CN114487418A - Application of TGFb RIII protein as a serum biomarker in gastric cancer - Google Patents

Abstract

The present application provides an application of a TGFb RIII protein as a serum biomarker for gastric cancer, and the benefits of the present application are that it provides an application of a TGFb RIII protein as a serum biomarker for gastric cancer.

Description

Application of TGFb RIII protein as a serum biomarker in gastric cancer

technical field

The present application relates to the application of a TGFb RIII protein as a serum biomarker for gastric cancer.

Background technique

At present, the main methods of gastric cancer screening are upper gastrointestinal endoscopy. However, upper gastrointestinal endoscopy requires advanced equipment and specialized operators, high technical requirements, high cost, and the subjects are more painful and have poor compliance, which is not suitable for repeated inspections and population censuses. In addition, there are some other methods for screening gastric cancer or its precursor lesions, such as Helicobacter pylori (Hp) detection, serum pepsinogen (PG) detection, gastrin-17, G-17 ) detection, etc., but due to its high false positive rate and low sensitivity, further research is still needed.

Biomarkers refer to biomolecules used for disease diagnosis, risk assessment and prognosis judgment. Through proteomic analysis of plasma/serum and urine of patients with different types of cancer, a series of protein markers related to tumorigenesis and development have been found, which can be used for cancer screening, diagnosis and prognosis monitoring. The lack of clinical biomarkers for the diagnosis of gastric cancer, especially the discovery of non-invasive biomarkers, is of great significance.

SUMMARY OF THE INVENTION

The purpose of this application is to provide the application of a TGFb RIII protein as a serum biomarker for gastric cancer.

The purpose of this application is achieved through the following technical solutions:

The first aspect of the present application discloses the use of a TGFb RIII protein as a serum biomarker for gastric cancer.

Further, the TGFb RIII protein is human TGFb RIII protein.

The second aspect of the present application discloses a gastric cancer serum biomarker, including TGFb RIII protein.

The optional solutions of the present application can be used as independent solutions or combined with each other. The structure in any one of the solutions of the present application can be used as an independent technical solution, or can be combined with other technical solutions.

The benefit of the present application is that it provides an application of a TGFb RIII protein as a serum biomarker for gastric cancer.

Description of drawings

Fig. 1 is the protein content and difference of TGFb RIII in various samples in Example 1 of the application;

Fig. 2 is the ROC curve of TGFb RIII protein of healthy-advanced gastric cancer patients in Example 1 of the application;

Fig. 3 is the protein content of TGFb RIII and the difference between groups in Example 2 of the application;

FIG. 4 is the ROC curve of TGFb RIII protein in healthy-gastric cancer patients in Example 2 of the application.

Detailed ways

As used herein, the term "ROC curve" or "ROC plot" refers to a graphical curve showing the performance of a binary classifier system as a function of its discrimination threshold. This curve was created by plotting the true positive rate against the false positive rate at various threshold settings. The true positive rate is also known as the sensitivity. The false positive rate was calculated as 1-specificity. Thus, the ROC curve is a graphical display of the true positive rate versus the false positive rate (sensitivity vs (1-specificity)) over a range of cutoff values and a way to select the best cutoff for clinical use. Expressing accuracy as the area under the ROC curve (AUC) provides a useful parameter for comparing test performance. An AUC close to 1 indicates that the test is highly sensitive and highly specific, while an AUC close to 0.5 indicates that the test is neither sensitive nor specific.

When referring to a difference between a test sample and a control or reference sample, the terms "statistically different" or "statistically significant" refer to the probability that the groups are the same by less than 5% when using an appropriate statistical analysis (eg, p<0.05 )Case. In other words, the probability of getting the same result on a completely random basis is less than 5 out of 100 attempts. Those skilled in the art know how to choose an appropriate statistical analysis. Typically, appropriate statistical analysis is determined based on whether the variable under study has a normal distribution (eg, by using the Kolmogorov-Smirnov test) and whether there is homoscedasticity (eg, using the Levene test). Preferably, in the presence of normal distribution and homoscedasticity, a parametric model such as a t-test or an ANOVA test is used; and in cases where at least one of these two requirements is not met, a non-parametric model, such as MannWhitney U test or Kruskal-Wallis test.

