CN108330192A - Smell mediates application of the element -4 as non-small cell lung carcinoma marker - Google Patents
Smell mediates application of the element -4 as non-small cell lung carcinoma marker Download PDFInfo
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Abstract
The invention discloses smell to mediate application of the element 4 as Diagnosis of Non-Small Cell Lung marker.Present invention research is found, Patients with Non-small-cell Lung smell mediates the expression of plain 4 genes or albumen compared with Healthy People or cancer beside organism, expression significantly increases, therefore can be used as Diagnosis of Non-Small Cell Lung marker, is used to prepare Diagnosis of Non-Small Cell Lung preparation and reagent kit product.The present invention provides a kind of non-small cell lung cancer biomarker smell of new specificity to mediate element 4, and gives the detection method that the smell with reference value mediates element 4.The non-small cell lung cancer biomarker smell mediates screening/diagnosis effect of the element 4 for non-small cell lung cancer good, and accuracy is high, has good application prospect, is worth a wide range of and promotes.
Description
Technical field
The present invention relates to screening/detection technique fields of non-small cell lung cancer, are mediated more particularly, to a kind of smell
Element -4 is used as the application of non-small cell lung carcinoma marker.
Background technology
Lung cancer is that morbidity and mortality growth is most fast, to one of population health and the maximum malignant tumour of life threat.
The morbidity and mortality of many countries all report lung cancer obviously increase in the past 50 years, and male lung cancer morbidity and mortality are equal
First of all malignant tumours is accounted for, women incidence accounts for second, and the death rate accounts for second.The cause of disease of lung cancer is still endless so far
Complete clear, the incidence of city dweller's lung cancer is higher than rural area, this may contain carcinogen with urban atmospheric pollution and flue dust
It is related.And since China's air pollution is serious, cause lung cancer morbidity persistently to increase.Lung cancer is still most universal and most fatal at present
Cancer, about newly-increased 1,800,000 patients in 2012 simultaneously cause 1,590,000 people dead.Wherein China accounts for about 1/3 or more of such case.Such as
Effective control measure are taken not in time, it is contemplated that by 2025, patient numbers were up to 1,000,000, and it is big to become the first in the world lung cancer
State.
Lung cancer can be divided into Small Cell Lung Cancer and non-small cell lung cancer, and latter of which accounts for about 80%.Non-small cell lung cancer is again
It can be divided into three classes：（i）Squamous cell carcinoma starts in squamous cell, and the squamous cell is look like fish scale thin
, pinacocyte.Squamous cell carcinoma is also referred to as epidermoid carcinoma；（ii）Large cell carcinoma is opened in the big pneumonocyte of several types
Begin；（iii）Gland cancer starts in the alveolar and preparing of liner lung in the cell of substance such as mucus.Other less common classes
The NSCLC of type includes pleomorphic carcinoma, carcinoid tumor and non-classified cancer.Cause lung cancer to occur many because being known as, smoking, heredity because
Element, radioactive radon gas, asbestos etc. are all risk factors.
The death rate of lung cancer is high, and five year survival rate is less than 15%.One important reason is that early diagnostic rate is low, less than 2%,
When 80% or more patient assessment late.Current diagnostic method has X-ray inspection, CT scan, bronchoscopy, phlegm cell
Learn inspection and lung cancer biological marker analyte detection etc..All there is respective defect in these methods, as Imaging Technology is difficult to find
The smaller tumour of knurl, the early stage omission factor of generaI investigation are higher.Existing lung cancer marker is wide spectrum, can not make a definite diagnosis lung cancer, because
This lung cancer biomarker for finding specificity has great importance.
Up to the present, it is related not have the occurrence and development that 10 kinds of gene unconventionality expression is determined with lung cancer, but
Unconventionality expression rate of the determining lung cancer related gene in lung cancer be not high, and the pathogenesis of lung cancer does not illustrate yet so far, lung cancer
Early diagnostic rate it is still to be improved.In addition, the operation of lung cancer traditional in China adds chemotherapy and is used cooperatively in recent years more
Kind gene therapy method finds new lung cancer related gene still without the survival rate for significantly improving patients with lung cancer, especially
Lung cancer cance high-expression gene is of great significance for the pathogenesis and early diagnosis of inquiring into lung cancer.
Invention content
The purpose of the invention is to overcome the deficiencies of the prior art and provide a species specific non-small cell lung cancer mark
Object.
The first purpose of the invention is to provide smell to mediate application of the element -4 as Diagnosis of Non-Small Cell Lung marker.
Second object of the present invention is to provide smell and plain -4 protein fragments is mediated to mediate plain -4 protein quantifications inspection in smell
Application in survey.
Third object of the present invention is to provide smell, and plain -4 albumen to be mediated to prepare non-small cell lung cancer screening/diagnosis
Application in kit.
Fourth object of the present invention is to provide smell and plain -4 albumen is mediated to prepare non-small cell lung cancer screening/diagnosis
Application in reagent.
Fifth object of the present invention is to provide smell, and plain -4 protein fragments to be mediated to prepare non-small cell lung cancer screening/examine
Application in disconnected kit.
Sixth object of the present invention is to provide smell, and plain -4 protein antibodies to be mediated to prepare non-small cell lung cancer screening/examine
Application in disconnected kit.
The 7th purpose of the present invention is to provide a kind of kit for non-small cell lung cancer screening/diagnosis.
To achieve the goals above, the present invention is achieved by the following technical programs：
Smell mediates element -4（Olfactomedin-4）, HGNC accession number is 17190；Entrez Gene accession number is 10562；
Ensembl accession number is ENSG00000102837；OMIM accession number is 614061；UniProtKB accession number is Q6UX06, is
A kind of new secreting type glycoprotein is one of the important member that smell mediates plain family, is mainly expressed in human marrow, stomach, small
The tissues such as intestines, colon, pancreas and prostate.To safeguarding that the stabilization of cell various biological function plays an important role, including
The generation of nerve, neural crest is formed and cell-cell adhesion, the adjusting of cell cycle and Apoptosis etc..But up to now, do not have also
There is smell to mediate the relevant report of element -4 and non-small cell lung cancer.
The present invention is the study found that Patients with Non-small-cell Lung smell mediates element -4（Olfactomedin-4）Gene or albumen
Expression compared with Healthy People or cancer beside organism, expression is high, therefore can be used for non-small cell lung diagnosis.
Wherein, smell mediates the nucleotide sequence of plain -4 genes as shown in SEQ ID NO.1, and smell mediates plain -4 albumen
Amino acid sequence is as shown in SEQ ID NO.2.
Therefore, following application should all be within protection scope of the present invention：
Smell mediates application of the element -4 as Diagnosis of Non-Small Cell Lung marker.
Smell mediates application of plain -4 protein fragments in smell mediates plain -4 protein quantifications detection, the smell to mediate
Amino acid sequence such as NO.3~26 SEQ ID of plain -4 protein fragments are any shown.
Smell mediates application of plain -4 albumen in the kit for preparing non-small cell lung cancer screening/diagnosis.
Smell mediates application of plain -4 albumen in the reagent for preparing non-small cell lung cancer screening/diagnosis.
Smell mediates application of plain -4 protein fragments in the kit for preparing non-small cell lung cancer screening/diagnosis, described
Smell mediates amino acid sequence such as NO.3~26 SEQ ID of plain -4 protein fragments any shown.
Smell mediates application of plain -4 protein antibodies in the kit for preparing non-small cell lung cancer screening/diagnosis.
In addition, a kind of kit for non-small cell lung cancer screening/diagnosis, the kit, which contains, can detect smell
The reagent for mediating the expression of plain -4 genes or protein, also belongs to protection scope of the present invention.
Preferably, the reagent includes one or several in peptide fragment shown in SEQ ID NO. 3~26.
Preferably, the reagent includes that smell mediates plain -4 protein antibodies.
Preferably, the antibody is monoclonal antibody or polyclonal antibody.
The application method of the kit, includes the following steps：
S1. the doubtful cancerous tissue of individual is obtained；
S2. the expression that smell therein mediates element -4 is measured；
S3. the same tissue of normal individual or the normal structure of same individual are obtained；
S4. it measures smell therein and mediates the expression of element -4 expression as a contrast；
What S5. the expression of the smell obtained in S2 mediation element -4 was mediated to element -4 with the smell obtained in S4 compares expression
Level is compared, when the smell obtained in S2 mediates the expression of element -4 to mediate compareing for element -4 with the smell obtained in S4
When expression is compared to improving, determine that the individual suffers from non-small cell lung cancer.
Alternatively, the application method of the kit, includes the following steps：
S1. the serum of individual is obtained；
S2. the expression that smell therein mediates element -4 is measured；
S3. the serum of normal individual is obtained；
S4. it measures smell therein and mediates the expression of element -4 expression as a contrast；
What S5. the expression of the smell obtained in S2 mediation element -4 was mediated to element -4 with the smell obtained in S4 compares expression
Level is compared, when the smell obtained in S2 mediates the expression of element -4 to mediate compareing for element -4 with the smell obtained in S4
When expression is compared to improving, determine that the individual suffers from non-small cell lung cancer.
