CN105954517B - A kind of screening lung cancer kit - Google Patents
A kind of screening lung cancer kit Download PDFInfo
- Publication number
- CN105954517B CN105954517B CN201610520573.6A CN201610520573A CN105954517B CN 105954517 B CN105954517 B CN 105954517B CN 201610520573 A CN201610520573 A CN 201610520573A CN 105954517 B CN105954517 B CN 105954517B
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- lck
- reagent
- detection
- expression levels
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of screening lung cancer kit, it includes the optional reagent for detecting LCK protein expression levels.The invention also discloses purposes of the reagent of detection LCK protein expression levels in preparing screening lung cancer reagent.The expression that kit of the present invention passes through detection LCK albumen, it can be determined that person to be checked suffers from the risk of lung cancer, can be used for the auxiliary diagnosis of clinical lung cancer, takes relevant remedy measures or decision provides effective foundation, potential applicability in clinical practice good for patient.
Description
Technical field
The present invention relates to a kind of screening lung cancer kits.
Background technology
Lung cancer is one of most common malignant tumour in the world, and morbidity and mortality are in ascendant trend year by year, at present
Incidence 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
It repaiies；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 familial inheritance, immunity function reduce, endocrine function is lacked of proper care.
Meanwhile lung cancer is a kind of disease for being good at concealment, and often developing to late period in disease just shows clinical symptoms, and 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 been spread, and is missed and is most preferably controlled
More opportunity, five year survival rate are low.For the patients with lung cancer of early stage, it is greatly improved 5 years or more of patient by treating in time
Survival rate and life quality.Therefore the early diagnosis and the effective screening of progress of lung cancer are most important.
The screening of lung cancer refers to not having the related indication crowd of lung cancer to carry out routine physical examination those, before there is symptom
Lung cancer is found in time.It is relevant for prompting clinician to take patient early stage if lung cancer molecular marker can be found
Remedy measures or decision have great importance.
Invention content
To solve the above-mentioned problems, lung cancer is studied in detail in inventor, it was found that LCK albumen (non-
Phosphorylated LCK) its molecular marker can be used as.Wherein, expressions of the LCK in lung tissue is in lung cancer
Positive correlation.Therefore, by detecting the expression of LCK in lung tissue, the risk that person to be checked suffers from lung cancer can be predicted.
Accordingly, the present invention provides a kind of screening lung cancer kits, and the reagent of the expression of detection LCK albumen to exist
Prepare the purposes in screening lung cancer reagent.
The screening lung cancer kit of the present invention, it includes the optional reagent for detecting LCK protein expression levels.Its
In, the reagent is the reagent for detecting LCK protein expression levels in lung tissue.
Wherein, the reagent of the detection LCK protein expression levels is method for immunohistochemical detection reagent.
Wherein, the reagent of the detection LCK protein expression levels is Western Blot or ELISA detection method is tried
Agent.
The present invention also provides purposes of the reagent of detection LCK protein expression levels in preparing screening lung cancer reagent.
Wherein, the reagent is the reagent for detecting LCK protein expression levels in lung tissue.
Wherein, the reagent of the detection LCK protein expression levels is method for immunohistochemical detection reagent.
Wherein, the reagent of the detection LCK protein expression levels is Western Blot or ELISA detection method is tried
Agent.
LCK (lymphocyte cell-specific protein-tyrosine kinase), Chinese is lymph
Cell-specific proteins tyrosine kinase is one of Src kinase families members, is present in T lymphocytes and some other non-T leaching
It is bar intracellular, the Phosphorylation events of protein molecule during signal transduction are participated in, during being the key that cell signalling
One of signal transducers.
Currently, the document of Skrzypski M etc., Main histologic are shown in the research about LCK and relationship between lung cancer
types of non-small-cell lung cancer differ in expression of prognosis-related
Genes, Clin Lung Cancer, 2013,14：666-673 points out that LCK mRNA are in Non-Small Cell Lung Carcinoma in document
There is no significant difference with the content in normal structure.Therefore, according to existing literature, the LCK eggs in lung cancer suspect tissue are detected
White level is unable to reach the effect of screening lung cancer.
Kit of the present invention, can by the LCK expressions at the pulmonary abnormalities position and normal portions of detection lung exception person
To judge the LCK expression differences of lung exception person, and then judge the risk that person to be checked suffers from lung cancer：If the expression of LCK is high,
The risk for then suffering from lung cancer is high, if the expression of LCK is low, the risk for suffering from lung cancer is low, can be used for the auxiliary diagnosis of clinical lung cancer,
Potential applicability in clinical practice is good.
Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific implementation mode of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Specific implementation mode
The relationship of embodiment 1 LCK expressions and lung cancer
One, experimental method
1, clinical data
Patients with lung cancer paraffin section 30 is chosen, distal end control 20 (refers to the distal end normal lung tissue paraffin of patients with lung cancer
Slice, apart from cancerous tissue 5cm), essential information is shown in Table 1.
1 clinical elementary information of table
*Note：Other include large cell carcinoma, adenosquamous carcinoma, small cell carcinoma.
2, the detection of LCK expressions
Paraffin section is compareed to patients with lung cancer paraffin section, remote organization, is carried out using LCK as the immunohistochemistry of index
(IHC)。
LCK antibody：Purchased from affinity companies of the U.S., article No. AF6101 detects total LCK albumen；
EnVisionTMHRP secondary antibodies, antibody diluent, DAB color developing agents, antigen retrieval buffers, Wash Buffer, immune group
Change pen：It is purchased from Dako companies of Denmark.
The expression of LCK is detected as follows：
(1) peroxidase is closed：Paraffin section is taken to dewax, (dewaxing process is as follows to aquation for dewaxing：Dimethylbenzene I
10min → II 10min of the dimethylbenzene → ethyl alcohol of absolute ethyl alcohol 5min → 95% ethyl alcohol of 5min → 85% 5min → 75% ethyl alcohol 5min)
Afterwards, 3%H is used2O2Solution closes endogenous peroxydase in dark place, is incubated at room temperature 15min；
(2) distilled water rinses：Slice is placed in distilled water, is washed 5min/ times on shaking table, altogether three times；
(3) antigen retrieval：The sodium citrate solution of the Tris-EDTA or pH 6.0 of pH 8.0 is added, in 95 DEG C of water-baths
Middle water-bath 50min carries out antigen retrieval；
(4) distilled water rinses：It takes out and is sliced, after cooled to room temperature, washed three times with distillation, then use Wash
Buffer rinses are primary；
(5) it is incubated primary antibody：Tissue is enclosed with immunohistochemistry pen, LCK antibody liquids are added dropwise in circle and (for LCK antibody and resist
Body dilution mixed preparing, the two ratio are 1:100), 4 DEG C of overnight incubations；
(6) distilled water rinses：With distillation washing three times, Wash Buffer rinses are primary；
(7) secondary antibody is added dropwise：The secondary antibody working solution that Dako HRP labels are added dropwise (mixes with antibody diluent for HRP secondary antibodies and matches
System, the two ratio are 1:1), it is incubated at room temperature 60min；
(8) distilled water rinses：Distillation washing 3 times, each 5min；
(9) DAB develops the color：Chromogenic substrate DAB is added dropwise, observation colour developing situation, terminates reaction in distilled water under the microscope；
(10) haematoxylin is redyed：Slice 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, it is dry in ventilating kitchen；
(12) mounting：It with neutral Instant cement mounting, observes under an optical microscope, DP Controller Image Acquisition system
System adopts figure.After adopting figure, immunohistochemistry is given according to the tumour cell staining power and positive area of patients with lung cancer and remote organization
Scoring.
3, immunohistochemistry standards of grading
Tumour cell staining power * positive tumor cells area=0-9 points,
As a result it counts：It is negative：0 point；It is low positive：1-3 points；High positive>3 points.
Wherein, tumour cell staining power, the scoring of positive area are as follows：
@
Note：It is independently scored tumour cell staining power result by two veteran pathologists
4, interpretation of result
Statistical analysis is carried out using SPSS17.0 cancerous lung tissues group and remote organization's control group.
Two, experimental result
The expression testing result of LCK is shown in Table 2 during cancerous lung tissue is compareed with remote organization.
The expression of 2 LCK of table
As can be seen from Table 2, the positive expression rate of LCK is 60% in the normal structure of distal end, High positive expression rate is 10%；Lung
LCK positive expression rates in cancerous tissue are 86.7%, and High positive expression rate is 63.3%；LCK significantly rises in cancerous lung tissue
Height, compared with the normal structure of distal end, LCK expression differences have statistical significance (P<0.001).
