is impossible to statistical presentation situation, such point is abandoned.
2) opening 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 calculated average accumulated optical density value in multiple regions from same sample is further averaged, and this value represents the expression 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.Being easy to analyze for simplifying classification, the expression in cancer of the Spondin-2 albumen plus 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, checks through T, it has been found that the expression in colon cancer sample of the Spondin-2 albumen 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, and effective cancer point is 88 examples, checks through T, it has been found that the expression in colon cancer sample of the Spondin-2 albumen 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 the sample of the other Spondin-2 positive expression of cancer is shown in that the sample that figure-3B, colon cancer Spondin-2 feminine gender is expressed is shown in that the sample of the basic, normal, high expression of figure-3C, colon cancer Spondin-2 is shown in Fig. 3 D, E, F.
These results prove that Spondin-2 albumen is that significant difference is expressed by colon cancer and cancer.
Embodiment 5
Spondin-2 expresses the relation with colon cancer patient clinical parameter
(obtaining data method the same) is analyzed with Oncomine gene expression data base, colon cancer sample is divided into 2 groups according to the median of the expression of Spondin-2 gene, one group is the high expressed group more than median, one group is the low expression group 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 expression and the (BittnerColon by stages of Spondin-2 gene, χ 2=16.96, p=0.001), T (BittnerColon, χ 2=13.852, p=0.003) by stages, M (BittnerColon 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 significant relation (table 1).Generally, Spondin-2 gene expression rise and tumour progression are proportionate, and male patient Spondin-2 gene upregulation becomes apparent from.
The expression of table 1.Spondin-2 gene and the relation of colon cancer patient Clinical symptoms
Integrate the analysis result from 2 organization chips and SABC, it has been found that the expression of Spondin-2 albumen and age (χ2=9.65, p=0.0468), M (χ by stages2=4.245, p=0.039) and transfer (χ2=4.332, p=0.037) there is significant relation, notable (χ close with relation by stages2=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.
Embodiment 6
The relation of Kaplan-Meier methods analyst Spondin-2 gene and albumen and colon cancer patient prognosis.
The gene expression dataset SmithColorectal2 utilizing Oncomine data base analyzes the relation of Spondin-2 gene expression height and the life span of Post operation colon cancer patient, and the expression of Spondin-2 gene is divided into more than median (28 example) with less than median (27 example) two groups.It is figure 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 gene expression is high, its overall life cycle (Overallsurvival) significantly shortens (Log-rank inspection, p=0.011) (figure-4A), mean survival time during low expression is 93.489 months (predictive values, standard error 6.604, confidence interval 80.545-106.433), and mean survival time during high expressed is 42.486 months (predictive values, standard error 5.497, confidence interval 31.712-53.260).
If it is considered that the factor of recurrence, statistics disease free survival rate (Diseasefreesurvival), one group of patient that then same Spondin-2 gene expression is high, its disease free survival phase also significantly shortens (Log-rank checks, p=0.0263) (figure-4B).Average disease-free life span during low expression is 82.138 months (predictive values, standard error 8.216, confidence interval 66.035-98.241), and average disease-free life span during high expressed is 41.507 months (predictive values, standard error 5.384, confidence interval 30.955-52.059).
Organization chip is utilized to carry out immunohistochemical analysis, the relation that research Spondin-2 albumen is survived with colon cancer patient prognosis.The patient that Spondin-2 protein expression is positive, significantly shortens (Log-rank checks, p=0.0363) (figure-4C) its life cycle than the patient of Spondin-2 protein expression feminine gender.Overall life span when Spondin-2 protein negative is expressed is 68.087 months (predictive values, standard error 5.354, confidence interval 57.592-78.581), and average disease-free life span during Spondin-2 protein positive expression is 50.800 months (predictive values, standard error 4.134, confidence interval 42.697-58.903)
These the results shows, the high expressed of Spondin-2 gene and albumen shortens the postoperative life span of colon cancer patient, and the expression of Spondin-2 gene and albumen can as the judge index of colon cancer prognosis.