The TGFb RIII protein in this application is the human protein TGFb RIII (type III Transforminggrowth factorβreceptor, also known as betaglycan), which is encoded by the TGFbRIII gene on chromosome 1 1p31-32, which consists of 16 exons , there are two promoters, a proximal promoter and a distal promoter, which produce two mRNAs. TGFb RIII is an 851 amino acid transmembrane proteoglycan containing a large 766 amino acid extracellular domain, a hydrophobic transmembrane domain, and a short 42 amino acid cytoplasmic domain. TGFb RIII is expressed on the cell surface as a non-covalently linked homodimer.

The preferred modes of the present application will be described in detail below with reference to the accompanying drawings. In order to enable those skilled in the art to better understand the solutions of the present application, the following will combine the drawings in the embodiments of the present application to describe the techniques in the embodiments of the present application. The plan is clearly and completely described. The following examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.

Example 1:

1. Obtain serum samples

A total of 330 serum samples were obtained through recruitment, including 39 healthy individuals, 128 early cancer patients, 80 advanced gastric cancer patients, 13 gastritis patients, 22 liver cancer patients, 32 colorectal cancer patients, and 19 breast cancer patients. .

2. Serum testing

In this example, the serum samples of each subject were detected using the QAH-CUST kit to obtain the protein content of TGFb RIII in each serum sample. The QAH-CUST kit includes a quantitative chip (Quantibody ArxaxGlass Chip). , Sample Diluent, 20× Wash Buffer I, 20× Wash Buffer II, Lyophilized cytokine standard mix*, Detection antibody cocktail), Cy3-streptavidin (Cy3equivalent dye-conjugated Streptavidin), slide washer/Dryer, Adhesivedevice sealer; the specific detection method includes the following steps:

S1. Quantitative chip drying

Take the quantitative chip out of the box and leave it at room temperature for 20-30min, open the packaging bag, peel off the sealing strip, and then put the quantitative chip in a desiccator or dry at room temperature for 1-2 hours.

S2, the configuration of standard products

S21. Add 500 μL of the sample diluent to the small tube of the standard mixture, and re-dissolve the standard mixture. Before opening the vial, centrifuge quickly and gently pipette up and down to dissolve the powder, label this vial as Std 1.

S22. Label 6 clean centrifuge tubes as Std2, Std3, Std4, Std5, Std6, and Std7 respectively, and add 200 μL of sample diluent to each centrifuge tube.

S23. Take 100 μL of sample diluent from the centrifuge tube marked Std 1 and add it to the centrifuge tube marked Std2 and mix it gently, then take 100 μL of sample dilution from the centrifuge tube marked Std 2 and add it to the centrifuge tube marked Std3 In the centrifuge tube, and so on, the serial dilution was carried out into the centrifuge tube marked Std7 to obtain the standard solution.

S24. Draw 100 μL of the standard solution from the centrifuge tube labeled Std7 into another new centrifuge tube, labeled CNTRL, as a negative control.

S3. Operation process of quantitative chip

S31. Add 100 μL of sample diluent to each well of the quantitative chip, incubate for 1 h on a shaker at room temperature, and seal the quantitative chip.

S32. Aspirate the sample diluent in each well, and add 80 μL of the standard solution from the centrifuge tube marked with CNTRL to the well together with the serum sample (sample 2-fold dilution and loading), and incubate at 4°C overnight.

S33. Clean the quantitative chip (glass slide). In this example, a Thermo Scientific Wellwash Versa chip washer is used, which is divided into two steps. First, wash with 1× Wash Solution I, and wash with 250 μL of 1× Wash Solution I in each well. 10 times, each shaking for 10 s, the shaking intensity is selected to be high, and 20× washing solution I is diluted with deionized water. Then use 1× Washing Solution II to clean the channel, 250 μL of 1× Washing Solution II per well, wash 6 times, shake for 10 s each time, choose a high vibration intensity, and dilute 20× Washing Solution II with deionized water.

