CN106405086A - 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 TXK. The invention also discloses the use of the reagent for detecting the expression level of TXK in preparing the reagent for lung cancer screening. By detecting the expression level of TXK, the kit of the present invention can judge the risk of lung cancer of the subject, can be used for auxiliary diagnosis of clinical lung cancer, and provides effective basis for patients to take relevant treatment measures or decision-making, and has 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.

TXK, also known as RLK (Resting lymphocyte kinase), is a member of the non-receptor protein tyrosine kinase Tec family, mainly expressed in T cells, involved in cytokine-activated T cell intracellular signal transduction, and plays a role in immune regulation important role. There is no report on the expression of TXK in lung cancer.

Contents of the invention

In order to solve the above problems, the inventors conducted a detailed study on lung cancer and found that TXK can be used as its molecular marker. Among them, the expression level of TXK in lung tissue was positively correlated with lung cancer. Therefore, by detecting the expression level of TXK 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 TXK 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 TXK.

Wherein, the reagent is a reagent for detecting the expression level of TXK in lung tissue.

Wherein, the reagent for detecting the expression level of TXK is a reagent for immunohistochemical detection method.

Wherein, the reagent for detecting the expression level of TXK 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 TXK in preparing the reagent for lung cancer screening.

Wherein, the reagent is a reagent for detecting the expression level of TXK in lung tissue.

Wherein, the reagent for detecting the expression level of TXK is a reagent for immunohistochemical detection method.

Wherein, the reagent for detecting the expression level of TXK is a reagent for Western Blot or ELISA detection method.

The kit of the present invention can determine the difference in the expression level of TXK in patients with abnormal lungs by detecting the expression levels of TXK in abnormal parts and normal parts of the lungs of patients with abnormal lungs, and then determine the risk of lung cancer of the subject to be tested: if the expression level of TXK is high, The risk of lung cancer is high, and if the expression level of TXK is low, the risk of lung cancer is low. It can be used for auxiliary diagnosis of clinical lung cancer, and the clinical application prospect is good.

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 embodiment 1 TXK expression level and lung cancer

1. Experimental method

1. Clinical data

20 cases of paraffin sections of lung cancer patients and 20 cases of remote controls (referring to paraffin sections of distal normal lung tissue of lung cancer patients, 5 cm away from 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 TXK expression level

Immunohistochemistry (IHC) using TXK as an index was performed on paraffin sections of lung cancer patients and control paraffin sections of distant tissues.

TXK antibody: purchased from Abcam, Cat. No. ab37818;

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 TXK 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, add TXK antibody diluent (mixed and prepared for TXK antibody and antibody diluent, the ratio of the two is 1:100) in the circle, and 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 ratio of the two is 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: The results of immunohistochemical staining were scored independently by two experienced pathologists

4. Result analysis

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

2. Experimental results

The detection results of the expression level of TXK in lung cancer tissue and remote tissue control are shown in Table 2.

The expression level of table 2TXK

It can be seen from Table 2 that the positive expression rate of TXK in distal normal tissues is 30%, and the highly positive expression rate is 5%; the positive expression rate of TXK in lung cancer tissues is 90%, and the highly positive expression rate is 65%; Compared with the distant normal tissue, the expression level of TXK was significantly different (P<0.001).

From the above results, it can be seen that the expression level of TXK 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 lung cancer and TXK expression level, and high expression of TXK will significantly increase the risk of lung cancer. possibility. Since the TXK level of normal lung tissue adjacent to cancer can reflect the TXK level of normal human lung tissue, the susceptible population of lung cancer can be screened out by detecting the expression level of TXK in the subject.

Embodiment 2 The present invention detects the composition of the kit of expression level of TXK and its application method

1. The composition of the immunohistochemical detection kit

Detection kit (50 copies):

components volume TXK 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 TXK was detected according to the following method:

(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, add TXK antibody diluent (mixed and prepared for TXK antibody and antibody diluent, the ratio of the two is 1:100) in the circle, and 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 ratio of the two is 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.

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

In summary, by detecting the expression level of TXK, the kit of the present invention can determine the difference in the expression level of TXK 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 in the candidate: if the expression level of TXK is high , the risk of lung cancer is high, and if the expression level of TXK is low, the risk of lung cancer is low. It can be used for auxiliary diagnosis of clinical lung cancer, and provide effective basis for patients to take relevant treatment measures or decision-making. The clinical application prospect is good.

Claims (8)

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