CN107817350B - A 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 SOST protein. The invention also discloses the use of the reagent for detecting the expression level of SOST protein in preparing the reagent for lung cancer screening. The kit of the present invention can judge the risk of lung cancer of a subject by detecting the expression level of the SOST 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

Among the morbidity and mortality of malignant tumors, lung cancer accounts for 13.0% and 19.4% respectively, ranking first among malignant tumors. Moreover, due to factors such as the intensification of China's aging population and the rejuvenation of the smoking population, the population base of lung cancer patients is still on the rise. Due to the heterogeneity of lung cancer, the treatment effect of the same treatment method on lung cancer is often different. Therefore, in the clinical research and clinical treatment of lung cancer, an important part is to classify and stage lung cancer to minimize its heterogeneity. Improve treatment efficiency.

At present, according to the degree of differentiation and morphological characteristics of various types of lung cancer, lung cancer can be divided into small cell lung cancer, adenocarcinoma, and squamous cell carcinoma. Among them, squamous cell carcinoma and adenocarcinoma are high-incidence types, accounting for 50% and 25% of primary lung cancers respectively, and are the focus of lung cancer prevention and research. Squamous cell carcinoma is more common in central lung cancer, and has a tendency to grow into the lumen, often causing bronchial stenosis in the early stage, causing atelectasis, or obstructive pneumonia. Cancerous tissue degeneration, necrosis, formation of cavity or cancerous lung abscess. Adenocarcinoma mostly grows in the mucous glands of the small bronchi at the edge of the lung, and is most common in peripheral lung cancer. Adenocarcinoma tends to grow outside the tube, but it can also spread along the alveolar wall, often forming a mass with a diameter of 2 to 4 cm at the edge of the lung. Adenocarcinoma contains a lot of blood vessels, so local invasion and hematogenous metastasis are earlier than squamous cell carcinoma. It is easy to transfer to the liver, brain and bone, and it is more likely to involve the pleura and cause pleural effusion.

Like other malignant tumors, early detection and early treatment are the key to reducing the mortality of lung cancer, including squamous cell carcinoma and adenocarcinoma. However, lung cancer does not have any special symptoms in the early stage, and the symptoms of lung cancer often appear when the disease develops to an advanced stage, and the best timing for surgery is missed. Even if surgery is performed, most patients will still die of lung cancer recurrence, invasion or metastasis. Therefore, early diagnosis and effective screening of lung cancer are very important.

The molecular biological mechanism of the occurrence and development of lung cancer is still unclear. Exploring effective molecular biological detection markers of lung cancer will not only provide a basis for elucidating the molecular mechanism of lung cancer cell invasion and metastasis, but also provide a basis for the pathological diagnosis and treatment options of lung cancer. Important theoretical significance and clinical application value.

Sclerostin (SOST) is a secreted protein with a CTCK domain at the C-terminus, which is encoded by the SOST gene and is generally considered to be specifically expressed by bone cells. Studies have shown that SOST can bind to LRP5/6 receptors. body and inhibit the Wnt signaling pathway, thereby inhibiting osteoblast differentiation and bone formation. At present, there is no report on the expression of SOST in lung cancer.

Contents of the invention

The inventors conducted a detailed study on lung cancer and found that SOST protein can be used as its molecular marker, and the expression level of SOST protein in lung tissue is related to lung squamous cell carcinoma and adenocarcinoma. Therefore, by detecting the expression level of SOST protein in the lung tissue, not only the risk of the subject suffering from lung cancer can be predicted, but also the type of lung cancer 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 SOST protein in preparing the lung cancer screening reagent.

The invention discloses a lung cancer screening kit, which includes optional reagents for detecting the expression level of SOST protein.

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

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

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

Wherein, the lung cancer is lung adenocarcinoma and/or lung squamous cell carcinoma.

The invention also provides the use of the reagent for detecting the expression level of SOST protein in the preparation of reagents for lung cancer screening.

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

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

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

Wherein, the lung cancer is lung adenocarcinoma and/or lung squamous cell carcinoma.