In addition, The present invention gives the detection methods that the smell with reference value mediates element -4.
The segment of plain -4 albumen is mediated using any shown smell in peptide fragment such as NO.3~26 SEQ ID（Table 1）, the segment
It should be with the albumen or its process enzymolysis in sample（Such as trypsin digestion）The sequence of the segment obtained afterwards is completely the same.It is described
Segment preferably passes through heavy isotope（Such as 13C, 14N）Label, mutually to distinguish itself and un-marked sample, to be smelt
Feel and mediates plain -4 protein quantifications detection.In addition, the best Transition of the segment should have preferable signal-to-noise ratio.
By a certain amount of segment by heavy label（Shown in table 1）It is added through enzyme（Such as trypsase）The trouble of digestion
In person's blood serum sample, the level of segment described in the technology detection patients serum with quantitative proteomics, so that it is determined that serum
Middle smell mediates element -4 horizontal；By measured level compared with the critical value determined according to the level of normal person, if surveyed
The level obtained is higher than critical value, then it represents that gastric cancer probability is big.
Table 1：
The critical value can be determined by those skilled in the art according to conventional means.For example, those skilled in the art can be with
According to the normal person measured（Group）Serum protein content data draw Receiver operating curve（ROC curve）, with
Critical value is determined afterwards（cut-off）.
It can be with the absolute quantitation technology based on section of synthesized peptide by quantitative proteomics technology（absolute
Quantification analysis, AQUA）It is combined, thus can element -4 directly be mediated to the smell in multiple samples
Directly carry out the detection of absolute content.By measured level compared with the level of normal control serum, if measured water
The flat level higher than normal control serum, then it represents that the object suffers from gastric cancer.
It is detected using the antibody for mediating plain -4 protein-specifics to be combined with people's smell.Term " antibody " herein has
Most broadly meaning includes specifically:Monoclonal antibody（Monoclonal antibody including overall length）, it is polyclonal antibody, mostly special
Property antibody（Such as bispecific antibody）.Term " antibody " further includes the segment of entire molecule, such as being capable of combining response antigen
Fab and F（ab' ）2.Fab and F（ab' ）2 segments lack the Fe segments of complete antibody, can be more quickly from the circulatory system
Middle removing, and with lower non-specific tissue binding compared with complete antibody.Useful Fab and F in the present invention
（ab' ）2 and other antibody fragments can also be used for detect and quantitative determine people's smell mediate element -4, as long as they show institute
The activity of the specific binding needed.The usually available papain of these segments（Generate Fab segments）Or pepsin（Generate F（ab'
）2 segments）It is cut and is generated by protease hydrolytic.
Wherein, the polyclonal antibody and monoclonal antibody for being used for the present invention can be made with conventional method.In general, using egg first
It is white that suitable animal is immunized, it is preferred that mouse, rat, rabbit or goat.Since obtainable serum volume is more, can obtain
The anti-rabbit of label and anti-goat antibody, therefore for preparing for polyclonal antiserum, rabbit and goat are preferable.It is immune
Usually carry out in this way：By albumen with brine（Preferably with adjuvant such as Freund's complete adjuvant）Mixing emulsifies, then parenteral（It is logical
It is often subcutaneously or intramuscularly）Inject the mixture or emulsion.Brine is used after 2-6 weeks（Preferably use incomplete Freund's adjuvant）Match
Protein injection is one or many with reinforced immunological.Animal blood after will be immune is drawn into glass or plastic containers, 25 DEG C
It cultivates the blood I hours, then cultivates 2-18 hours for 4 DEG C, obtain polyclonal antiserum.Monoclonal antibody can use Kohler and
Standard method [the Nature of Milstein（1975）256:495-96] or its improved method be made.In general, as described above to small
Mouse or rat immunity.However, being not to take blood then to extract serum animal, but take out spleen（And it optionally takes out several
A big lymph node）, it is dispersed into unicellular.If desired, can be by cell suspension（In the cell for removing non-specific adhesion
Afterwards）Addition has been coated in plate or the hole of proteantigen, is screened to splenocyte.The film combination for expressing antigentic specificity is immune
The B cell of globulin is attached on plate, is washed away not as suspension other materials.Then to gained B cell or all dissociation
Splenocyte induced, make it merge to form hybridoma with myeloma cell, cultivate in selective medium（It is as follows yellow fast
Purine, aminopterin, thymidine medium, " HAT "）In.The hybridoma as obtained by limiting dilution inoculation, and measure specific binding and exempt from
Epidemic disease antigen（And do not combine irrelevant antigen）Antibody generation.Then, in vitro（Such as it is reacted in tissue culture flasks or doughnut
In device）Or in vivo（In mouse ascites）The hybridoma of the selected secrete monoclonal antibody of culture.
With antigen in antibody determination serum can use immunofluorescence technique, using fluorescent marker antibody and combine optical microphotograph
Art, flow cytometry or fluorescence metering art are detected and are realized.Test method is for example including making biological sample（Such as biofluid
Such as serum）It is cultivated in the presence of can differentiate detectable label antibody, then in numerous methods well known in the art
Any method detects antibody.
Biological sample can use solid support or carrier（Such as nitrocellulose）Or can fix cell, cell granulations or
Other solid supports or carrier of soluble protein are handled.Then support or carrier, then root are washed with suitable buffer solution
It is handled like that with the antibody of detectable label according to the present invention is above-mentioned.Again with second of washing solid support of buffer solution or load
Body removes unbonded antibody.Then conventional method can be used to detect the label combined on the solid support or carrier
Amount.Well known support or carrier include:It is glass, polystyrene, polypropylene, polyethylene, glucan, nylon amylases, natural
And modified cellulose, polyacrylamide, gabbro and magnetite.The structure of support or carrier can be spherical（Such as exist
In bead）, it is cylindrical（Such as the inner surface of test tube or the outer surface of stick）.In addition, surface can be flat, such as plate, test
Band etc..Preferable support or carrier include polystyrene bead.Skilled person will appreciate that road is for binding antibody or resists
Other former many suitable carriers, or can determine these carriers by routine experiment.
In the present invention, antibody can be connected with enzyme, is subsequently used for enzyme and connects immunoassay.Then, when the enzyme and suitably
When substrate contacts, enzyme meeting and substrate reactions can be detected to generate（Such as pass through spectrophotometric analysis, fluorescence analysis or meat
Eye observation）Chemicals.The enzyme that can be used for detectably labelled antibody includes（But it is not limited to）：Malic dehydrogenase, grape
It is gonorrhoeae nucleic acid enzyme, S-5- steroid isomeras, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, phosphotriose isomerase, peppery
Root peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribalgilase, urea
Enzyme, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.Using the chromophore bottom of enzyme
Object can be completed to detect using colorimetric method.Detection can also pass through the degree for reacting enzyme-to-substrate and similar preparation
Reference substance carries out naked eyes and relatively realizes.
Detection can also be realized with other various immunity tests.For example, passing through radiolabelled antibody, so that it may with by using
Radiommunoassay（RIA）To detect.Radioactive isotope can with the equipment such as scintillation counter or by radioactive automatic developing come
It is detected.
The antibody of the present invention can also be marked with fluorescent chemicals.When the antibody of fluorescent marker is exposed to appropriate wavelength
Light when, its presence can be detected because of fluorescence.Most common fluorescent labelling compound has fluorescein isothiocynate, Luo Dan
Bright, phycoerythrin, phycocyanin, allophycocyanin, phthalic aldehyde and fluorescamine.The metal for sending out fluorescence also can be used in antibody（Such as
152E or other lanthanide series metals）Detectably marked.These metals can pass through the chelation group of these metals（Such as diethyl three
Triamine pentaacetic acid（ETPA））It is connect with antibody.Can also antibody coupling chemiluminescence compound be subjected to detectable mark to antibody
Note.It is then detected that the presence to shine in chemical reaction process detects the presence of the antibody with chemiluminescent labeling.Especially
The example of useful chemiluminescent labeling compound is luminol, different luminol, hot A Ding Gun esters, imidazoles, A Ding Gun salt and grass
Acid esters.Same way, it is also possible to mark antibody of the present invention with bioluminescent compound.Bioluminescence is found in biosystem
A kind of chemiluminescence, wherein catalytic protein improve the efficiency of chemiluminescence reaction.The presence of bioluminescent protein can pass through
Detection shines to determine.Important bioluminescent compound for labeling purposes is luciferin, luciferase and aequorin
Albumen.