Embodiment 7
Single argument and Multivariate Cox Regression analysis.
We use single argument and Multivariate Cox Regression method, analyze Spondin-2 protein expression and the impact on colon cancer patient life span of other clinical parameters further.Univariate analysis result shows, the T of colon cancer patient (p=0.018 by stages, Hazard ratio 2.2,95% confidence interval 1.144-4.231), N (Hazard ratio=2.766 by stages, 95% confidence interval=1.8-4.249, p < 0.001), classification (Hazard ratio=1.832,95% confidence interval=1.035-3.242, and Spondin-2 protein expression (Hazard ratio=2.229 p=0.038), 95% confidence interval=1.028-4.832, p=0.042) and colon cancer prognosis significant correlation (table 3)；
Multivariate regression analysis shows, Spondin-2 protein expression (Hazard ratio 2.197,95% confidence interval=1.013-4.768, and N (Hazard ratio 2.791 by stages p=0.046), 95% confidence interval=1.801-4.326, p < 0.001) prognosis of appreciable impact 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 life span
Result above shows, Spondin-2 protein expression be independent, affect the factor of colon cancer patient life span.By detecting the expression of the Spondin-2 albumen in colon cancer patient cancerous tissue, it is possible to the prognosis survival risk situation of prediction colon cancer patient.
Embodiment 8
The expression of the Spondin-2 albumen in detection colon cancer patient blood and the ability of discriminating colorectal carninomatosis people and normal person thereof.
Operating process:
Spondin-2 enzyme linked immunological kit reagent box (Cloud-CloneCorp Products) is purchased from Shanghai Wu Hao company.
1, each 20 examples of the blood plasma of colon cancer patient and normal person, 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, and reverse/rubbing is with hydrotropy solution repeatedly simultaneously, and its concentration is 10,000pg/mL (storage liquid).Then carried out gradient dilution again, 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, take 20 μ L serum or blood plasma adds 180 μ LPBS.
3) detection solution A and detection solution B preparation: DetectionA and DetectionB hands before use get rid of several under so that the liquid deposition of tube wall or bottle cap is at the bottom of pipe.Before use respectively with deionized water with 1:100 dilute (as: 10 μ L detect solution A/990 μ L deionized waters), fully mixing, according to precalculated total amount preparation (100 μ L/ hole) that experiment is required every time before dilution, during actual preparation, 0.1-0.2mL should be prepared more.
4) cleaning mixture: dense for 20mL cleaning mixture is diluted to 600mL with 580mL deionized water, carries out 30 times of dilutions.
3, operating procedure:
1) application of sample: set gauge orifice, testing sample hole, blank well respectively.If gauge orifice 7 hole, it is sequentially added into the standard substance of 100 μ L variable concentrations.Blank well adds 100 μ L standard dilutions, and remaining hole adds testing sample 100 μ L, and ELISA Plate adds overlay film, 37 DEG C of incubations 2 hours.
2) discard liquid, dry, need not wash.
3) every hole adds detection solution A working solution 100 μ L (prepared before use), and ELISA Plate adds overlay film, 37 DEG C of incubations 1 hour.
4) discarding liquid in hole, every hole cleaning mixture of 350 μ L washs, and soaks 1-2 minute, gets rid of the liquid in ELISA Plate, which floor absorbent paper of place mat in laboratory table, and ELISA Plate is firmly clapped several times down, repeats to wash plate 3 times.After last washing, the cleaning mixture in hole is dried completely.
5) every hole adds detection solution B working solution (prepared before use) 100 μ L, adds overlay film, 37 DEG C of incubations 30 minutes.
6) discarding liquid in hole, dry, wash plate 5 times, method is with step 4.
7) every hole adds substrate solution 90 μ L, and ELISA Plate adds overlay film, and 37 DEG C of lucifuges develop the color 20 minutes
8) every hole adds stop bath 50 μ L, terminates reaction.
