CN110763845B

CN110763845B - Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit

Info

Publication number: CN110763845B
Application number: CN201810844452.6A
Authority: CN
Inventors: 刘蕾; 郭元彪; 贺巧; 李砚
Original assignee: No 3 Peoples Hospital of Chengdu
Current assignee: No 3 Peoples Hospital of Chengdu
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2022-10-28
Anticipated expiration: 2038-07-27
Also published as: CN110763845A

Abstract

The present disclosure relates to the use of a ligand for detecting a protein marker in the preparation of a product for diagnosing colon cancer and a kit. According to the method, through detecting the SYPL1 protein in the sample, a patient suspected of suffering from colon cancer can be effectively diagnosed, the accuracy and specificity of the method are far higher than those of a detection result of a traditional tumor marker, and the method is favorable for finding colon cancer early in clinic so as to improve the survival quality and survival rate of the patient.

Description

Application of ligand for detecting protein marker in preparation of product for diagnosing colon cancer and kit

Technical Field

The disclosure relates to the field of medical biotechnology, in particular to application of a ligand for detecting a protein marker in preparation of a product for diagnosing colon cancer and a kit for diagnosing colon cancer.

Background

Colon cancer is one of the most common malignancies, and the incidence and mortality of colon cancer has increased year by year in recent years. Because early symptoms of the colon cancer are hidden, most colon cancer patients reach late stage when the diagnosis is confirmed, and the survival rate is below 30.0 percent, the clinical early discovery and early diagnosis have important significance for improving the survival quality and the survival rate of the patients.

The tumor marker refers to a substance which is released into blood and body fluid after being generated or secreted by tumor cells in the processes of generating and proliferating tumors and can reflect the existence and growth of the tumors. Due to the characteristics of economy, convenience, rapidness and the like, the serum tumor marker plays an important role in screening, auxiliary diagnosis, disease condition evaluation and diagnosis and treatment effect evaluation of cancers. At present, the serum tumor markers commonly used for auxiliary diagnosis of colon cancer comprise carcinoembryonic antigen (CEA), carbohydrate chain antigen 19-9 (CAl 9-9), carbohydrate chain antigen 242 (CA 242), carbohydrate chain antigen 50 (CA 50) and the like, but the currently discovered serum tumor markers generally have the limitations of lower sensitivity and specificity, and a single marker has obvious defects in the diagnosis of malignant tumors.

For example, CEA is the most commonly used serum tumor marker for assisting colon cancer screening and diagnosis, but more and more studies report that CEA is a very limited serum marker for colon cancer. CEA is a nonspecific marker, and the increase of serum level can be found in various malignant tumors, including breast cancer, lung cancer, gastrointestinal tumors, etc.; in addition, many non-malignant diseases can also lead to elevated CEA levels, such as cirrhosis, gastritis, inflammatory bowel disease, etc., and even serum CEA levels in smokers (especially in males) can be elevated, so the specificity of CEA remains to be improved. CEA has no role in colorectal cancer screening in asymptomatic patients, and thus it lacks sensitivity and specificity for early disease.

Disclosure of Invention

The purpose of the present disclosure is to overcome the defect of low sensitivity and specificity of the existing molecular marker for colon cancer diagnosis, and to improve the sensitivity and specificity of the molecular marker for colon cancer diagnosis.

In order to achieve the above objects, in one aspect, the present disclosure provides a use of a ligand for detecting a protein marker in the preparation of a product for diagnosis of colon cancer, wherein the protein marker is SYPL1 protein.

In another aspect, the present disclosure also provides a kit for colon cancer diagnosis, wherein the kit comprises a ligand for detecting a protein marker including SYPL1 protein.

By the technical scheme, the SYPL1 protein in the sample is detected, so that a patient suspected of suffering from colon cancer can be effectively diagnosed, the accuracy and specificity of the detection are far higher than those of the detection result of the traditional tumor marker, the colon cancer can be found early in clinic to improve the survival quality and survival rate of the patient, and therefore the serum tumor marker or the marker combination for assisting in judging the colon cancer can be developed, and the colon cancer can be effectively predicted in clinic.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 is the concentration of SYPL1 in the serum of healthy persons and colon cancer patients in an embodiment of the present disclosure.