The antibody of the present invention can also be used for immunoassay test, such as sandwich assay.It, will in typical immunoassay test
A certain amount of unmarked antibody（Or antibody fragment）It is incorporated on solid support or carrier, the detectable mark of a certain amount of band is added
The solvable antibody of note, so as to the ternary detected and/or quantitative analysis is formed between solid phase antibody, antigen and labelled antibody
Compound.Typical and preferable immunometric assays are tested including " forward direction ", first with the antibody of solid phase binding in this experiment
It is contacted with sample to be tested, by forming binary solid matrix antibody-antigen compound to extract antigen from sample.It is training
After educating the suitable time, solid support or carrier are washed to remove fluid sample residue（Including unreacted antigen, if
If having）, then contacted again with the solution of the labelled antibody containing unknown quantity.Being cultivated at second, which makes labelled antibody pass through, does not mark
After note antibody and the antigen being incorporated on solid support or carrier are compound, second of washing solid support or carrier remove
Remove unreacted labelled antibody.
Compared with prior art, the present invention has the advantages that：
The present invention provides a kind of non-small cell lung cancer biomarker Olfactomedin-4 of new specificity, and give
The detection method of Olfactomedin-4.Non-small cell lung cancer biomarker Olfactomedin-4 is used for non-small cell lung
Cancer screening/diagnosis effect is good, and accuracy is high, has good application prospect, is worth a wide range of and promotes.
Description of the drawings
Fig. 1 is to mediate the immunoblotting collection of illustrative plates of element -4, Olfactomedin-4 to carry out the egg that data analysis obtains smell
White matter expression quantity Relative distribution figure.
Fig. 2 is to mediate element -4, Olfactomedin-4 immunohistochemical assay results to carry out data analysis smell to obtain
The protein expression amount Relative distribution figure gone out.
Fig. 3 is to mediate element -4, Olfactomedin-4 Proteins in Serum content experimental results to carry out data point smell
Phase separation is to distribution map.
Specific implementation mode
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as Sambrook et al.,
Molecular cloning:Laboratory manual（New York:Cold Spring Harbor Laboratory Press, 1989）Described in
Condition, or according to the normal condition proposed by manufacturer.
In following embodiments of the present invention, urea, 3- ［ 3-（Courage amido propyl）Dimethylamino ］ propane sulfonic acid inner salt
（CHAPS）, dodecyl sodium sulfate（SDS）, dithiothreitol (DTT)（DTT）, three（Methylol）Aminoethane（Tris）, iodoacetamido
Amine（IAA）Purchased from Bio-Rad companies；The chemical reagent such as beta -mercaptoethanol are purchased from Sigma companies；Trypsase（Trypsin,
sequencing grade）Purchased from Promega companies；iTRAQ（isobaric tags for relative and
absolute quantitation）Reagent is purchased from AB SCIEX；SCX columns and SAX columns and pH buffer kits are purchased from
Waters companies.
In following embodiments of the present invention, the solution formula used is as follows：
Lysate：8mol/L urea, 4% CHAPS, 40mmol/L Tris and 65mmol/L DTT.
TBST：Tris 2.42g/L, sodium chloride 8g/L, Tween-20 1ml/L adjust pH to 7.6 with HCl.
The expression contents of 1 iTRAQ Different Proteomics mass spectrum quantitative analysis Olfactomedin-4 albumen of embodiment
1, sample overview
The cancerous tissue of 4 Patients with Non-small-cell Lung and cancer beside organism, 4 patients are made a definite diagnosis by 2 Pathologis, are suffered from
There are non-small cell lung cancer, the pathological data of 4 patients as shown in table 2.
Table 2：The pathological data of 4 Patients with Non-small-cell Lung.
| No. | Gender | Age | Grade | Tumor classification | Tumor size（CM） |
| P1 | Male | 55 | Ⅲ | Non-small cell lung cancer | 4.8 × 5.3 |
| P2 | Male | 59 | Ⅲ | Non-small cell lung cancer | 5.5 × 6.7 |
| P3 | Male | 57 | Ⅲ | Non-small cell lung cancer | 3.8 × 6.2 |
| P4 | Male | 60 | Ⅲ | Non-small cell lung cancer | 8.3 × 6.5 |
2, sample preparation
The cancerous tissue and cancer beside organism's albumen of the non-small cell lung cancer of above-mentioned 4 patients are prepared with enzymolysis sample the preparation method in solution
Quality sample（Cancerous tissue used and cancer beside organism's sample are the paired samples for being derived from same Patients with Non-small-cell Lung）, specific mistake
Journey is as follows：
The flesh tissue block of operation excision is immediately placed on ice, is quickly cut into that several naked eyes are visible, fritter without necrotic zone.With
After the PBS solution of precooling washs tissue fritter for several times, it is quickly ground into cell precipitation in liquid nitrogen, then distinguishes cell precipitation
It is dissolved in lysate, then under condition of ice bath, uses ultrasonic cell disintegration instrument（Soniprep 150, Britain, MSE companies）Between
It has a rest after ultrasonication 2min, in 15000g/min, 4 DEG C of centrifugation 1h, supernatant is taken, with the Bradford methods of improvement（See Bio-Rad public affairs
Take charge of product description）It is quantitative to carry out gross protein.
3, Olfactomedin-4 detections
Using iTRAQ technologies（With reference to 2010 Aug 1 of Pichler P et al. Anal Chem.;82（15）:6549-
58.）Zymolysis technique in binding soln（With reference to William M. Old et al. Mol Cell Proteomics. 2005；
4:1487-1502）With negative and positive multidimensional Liquid Chromatography-Tandem Mass Spectrometry technology（With reference to Jie Dai et al. J ProteomeRes.
2007；6（1）：250-262）The screening of differentially expressed protein is carried out, detailed process is as follows：
The 4 pairs of Non-Small Cell Lung Carcinomas and each 200ug of cancer beside organism's protein example that previous step obtains are taken, with enzyme in solution
Solution technology is digested into peptide fragment, is then replaced as iTRAQ reaction reagents, wherein uses peptide fragment such as NO.3~26 times SEQ ID
Shown in one.It respectively takes 100ug to be mixed respectively with iTRAQ reagents, is stored at room temperature 1h.8uL azanols are added, are stored at room temperature 15min,
Terminate reaction.Merge 8 samples, concussion makes to be sufficiently mixed, and removes the impurity such as the salt in sample.
The peptide fragment mixture that zymolysis technique obtains in solution is analyzed with SCX/SAX columns first, then with fully automated
The multidimensional Liquid Chromatography-Tandem Mass Spectrometry instrument LTQ Q-Exactive of change（Purchased from Thermo Fisher companies）It is analyzed,
Obtained initial data uses Maxquant softwares（German Ma Pu research institutes）Carry out database search, and the albumen to identifying
Matter carries out quantitative analysis.
4, as a result
Quantitative result is as shown in table 3.
Table 3：The quantitative result of Olfactomedin-4 in Non-Small Cell Lung Carcinoma and cancer beside organism.
| No. | Ratio（Tumor tissues/cancer beside organism） |
| P1 | 3.27 |
| P2 | 4.15 |
| P3 | 3.88 |
| P4 | 2.96 |
According to the result of table 3, smell mediates element -4（Olfactomedin-4）Expression in Non-Small Cell Lung Carcinoma
Content is compared with cancer beside organism, and high 3 times of expression or more, is expressed bright in Non-Small Cell Lung Carcinoma in Non-Small Cell Lung Carcinoma
Aobvious up-regulation.
The expression contents of 2 immunoblotting quantitative analysis Olfactomedin-4 albumen of embodiment
1, sample overview
Take 5 pairs of Non-Small Cell Lung Carcinomas and the protein example of corresponding cancer beside organism, pathological data as shown in table 4.
Table 4：The pathological data of 5 non-small cell lung cancer samples.
| No. | Gender | Age | Grade | Tumor classification | Tumor size（CM） |
| P1 | Male | 48 | Ⅲ | Non-small cell lung cancer | 3.7 × 8.3 |
| P2 | Male | 69 | Ⅲ | Non-small cell lung cancer | 5.9 × 8.2 |
| P3 | Male | 53 | Ⅲ | Non-small cell lung cancer | 6.6 × 5.5 |
| P4 | Male | 62 | Ⅲ | Non-small cell lung cancer | 7.2 × 4.7 |
| P5 | Male | 65 | Ⅲ | Non-small cell lung cancer | 6.8 × 5.1 |
2, sample preparation
2 in embodiment 2 simultaneously, sample preparation.
3, Olfactomedin-4 detections
Element -4 is mediated using the anti-smell of purchase（Olfactomedin-4）Antibody to above-mentioned 5 pairs of Non-Small Cell Lung Carcinomas and
The protein example of corresponding cancer beside organism carries out immunoblotting assay, and process is as follows：
Each sample takes 20ug protein examples to be detached with 12% SDS-PAGE, is transferred to pvdf membrane（Purchased from GE Healthcare
Company）On；
Primary antibody mediates element -4 using rabbit-anti people's smell（Olfactomedin-4）Polyclonal antibody（Purchased from Abcam companies, 1:
1000 dilutions）, 4 DEG C are incubated overnight, and are washed three times with TBST, 5 minutes every time；
Secondary antibody is anti-rabbit antibody（Purchased from Santa Cruz companies, I:10000 dilutions）, it is incubated at room temperature I hours, then washed with TBST
It washs three times, 10 minutes every time；
ECL plus reagents are added（Purchased from GE Healthcare companies）It reacts after five minutes, with X- mating plate exposure tests.
