CN106153920A - Lung cancer screening kit - Google Patents

Abstract

The invention discloses a lung cancer screening kit, which includes optional reagents for detecting the expression level of p-EphB1 protein. The invention also discloses the use of the reagent for detecting the expression level of p-EphB1 protein in preparing the reagent for lung cancer screening. The kit of the present invention can determine the risk of lung cancer of the subject by detecting the expression level of p-EphB1 protein, can be used for auxiliary diagnosis of clinical lung cancer, and provides an effective basis for patients to take relevant treatment measures or decision-making, and has a good clinical application prospect .

Description

A lung cancer screening kit

technical field

The invention relates to a lung cancer screening kit.

Background technique

Lung cancer is one of the most common malignant tumors in the world. Its morbidity and mortality are increasing year by year. Currently, the incidence rate ranks first in the world, which seriously threatens human health and life.

The etiology of lung cancer is complex, and it is generally believed that the influencing factors include: ① smoking; ② environmental pollution: such as smog, interior decoration; ③ unhealthy lifestyle: such as poor eating habits, high life pressure; Silicosis, chronic bronchitis; ⑤ Intrinsic factors in the human body: such as family inheritance, decreased immune function, endocrine dysfunction, etc.

At the same time, lung cancer is a disease that is good at hiding. It often shows clinical symptoms when the disease develops to an advanced stage. 70-80% of lung cancer patients are already in the middle or late stage when they are diagnosed with lung cancer symptoms. The optimal time for cure is lost, and the five-year survival rate is low. For patients with early lung cancer, timely treatment can greatly improve the 5-year survival rate and quality of life of the patients. Therefore, early diagnosis and effective screening of lung cancer are very important.

Screening for lung cancer refers to routine physical examinations for those without lung cancer-related symptoms to detect lung cancer in time before symptoms appear. If molecular markers of lung cancer can be found, it is of great significance to prompt clinicians to take relevant treatment measures or decision-making for patients in the early stage.

Contents of the invention

In order to solve the above problems, the inventors conducted a detailed study on lung cancer and found that phosphorylated EphB1 protein (phosphorylated EphB1, p-EphB1) can be used as its molecular marker. Among them, the expression level of p-EphB1 protein in lung tissue was positively correlated with lung cancer. Therefore, by detecting the expression level of p-EphB1 protein in lung tissue, the risk of lung cancer of the subject can be predicted.

Accordingly, the present invention provides a lung cancer screening kit and the use of the reagent for detecting the expression level of p-EphB1 protein in preparing the lung cancer screening reagent.

The lung cancer screening kit of the present invention includes optional reagents for detecting the expression level of p-EphB1 protein.

Wherein, the reagent is a reagent for detecting the expression level of p-EphB1 protein in lung tissue.

Wherein, the reagent for detecting the expression level of p-EphB1 protein is a reagent for immunohistochemical detection method.

Wherein, the reagent for detecting the expression level of p-EphB1 protein is a reagent for Western Blot or ELISA detection method.

The invention also provides the use of the reagent for detecting the expression level of p-EphB1 protein in the preparation of the reagent for lung cancer screening.

Wherein, the reagent is a reagent for detecting the expression level of p-EphB1 protein in lung tissue.

Wherein, the reagent for detecting the expression level of p-EphB1 protein is a reagent for immunohistochemical detection method.

Wherein, the reagent for detecting the expression level of p-EphB1 protein is a reagent for Western Blot or ELISA detection method.

EphB1, Eph receptor B1, is one of the members of the Eph receptor family, a tyrosine kinase receptor involved in the phosphorylation of protein molecules during signal transduction. At present, the research on EphB1 is mostly focused on the development of the nervous system and the transduction of pain signals. For the research on the relationship between EphB1 and lung cancer, see the literature of Hafner C, Differential Gene Expression of Eph Receptors and Ephrins in Benign Human Tissues and Cancers, It is pointed out in the literature that the content of total EphB1mRNA in lung squamous cell carcinoma tissues is significantly lower than that in normal controls. Moreover, there is currently no report on the relationship between EphB1 protein and lung cancer, let alone the report on the relationship between phosphorylated EphB1 protein and lung cancer (p-EphB1).

The kit of the present invention can determine the difference in the expression level of p-EphB1 protein in the lung abnormality by detecting the p-EphB1 protein expression level in the lung abnormality part and the normal part of the lung abnormality, and then judge the risk of lung cancer of the candidate: if If the expression level of p-EphB1 protein is high, the risk of lung cancer is high. If the expression level of p-EphB1 protein is low, the risk of lung cancer is low. It can be used for auxiliary diagnosis of clinical lung cancer, and has a good clinical application prospect.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

detailed description

The relationship between the expression level of embodiment 1p-EphB1 and lung cancer

1. Experimental method

1. Clinical data

25 cases of paraffin sections of lung cancer patients and 16 cases of remote controls (refers to the paraffin sections of distal normal lung tissue of lung cancer patients, 5 cm away from the cancer tissue) were selected. The basic information is shown in Table 1.