FIG. 2 is a ROC curve of serum SYPL1 concentration for diagnosing colon cancer in embodiments of the present disclosure.

FIG. 3 shows the expression of SYPL1 molecules in colon cancer tissue and paracarcinoma tissue in an embodiment of the present disclosure.

FIG. 4 is a ROC curve for tissue SYPL1 score diagnosis of colon cancer in embodiments of the present disclosure.

FIG. 5 is SYPL1mRNA expression levels in colon cancer tissue and paracarcinoma tissue in embodiments of the present disclosure.

FIG. 6 shows the expression of SYPL1 molecules in colon epithelial cell lines and colon cancer cell lines in an embodiment of the present disclosure.

Detailed Description

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

In one aspect, the present disclosure provides a use of a ligand for detecting a protein marker in the preparation of a product for determining prognosis of colon cancer, wherein the protein marker is SYPL1 protein

The SYPL1 protein is a synaptophysin-like protein 1 (SYPL1), also known as a pantophysin, whose gene is located at 7q22.3 and has two transcripts, of which transcript 1 contains 6 exons and encodes 259 amino acids; transcript 2 contains 5 exons, encoding 241 amino acids; both protein isoforms contain MARVEL domains. SYPL1 belongs to a member of the synaptophysin/synaptophysin family, has transporter activity and is generally expressed in the cell membrane. SYPL1, although originally reported as a protein associated with neuroendocrine-specific synaptophysin, was recently found to be present in non-neural tissues as well. The SYPL1 protein is referred to in NCBI databases under the reference numbers NP-006745.1, NP _874384.1; the reference number of the SYPL1 gene in NCBI database is 6856.

According to the present disclosure, the SYPL1 protein has the following characteristics:

the concentration in the serum of colon cancer patients is relatively high;

the expression level in tumor tissue of a colon cancer patient is higher than that in para-carcinoma tissue;

the expression level in colon cancer cell strain is higher than that of colon epithelial cell strain.

Optionally, wherein the ligand is a ligand that specifically binds to a SYPL1 protein for detection of the protein, e.g., may be a nucleic acid and/or polypeptide. Wherein the polypeptide comprises an antibody, such as at least one selected from the group consisting of a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, and a polyclonal antibody. Antibodies can be produced using standard techniques well known to those skilled in the art, or commercially available antibodies can be used. Further, the monoclonal antibody may be a monoclonal antibody of abcam, cat # ab184176.

Preferably, wherein the monoclonal antibody is a monoclonal antibody of abcam, cat # ab184176.

According to the present disclosure, detecting a protein marker is performed by:

1) Obtaining a serum sample or a colon tissue sample of a patient to be tested; and

2) Determining the protein expression level or mRNA expression level of the protein marker in the sample.

Wherein, the method in the step 2) is an enzyme-linked immunosorbent assay method or a colloidal gold test paper assay method or a PCR assay method.

The presence of a protein marker can be quantitatively detected, for example, using immunoassay methods. The immunoassay typically involves incubating the biological sample with an antibody and detecting the bound antibody by techniques well known to those skilled in the art, such as immunohistochemical experiments (IHC).

In one embodiment of the present disclosure, the method of preparing a sample, the IHC method and the positive judgment criteria are as follows:

(1) Preparing a sample:

putting the in vitro surgical tissue within 30min into neutral formalin fixing solution for fixing overnight, washing with running water, dehydrating, transparentizing and waxing to prepare tissue wax block. The histopathology was judged as a case of stage II colon cancer. 3 typical cancer tissue spots and 2 normal paracancerous tissue spots were each selected, a tissue microarray was prepared, and 4 μm sections were prepared.

