CN109212214B - A kind of screening lung cancer kit - Google Patents

A kind of screening lung cancer kit Download PDF

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Publication number
CN109212214B
CN109212214B CN201811116158.XA CN201811116158A CN109212214B CN 109212214 B CN109212214 B CN 109212214B CN 201811116158 A CN201811116158 A CN 201811116158A CN 109212214 B CN109212214 B CN 109212214B
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srms
phosphorylating
lung cancer
reagent
detection
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CN109212214A (en
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张立
步宏
李为民
李丽
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West China Hospital of Sichuan University
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West China Hospital of Sichuan University
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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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Abstract

The invention discloses a kind of screening lung cancer kits, it includes optional for detecting the reagent of non-phosphorylating SRMS protein expression level.The invention also discloses the reagents of detection non-phosphorylating SRMS protein expression level to prepare the purposes in screening lung cancer reagent.The expression that kit of the present invention passes through detection non-phosphorylating SRMS, 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 to provide effective foundation for patient, potential applicability in clinical practice is good.

Description

A kind of screening lung cancer kit
Technical field
The present invention relates to a kind of screening lung cancer kits.
Background technique
Lung cancer is that one of most common malignant tumour, morbidity and mortality are in rise year by year trend in the world, at present Disease 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. to smoke；2. environmental pollution: such as haze, indoor dress It repairs；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, often develops to advanced stage 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 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, referring to does not have the related indication crowd of lung cancer to carry out routine physical examination those, before there is symptom Discovery lung cancer 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.
Summary of the invention
To solve the above-mentioned problems, lung cancer is studied in detail in inventor, it was found that non-phosphorylating SRMS albumen (non-phosphorylated SRMS) can be used as its molecular marker.Wherein, table of the non-phosphorylating SRMS in lung tissue It is positively correlated up to level with lung cancer.Therefore, it by the expression of non-phosphorylating SRMS in detection lung tissue, can predict to be checked Person suffers from the risk of lung cancer.
Accordingly, the present invention provides a kind of screening lung cancer kits, and the expression water of detection non-phosphorylating SRMS albumen Flat reagent is preparing the purposes in screening lung cancer reagent.
Screening lung cancer kit of the invention, it includes optional for detecting non-phosphorylating SRMS protein expression level Reagent.
Wherein, the reagent is the reagent for detecting non-phosphorylating SRMS protein expression level in lung tissue.
Wherein, the reagent of the detection non-phosphorylating SRMS protein expression level is method for immunohistochemical detection reagent.
Wherein, the reagent of the detection non-phosphorylating SRMS protein expression level is Western Blot or ELISA detection Method reagent.
The present invention also provides the reagents of detection non-phosphorylating SRMS protein expression level to prepare screening lung cancer reagent In purposes.
Wherein, the reagent is the reagent for detecting non-phosphorylating SRMS protein expression level in lung tissue.
Wherein, the reagent of the detection non-phosphorylating SRMS protein expression level is method for immunohistochemical detection reagent.
Wherein, the reagent of the detection non-phosphorylating SRMS protein expression level is Western Blot or ELISA detection Method reagent.
SRMS(src-related kinase lacking C-terminal regulatory tyrosine and N- Terminal myristylation sites), Chinese is that the relevant shortage C-terminal of src adjusts tyrosine and N-terminal ten Tetra-alkylation site kinases is present in T lymphocyte and some other non-T lymphocyte, participates in egg during signal transduction The Phosphorylation events of white matter molecule are one of the key signal transduction molecules during cell signalling.
Non-phosphorylating SRMS is not disclosed in the expression situation of cancerous lung tissue and normal control in existing literature.