Table 1 Basic clinical information

^* Note: Others include large cell carcinoma, adenosquamous carcinoma, and small cell carcinoma.

2. Detection of p-EphB1 expression level

Immunohistochemistry (IHC) using p-EphB1 as an indicator was performed on paraffin sections of lung cancer patients and control paraffin sections of distant tissues.

p-EphB1 antibody: purchased from Beijing Boaosen Biotechnology Co., Ltd., product number bs-10781R;

Please confirm whether the following reagents are still available:

EnVision ^TM HRP secondary antibody, antibody diluent, DAB chromogenic reagent, antigen retrieval solution, Wash Buffer, immunohistochemical pen: all were purchased from Dako Company in Denmark.

The expression level of p-EphB1 was detected as follows:

(1) Blocking peroxidase: Take paraffin sections for dewaxing, dewaxing until hydration (the dewaxing process is as follows: xylene I 10 min → xylene II 10 min → absolute ethanol 5 min → 95% ethanol 5 min → 85% ethanol 5 min → After 75% ethanol for 5 min), block endogenous peroxidase with 3% H ₂ O ₂ solution in the dark, and incubate at room temperature for 15 min;

(2) Distilled water rinsing: slices were placed in distilled water and washed on a shaker for 5 min/time, a total of three times;

(3) Antigen retrieval: add Tris-EDTA with pH 8.0 or sodium citrate solution with pH 6.0, and bathe in a water bath at 95°C for 50 minutes to carry out antigen retrieval;

(4) Rinse with distilled water: take out the slices, cool to room temperature naturally, wash with distilled water three times, and then rinse once with WashBuffer;

(5) Incubate the primary antibody: circle the tissue with an immunohistochemical pen, and add p-EphB1 antibody diluent (prepared by mixing p-EphB1 antibody and antibody diluent, the ratio of which is 1:100) in the circle, Incubate overnight at 4°C;

(6) Rinse with distilled water: wash three times with distilled water, rinse once with Wash Buffer;

(7) Add secondary antibody dropwise: add Dako HRP-labeled secondary antibody working solution (prepared by mixing HRP secondary antibody and antibody diluent, the two are mixed at 1:1), and incubate at room temperature for 60 minutes;

(8) Rinse with distilled water: wash with distilled water 3 times, 5 minutes each time;

(9) DAB color development: drop the color development substrate DAB, observe the color development under a microscope, and terminate the reaction in distilled water;

(10) Hematoxylin counterstaining: Put the slices into the hematoxylin staining solution for 2 minutes, and after differentiation with 1% hydrochloric acid alcohol, rinse with running water for 10 minutes;

(11) Dehydration and transparency: through 80%, 95%, 100% gradient alcohol dehydration, xylene transparency, dry in a fume hood;

(12) Seal the slide: seal the slide with neutral quick-drying glue, observe under an optical microscope, and collect pictures with the DP Controller image acquisition system. Immunohistochemical scores were given according to the staining intensity and positive area of tumor cells in lung cancer patients and distant tissues after the pictures were taken.

3. Immunohistochemical scoring criteria

Tumor cell staining intensity * tumor cell positive area = 0-9 points,

Results statistics: Negative: 0 points; Low positive: 1-3 points; Highly positive > 3 points.

Among them, the scores of tumor cell staining intensity and positive area are as follows:

@

Note: Two experienced pathologists independently scored the staining intensity of tumor cells

4. Result analysis

SPSS17.0 lung cancer tissue group and remote tissue control group were used for statistical analysis.

2. Experimental results

Table 2 shows the detection results of p-EphB1 expression level in lung cancer tissue and remote tissue control.

Table 2p-Expression levels of EphB1

It can be seen from Table 2 that the positive expression rate of p-EphB1 in remote normal tissues was 56.3%, and the highly positive expression rate was 0; the positive expression rate of p-EphB1 in lung cancer tissues was 92%, and the highly positive expression rate was 72%; p-EphB1 was significantly elevated in lung cancer tissues, and there was a statistically significant difference in the expression level of p-EphB1 compared with distant normal tissues (P<0.001).

From the above results, it can be seen that the expression level of p-EphB1 in lung cancer tissue is significantly higher than that in adjacent normal lung tissue (P<0.001), indicating that there is a positive correlation between the expression level of p-EphB1 in lung cancer and the high expression of p-EphB1 Significantly increases the risk of developing lung cancer. Since the p-EphB1 level of normal adjacent lung tissue can reflect the p-EphB1 level of normal human lung tissue, the susceptible population of lung cancer can be screened out by detecting the expression level of p-EphB1 in the examinee.