(2) And (3) IHC: the specific detection method of the protein is as follows:

placing the tissue chip in a 65 ℃ baking oven facing the wind on the front side for baking for 40min to enable the specimens to be better attached, soaking the tissue chip in xylene washing cylinders I, II and III, respectively placing the tissue chip in a 25 ℃ constant-temperature water bath for 10min, transferring the tissue chip into 100%, 85% and 75% ethanol for gradient hydration for 3min respectively, then transferring the tissue chip into PBS buffer solutions I, II and III, placing the tissue chip in a 3% hydrogen peroxide washing cylinder for 15min every 5min, and sealing endogenous peroxidase. Soaking in heated sodium citrate buffer solution, and microwave drying for 20min. Immersing in PBS buffer I, II, III for 3min each time. The range of the PAP Pen is drawn along the edge of the tissue chip by the PAP Pen, the diluted primary antibody solution or the diluted antibody working solution is paved on the surface of the tissue array within the drawn range by a 200 mu L microsyringe and is placed in a wet box, and the tissue array is incubated in a constant temperature incubator at 37 ℃ for 2h or overnight in a refrigerator at 4 ℃. Excess primary antibody was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Adding reagent II in the two-step immunohistochemical detection kit to ensure that the surface of the tissue array in the drawn range is fully paved and placed in a wet box, and incubating for 15min in a constant temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Adding reagent III (Polyperoxidase-anti-mouse/rabbitIgG) in the two-step immunohistochemical detection kit, ensuring that the surface of the tissue array within the drawn range is fully paved, placing the tissue array in a wet box, and incubating for 30min in a constant-temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Taking a proper amount of reagent from the DAB kit, and uniformly mixing according to the proportion of 1mLDAB diluent to 50 mu LDAB concentrated solution; spreading the diluted DAB solution on the surface of the tissue array within the drawn range by using a 200 mu L microsyringe for color development, and keeping the color development time of the same chip consistent; observing the color development effect under a mirror until the interior of the same chip and different chips form obvious contrast, removing the redundant DAB solution on a paper towel and placing the paper towel in distilled water; the development time was 60s, and the development time was different between different antibodies. Spreading hematoxylin staining solution on the surface of the tissue array within the range to be drawn rapidly by a 1000 mu L microsyringe, and immediately flushing the surface by slow running water after 10 s; the stained chip was placed in 1% ammonia water. The tissue chip is immersed in 75%, 85% and 100% ethanol for 3min in sequence for gradient dehydration. Immersing in xylene washing jar, washing for 10min, removing the handwriting drawn by PAP Pen immunohistochemistry fossil wax Pen, air drying the tissue chip in ventilation place, and sealing with quick-drying sealing agent.

(3) Positive judgment standard:

20 high-power fields of immunohistochemical stained cells were counted, and the staining intensity was integrated as: no dyeing is divided into 0, weak dyeing is divided into 1, medium dyeing is divided into 2, and strong dyeing is divided into 3; the integrated dyeing area is as follows: the coloring range is less than or equal to 10% and is 0, more than 10% -25% and 1, more than 25% -50% and 2, more than 50% -75% and 3, and more than 75% and 4. If the sum of the integral of the two is more than or equal to 3 points, the product is positive, and if the sum is less than 3 points, the product is negative.

When the test result is positive, the patient is indicated to have a better prognosis.

In another aspect, the present disclosure also provides a kit for diagnosis of colon cancer, wherein the kit comprises a ligand that detects a protein marker comprising SYPL1 protein.

According to the present disclosure, the ligand is a ligand that can specifically bind to a SYPL1 protein for detection of the protein, e.g., may be a nucleic acid and/or a polypeptide. Wherein the polypeptide comprises an antibody, such as at least one selected from the group consisting of a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, and a polyclonal antibody. Antibodies can be produced using standard techniques well known to those skilled in the art, or commercially available antibodies can be used. Further, the monoclonal antibody may be a monoclonal antibody of abcam, cat # ab184176.