Gel-Pro Analyzer quantitative gel analysis softwares are used to immunoblotting collection of illustrative plates（Media Cybernetic are public
Department）Data analysis is carried out, obtains the Relative distribution figure of protein expression amount.
4, as a result
The results are shown in Figure 1.Fig. 1 is the results show that the expression quantity in Non-Small Cell Lung Carcinoma is apparently higher than by corresponding cancer
Tissue tissue, mean ratio 3.11, P values（Paired t-test）It is 0.00019.According to Fig. 1's as a result, smell mediates element -4
（Olfactomedin-4）There is high expression in Non-Small Cell Lung Carcinoma, the result is consistent with mass spectral results.
3 Tissue Microarray in Non small Cell Lung Cancer immunohistochemistry research quantitative analysis Olfactomedin-4 eggs of embodiment
White expression contents
In order to further confirm that smell mediates element -4（Olfactomedin-4）Non-Small Cell Lung Carcinoma and cancer beside organism it
Between differential expression, use Tissue Microarray in Non small Cell Lung Cancer, carry out immunohistochemistry research.
1, sample overview
Randomly select the cancerous tissue of 67 pairs of non-small cell lung cancers and corresponding cancer beside organism's sample, wherein the tool of the sample of selection
Body data is as shown in table 5.
Table 5：Tissue Microarray in Non small Cell Lung Cancer data, 0 represents non-smoking, and 1 represents smoking.
| Point position | Organization type | Coring number | Gender | Age | Diameter | Grade | Life span | Smoking |
| A1 | Cancer | 1 | Man | 53 | 3.5 | 3 | 25.5 | 0 |
| A2 | Cancer | 1 | Man | 66 | 4 | 3 | 62.3 | 0 |
| A3 | Cancer | 1 | Female | 67 | 2.5 | 3 | 60.5 | 0 |
| A4 | Cancer | 1 | Female | 55 | 2 | 3 | 53.7 | 0 |
| A5 | Cancer | 1 | Man | 64 | 2 | 3 | 56.3 | 0 |
| A6 | Cancer | 1 | Female | 56 | 1.8 | 3 | 64.4 | 0 |
| A7 | Cancer | 1 | Female | 53 | 2 | 3 | 51.4 | 0 |
| A8 | Cancer | 1 | Female | 62 | 7.5 | 3 | 28.4 | 0 |
| A9 | Cancer | 1 | Man | 62 | 4 | 3 | 48.5 | 0 |
| A10 | Cancer | 1 | Man | 67 | 3 | 3 | 47.3 | 0 |
| A11 | Cancer | 1 | Man | 53 | 3.5 | 3 | 25.5 | 0 |
| A12 | Cancer | 1 | Man | 77 | 7 | 3 | 26.3 | 0 |
| A13 | Cancer | 1 | Man | 54 | 1.5 | 3 | 17.9 | 0 |
| A14 | Cancer | 1 | Female | 41 | 3 | 3 | 55.7 | 0 |
| A15 | Cancer | 1 | Man | 55 | 2.5 | 3 | 55.9 | 1 |
| A16 | Cancer | 1 | Man | 59 | 4 | 3 | 31.6 | 0 |
| A17 | Cancer | 1 | Female | 63 | 3 | 3 | 15.3 | 0 |
| A18 | Cancer | 1 | Man | 67 | 2.5 | 3 | 30.4 | 0 |
| A19 | Cancer | 1 | Female | 67 | 3.5 | 3 | 48.3 | 0 |
| A20 | Cancer | 1 | Man | 60 | 1.5 | 3 | 41.2 | 1 |
| A21 | Cancer | 1 | Man | 64 | 3 | 3 | 64.8 | 0 |
| A22 | Cancer | 1 | Female | 43 | 4 | 3 | 62.2 | 0 |
| A23 | Cancer | 1 | Man | 58 | 3.5 | 3 | 60.4 | 1 |
| A24 | Cancer | 1 | Man | 69 | 2.5 | 3 | 55.8 | 0 |
| A25 | Cancer | 1 | Man | 76 | 3 | 3 | 55.8 | 0 |
| A26 | Cancer | 1 | Female | 72 | 3 | 3 | 53 | 0 |
| A27 | Cancer | 1 | Man | 67 | 3.5 | 3 | 15.6 | 1 |
| A28 | Cancer | 1 | Man | 69 | 4.5 | 3 | 27.9 | 1 |
| A29 | Cancer | 1 | Female | 62 | 2 | 3 | 48.3 | 0 |
| A30 | Cancer | 1 | Female | 44 | 2 | 3 | 47.1 | 0 |
| A31 | Cancer | 1 | Man | 64 | 3 | 3 | 64.8 | 0 |
| A32 | Cancer | 1 | Female | 56 | 3 | 3 | 62 | 0 |
| A33 | Cancer | 1 | Female | 56 | 1.8 | 3 | 58.9 | 0 |
| A34 | Cancer | 1 | Man | 73 | 3 | 3 | 57.5 | 0 |
| A35 | Cancer | 1 | Man | 64 | 3 | 3 | 46.8 | 0 |
| A36 | Cancer | 1 | Female | 54 | 3 | 3 | 29 | 0 |
| A37 | Cancer | 1 | Man | 64 | 0.7 | 3 | 36.3 | 0 |
| A38 | Cancer | 1 | Female | 44 | 4 | 3 | 20.1 | 1 |
| A39 | Cancer | 1 | Female | 71 | 3 | 3 | 48.3 | 0 |
| A40 | Cancer | 1 | Man | 49 | 1 | 3 | 46.9 | 0 |
| A41 | Cancer | 1 | Man | 62 | 4 | 3 | 45.1 | 0 |
| A42 | Cancer | 1 | Man | 69 | 4.5 | 3 | 27.3 | 1 |
| A43 | Cancer | 1 | Female | 57 | 1.5 | 3 | 58.8 | 0 |
| A44 | Cancer | 1 | Female | 70 | 3.5 | 3 | 22.3 | 0 |
| A45 | Cancer | 1 | Female | 58 | 7 | 3 | 55.4 | 0 |
| A46 | Cancer | 1 | Man | 56 | 1 | 3 | 24.8 | 0 |
| A47 | Cancer | 1 | Female | 50 | 3 | 3 | 50.4 | 0 |
| A48 | Cancer | 1 | Man | 57 | 4 | 3 | 9.8 | 0 |
| A49 | Cancer | 1 | Man | 75 | 6 | 3 | 48.1 | 0 |
| A50 | Cancer | 1 | Female | 73 | 6.5 | 3 | 42.8 | 0 |
| A51 | Cancer | 1 | Female | 60 | 3 | 3 | 30.8 | 0 |
| A52 | Cancer | 1 | Man | 60 | 5 | 3 | 61.1 | 0 |
| A53 | Cancer | 1 | Female | 55 | 2.5 | 3 | 57.1 | 0 |
| A54 | Cancer | 1 | Man | 52 | 3 | 3 | 27.7 | 0 |
| A55 | Cancer | 1 | Female | 39 | 4 | 3 | 31.7 | 0 |
| A56 | Cancer | 1 | Female | 67 | 2.5 | 3 | 49.7 | 0 |
| A57 | Cancer | 1 | Female | 60 | 2.5 | 3 | 48.8 | 0 |
| A58 | Cancer | 1 | Female | 73 | 2 | 3 | 47.8 | 0 |
| A59 | Cancer | 1 | Female | 75 | 4.5 | 3 | 18.8 | 0 |
| A60 | Cancer | 1 | Female | 69 | 1 | 3 | 62.3 | 0 |
| A61 | Cancer | 1 | Female | 58 | 1.5 | 3 | 61 | 0 |
| A62 | Cancer | 1 | Man | 63 | 4 | 3 | 57.9 | 0 |
| A63 | Cancer | 1 | Female | 49 | 2 | 3 | 30.8 | 0 |
| A64 | Cancer | 1 | Man | 69 | 2.8 | 3 | 54.6 | 0 |
| A65 | Cancer | 1 | Female | 56 | 2.5 | 3 | 52.6 | 0 |
| A66 | Cancer | 1 | Man | 69 | 3.5 | 3 | 49.6 | 0 |
| A67 | Cancer | 1 | Female | 39 | 4 | 3 | 48.5 | 0 |
| B1 | By cancer | 1 | Man | 53 | 3.5 | 3 | 25.5 | 0 |
| B2 | By cancer | 1 | Man | 66 | 4 | 3 | 62.3 | 0 |
| B3 | By cancer | 1 | Female | 67 | 2.5 | 3 | 60.5 | 0 |
| B4 | By cancer | 1 | Female | 55 | 2 | 3 | 53.7 | 0 |
| B5 | By cancer | 1 | Man | 64 | 2 | 3 | 56.3 | 0 |
| B6 | By cancer | 1 | Female | 56 | 1.8 | 3 | 64.4 | 0 |
| B7 | By cancer | 1 | Female | 53 | 2 | 3 | 51.4 | 0 |
| B8 | By cancer | 1 | Female | 62 | 7.5 | 3 | 28.4 | 0 |
| B9 | By cancer | 1 | Man | 62 | 4 | 3 | 48.5 | 0 |
| B10 | By cancer | 1 | Man | 67 | 3 | 3 | 47.3 | 0 |