Example 2 The present invention detects the composition of the kit for the expression level of p-EphB1 and its application method

1. The composition of the immunohistochemical detection kit

Detection kit (50 copies):

components volume p-EphB1 antibody 30μl EnVision ^™ HRP Secondary Antibody 100μl Antibody Diluent 10ml DAB developer 10ml Antigen Retrieval Solution 500ml Wash Buffer ^* 500ml immunohistochemistry pen 1 stick

Note: This kit is calculated as 100 μl of various experimental reagents per slice, and the specific dosage can be appropriately increased or decreased according to the size of the tissue.

2. How to use the kit

The lung tissue of the sample to be tested was prepared as a paraffin section as a test specimen, and the expression level of p-EphB1 was detected according to the following method:

The expression level of p-EphB1 was detected as follows:

(1) Blocking peroxidase: Take paraffin sections for dewaxing, dewaxing until hydration (the dewaxing process is as follows: xylene I 10 min → xylene II 10 min → absolute ethanol 5 min → 95% ethanol 5 min → 85% ethanol 5 min → After 75% ethanol for 5 min), block endogenous peroxidase with 3% H ₂ O ₂ solution in the dark, and incubate at room temperature for 15 min;

(2) Distilled water rinsing: slices were placed in distilled water and washed on a shaker for 5 min/time, a total of three times;

(3) Antigen retrieval: add Tris-EDTA with pH 8.0 or sodium citrate solution with pH 6.0, and bathe in a water bath at 95°C for 50 minutes to carry out antigen retrieval;

(4) Rinse with distilled water: take out the slices, cool to room temperature naturally, wash with distilled water three times, and then rinse once with WashBuffer;

(5) Incubate the primary antibody: circle the tissue with an immunohistochemical pen, and add p-EphB1 antibody diluent (prepared by mixing p-EphB1 antibody and antibody diluent, the ratio of which is 1:100) in the circle, Incubate overnight at 4°C;

(6) Rinse with distilled water: wash three times with distilled water, rinse once with Wash Buffer;

(7) Add secondary antibody dropwise: add Dako HRP-labeled secondary antibody working solution (prepared by mixing HRP secondary antibody and antibody diluent, the two are mixed at 1:1), and incubate at room temperature for 60 minutes;

(8) Rinse with distilled water: wash with distilled water 3 times, 5 minutes each time;

(9) DAB color development: drop the color development substrate DAB, observe the color development under a microscope, and terminate the reaction in distilled water;

(10) Hematoxylin counterstaining: Put the slices into the hematoxylin staining solution for 2 minutes, and after differentiation with 1% hydrochloric acid alcohol, rinse with running water for 10 minutes;

(11) Dehydration and transparency: through 80%, 95%, 100% gradient alcohol dehydration, xylene transparency, dry in a fume hood;

(12) Seal the slide: seal the slide with neutral quick-drying glue, observe under an optical microscope, and collect pictures with the DP Controller image acquisition system. Immunohistochemical scores were given according to the staining intensity and positive area of tumor cells in lung cancer patients and distant tissues after the pictures were collected.

For those with abnormal lungs, tissues from abnormal parts and normal parts can be taken respectively, and the expression level of p-EphB1 can be compared to evaluate the possibility of lung cancer, which can be used as an auxiliary diagnostic method for clinical lung cancer.

To sum up, by detecting the expression level of p-EphB1, the kit of the present invention can determine the difference in the expression level of p-EphB1 between the abnormal part of the lung and the normal part of the lung of the person with abnormal lung, and then screen the risk of lung cancer of the candidate: if p If the expression level of -EphB1 is high, the risk of lung cancer is high; if the expression level of p-EphB1 is low, the risk of lung cancer is low. According to the basis, the prospect of clinical application is good.

Claims (8)

1. A lung cancer screening kit, characterized in that: it includes optional reagents for detecting the expression level of phosphorylated EphB1 protein. 2. The screening kit according to claim 1, wherein the reagent is a reagent for detecting the expression level of phosphorylated EphB1 protein in lung tissue. 3. The screening kit according to claim 1 or 2, characterized in that: the reagent for detecting the expression level of phosphorylated EphB1 protein is a reagent for immunohistochemical detection method. 4. The screening kit according to claim 1 or 2, characterized in that: the reagent for detecting the expression level of phosphorylated EphB1 protein is a reagent for Western Blot or ELISA detection method. 5. Use of the reagent for detecting the expression level of phosphorylated EphB1 protein in the preparation of a reagent for lung cancer screening. 6. The use according to claim 5, characterized in that: the reagent is a reagent for detecting the expression level of phosphorylated EphB1 protein in lung tissue. 7. The use according to claim 5 or 6, characterized in that: the reagent for detecting the expression level of phosphorylated EphB1 protein is a reagent for immunohistochemical detection method. 8. The use according to claim 5 or 6, characterized in that: the reagent for detecting the expression level of phosphorylated EphB1 protein is a reagent for Western Blot or ELISA detection method.