Kit of the present invention passes through the pulmonary abnormalities position of detection lung exception person and the non-phosphorylating SRMS table of normal portions Up to level, it can be determined that the non-phosphorylating SRMS expression difference of lung exception person, and then judge the risk that person to be checked suffers from lung cancer: If the expression of non-phosphorylating SRMS is high, the risk for suffering from lung cancer is high, if the expression of non-phosphorylating SRMS is low, suffers from lung The risk of cancer is low, can be used for the auxiliary diagnosis of clinical lung cancer, and potential applicability in clinical practice is good.
Obviously, above content 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 embodiment of form by the following examples remakes further specifically above content of the 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 above content of the present invention The technology realized all belongs to the scope of the present invention.
Specific embodiment
The relationship of embodiment 1 non-phosphorylating SRMS expression and lung cancer
One, experimental method
1, clinical data
Patients with lung cancer paraffin section 37 are chosen, distal end control 30 (refers to the distal end normal lung tissue paraffin of patients with lung cancer Slice, is confirmed as normal tissue apart from cancerous tissue 5cm, and through pathology), 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 non-phosphorylating SRMS expression
Paraffin section is compareed to patients with lung cancer paraffin section, remote organization, is carried out using SRMS as the immunohistochemistry of index (IHC)。
Non-phosphorylating SRMS antibody: U.S. ProteinTech company, article No. 26447-1-AP are purchased from；
EnVisionTMHRP secondary antibody, antibody diluent, DAB color developing agent, antigen retrieval buffers, Wash Buffer, immunohistochemistry Pen: it is purchased from Dako company, Denmark.
The expression of non-phosphorylating SRMS is detected as follows:
(1) it closes peroxidase: paraffin section being taken to dewax, (dewaxing process is as follows: dimethylbenzene I to aquation for dewaxing 10min → II 10min of the dimethylbenzene → ethyl alcohol of dehydrated 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: taking out and is sliced, after cooled to room temperature, is washed three times with distillation, then use Wash Buffer rinse is primary；
(5) it is incubated for primary antibody: being enclosed tissue with immunohistochemistry pen, non-phosphorylating SRMS antibody diluent is added dropwise in circle (being mixed with antibody diluent 1:100i), 4 DEG C of overnight incubations；
(6) distilled water rinses: with distillation washing three times, Wash Buffer rinse is primary；
(7) secondary antibody is added dropwise: the secondary antibody working solution that Dako HRP is marked is added dropwise, and (HRP secondary antibody is mixed with antibody diluent 1:1 matches System), 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, and 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) it mounting: 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
First according to the form below provides the scoring of tumour cell staining power, positive area:
@Note: it is independently scored tumour cell staining power result by two veteran pathologists
Calculate the product (0-9 point) of the scoring of tumour cell staining power and positive area scoring again, it is specified that:
Non-phosphorylating SRMS expression is negative: 0 point；
Non-phosphorylating SRMS expression is low positive: 1-3 points；
Non-phosphorylating SRMS expresses High positive > 3 point.
4, interpretation of result
Statistical analysis is carried out using SPSS17.0 cancerous lung tissue group and remote organization's control group.
Two, experimental result
Cancerous lung tissue compareed with remote organization in the expression testing result of non-phosphorylating SRMS be shown in Table 2.
The expression of 2 non-phosphorylating SRMS of table
As can be seen from Table 2, the positive expression rate of non-phosphorylating SRMS is 60% in the normal tissue of distal end, High positive expression rate It is 10%；Non-phosphorylating SRMS positive expression rate in cancerous lung tissue is 86.7%, and High positive expression rate is 63.3%；It is non-phosphorus Acidification SRMS is significantly increased in cancerous lung tissue, and compared with the normal tissue of distal end, non-phosphorylating SRMS expression difference has Statistical significance (P < 0.001).