The kit of the present invention can judge the difference in the expression level of SOST in the lung abnormality by detecting the expression level of SOST in the abnormal part of the lung and the normal part of the lung of the person with abnormal lung, and then judge the risk of lung cancer of the candidate: if the expression level of SOST is high, The risk of lung adenocarcinoma is low, and the risk of lung squamous cell carcinoma is high; if the expression level of SOST is low, the risk of the two types of lung cancer is opposite. 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.

Description of drawings

Figure 1 SOST expression in lung cancer tissue samples.

Fig. 2 The expression level of SOST, where ** represents P<0.01; * represents P<0.05.

Detailed ways

Example 1 Detection of the expression level of SOST in lung cancer

1. Experimental materials

Lung cancer tissue microarrays (HLugS060CD01 and HLug-Ade050CD-01) were purchased from Shanghai Xinchao Biology. The microarray contains histopathological sections of lung cancer, in which:

HLugS060CD01 tissue chip is lung squamous cell carcinoma disease course chip: including 5 cases of normal human lungs, 22 cases of paracancerous and primary lung squamous cell carcinoma (clinical stage 1, 2, 3, 4) and 3 metastatic lung squamous cell carcinoma 4 cases and 4 cases of negative and positive lymph nodes of lung squamous cell carcinoma.

HLug-Ade050CD-01 tissue chip is a lung adenocarcinoma disease course chip: including 5 cases of normal human lung, 14 cases of paracancerous/primary lung adenocarcinoma (clinical stage 1, 2, 3, 4) each, lung adenocarcinoma There were 4 cases of cancer metastasis, 7 cases of negative lymph nodes and 6 cases of positive lymph nodes.

Anti-SOST polyclonal antibody was obtained from ProteinTech Group, Inc (Wuhan, China).

All other kits or reagents were purchased from Biyuntian Biotechnology Co., Ltd. (Shanghai, China).

2. Detection of SOST expression level

Detection was performed using immunohistochemical methods, as follows:

1. Paraffin sections are dewaxed to water: (Paraffin sections should be placed at 60°C for 1 hour before staining).

(1) Xylene I, II, 10 minutes each.

(2) Gradient alcohol: 100%, 2 minutes → 95%, 2 minutes → 80%, 2 minutes → 70% for 2 minutes.

(3) Washing with distilled water: 5 minutes, twice (on a shaker).

2. Block endogenous peroxidase with hydrogen peroxide: 3% H2O2, 10 minutes at room temperature (protect from light).

3. Washing with distilled water: 5 minutes, 2 times (on a shaker).

4. Antigen retrieval: High-pressure thermal restoration method: Sodium citrate buffer (10mM, PH6.0), submerge the slices, cover the pot, boil in the pressure cooker, and slowly cool down after 3 minutes.

Attachment: Preparation of Antigen Retrieval Solution (10mM pH 6.0 Sodium Citrate Buffer)

(1) Preparation of stock solution:

Solution A: Trisodium citrate-2H2O 29.41g+ distilled water 1000ml

Solution B: citric acid 21g + distilled water 1000ml

(2) Preparation of working solution: A solution 82ml + B solution 18ml + distilled water 900ml

Methods of antigen retrieval:

5. PBS: 5 minutes, 2 times (on a shaker).

6. Normal serum sealing: take out the slices from the staining tank, wipe off the water on the back of the slices and the tissue on the front of the slices

Surrounding moisture (keep the tissue in a moist state), add normal rabbit serum dropwise for treatment, 37°C, 15 minutes.

Attachment: Normal serum preparation (or preparation according to the concentration specified in the kit)

According to the ratio of 1:20, it is prepared with PBS, and the required amount of each slice is calculated according to 50μl+5μl (10% sprinkled amount).

7. Add the primary antibody dropwise: absorb the serum with filter paper, without washing, directly add anti-SOST (antibody diluted 1:200 with PBS), and freeze overnight at 4°C.