S34. Incubation of the detection antibody mixture

Centrifuge the small tube containing the detection antibody mixture, then add 1.4 ml of sample diluent, mix well and centrifuge again quickly, then add 80 μL of the detection antibody mixture to each well, and incubate for 2 hours on a RT shaker.

S35, cleaning, the same as step S33.

Incubation of S36, Cy3-streptavidin

Centrifuge the vial containing Cy3-streptavidin, then add 1.4 ml of sample diluent, mix well and centrifuge again quickly, add 80 μL of Cy3-streptavidin to each well, wrap with aluminum foil Quantitative chips (glass slides) were incubated in the dark for 1 hour on a RT shaker.

S37, cleaning, the same as step S33.

S38. Fluorescence detection

Scan the signal with a laser scanner, using Cy3 or green channel (excitation frequency = 532 nm)

In this example, InnoScan 300 was used, instrument model: InnoScan 300 MicroarrayScanner; manufacturer: Innopsys; place of origin: Parc d'Activités Activestre; 31 390 Carbonne-France; scanning parameters: WaveLengh: 532 nm; Resolution: 10 μm.

S39, using the data analysis software of QAH-CUST to perform data analysis to obtain the content of TGFbRIII protein in each serum sample.

3. Experimental results

As shown in Figure 1, the level of TGFb RIII protein in the advanced gastric cancer group was increased, and it was statistically significant with other groups (wherein, the "***" between the healthy group and the advanced gastric cancer group in Figure 1 indicates that Statistical difference P value is less than 0.001), further analysis of the data, the results obtained by ROC curve (Receiver Operating Characteristic curve, receiver operating characteristic curve) are shown in Figure 2, the AUC (Area Under rocCurve, the AUC value is the area covered by the ROC curve, the larger the AUC, the better the effect of distinguishing the two groups) value is 0.797.

Example 2:

To further demonstrate the reliability of the results, another 60 subjects were recruited: 10 healthy, 10 early cancer, and 40 late cancer. After the subject serum was collected, an Elisa test was performed.

In this embodiment, the serum samples of each subject are detected by using a TGFb RIII detection kit to obtain the TGFb RIII protein content in each serum sample, and the kit includes the following components:

TGFb RIII Microplate (Item A): 96-well plate coated with anti-Human TGFb RIII antibody

Wash Buffer Concentrate (20x) (Item B): 25ml 20x concentrated wash

Standards (Item C): Recombinant Human TGFb RIII Standard

Assay Diluent (Item E2): 15ml, 5x concentrate. For standard and sample dilution

Detection Antibody TGFb RIII (Item F): Biotinylated anti-TGFb RIII antibody

HRP-Streptavidin concentrate (Item G): 200μL 200x concentrated HRP-streptavidin

TMB One-Step Substrate Reagent(Item H): 12ml of 3,3',5,5'-tetramethylbenzidine(TMB)

Stop Solution (Item I): 8ml of 0.2M sulfuric acid

1. Reagent preparation

1) Equilibrate the kit and serum samples to room temperature (18-25°C);

2) According to the pre-experimental serum sample diluted 2 times and loaded

3) Diluent (Item E2) is diluted 5 times with deionized water for use;

4) Standard (Item C) preparation: centrifuge the vial containing the standard (Item C), then add 400 μL of 1x dilution (Item E2) to the vial containing the standard (Item C), and mix well. 50000pg/ml standard stock solution; pipette 100μL of standard stock solution into a centrifuge tube containing 400μL of 1x diluent (Item E2), mix well, mark it as STD1; prepare seven 1.5ml small centrifuge tubes and add them to each of the seven tubes 300μL of 1x dilution (Item E2) was used as buffer, then seven 1.5ml small centrifuge tubes were labeled as STD2, STD3, STD4, STD5, STD6, STD7, STD8; Carry out gradient dilution: extract 200μL of 10000pg/ml standard stock solution (i.e. STD1) and add it to STD2 vial. After mixing, extract 200μL of STD2 vial and add it to STD3 vial and mix well. Follow the method until STD7 is prepared; STD8 is 300μL 1x diluent (Item E2) is standard 0pg/ml;