As can be seen from the above results, compared with Ai Pang normal lung tissues, the LCK expressions of cancerous lung tissue significantly increase
(P<0.0001), illustrate that lung cancer is proportionate with LCK expressions, the high expression of LCK can significantly improve the possibility for suffering from lung cancer.
Since the LCK levels of Ai Pang normal lung tissues can reflect that the LCK of Normal Lung tissue is horizontal, it can be to be checked by detecting
The expression of the LCK of person comes out Susceptible population's screening of lung cancer.
The kit forms and its application method of 2 present invention detection LCK expressions of embodiment
One, the composition of immunologic combined detection reagent kit
Detection kit (50 person-portion)：
| Component | Volume |
| LCK antibody | 30μl |
| EnVisionTMHRP secondary antibodies | 100μl |
| Antibody diluent | 10ml |
| DAB color developing agents | 10ml |
| Antigen retrieval buffers | 500ml |
| Wash Buffer* | 500ml |
| Immunohistochemistry pen | 1 |
Note：It is based on 100 μ l that all kinds of experiment reagents, which are added dropwise, by every slice in this kit, and specific dosage can be according to tissue size
Appropriate increase and decrease.
Two, the application method of kit
By sample lung tissue to be checked, paraffin section is prepared, as detection sample, detects the expression of LCK as follows
It is horizontal：
(1) peroxidase is closed：Paraffin section is taken to dewax, (dewaxing process is as follows to aquation for dewaxing：Dimethylbenzene I
10min → II 10min of the dimethylbenzene → ethyl alcohol of absolute ethyl alcohol 5min → 95% ethyl alcohol of 5min → 85% 5min → 75% ethyl alcohol 5min)
Afterwards, 3%H is used2O2Solution closes endogenous peroxydase in dark place, is incubated at room temperature 15min；
(2) distilled water rinses：Slice is placed in distilled water, is washed 5min/ times on shaking table, altogether three times；
(3) antigen retrieval：The sodium citrate solution of the Tris-EDTA or pH 6.0 of pH 8.0 is added, in 95 DEG C of water-baths
Middle water-bath 50min carries out antigen retrieval；
(4) distilled water rinses：It takes out and is sliced, after cooled to room temperature, washed three times with distillation, then use Wash
Buffer rinses are primary；
(5) it is incubated primary antibody：Tissue is enclosed with immunohistochemistry pen, LCK antibody liquids are added dropwise in circle and (for LCK antibody and resist
Body dilution mixed preparing, the two ratio are 1:100), 4 DEG C of overnight incubations；
(6) distilled water rinses：With distillation washing three times, Wash Buffer rinses are primary；
(7) secondary antibody is added dropwise：The secondary antibody working solution that Dako HRP labels are added dropwise (mixes with antibody diluent for HRP secondary antibodies and matches
System, the two ratio are 1:1), it is incubated at room temperature 60min；
(8) distilled water rinses：Distillation washing 3 times, each 5min；
(9) DAB develops the color：Chromogenic substrate DAB is added dropwise, observation colour developing situation, terminates reaction in distilled water under the microscope；
(10) haematoxylin is redyed：Slice 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, it is dry in ventilating kitchen；
(12) mounting：It with neutral Instant cement mounting, observes under an optical microscope, DP Controller Image Acquisition system
System adopts figure.After adopting figure, immunohistochemistry is given according to the tumour cell staining power and positive area of patients with lung cancer and remote organization
Scoring.
To pulmonary abnormalities person, abnormal position, normal portions tissue can be taken respectively, compares LCK expressions, and then evaluate it
The possibility for suffering from lung cancer, the auxiliary diagnosis means as clinical lung cancer.
To sum up, the expression that kit of the present invention passes through detection LCK, it can be determined that the pulmonary abnormalities position of lung exception person
With the LCK expression differences of normal portions, and then screening person to be checked suffers from the risk of lung cancer：If the expression of LCK is high,
The risk for suffering from lung cancer is high, if the expression of LCK is low, the risk for suffering from lung cancer is low, can be used for the auxiliary diagnosis of clinical lung cancer, is
Patient takes relevant remedy measures or decision provides effective foundation, potential applicability in clinical practice good.
Claims (3)
1. detecting purposes of the reagent of LCK protein expression levels in preparing screening lung cancer reagent；Wherein, the reagent is to use
In the reagent for detecting LCK protein expression levels in lung tissue.