8. PBS: 5 minutes, 2 times (on a shaker).

9. Add biotinylated secondary antibody dropwise (dilute the secondary antibody with PBS at 1:5000), 37°C, 40 minutes.

10. PBS: 5 minutes, 2 times (on a shaker).

11. Add the third antibody (SAB complex) dropwise at 37°C for 40 minutes.

12. PBS: 5 minutes, 2 times (on a shaker).

13. DAB color development, observe under the microscope, stop in time (stop with tap water).

Attachment: DAB preparation

Preparation of stock solution (DAB 25mg/ml): DAB 250mg+PBS 10ml, after complete dissolution, aliquot into 1ml, 100μl, 50μl, 20μl, etc., and freeze at -20°C.

(2) Working solution: DAB stock solution 20μl+PBS 1000μl+3%H2O2 5μl

14. Rinse thoroughly with tap water (fine water).

15. Hematoxylin counterstaining, room temperature, 30 seconds, rinse with tap water.

16. Rinse with tap water for 15 minutes.

17. Gradient alcohol dehydration:

80%, 2 minutes → 95%, 2 minutes → 100%, 2 times, 5 minutes.

18. Xylene transparent:

I, II (Xylene) 5 minutes each

19. Sealing: Canadian gum (or neutral gum) sealing.

20. Take pictures under a microscope, and perform semi-quantitative analysis on SOST immunohistochemical staining after taking pictures, among which,

The fluorescence intensity is: negative: 0; weak positive: 1; positive: 2; strong positive: 3;

The positive percentages are: 0%: 0; 0-25%: 1; 25%-50%: 2; 50%-75%: 3; 75%-100%: 4.

Semi-quantitative score = fluorescence intensity X positive percentage,

The semi-quantitative score is 0-12.

Graphpad 5.0 was used to conduct statistical analysis on the semi-quantitative scores of each group, and P<0.05 was considered statistically significant.

2. Experimental results

The detection results of SOST expression in lung cancer tissue and normal tissue control are shown in Figure 1-2.

It can be seen from Figure 1-2 that in normal lung tissue, the average score of SOST immunohistochemistry is 8.00±0.58, and in lung squamous cell carcinoma tissue, the average score of SOST is 11.67±0.33. Compared with normal lung tissue, the expression level difference is significant Statistically significant (P<0.01), SOST was significantly increased in lung squamous cell carcinoma tissue; in lung adenocarcinoma tissue, the average score of SOST was 3.00±1.73, compared with normal lung tissue, the expression level difference was statistically significant ( P<0.05), SOST was significantly decreased in lung adenocarcinoma tissue.

It can be seen that compared with normal lung tissue, the expression level of SOST in lung adenocarcinoma tissue was significantly reduced, and the expression level of SOST in lung squamous cell carcinoma tissue was significantly increased, indicating that lung cancer is related to the expression level of SOST. Since the SOST level of normal lung tissue can reflect the SOST level of normal human lung tissue, for those with abnormal lung tissue, the abnormal part and normal part tissue can be taken respectively, and the expression level of SOST can be compared to evaluate the risk of lung adenocarcinoma and squamous cell carcinoma. Possibility, as an auxiliary diagnostic tool for clinical lung cancer.

In summary, by detecting the expression level of SOST, the kit of the present invention can determine the difference in the expression level of SOST 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 SOST is low , the risk of lung adenocarcinoma is high, and the risk of lung squamous cell carcinoma is high. On the contrary, the conclusion is opposite. 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 (3)

1. the reagent of detection SOST protein expression level is preparing the purposes in screening lung cancer reagent, it is characterised in that:

The reagent is the reagent for detecting SOST protein expression level in lung tissue；

The lung cancer is adenocarcinoma of lung and/or lung squamous cancer；

Compared with normal lung tissue, SOST protein expression level is increased in lung squamous cell carcinoma cancers, is reduced in pulmonary adenocarcinoma.

2. purposes according to claim 1, it is characterised in that: the reagent of the detection SOST protein expression level is immune Groupization detection method reagent.

3. purposes according to claim 1, it is characterised in that: it is described detection SOST protein expression level reagent be WesternBlot or ELISA detection method reagent.