| B11 | By cancer | 1 | Man | 53 | 3.5 | 3 | 25.5 | 0 |
| B12 | By cancer | 1 | Man | 77 | 7 | 3 | 26.3 | 0 |
| B13 | By cancer | 1 | Man | 54 | 1.5 | 3 | 17.9 | 0 |
| B14 | By cancer | 1 | Female | 41 | 3 | 3 | 55.7 | 0 |
| B15 | By cancer | 1 | Man | 55 | 2.5 | 3 | 55.9 | 1 |
| B16 | By cancer | 1 | Man | 59 | 4 | 3 | 31.6 | 0 |
| B17 | By cancer | 1 | Female | 63 | 3 | 3 | 15.3 | 0 |
| B18 | By cancer | 1 | Man | 67 | 2.5 | 3 | 30.4 | 0 |
| B19 | By cancer | 1 | Female | 67 | 3.5 | 3 | 48.3 | 0 |
| B20 | By cancer | 1 | Man | 60 | 1.5 | 3 | 41.2 | 1 |
| B21 | By cancer | 1 | Man | 64 | 3 | 3 | 64.8 | 0 |
| B22 | By cancer | 1 | Female | 43 | 4 | 3 | 62.2 | 0 |
| B23 | By cancer | 1 | Man | 58 | 3.5 | 3 | 60.4 | 1 |
| B24 | By cancer | 1 | Man | 69 | 2.5 | 3 | 55.8 | 0 |
| B25 | By cancer | 1 | Man | 76 | 3 | 3 | 55.8 | 0 |
| B26 | By cancer | 1 | Female | 72 | 3 | 3 | 53 | 0 |
| B27 | By cancer | 1 | Man | 67 | 3.5 | 3 | 15.6 | 1 |
| B28 | By cancer | 1 | Man | 69 | 4.5 | 3 | 27.9 | 1 |
| B29 | By cancer | 1 | Female | 62 | 2 | 3 | 48.3 | 0 |
| B30 | By cancer | 1 | Female | 44 | 2 | 3 | 47.1 | 0 |
| B31 | By cancer | 1 | Man | 64 | 3 | 3 | 64.8 | 0 |
| B32 | By cancer | 1 | Female | 56 | 3 | 3 | 62 | 0 |
| B33 | By cancer | 1 | Female | 56 | 1.8 | 3 | 58.9 | 0 |
| B34 | By cancer | 1 | Man | 73 | 3 | 3 | 57.5 | 0 |
| B35 | By cancer | 1 | Man | 64 | 3 | 3 | 46.8 | 0 |
| B36 | By cancer | 1 | Female | 54 | 3 | 3 | 29 | 0 |
| B37 | By cancer | 1 | Man | 64 | 0.7 | 3 | 36.3 | 0 |
| B38 | By cancer | 1 | Female | 44 | 4 | 3 | 20.1 | 1 |
| B39 | By cancer | 1 | Female | 71 | 3 | 3 | 48.3 | 0 |
| B40 | By cancer | 1 | Man | 49 | 1 | 3 | 46.9 | 0 |
| B41 | By cancer | 1 | Man | 62 | 4 | 3 | 45.1 | 0 |
| B42 | By cancer | 1 | Man | 69 | 4.5 | 3 | 27.3 | 1 |
| B43 | By cancer | 1 | Female | 57 | 1.5 | 3 | 58.8 | 0 |
| B44 | By cancer | 1 | Female | 70 | 3.5 | 3 | 22.3 | 0 |
| B45 | By cancer | 1 | Female | 58 | 7 | 3 | 55.4 | 0 |
| B46 | By cancer | 1 | Man | 56 | 1 | 3 | 24.8 | 0 |
| B47 | By cancer | 1 | Female | 50 | 3 | 3 | 50.4 | 0 |
| B48 | By cancer | 1 | Man | 57 | 4 | 3 | 9.8 | 0 |
| B49 | By cancer | 1 | Man | 75 | 6 | 3 | 48.1 | 0 |
| B50 | By cancer | 1 | Female | 73 | 6.5 | 3 | 42.8 | 0 |
| B51 | By cancer | 1 | Female | 60 | 3 | 3 | 30.8 | 0 |
| B52 | By cancer | 1 | Man | 60 | 5 | 3 | 61.1 | 0 |
| B53 | By cancer | 1 | Female | 55 | 2.5 | 3 | 57.1 | 0 |
| B54 | By cancer | 1 | Man | 52 | 3 | 3 | 27.7 | 0 |
| B55 | By cancer | 1 | Female | 39 | 4 | 3 | 31.7 | 0 |
| B56 | By cancer | 1 | Female | 67 | 2.5 | 3 | 49.7 | 0 |
| B57 | By cancer | 1 | Female | 60 | 2.5 | 3 | 48.8 | 0 |
| B58 | By cancer | 1 | Female | 73 | 2 | 3 | 47.8 | 0 |
| B59 | By cancer | 1 | Female | 75 | 4.5 | 3 | 18.8 | 0 |
| B60 | By cancer | 1 | Female | 69 | 1 | 3 | 62.3 | 0 |
| B61 | By cancer | 1 | Female | 58 | 1.5 | 3 | 61 | 0 |
| B62 | By cancer | 1 | Man | 63 | 4 | 3 | 57.9 | 0 |
| B63 | By cancer | 1 | Female | 49 | 2 | 3 | 30.8 | 0 |
| B64 | By cancer | 1 | Man | 69 | 2.8 | 3 | 54.6 | 0 |
| B65 | By cancer | 1 | Female | 56 | 2.5 | 3 | 52.6 | 0 |
| B66 | By cancer | 1 | Man | 69 | 3.5 | 3 | 49.6 | 0 |
| B67 | By cancer | 1 | Female | 39 | 4 | 3 | 48.5 | 0 |
2, sample preparation and Olfactomedin-4 are detected
（1）Histotomy is placed in 60 DEG C of insulating boxs and toasts about 3 hours, uses dimethylbenzene, dimethylbenzene/ethyl alcohol after taking-up successively
（1：1）, 100% ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol, 70% ethyl alcohol, 50% second alcohol and water, carry out dewaxing hydration process；
（2）PBS is washed 3 times, every time 5 minutes；0.3% H2O2 （Methanol dilution）Half an hour is impregnated, PBS is washed 3 times, every time 5 minutes；
（3）Pressure method antigen, distilled water are washed 2 times, every time 5 minutes；PBS is washed 2 times, every time 5 minutes；10% serum room temperature is closed
20 minutes；
（4）Rabbit-anti people's smell is added and mediates element -4（Olfactomedin-4）Polyclonal antibody（Purchased from Abcam companies, 1：50 is dilute
It releases）, 4 DEG C overnight, and PBST is washed 3 times, every time 5 minutes；
（5）The goat anti-rabbit antibody of biotin labeling is added（From ABC kits, it is purchased from VECTOR companies）, it is incubated at room temperature 30 points
Clock, PBST are washed 3 times, every time 5 minutes；PBS is washed 2 times, every time 5 minutes；
（6）ABC solution is added（From ABC kits, it is purchased from VECTOR companies）, it is incubated at room temperature 30 minutes, PBS washes 3 times, every time
5 minutes；
（7）DAB solution（Purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd）Colour developing；Hematoxylin（Purchased from VECTOR public affairs
Department, H3404）Dyeing 20 seconds；
（8）Histotomy uses successively：50 % ethyl alcohol, 70 % ethyl alcohol, 80 % ethyl alcohol, 90 % ethyl alcohol, 100 % ethyl alcohol, dimethylbenzene/
Ethyl alcohol（1：1）, dimethylbenzene, carry out dehydration transparent processing.
（9）Then organization chip is assessed in micro- sem observation using resinene mounting.
3, result
As a result it shows：Totally 67 pairs of effective samples in organization chip, and given a mark according to staining power and positive rate, wherein dyeing is strong
Spending scoring criterion is：Feminine gender is 0 point, weakly positive is 1 point, moderate positive is 2 points, strong positive is 3 points；Positive rate scoring criterion
For：It is 0 point less than 5%, 5% ~ 30% is 1 point, and 31% ~ 60% is 2 points, and 60% or more is 3 points.By the score and positive rate of staining power
Score be added, obtain the comprehensive score of histotomy, scoring event is as shown in table 6.