As can be seen from the above results, compared with Ai Pang normal lung tissue, the non-phosphorylating SRMS of cancerous lung tissue expresses water Head up display, which writes, increases (P < 0.0001), illustrates that lung cancer is positively correlated with non-phosphorylating SRMS expression, the height of non-phosphorylating SRMS Expression can significantly improve a possibility that suffering from lung cancer.Since the non-phosphorylating SRMS level of Ai Pang normal lung tissue can reflect normal person The non-phosphorylating SRMS of lung tissue is horizontal, therefore, can be by the expression of the non-phosphorylating SRMS of detection person to be checked, by lung Susceptible population's screening of cancer comes out.
The kit forms and its application method of 2 present invention detection non-phosphorylating SRMS expression of embodiment
One, the composition of immunologic combined detection reagent kit
Detection kit (50 person-portion):
Component Volume
Non-phosphorylating SRMS antibody 30 μ l (please be provided)
EnVisionTMHRP secondary antibody 100μl
Antibody diluent 10ml
DAB color developing agent 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 to be examined lung tissue, paraffin section is prepared, as detection sample, detects non-phosphorylating as follows The expression of SRMS:
(1) it closes peroxidase: paraffin section being taken to dewax, (dewaxing process is as follows: dimethylbenzene I to aquation for dewaxing 10min → II 10min of the dimethylbenzene → ethyl alcohol of dehydrated alcohol 5min → 95% ethyl alcohol of 5min → 85% 5min → 75% ethyl alcohol 5min) Afterwards, endogenous peroxydase is closed in dark place with 3%H2O2 solution, 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: taking out and is sliced, after cooled to room temperature, is washed three times with distillation, then use Wash Buffer rinse is primary；
(5) it is incubated for primary antibody: being enclosed tissue with immunohistochemistry pen, it is (dilute with antibody that SRMS antibody diluent is added dropwise in circle Release liquid 1:100i mixing), 4 DEG C of overnight incubations；
(6) distilled water rinses: with distillation washing three times, Wash Buffer rinse is primary；
(7) secondary antibody is added dropwise: the secondary antibody working solution that Dako HRP is marked is added dropwise, and (HRP secondary antibody is mixed with antibody diluent 1:1 matches System), 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, and 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) it mounting: 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, methods of marking are shown in the Part III of embodiment 1.
To pulmonary abnormalities person, abnormal position, normal portions tissue can be taken respectively, compare non-phosphorylating SRMS expression, And then a possibility that it suffers from lung cancer is evaluated, the auxiliary diagnosis means as clinical lung cancer.
To sum up, the expression that kit of the present invention passes through detection non-phosphorylating SRMS, it can be determined that the lung of lung exception person The non-phosphorylating SRMS expression difference of portion's abnormal position and normal portions, and then screening person to be checked suffers from the risk of lung cancer: If the expression of non-phosphorylating SRMS is high, the risk for suffering from lung cancer is high, if the expression of non-phosphorylating SRMS is low, suffers from lung The risk of cancer is low, can be used for the auxiliary diagnosis of clinical lung cancer, takes relevant remedy measures or decision to provide effectively for patient Foundation, potential applicability in clinical practice is good.

Claims (3)

1. the reagent of detection non-phosphorylating SRMS protein expression level is preparing the purposes in screening lung cancer reagent；
The reagent is the reagent for detecting non-phosphorylating SRMS protein expression level in lung tissue.
2. purposes according to claim 1, it is characterised in that: the examination of the detection non-phosphorylating SRMS protein expression level Agent is method for immunohistochemical detection reagent.
3. purposes according to claim 1, it is characterised in that: the examination of the detection non-phosphorylating SRMS protein expression level Agent is Western Blot or ELISA detection method reagent.
CN201811116158.XA 2018-09-25 2018-09-25 A kind of screening lung cancer kit Active CN109212214B (en)

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CN106918698B (en) * 2015-12-25 2019-11-22 广州瑞博奥生物科技有限公司 A kind of phospho-AB chip agent box detecting human receptor tyrosine kinase enzyme
WO2017222285A1 (en) * 2016-06-20 2017-12-28 재단법인 대구경북첨단의료산업진흥재단 Novel imidazopyridine derivative, method for preparing same, and pharmaceutical composition containing same as active ingredient for preventing or treating cancer
JP2020533269A (en) * 2016-12-14 2020-11-19 デベロップメント センター フォー バイオテクノロジー Antibody-drug conjugates and their use

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