9) guarantee without after bubble-free in water droplet and hole at the bottom of ELISA Plate, immediately by the microplate reader optical density (O.D. value) in each hole of 450nm wavelength measurement.
10) calculate: with the concentration of standard substance for vertical coordinate, O.D. value is abscissa, draws standard curve.O.D. value (sample has done two multiple holes, averages), is found corresponding concentration by standard curve, is multiplied by extension rate (10 times), be the actual concentrations (pg/ml) of sample per sample.
4, result
1) expression of the Spondin-2 albumen in colon cancer patient and normal human blood
Enzyme-linked immuno-sorbent assay determines the expression of the Spondin-2 albumen in colon cancer patient and normal human blood, it was demonstrated that Spondin-2 albumen level in colon cancer patient (20 example) blood plasma is significantly higher than normal person (20 example) (p=3.2E-9) (figure-5A).In colon cancer patient blood plasma, the mean concentration of Spondin-2 albumen is 61.8ng/ml, and normal person is then 17.4ng/ml, and patient is far above normal person.
2) ability of the expression discriminating colorectal carninomatosis people of Spondin-2 albumen and normal person in blood plasma
Result according to 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), illustrating that colon cancer patient and normal healthy people can be made a distinction well by Spondin-2 albumen as blood plasma marker thing, Spondin-2 albumen can as the blood plasma index of diagnosis of colon cancer.
3) sensitivity of blood plasma Spondin-2 Protein Detection colon cancer and specificity
4) result according to enzyme-linked immunosorbent assay, sensitivity and the specificity of blood plasma Spondin-2 Protein Detection colon cancer can be known, when blood plasma Spondin-2 protein concentration is 40.7ng/ml, the sensitivity of colon cancer detection is 85%, and specificity is 100% (table 5).
The area under a curve (AUC) that table 4. experimenter's characteristic curve is analyzed
A. under nonparametric hypothesis.
B. null hypothesis: solid area=0.5.
Table 5. receiver operating characteristic curve analyzes the expression of Spondin-2 in colon cancer patient and human normal plasma
| If greater than or equal to being then justa(ng/ml) | Sensitivity | 1-specificity | Specificity | Sensitivity+specificity |
| 2.5200 | 1.000 | 1.00 | 0.000 | 1.000 |
| 4.0450 | 1.000 | 0.95 | 0.050 | 1.050 |
| 6.6450 | 1.000 | 0.90 | 0.100 | 1.100 |
| 9.1450 | 1.000 | 0.85 | 0.150 | 1.150 |
| 9.6700 | 1.000 | 0.80 | 0.200 | 1.200 |
| 10.6450 | 1.000 | 0.75 | 0.250 | 1.250 |
| 11.6950 | 1.000 | 0.70 | 0.300 | 1.300 |
| 12.1700 | 1.000 | 0.65 | 0.350 | 1.350 |
| 12.5950 | .950 | 0.65 | 0.350 | 1.300 |
| 13.8950 | .950 | 0.60 | 0.400 | 1.350 |
| 15.6200 | .950 | 0.55 | 0.450 | 1.400 |
| 16.6700 | .950 | 0.50 | 0.500 | 1.450 |
| 17.6200 | .950 | 0.45 | 0.550 | 1.500 |
| 18.6700 | .950 | 0.40 | 0.600 | 1.550 |
| 19.3200 | .950 | 0.35 | 0.650 | 1.600 |
| 19.8700 | .900 | 0.35 | 0.650 | 1.550 |
| 22.4200 | .900 | 0.30 | 0.700 | 1.600 |
| 24.9950 | .850 | 0.30 | 0.700 | 1.550 |
| 25.9950 | .850 | 0.25 | 0.750 | 1.600 |
| 27.0450 | .850 | 0.20 | 0.800 | 1.650 |
| 27.7450 | .850 | 0.15 | 0.850 | 1.700 |
| 28.1450 | .850 | 0.10 | 0.900 | 1.750 |
| 31.1700 | .850 | 0.05 | 0.950 | 1.800 |
| 40.6950 | .850 | 0.00 | 1.000 | 1.850 |
| 47.5950 | .800 | 0.00 | 1.000 | 1.800 |
| 49.2700 | .750 | 0.00 | 1.000 | 1.750 |
| 51.5450 | .700 | 0.00 | 1.000 | 1.700 |
| 52.4200 | .650 | 0.00 | 1.000 | 1.650 |
| 58.0450 | .600 | 0.00 | 1.000 | 1.600 |
| 63.5450 | .550 | 0.00 | 1.000 | 1.550 |
| 27.7450 | .850 | 0.15 | 0.850 | 1.700 |
| 66.3200 | .500 | 0.00 | 1.000 | 1.500 |
| 70.3200 | .450 | 0.00 | 1.000 | 1.450 |
| 72.1450 | .400 | 0.00 | 1.000 | 1.400 |
| 72.9700 | .350 | 0.00 | 1.000 | 1.350 |
| 74.8700 | .300 | 0.00 | 1.000 | 1.300 |
| 76.9950 | .250 | 0.00 | 1.000 | 1.250 |
| 79.2200 | .200 | 0.00 | 1.000 | 1.200 |
| 81.0700 | .150 | 0.00 | 1.000 | 1.150 |
| 81.5950 | .100 | 0.00 | 1.000 | 1.100 |
| 99.1200 | .050 | 0.00 | 1.000 | 1.050 |
| 117.2200 | .000 | 0.00 | 1.000 | 1.000 |
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 meansigma methodss of the sight control value of two vicinities.
Above content described in this specification is only illustration made for the present invention.Described specific embodiment can be done various amendment or supplements or adopt similar mode to substitute by those skilled in the art; without departing from the content of description of the present invention or surmount the scope that present claims book is defined, protection scope of the present invention all should be belonged to.
SEQUENCELISTING
<110>Fudan University
<120>application process of a kind of human colon carcinoma protein markers Spondin-2
<130>20141212
<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. human colon carcinoma Protein S pondin-2 application in preparing diagnosis of colon cancer preparation.
2. apply as claimed in claim 1, it is characterised in that described diagnosis of colon cancer preparation be diagnosis or prediction colon cancer transfer, colorectal cancer clinical by stages, the preparation of colorectal cancer patients prognosis.
3. apply as claimed in claim 1, it is characterised in that described diagnosis of colon cancer preparation be diagnosis or prediction colon cancer transfer, colorectal cancer clinical by stages, the test kit of colorectal cancer patients prognosis.
4. apply as claimed in claim 1, it is characterised in that described diagnosis of colon cancer preparation be the propagation of examination colon cancer cell, transfer, colorectal cancer clinical by stages or the preparation of colon cancer patient prognosis.
5. apply as claimed in claim 1, it is characterised in that human colon carcinoma Protein S pondin-2 is the diagnosis marker of human colon carcinoma.
6. the test kit detecting colon cancer, it is characterised in that containing the reagent detecting human colon carcinoma Protein S pondin-2 expression in described test kit.
7. test kit as claimed in claim 6, it is characterised in that possibly together with human colon carcinoma Protein S pondin-2 standard sample in described test kit.
8. test kit as claimed in claim 6, it is characterised in that containing the primer detecting human colon carcinoma Protein S pondin-2 in described test kit.
9. test kit as claimed in claim 8, it is characterised in that the sequence of described primer is such as shown in SEQIDNO1 and SEQIDNO2.
10. the purposes of test kit described in claim 6, it is characterized in that, utilize this test kit detection human colon carcinoma Protein S pondin-2 expression, further for the propagation of colon cancer cell, transfer, colorectal cancer clinical by stages, colon cancer patient prognosis or colon cancer canceration process evidence is provided.
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