5) Dilution of concentrated washing solution (Item B): dilute the concentrated washing solution (Item B) by 20 times with deionized water into washing buffer for later use;

6) Centrifuge the TGFb RIII detection antibody (Item F), add 100L 1x diluent (Item E2) to fully dissolve, gently pipette up and down with a pipette, and then dilute 80 times with 1x diluent (Item E2) for later use;

7) Centrifuge HRP-streptavidin (Item G), then dilute it 200 times with 1x diluent (Item E2) for later use;

2. Operation steps

1) After equilibrating the antibody-coated ELISA plate (Item A) to room temperature, add 100 μL of the prepared standard stock solution (STD7) and serum samples to the corresponding wells, and seal the entire plate with a sealing film. 4 Incubate overnight at ℃;

2) Add the prepared 1x washing buffer to the plate washer, and use the plate washer to wash the strips 4 times. When washing, add 300 μL of washing buffer to each well;

3) After the strips are cleaned, add 100 μL of the prepared TGFb RIII detection antibody (Item F) to each well, and incubate at room temperature for 1 h;

4) cleaning, step is the same as 2);

5) Add 100 μL of prepared HRP-streptavidin (Item G) to each well and incubate at room temperature for 45 minutes;

6) cleaning, step is the same as 2);

7) Add 100 μL of TMB chromogenic solution (Item H) to each well, and incubate at room temperature for 30 minutes in the dark;

8) Add 50 μL of stop solution (Item I) to each well and read immediately on a microplate reader (450 nm).

9) Use sigmaplot 12.0 software to calculate the concentration value.

The obtained results are shown in Figure 3, and it can be seen that there are statistical differences in the expression levels of TGFb RIII protein among the three populations (wherein, the "**" between the healthy group and the early gastric cancer group in Figure 3 represents the statistical difference P value. less than 0.01; "**" between the early gastric cancer group and the advanced gastric cancer group means the statistical difference P value is less than 0.01; "***" between the healthy group and the advanced gastric cancer group means the statistical difference P value is less than 0.001), The ROC diagram of gastric cancer population and healthy population was further drawn, and the AUC value for distinguishing gastric cancer from healthy population can reach 0.984, and the serum protein concentration with serum value greater than 5752.909 pg/ml can be used as the critical value to distinguish the two populations, that is, this Elisa When the kit detects serum, the population with the expression level of TGFb RIII higher than 5752.909 pg/ml is the gastric cancer population.

Any embodiment of the present application can be used as an independent technical solution, or can be combined with other embodiments. All patents and publications mentioned in the specification of this application indicate that they are disclosed technology in the art and can be used in this application. All patents and publications cited herein are also incorporated by reference as if each publication was specifically and individually incorporated by reference. The application herein may be practiced in the absence of any element or elements, limitation or limitations, such limitation not specifically stated herein. The terms and expressions used herein are by way of description, not limitation, and there is no intention here to indicate that these terms and interpretations described in this book exclude any equivalent features, but it is understood that the make any suitable changes or modifications to the extent required. It can be understood that the embodiments described in this application are all embodiments and features in some embodiments, and any person of ordinary skill in the art can make some changes and changes according to the essence described in this application, and these changes and changes also It is considered to be within the scope of this application and as limited by the independent and dependent claims.

Claims (5)

1. An application of the immunoglobulin G antibody of TGFb RIII protein in preparing the diagnostic product of serum biomarker of gastric cancer.

2. Use of immunoglobulin G antibodies of TGFb RIII protein according to claim 1 in the preparation of gastric cancer serum biomarker diagnostic products characterized in that: the TGFb RIII protein is a human TGFb RIII protein.

3. Use of immunoglobulin G antibodies of TGFb RIII protein according to claim 1 in the preparation of gastric cancer serum biomarker diagnostic products characterized in that: the diagnostic product is a detection kit.

4. An application of TGFb RIII protein as a gastric cancer serum biomarker.

5. An application of TGFb RIII protein with non-diagnosis purpose as gastric cancer serum biomarker in gastric cancer judgment.

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