2. purposes according to claim 1, it is characterised in that：The reagent of the detection LCK protein expression levels is immune
Groupization detection method reagent.
3. purposes according to claim 1, it is characterised in that：It is described detection LCK protein expression levels reagent be
Western Blot or ELISA detection method reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610520573.6A CN105954517B (en) | 2016-06-30 | 2016-06-30 | A kind of screening lung cancer kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610520573.6A CN105954517B (en) | 2016-06-30 | 2016-06-30 | A kind of screening lung cancer kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105954517A CN105954517A (en) | 2016-09-21 |
| CN105954517B true CN105954517B (en) | 2018-09-18 |
Family
ID=56902330
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610520573.6A Active CN105954517B (en) | 2016-06-30 | 2016-06-30 | A kind of screening lung cancer kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105954517B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107817350B (en) * | 2017-10-26 | 2019-09-06 | 成都医学院第一附属医院 | A kind of screening lung cancer kit |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7955800B2 (en) * | 2002-06-25 | 2011-06-07 | Advpharma Inc. | Metastasis-associated gene profiling for identification of tumor tissue, subtyping, and prediction of prognosis of patients |
| CN103834729B (en) * | 2007-06-01 | 2016-09-14 | 加利福尼亚大学董事会 | The multigene prognostic assay of pulmonary carcinoma |
| EP3722810A3 (en) * | 2009-02-11 | 2021-01-13 | Caris MPI, Inc. | Molecular profiling of tumors |
| CN103998622A (en) * | 2011-07-01 | 2014-08-20 | 加利福尼亚大学董事会 | Multigene prognostic assay for lung cancer |
-
2016
- 2016-06-30 CN CN201610520573.6A patent/CN105954517B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| Main Histologic Types of Non–Small-Cell Lung Cancer Differ in Expression of Prognosis-related Genes;Marcin Skrzypski et al;《Clinical Lung Cancer》;20131130;第4卷(第6期);666-673e2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105954517A (en) | 2016-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dhingra et al. | Clinicopathologic significance of putative stem cell markers, CD44 and nestin, in gastric adenocarcinoma | |
| CN106771248B (en) | High-level serous ovarian cancer diagnosis and/or the marker of Index for diagnosis | |
| CN105861692A (en) | Method for studying on prostate cancer reoccurrence and metastasis | |
| CN110187110A (en) | A kind of cardia cancer prognosis prediction marker and its application | |
| CN107255711A (en) | Osteopontin is used for the purposes for preparing or screening acute-on-chronic liver failure diagnostic reagent | |
| CN105954517B (en) | A kind of screening lung cancer kit | |
| CN107807243B (en) | Biomarker of esophageal cancer and application thereof | |
| CN105929164B (en) | A kind of screening lung cancer kit | |
| CN109212214B (en) | A kind of screening lung cancer kit | |
| Jin et al. | Increased expression of metastasis-associated in colon cancer-1 in renal cell carcinoma is associated with poor prognosis | |
| CN110244058A (en) | ENPP1 is preparing the application in high-level serous ovarian cancer diagnosis and prognosis kit | |
| CN108088998A (en) | A kind of screening lung cancer kit | |
| CN106153920B (en) | A kind of screening lung cancer kit | |
| CN106645722B (en) | A kind of screening lung cancer kit | |
| CN105548548B (en) | A kind of screening lung cancer kit | |
| US8148059B2 (en) | Method of detecting malignancy of nasopharyngeal carcinoma and a nasopharyngeal carcinoma malignancy biomarker | |
| CN106383230B (en) | A kind of pulmonary cancer diagnosis kit | |
| Lu et al. | Diagnostic utility of serum Golgi phosphoprotein 3 in bladder cancer patients | |
| Elnady et al. | Diagnostic accuracy of SATB2 in identifying primary and metastatic colorectal carcinoma: a comparative immunohistochemical study | |
| CN106405086A (en) | Lung cancer screening kit | |
| CN110095605B (en) | Lung cancer screening kit | |
| CN116183935B (en) | Molecular marker for predicting prognosis of hepatic portal cholangiocarcinoma and application thereof | |
| CN110244057B (en) | Application of ADORA3 in preparation of high-grade serous ovarian cancer diagnosis and prognosis kit | |
| CN110095606A (en) | A kind of kit for screening of lung cancer | |
| CN106442995A (en) | Lung cancer screening check test kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |