CN105548548B - A kind of screening lung cancer kit - Google Patents
A kind of screening lung cancer kit Download PDFInfo
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- CN105548548B CN105548548B CN201610108030.3A CN201610108030A CN105548548B CN 105548548 B CN105548548 B CN 105548548B CN 201610108030 A CN201610108030 A CN 201610108030A CN 105548548 B CN105548548 B CN 105548548B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
- G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
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Abstract
The invention discloses a kind of screening lung cancer kit, it includes the optional reagent for being used to detect FRK expressions.The invention also discloses purposes of the reagent of detection FRK expressions in screening lung cancer reagent is prepared.Kit of the present invention is by detecting FRK expression, it can be determined that person to be checked suffers from the risk of lung cancer, available for the auxiliary diagnosis of clinical lung cancer, takes the remedy measures of correlation or decision-making to provide effective foundation for patient, potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to a kind of screening lung cancer kit.
Background technology
Lung cancer is one of most common malignant tumour in the world, and its morbidity and mortality is in ascendant trend year by year, at present
The incidence of disease occupies first place in the world, and seriously threatens human health and life.
The cause of disease of lung cancer is complicated, it is considered that influence factor includes：1. smoking；2. environmental pollution：Such as haze, indoor dress
Repair；3. bad life style：As eating habit is poor, life stress is big；4. chronic lung disease：Such as pulmonary tuberculosis, pneumoconiosis, silicosis,
Chronic bronchitis；5. human body internal factor：Such as reduction of familial inheritance, immunity function, endocrine function imbalance.
Meanwhile, lung cancer is a kind of disease for being good at concealment, often develops into late period in disease and just shows clinical symptoms, 70
~80% patients with lung cancer has been middle and advanced stage when being diagnosed to be with Lung Cancer Symptoms, and cancer cell has spread, and misses and most preferably controls
More opportunity, five year survival rate is low.For the patients with lung cancer of early stage, by treatment in time be greatly improved 5 years of patient and more than
Survival rate and life quality.Therefore the early diagnosis and the effective examination of progress of lung cancer are most important.
The examination of lung cancer, refers to do not have the related indication crowd of lung cancer to carry out routine physical examination those, before there is symptom
Lung cancer is found in time.If lung cancer molecular marker can be found, for pointing out clinician early stage to take patient correlation
Remedy measures or decision-making have great importance.
FRK (Fyn related kinase, Fyn related protein kinase), also known as GASK, PTK5 are Src kinase families into
One of member, has no the report related to lung cancer on FRK at present.
The content of the invention
In order to solve the above problems, lung cancer is studied in detail inventor, it was found that FRK can be used as its molecule mark
Will thing.Wherein, expressions of the FRK in lung tissue is proportionate with lung cancer.Therefore, by detecting the table of FRK in lung tissue
Up to level, the risk that person to be checked suffers from lung cancer can be predicted.
Accordingly, the invention provides a kind of screening lung cancer kit, and prepared by the reagent of detection FRK expression
Purposes in screening lung cancer reagent.
The screening lung cancer kit of the present invention, it includes the optional reagent for being used to detect FRK expressions.
Wherein, the reagent is the reagent for detecting FRK expressions in lung tissue.
Wherein, the reagent of the detection FRK expressions is method for immunohistochemical detection reagent.
Wherein, the reagent of the detection FRK expressions is Western Blot or ELISA detection method reagent.
Present invention also offers purposes of the reagent of detection FRK expressions in screening lung cancer reagent is prepared.
Wherein, the reagent is the reagent for detecting FRK expressions in lung tissue.
Wherein, the reagent of the detection FRK expressions is method for immunohistochemical detection reagent.
Wherein, the reagent of the detection FRK expressions is Western Blot or ELISA detection method reagent.
Kit of the present invention, can by detecting the pulmonary abnormalities position of lung exception person and the FRK expressions of normal portions
To judge the FRK expression differences of lung exception person, and then judge the risk that person to be checked suffers from lung cancer：If FRK expression is high,
The risk for then suffering from lung cancer is high, if FRK expression is low, the risk for suffering from lung cancer is low, available for the auxiliary diagnosis of clinical lung cancer,
Potential applicability in clinical practice is good.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other diversified forms can also be made, replaces or changes.
The embodiment of form, remakes further specifically to the above of the invention by the following examples
It is bright.But the scope that this should not be interpreted as to above-mentioned theme of the invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Embodiment
The relation of the FRK expressions of embodiment 1 and lung cancer
First, experimental method
1st, clinical data
Patients with lung cancer paraffin section 241 is chosen, remote organization compares 26 and (refers to the distal end normal lung tissue of patients with lung cancer
Paraffin section, apart from cancerous tissue 5cm), essential information is shown in Table 1.
The clinical elementary information of table 1
*Note：Other include large cell carcinoma, adenosquamous carcinoma, small cell carcinoma.
2nd, the detection of FRK expressions
To patients with lung cancer paraffin section, remote organization's control paraffin section, SABC (IHC) is carried out.