Table 6：Tissue Microarray in Non small Cell Lung Cancer comprehensive score, 0 represents non-smoking, and 1 represents smoking.
| Number | Gender | Age | Diameter | Grade | Life span（Month） | Smoking | By marking-cancer | Marking-cancer |
| 1 | Man | 53 | 3.5 | 3 | 25.5 | 0 | 1 | 3 |
| 2 | Man | 66 | 4 | 3 | 62.3 | 0 | 5 | 4 |
| 3 | Female | 67 | 2.5 | 3 | 60.5 | 0 | 1 | 6 |
| 4 | Female | 55 | 2 | 3 | 53.7 | 0 | 0 | 3 |
| 5 | Man | 64 | 2 | 3 | 56.3 | 0 | 2 | 1 |
| 6 | Female | 56 | 1.8 | 3 | 64.4 | 0 | 2 | 6 |
| 7 | Female | 53 | 2 | 3 | 51.4 | 0 | 1 | 5 |
| 8 | Female | 62 | 7.5 | 3 | 28.4 | 0 | 0 | 6 |
| 9 | Man | 62 | 4 | 3 | 48.5 | 0 | 1 | 2 |
| 10 | Man | 67 | 3 | 3 | 47.3 | 0 | 1 | 3 |
| 11 | Man | 53 | 3.5 | 3 | 25.5 | 0 | 2 | 3 |
| 12 | Man | 77 | 7 | 3 | 26.3 | 0 | 4 | 5 |
| 13 | Man | 54 | 1.5 | 3 | 17.9 | 0 | 0 | 3 |
| 14 | Female | 41 | 3 | 3 | 55.7 | 0 | 3 | 2 |
| 15 | Man | 55 | 2.5 | 3 | 55.9 | 1 | 0 | 2 |
| 16 | Man | 59 | 4 | 3 | 31.6 | 0 | 3 | 4 |
| 17 | Female | 63 | 3 | 3 | 15.3 | 0 | 0 | 5 |
| 18 | Man | 67 | 2.5 | 3 | 30.4 | 0 | 0 | 6 |
| 19 | Female | 67 | 3.5 | 3 | 48.3 | 0 | 5 | 1 |
| 20 | Man | 60 | 1.5 | 3 | 41.2 | 1 | 1 | 2 |
| 21 | Man | 64 | 3 | 3 | 64.8 | 0 | 2 | 3 |
| 22 | Female | 43 | 4 | 3 | 62.2 | 0 | 2 | 3 |
| 23 | Man | 58 | 3.5 | 3 | 60.4 | 1 | 2 | 5 |
| 24 | Man | 69 | 2.5 | 3 | 55.8 | 0 | 2 | 6 |
| 25 | Man | 76 | 3 | 3 | 55.8 | 0 | 3 | 4 |
| 26 | Female | 72 | 3 | 3 | 53 | 0 | 3 | 5 |
| 27 | Man | 67 | 3.5 | 3 | 15.6 | 1 | 0 | 2 |
| 28 | Man | 69 | 4.5 | 3 | 27.9 | 1 | 0 | 1 |
| 29 | Female | 62 | 2 | 3 | 48.3 | 0 | 0 | 0 |
| 30 | Female | 44 | 2 | 3 | 47.1 | 0 | 1 | 3 |
| 31 | Man | 64 | 3 | 3 | 64.8 | 0 | 1 | 3 |
| 32 | Female | 56 | 3 | 3 | 62 | 0 | 3 | 4 |
| 33 | Female | 56 | 1.8 | 3 | 58.9 | 0 | 0 | 2 |
| 34 | Man | 73 | 3 | 3 | 57.5 | 0 | 0 | 3 |
| 35 | Man | 64 | 3 | 3 | 46.8 | 0 | 2 | 4 |
| 36 | Female | 54 | 3 | 3 | 29 | 0 | 2 | 5 |
| 37 | Man | 64 | 0.7 | 3 | 36.3 | 0 | 3 | 5 |
| 38 | Female | 44 | 4 | 3 | 20.1 | 1 | 3 | 4 |
| 39 | Female | 71 | 3 | 3 | 48.3 | 0 | 0 | 3 |
| 40 | Man | 49 | 1 | 3 | 46.9 | 0 | 3 | 1 |
| 41 | Man | 62 | 4 | 3 | 45.1 | 0 | 1 | 0 |
| 42 | Man | 69 | 4.5 | 3 | 27.3 | 1 | 1 | 2 |
| 43 | Female | 57 | 1.5 | 3 | 58.8 | 0 | 5 | 5 |
| 44 | Female | 70 | 3.5 | 3 | 22.3 | 0 | 2 | 4 |
| 45 | Female | 58 | 7 | 3 | 55.4 | 0 | 1 | 3 |
| 46 | Man | 56 | 1 | 3 | 24.8 | 0 | 1 | 2 |
| 47 | Female | 50 | 3 | 3 | 50.4 | 0 | 2 | 4 |
| 48 | Man | 57 | 4 | 3 | 9.8 | 0 | 0 | 2 |
| 49 | Man | 75 | 6 | 3 | 48.1 | 0 | 0 | 3 |
| 50 | Female | 73 | 6.5 | 3 | 42.8 | 0 | 3 | 0 |
| 51 | Female | 60 | 3 | 3 | 30.8 | 0 | 0 | 0 |
| 52 | Man | 60 | 5 | 3 | 61.1 | 0 | 3 | 5 |
| 53 | Female | 55 | 2.5 | 3 | 57.1 | 0 | 1 | 6 |
| 54 | Man | 52 | 3 | 3 | 27.7 | 0 | 0 | 6 |
| 55 | Female | 39 | 4 | 3 | 31.7 | 0 | 2 | 4 |
| 56 | Female | 67 | 2.5 | 3 | 49.7 | 0 | 0 | 3 |
| 57 | Female | 60 | 2.5 | 3 | 48.8 | 0 | 2 | 3 |
| 58 | Female | 73 | 2 | 3 | 47.8 | 0 | 1 | 6 |
| 59 | Female | 75 | 4.5 | 3 | 18.8 | 0 | 1 | 3 |
| 60 | Female | 69 | 1 | 3 | 62.3 | 0 | 0 | 6 |
| 61 | Female | 58 | 1.5 | 3 | 61 | 0 | 2 | 2 |
| 62 | Man | 63 | 4 | 3 | 57.9 | 0 | 1 | 3 |
| 63 | Female | 49 | 2 | 3 | 30.8 | 0 | 1 | 6 |
| 64 | Man | 69 | 2.8 | 3 | 54.6 | 0 | 2 | 6 |
| 65 | Female | 56 | 2.5 | 3 | 52.6 | 0 | 3 | 4 |
| 66 | Man | 69 | 3.5 | 3 | 49.6 | 0 | 1 | 2 |
| 67 | Female | 39 | 4 | 3 | 48.5 | 0 | 1 | 6 |
Fig. 2 is calculated according to the result of table 6:The average aggregate of non-small cell lung cancer cell cancerous tissue is scored at 3.49,
The average aggregate of cancer beside organism is scored at 1.51, and it is 6.80E-12 that the two, which has pole significant difference, P values,.Statistics find, more than
86% （58 pairs）Sample centering, smell mediate element -4（Olfactomedin-4）The high table in non-small cell lung cancer cell cancer
It reaches.The result is consistent with the mass spectral results of front, immunoblot results.
The expression contents of 4 Enzyme-linked Immunosorbent Assay quantitative analysis Olfactomedin-4 albumen of embodiment
In order to be further discovered that smell mediates element -4（Olfactomedin-4）In serum of patients with non-small cell lung and normal human serum
Between differential expression, carry out enzyme linked immunosorbent assay (ELISA).
1, sample overview
Randomly select 30 Patients with Non-small-cell Lung serum and 30 normal human serum samples wherein, the tool of the sample of selection
Body data is respectively as shown in table 7 and table 8.
Table 7：Serum of patients with non-small cell lung sample information, 0 represents non-smoking, and 1 represents smoking.
| Number | Gender | Age | Grade | Smoking |
| 1 | Female | 56 | 3 | 0 |
| 2 | Man | 58 | 3 | 1 |
| 3 | Man | 47 | 3 | 0 |
| 4 | Man | 75 | 3 | 1 |
| 5 | Female | 59 | 3 | 1 |
| 6 | Man | 65 | 3 | 1 |
| 7 | Female | 58 | 3 | 1 |
| 8 | Female | 67 | 3 | 0 |
| 9 | Female | 74 | 3 | 0 |
| 10 | Female | 55 | 3 | 1 |
| 11 | Man | 69 | 3 | 1 |
| 12 | Female | 65 | 3 | 1 |
| 13 | Man | 61 | 3 | 0 |
| 14 | Man | 51 | 3 | 1 |
| 15 | Female | 58 | 3 | 0 |
| 16 | Man | 70 | 3 | 0 |
| 17 | Female | 68 | 3 | 1 |
| 18 | Man | 62 | 3 | 1 |
| 19 | Female | 53 | 3 | 1 |
| 20 | Man | 54 | 3 | 1 |
| 21 | Man | 60 | 3 | 1 |
| 22 | Female | 68 | 3 | 0 |
| 23 | Man | 62 | 3 | 1 |
| 24 | Man | 64 | 3 | 1 |
| 25 | Man | 57 | 3 | 0 |
| 26 | Female | 56 | 3 | 0 |
| 27 | Man | 71 | 3 | 1 |
| 28 | Man | 53 | 3 | 1 |
| 29 | Man | 67 | 3 | 1 |
| 30 | Female | 49 | 3 | 1 |
Table 8：Normal serum sample information.
| Number | Gender | Age |
| 1 | Man | 70 |
| 2 | Female | 62 |
| 3 | Female | 59 |
| 4 | Female | 63 |
| 5 | Female | 66 |
| 6 | Man | 67 |
| 7 | Man | 63 |
| 8 | Man | 54 |
| 9 | Man | 57 |
| 10 | Female | 55 |
| 11 | Female | 64 |
| 12 | Female | 50 |
| 13 | Man | 63 |
| 14 | Female | 66 |
| 15 | Female | 65 |
| 16 | Man | 64 |
| 17 | Man | 54 |
| 18 | Man | 53 |
| 19 | Man | 59 |
| 20 | Man | 61 |
| 21 | Female | 71 |
| 22 | Female | 49 |
| 23 | Female | 66 |
| 24 | Man | 55 |
| 25 | Man | 55 |
| 26 | Female | 61 |
| 27 | Man | 70 |
| 28 | Female | 44 |
| 29 | Man | 52 |
| 30 | Female | 68 |
2, sample preparation and Olfactomedin-4 are detected
Serum passes through the incubation of a hour with antibody, then board-washing, and after adding substrate, half an hour to be protected from light plus terminate liquid is complete
At reaction.
3, as a result
Fig. 3 is obtained according to the result of enzyme linked immunosorbent assay (ELISA)：Statistics finds that smell mediates element -4（Olfactomedin-4）
The high expression in the patients serum of non-small cell lung cancer.The mass spectral results of the result and front, immunoblot results and immune
Groupization result is consistent.
In conclusion smell mediates element -4（Olfactomedin-4）The group by the cancerous tissue of non-small cell lung cancer and cancer
There are apparent differential expressions in knitting, and express and increase in the serum of Patients with Non-small-cell Lung, it is clear that with non-small cell
The occurrence and development of lung cancer have close correlation, therefore its expression quantity can be used for detecting non-small cell lung cancer.Correspondingly,
Specific smell mediates element -4（Olfactomedin-4）Antibody, including various smell mediate element -4（Olfactomedin-
4）Monoclonal antibody and polyclonal antibody, due to it can be used to detect smell mediate element -4（Olfactomedin-4）'s
Expression quantity, thus can be used for detecting non-small cell lung cancer, or it is used to prepare the preparation or reagent of detection non-small cell lung cancer
Box.
Although related smell mediates element -4（Olfactomedin-4）Dynamic biological function and tumour related mechanism are also
Need further to be studied, but is affirmative using it as the marker of detection non-small cell lung cancer.
Sequence table
<110>Dongguan University of Technology
<120>Smell mediates application of the element -4 as non-small cell lung carcinoma marker
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1533
<212> DNA
<213>People (Homo sapiens)
<400> 1
atgaggcccg gcctctcatt tctcctagcc cttctgttct tccttggcca agctgcaggg 60
gatttggggg atgtgggacc tccaattccc agccccggct tcagctcttt cccaggtgtt 120
gactccagct ccagcttcag ctccagctcc aggtcgggct ccagctccag ccgcagctta 180
ggcagcggag gttctgtgtc ccagttgttt tccaatttca ccggctccgt ggatgaccgt 240
gggacctgcc agtgctctgt ttccctgcca gacaccacct ttcccgtgga cagagtggaa 300
cgcttggaat tcacagctca tgttctttct cagaagtttg agaaagaact ttccaaagtg 360
agggaatatg tccaattaat tagtgtgtat gaaaagaaac tgttaaacct aactgtccga 420
attgacatca tggagaagga taccatttct tacactgaac tggacttcga gctgatcaag 480
gtagaagtga aggagatgga aaaactggtc atacagctga aggagagttt tggtggaagc 540
tcagaaattg ttgaccagct ggaggtggag ataagaaata tgactctctt ggtagagaag 600
cttgagacac tagacaaaaa caatgtcctt gccattcgcc gagaaatcgt ggctctgaag 660
accaagctga aagagtgtga ggcctctaaa gatcaaaaca cccctgtcgt ccaccctcct 720
cccactccag ggagctgtgg tcatggtggt gtggtgaaca tcagcaaacc gtctgtggtt 780
cagctcaact ggagagggtt ttcttatcta tatggtgctt ggggtaggga ttactctccc 840
cagcatccaa acaaaggact gtattgggtg gcgccattga atacagatgg gagactgttg 900
gagtattata gactgtacaa cacactggat gatttgctat tgtatataaa tgctcgagag 960
ttgcggatca cctatggcca aggtagtggt acagcagttt acaacaacaa catgtacgtc 1020
aacatgtaca acaccgggaa tattgccaga gttaacctga ccaccaacac gattgctgtg 1080
actcaaactc tccctaatgc tgcctataat aaccgctttt catatgctaa tgttgcttgg 1140
caagatattg actttgctgt ggatgagaat ggattgtggg ttatttattc aactgaagcc 1200
agcactggta acatggtgat tagtaaactc aatgacacca cacttcaggt gctaaacact 1260
tggtatacca agcagtataa accatctgct tctaacgcct tcatggtatg tggggttctg 1320
tatgccaccc gtactatgaa caccagaaca gaagagattt tttactatta tgacacaaac 1380
acagggaaag agggcaaact agacattgta atgcataaga tgcaggaaaa agtgcagagc 1440
attaactata acccttttga ccagaaactt tatgtctata acgatggtta ccttctgaat 1500
tatgatcttt ctgtcttgca gaagccccag taa 1533
<210> 2
<211> 510
<212> PRT
<213>People (Homo sapiens)
<400> 2
Met Arg Pro Gly Leu Ser Phe Leu Leu Ala Leu Leu Phe Phe Leu Gly
1 5 10 15
Gln Ala Ala Gly Asp Leu Gly Asp Val Gly Pro Pro Ile Pro Ser Pro
20 25 30
Gly Phe Ser Ser Phe Pro Gly Val Asp Ser Ser Ser Ser Phe Ser Ser
35 40 45
Ser Ser Arg Ser Gly Ser Ser Ser Ser Arg Ser Leu Gly Ser Gly Gly
50 55 60
Ser Val Ser Gln Leu Phe Ser Asn Phe Thr Gly Ser Val Asp Asp Arg
65 70 75 80
Gly Thr Cys Gln Cys Ser Val Ser Leu Pro Asp Thr Thr Phe Pro Val
85 90 95
Asp Arg Val Glu Arg Leu Glu Phe Thr Ala His Val Leu Ser Gln Lys
100 105 110
Phe Glu Lys Glu Leu Ser Lys Val Arg Glu Tyr Val Gln Leu Ile Ser
115 120 125
Val Tyr Glu Lys Lys Leu Leu Asn Leu Thr Val Arg Ile Asp Ile Met
130 135 140
Glu Lys Asp Thr Ile Ser Tyr Thr Glu Leu Asp Phe Glu Leu Ile Lys
145 150 155 160
Val Glu Val Lys Glu Met Glu Lys Leu Val Ile Gln Leu Lys Glu Ser
165 170 175
Phe Gly Gly Ser Ser Glu Ile Val Asp Gln Leu Glu Val Glu Ile Arg
180 185 190
Asn Met Thr Leu Leu Val Glu Lys Leu Glu Thr Leu Asp Lys Asn Asn
195 200 205
Val Leu Ala Ile Arg Arg Glu Ile Val Ala Leu Lys Thr Lys Leu Lys
210 215 220
Glu Cys Glu Ala Ser Lys Asp Gln Asn Thr Pro Val Val His Pro Pro
225 230 235 240
Pro Thr Pro Gly Ser Cys Gly His Gly Gly Val Val Asn Ile Ser Lys
245 250 255
Pro Ser Val Val Gln Leu Asn Trp Arg Gly Phe Ser Tyr Leu Tyr Gly
260 265 270