FRK antibody：Purchased from affinity companies of the U.S., article No. DF3179；
EnVisionTMHRP secondary antibodies, antibody diluent, diaminobenzidine (Diaminobenzidine, DAB) developer,
Antigen retrieval buffers, Wash Buffer, SABC pen：It is purchased from Dako companies of Denmark.
FRK expression is detected as follows：
(1) peroxidase is closed：Paraffin section is taken to be dewaxed, (dewaxing process is as follows to aquation for dewaxing：Dimethylbenzene I
The ethanol 5min of the ethanol 5min of the ethanol 5min of 10min → II 10min of dimethylbenzene → absolute ethyl alcohol 5min → 95% → 85% → 75%)
Afterwards, 3%H is used2O2Solution closes endogenous peroxydase in dark place, is incubated at room temperature 15min；
(2) distilled water is rinsed：Section is placed in distilled water, is washed 5min/ times on shaking table, totally three times；
(3) antigen retrieval：PH 8.0 Tris-EDTA or pH 6.0 sodium citrate solution is added, in 95 DEG C of water-baths
Middle water-bath 50min, carries out antigen retrieval；
(4) distilled water is rinsed：Section is taken out, is naturally cooled to after room temperature, with distilled water flushing three times, then Wash is used
Buffer rinses are once；
(5) it is incubated primary antibody：Tissue is enclosed with SABC pen, FRK antibody liquids are added dropwise in circle (according to FRK antibody：It is anti-
Body dilution=1:200, be formulated), 4 DEG C of overnight incubations；
(6) distilled water is rinsed：With distillation washing three times, Wash Buffer rinses are once；
(7) secondary antibody is added dropwise：The secondary antibody working solution of Dako HRP marks is added dropwise (according to EnVisionTMHRP secondary antibodies：Antibody is dilute
Release liquid=1:50, be formulated), it is incubated at room temperature 60min；
(8) distilled water is rinsed：Flowing water rinses 3-5min, and distilled water is rinsed 3 times；Wash Buffer rinses are once.
(9) DAB develops the color：DAB developers are added dropwise, colour developing situation, the terminating reaction in distilled water are observed under the microscope；
(10) haematoxylin is redyed：Section is put into haematoxylin dye liquor and dyes 2min, after the differentiation of 1% hydrochloride alcohol, flowing water
Rinse 10min；
(11) it is dehydrated transparent：It is transparent through 80%, 95%, 100% gradient alcohol dehydration, dimethylbenzene, dried in ventilating kitchen；
(12) mounting：With neutral Instant cement mounting, observe under an optical microscope, DP Controller IMAQs system
System adopts figure.Adopt after figure, SABC is given according to the tumour cell staining power and positive area of patients with lung cancer and remote organization
Scoring.
3rd, SABC standards of grading
Tumour cell staining power * positive tumor cells area=0-9 points,
As a result count：It is negative：0 point；It is low positive：1-3 points；High positive>3 points.
Wherein, tumour cell staining power, positive area scoring it is as follows：
| Tumour cell staining power scores@ | Positive tumor cell area scores |
| It is negative 0 point | Area<10% 0 points |
| 1 point of weakly positive | 1 point of area 11-50% |
| Positives 2 points | 2 points of area 51-75% |
| 3 points of strong positive | Area>76% 3 points |
@
Note：Independently tumour cell staining power result is scored by two veteran pathologists
4th, interpretation of result
Statistical analysis is carried out using SPSS17.0 cancerous lung tissues group and remote organization's control group.
2nd, experimental result
FRK expression testing result is shown in Table 2 during cancerous lung tissue is compareed with remote organization.
The FRK of table 2 expression
FRK positive expression rate is the positive tables of FRK in 30.8%, cancerous lung tissue in table 2, distal end normal structure
It is that 72.2%, FRK is significantly raised in cancerous lung tissue up to rate, compared with the normal structure of distal end, FRK expressions difference has system
Meter learns meaning (P<0.0001).
As can be seen from the above results, compared with Ai Pang normal lung tissues, the FRK expressions of cancerous lung tissue are significantly raised
(P<0.0001), illustrate that lung cancer is proportionate with FRK expressions, FRK high expression can significantly improve the possibility for suffering from lung cancer.
Because the FRK levels of Ai Pang normal lung tissues can reflect the FRK levels of Normal Lung tissue, it therefore, it can be checked by detecting
The FRK of person expression, Susceptible population's examination of lung cancer is come out.
The kit forms and its application method of the present invention detection FRK expressions of embodiment 2
First, the composition of immunologic combined detection reagent kit
Detection kit (50 person-portion)：
| Component | Volume |
| FRK antibody | 30μl |
| EnVisionTMHRP secondary antibodies | 100μl |
| Antibody diluent | 10ml |
| DAB developers | 10ml |
| Antigen retrieval buffers | 500ml |
| Wash Buffer* | 500ml |
| SABC pen | 1 |
Note：All kinds of experiment reagents are added dropwise for based on 100 μ l, specific consumption can be according to tissue size by every section in this kit
Appropriate increase and decrease；
Wash Buffer*It is reusable 2-3 times.
2nd, the application method of kit
By sample lung tissue to be checked, paraffin section is prepared, as detection sample, FRK expression is detected as follows
Level：
(1) peroxidase is closed：Detection sample is taken to be dewaxed, (dewaxing process is as follows to aquation for dewaxing：Dimethylbenzene I
The ethanol 5min of the ethanol 5min of the ethanol 5min of 10min → II 10min of dimethylbenzene → absolute ethyl alcohol 5min → 95% → 85% → 75%)
Afterwards, 3%H is used2O2Solution closes endogenous peroxydase in dark place, is incubated at room temperature 15min；
(2) distilled water is rinsed：Section is placed in distilled water, is washed 5min/ times on shaking table, totally three times；
(3) antigen retrieval：PH 8.0 Tris-EDTA or pH 6.0 sodium citrate solution is added, in 95 DEG C of water-baths
Middle water-bath 50min, carries out antigen retrieval；
(4) distilled water is rinsed：Section is taken out, is naturally cooled to after room temperature, with distilled water flushing three times, then Wash is used
Buffer rinses are once；
(5) it is incubated primary antibody：Tissue is enclosed with SABC pen, FRK antibody liquids are added dropwise in circle (according to FRK antibody：It is anti-
Body dilution=1:200, be formulated), 4 DEG C of overnight incubations；
(6) distilled water is rinsed：With distillation washing three times, Wash Buffer rinses are once；
(7) secondary antibody is added dropwise：The secondary antibody working solution of Dako HRP marks is added dropwise (according to EnVisionTMHRP secondary antibodies：Antibody is dilute
Release liquid=1:50, be formulated), it is incubated at room temperature 60min；
(8) distilled water is rinsed：Flowing water rinses 3-5min, and distilled water is rinsed 3 times；Wash Buffer rinses are once.
(9) DAB develops the color：DAB developers are added dropwise, colour developing situation, the terminating reaction in distilled water are observed under the microscope；
(10) haematoxylin is redyed：Section is put into haematoxylin dye liquor and dyes 2min, after the differentiation of 1% hydrochloride alcohol, flowing water
Rinse 10min；
(11) it is dehydrated transparent：It is transparent through 80%, 95%, 100% gradient alcohol dehydration, dimethylbenzene, dried in ventilating kitchen；
(12) mounting：With neutral Instant cement mounting, observe under an optical microscope, DP Controller IMAQs system
System adopts figure.Adopt after figure, SABC scoring is carried out to detection sample.
To pulmonary abnormalities person, abnormal position, normal portions tissue can be taken respectively, compares FRK expressions, and then evaluate it
Suffer from the possibility of lung cancer, be used as the auxiliary diagnosis means of clinical lung cancer.
To sum up, kit of the present invention is by detecting FRK expression, it can be determined that the pulmonary abnormalities position of lung exception person
With the FRK expression differences of normal portions, and then examination person to be checked suffers from the risk of lung cancer：If FRK expression is high,
The risk for suffering from lung cancer is high, if FRK expression is low, the risk for suffering from lung cancer is low, available for the auxiliary diagnosis of clinical lung cancer, is
Patient takes the remedy measures of correlation or decision-making to provide effective foundation, and potential applicability in clinical practice is good.
Claims (4)
1. detect purposes of the reagent of FRK expressions in screening lung cancer reagent is prepared.
2. purposes according to claim 1, it is characterised in that：The reagent is to be used to detect that FRK to express water in lung tissue
Flat reagent.
3. purposes according to claim 1 or 2, it is characterised in that：The reagent of the detection FRK expressions is immune group
Change detection method reagent.
4. purposes according to claim 1 or 2, it is characterised in that：The reagent of the detection FRK expressions is
Western Blot or ELISA detection method reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610108030.3A CN105548548B (en) | 2016-02-26 | 2016-02-26 | A kind of screening lung cancer kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610108030.3A CN105548548B (en) | 2016-02-26 | 2016-02-26 | A kind of screening lung cancer kit |
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| Publication Number | Publication Date |
|---|---|
| CN105548548A CN105548548A (en) | 2016-05-04 |
| CN105548548B true CN105548548B (en) | 2017-08-25 |
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| CN201610108030.3A Active CN105548548B (en) | 2016-02-26 | 2016-02-26 | A kind of screening lung cancer kit |
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2016
- 2016-02-26 CN CN201610108030.3A patent/CN105548548B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Antagomir-1290 suppresses CD133 cells in non-small cell lung cancer by targeting fyn-related Src family tyrosine kinase;Bo Sun et al;《Tumor Biol.》;20150318;第36卷;第6223－6230页 * |
| The Rak/Frk tyrosine kinase associates with and internalizes the epidermal growth factor receptor;L Jin et al;《Oncogene》;20130114;第33卷;第330、332－333页 * |
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