Ala Trp Gly Arg Asp Tyr Ser Pro Gln His Pro Asn Lys Gly Leu Tyr
275 280 285
Trp Val Ala Pro Leu Asn Thr Asp Gly Arg Leu Leu Glu Tyr Tyr Arg
290 295 300
Leu Tyr Asn Thr Leu Asp Asp Leu Leu Leu Tyr Ile Asn Ala Arg Glu
305 310 315 320
Leu Arg Ile Thr Tyr Gly Gln Gly Ser Gly Thr Ala Val Tyr Asn Asn
325 330 335
Asn Met Tyr Val Asn Met Tyr Asn Thr Gly Asn Ile Ala Arg Val Asn
340 345 350
Leu Thr Thr Asn Thr Ile Ala Val Thr Gln Thr Leu Pro Asn Ala Ala
355 360 365
Tyr Asn Asn Arg Phe Ser Tyr Ala Asn Val Ala Trp Gln Asp Ile Asp
370 375 380
Phe Ala Val Asp Glu Asn Gly Leu Trp Val Ile Tyr Ser Thr Glu Ala
385 390 395 400
Ser Thr Gly Asn Met Val Ile Ser Lys Leu Asn Asp Thr Thr Leu Gln
405 410 415
Val Leu Asn Thr Trp Tyr Thr Lys Gln Tyr Lys Pro Ser Ala Ser Asn
420 425 430
Ala Phe Met Val Cys Gly Val Leu Tyr Ala Thr Arg Thr Met Asn Thr
435 440 445
Arg Thr Glu Glu Ile Phe Tyr Tyr Tyr Asp Thr Asn Thr Gly Lys Glu
450 455 460
Gly Lys Leu Asp Ile Val Met His Lys Met Gln Glu Lys Val Gln Ser
465 470 475 480
Ile Asn Tyr Asn Pro Phe Asp Gln Lys Leu Tyr Val Tyr Asn Asp Gly
485 490 495
Tyr Leu Leu Asn Tyr Asp Leu Ser Val Leu Gln Lys Pro Gln
500 505 510
<210> 3
<211> 7
<212> PRT
<213>People (Homo sapiens)
<400> 3
Ser Gly Ser Ser Ser Ser Arg
1 5
<210> 4
<211> 22
<212> PRT
<213>People (Homo sapiens)
<400> 4
Ser Leu Gly Ser Gly Gly Ser Val Ser Gln Leu Phe Ser Asn Phe Thr
1 5 10 15
Gly Ser Val Asp Asp Arg
20
<210> 5
<211> 18
<212> PRT
<213>People (Homo sapiens)
<400> 5
Gly Thr Cys Gln Cys Ser Val Ser Leu Pro Asp Thr Thr Phe Pro Val
1 5 10 15
Asp Arg
<210> 6
<211> 11
<212> PRT
<213>People (Homo sapiens)
<400> 6
Leu Glu Phe Thr Ala His Val Leu Ser Gln Lys
1 5 10
<210> 7
<211> 7
<212> PRT
<213>People (Homo sapiens)
<400> 7
Phe Glu Lys Glu Leu Ser Lys
1 5
<210> 8
<211> 7
<212> PRT
<213>People (Homo sapiens)
<400> 8
Leu Leu Asn Leu Thr Val Arg
1 5
<210> 9
<211> 14
<212> PRT
<213>People (Homo sapiens)
<400> 9
Asp Thr Ile Ser Tyr Thr Glu Leu Asp Phe Glu Leu Ile Lys
1 5 10
<210> 10
<211> 18
<212> PRT
<213>People (Homo sapiens)
<400> 10
Glu Ser Phe Gly Gly Ser Ser Glu Ile Val Asp Gln Leu Glu Val Glu
1 5 10 15
Ile Arg
<210> 11
<211> 7
<212> PRT
<213>People (Homo sapiens)
<400> 11
Arg Glu Ile Val Ala Leu Lys
1 5
<210> 12
<211> 35
<212> PRT
<213>People (Homo sapiens)
<400> 12
Asp Gln Asn Thr Pro Val Val His Pro Pro Pro Thr Pro Gly Ser Cys
1 5 10 15
Gly His Gly Gly Val Val Asn Ile Ser Lys Pro Ser Val Val Gln Leu
20 25 30
Asn Trp Arg
35
<210> 13
<211> 11
<212> PRT
<213>People (Homo sapiens)
<400> 13
Gly Phe Ser Tyr Leu Tyr Gly Ala Trp Gly Arg
1 5 10
<210> 14
<211> 9
<212> PRT
<213>People (Homo sapiens)
<400> 14
Asp Tyr Ser Pro Gln His Pro Asn Lys
1 5
<210> 15
<211> 13
<212> PRT
<213>People (Homo sapiens)
<400> 15
Gly Leu Tyr Trp Val Ala Pro Leu Asn Thr Asp Gly Arg
1 5 10
<210> 16
<211> 6
<212> PRT
<213>People (Homo sapiens)
<400> 16
Leu Leu Glu Tyr Tyr Arg
1 5
<210> 17
<211> 15
<212> PRT
<213>People (Homo sapiens)
<400> 17
Leu Tyr Asn Thr Leu Asp Asp Leu Leu Leu Tyr Ile Asn Ala Arg
1 5 10 15
<210> 18
<211> 28
<212> PRT
<213>People (Homo sapiens)
<400> 18
Ile Thr Tyr Gly Gln Gly Ser Gly Thr Ala Val Tyr Asn Asn Asn Met
1 5 10 15
Tyr Val Asn Met Tyr Asn Thr Gly Asn Ile Ala Arg
20 25
<210> 19
<211> 22
<212> PRT
<213>People (Homo sapiens)
<400> 19
Val Asn Leu Thr Thr Asn Thr Ile Ala Val Thr Gln Thr Leu Pro Asn
1 5 10 15
Ala Ala Tyr Asn Asn Arg
20
<210> 20
<211> 37
<212> PRT
<213>People (Homo sapiens)
<400> 20
Phe Ser Tyr Ala Asn Val Ala Trp Gln Asp Ile Asp Phe Ala Val Asp
1 5 10 15
Glu Asn Gly Leu Trp Val Ile Tyr Ser Thr Glu Ala Ser Thr Gly Asn
20 25 30
Met Val Ile Ser Lys
35
<210> 21
<211> 15
<212> PRT
<213>People (Homo sapiens)
<400> 21
Leu Asn Asp Thr Thr Leu Gln Val Leu Asn Thr Trp Tyr Thr Lys
1 5 10 15
<210> 22
<211> 20
<212> PRT
<213>People (Homo sapiens)
<400> 22
Gln Tyr Lys Pro Ser Ala Ser Asn Ala Phe Met Val Cys Gly Val Leu
1 5 10 15
Tyr Ala Thr Arg
20
<210> 23
<211> 5
<212> PRT
<213>People (Homo sapiens)
<400> 23
Thr Met Asn Thr Arg
1 5
<210> 24
<211> 14
<212> PRT
<213>People (Homo sapiens)
<400> 24
Thr Glu Glu Ile Phe Tyr Tyr Tyr Asp Thr Asn Thr Gly Lys
1 5 10
<210> 25
<211> 7
<212> PRT
<213>People (Homo sapiens)
<400> 25
Leu Asp Ile Val Met His Lys
1 5
<210> 26
<211> 12
<212> PRT
<213>People (Homo sapiens)
<400> 26
Val Gln Ser Ile Asn Tyr Asn Pro Phe Asp Gln Lys
1 5 10
Claims (9)
1. smell mediates application of the element -4 as Diagnosis of Non-Small Cell Lung marker.
2. smell mediates application of plain -4 protein fragments in smell mediates plain -4 protein quantifications detection, which is characterized in that described
Smell mediates amino acid sequence such as NO.3~26 SEQ ID of plain -4 protein fragments any shown.
3. smell mediates application of plain -4 albumen in the kit for preparing non-small cell lung cancer screening/diagnosis.
4. smell mediates application of plain -4 albumen in the reagent for preparing non-small cell lung cancer screening/diagnosis.
5. smell mediates application of plain -4 protein fragments in the kit for preparing non-small cell lung cancer screening/diagnosis, feature
It is, the smell mediates amino acid sequence such as NO.3~26 SEQ ID of plain -4 protein fragments any shown.
6. smell mediates application of plain -4 protein antibodies in the kit for preparing non-small cell lung cancer screening/diagnosis.
7. a kind of kit for non-small cell lung cancer screening/diagnosis, which is characterized in that the kit, which contains, to be detected
Smell mediates the reagent of the expression of plain -4 genes or protein.
8. kit according to claim 7, which is characterized in that the reagent includes peptide shown in SEQ ID NO. 3~26
Section in one or several.
9. kit according to claim 7, which is characterized in that the reagent includes that smell mediates plain -4 protein antibodies.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363813A (en) * | 2018-12-25 | 2020-07-03 | 中科院上海巴斯德研究所麒麟创新研究院 | Biomarker of non-small cell lung cancer, detection method and application |
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|---|---|---|---|---|
| WO2016026933A1 (en) * | 2014-08-21 | 2016-02-25 | Centre National De La Recherche Scientifique - Cnrs - | Adjuvant or neoadjuvant therapy for sensitizing cancer stem cells to chemotherapy |
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|---|---|---|---|---|
| WO2016026933A1 (en) * | 2014-08-21 | 2016-02-25 | Centre National De La Recherche Scientifique - Cnrs - | Adjuvant or neoadjuvant therapy for sensitizing cancer stem cells to chemotherapy |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363813A (en) * | 2018-12-25 | 2020-07-03 | 中科院上海巴斯德研究所麒麟创新研究院 | Biomarker of non-small cell lung cancer, detection method and application |
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