The SYPL1 protein in a sample is jointly detected by using the ligand, so that a patient suspected of suffering from colon cancer can be effectively diagnosed, and the accuracy and the specificity of the method are far higher than those of the detection result of the traditional tumor marker. The kit is favorable for discovering colon cancer early in clinic to improve the life quality and the survival rate of patients, so that a serum tumor marker or a marker combination which can be used for assisting in judging the colon cancer can be developed, and the colon cancer can be effectively predicted in clinic.

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

In the examples, an antibody test reagent for detecting SYPL1 protein was purchased from abcam, inc. under the trade name ab184176.

Examples

Example 1:

the levels of SYPL1 protein in the sera of the population were determined using ELISA on serum samples from 98 colorectal cancer patients and from 69 healthy volunteers and statistically analyzed.

The results show that the concentration of SYPL1 in the serum of colon cancer patients is 144.3 +/-5.72 ng/mL, the concentration in the serum of healthy control population is 42.53 +/-3.19 ng/mL, the content of SYPL1 in the serum of colon cancer patients is obviously higher than that of the healthy control group, and the difference has statistical significance (P < 0.0001), as can be seen in figure 1.

An ROC curve was plotted based on the serum SYPL1 levels of the colon cancer group and the healthy control group, and as can be seen with reference to FIG. 2, the area under the curve was 0.97, the cutoff value was 63.75ng/mL (95% by volume CI was 0.948 to 0.992, P < -0.001), the sensitivity for diagnosing colon cancer was 92.6%, and the specificity was 90%. The results show that SYPL1 is a colon cancer serum marker with high sensitivity and specificity, and can be developed into a serological screening and diagnosis marker of colon cancer.

Example 2:

the expression of SYPL1 molecules in two groups of tissues was examined by immunohistochemical staining of 344 tumor tissues from colon cancer patients and the corresponding paracarcinoma tissues from 308 colon cancer patients and scoring the staining.

The scoring method is as follows:

the staining positive rates are respectively marked as follows: no dyeing is 0min, less than or equal to 10% is 1 min, more than 10% -50% is 2 min, more than 50% is 3 min; according to the staining intensity, the staining intensity is respectively marked as: no dyeing is divided into 0, weak dyeing is divided into 1, medium dyeing is divided into 2, and strong dyeing is divided into 3; the final score is the positive rate of staining score multiplied by the intensity of staining score.

The results showed that the SYPL1 molecule scored 7.36. + -. 0.13 in tumor tissue and 4.19. + -. 0.16 in para-carcinoma tissue, the expression level of SYPL1 molecule was significantly higher in tumor tissue of colon cancer patients than in para-carcinoma tissue, with a statistically significant difference (P < 0.0001), as shown in FIG. 3.

An ROC curve was plotted based on the expression of SYPL1 in tumor tissues and paracancerous tissues, and as can be seen with reference to FIG. 4, the area under the curve was 0.789, the cutoff value was 5 points (95% by weight CI was 0.754 to 0.824, P- < -0.001), and the sensitivity for diagnosing colon cancer was 85.5% and the specificity was 59.1%.

The above results indicate that SYPL1 is not only a reliable serum marker for colon cancer, but also a more reliable biomarker for colon cancer histon.

Example 3:

SYPL1mRNA level was detected by qPCR in tumor tissue and corresponding paracancerous tissue of 15 patients with colon cancer surgery, with β -actin as an internal reference. As can be seen in FIG. 5, the mean expression level of mRNA of SYPL1 molecule in tumor tissues of colon cancer patients was significantly higher than that of the corresponding paracarcinoma tissues, and the paired t-test results showed that the two groups of differences were statistically significant (P < 0.05).

The experimental results show that SYPL1 may be a potential nucleic acid biomarker of colon cancer tissues.

Example 4:

expression of SYPL1 protein was detected by Western blotting in 7 colon cancer cell lines (including Colo205 cell line, HCT116 cell line, lovo cell line, RKO cell line, SW1116 cell line, SW480 cell line, SW620 cell line) and colon epithelial cell line (NCM 460 cell line). The results show that the SYPL1 molecule can not detect obvious protein expression in the colonic epithelial cell line NCM460, but has expression in a plurality of colon cancer cell lines, and as can be seen in reference to FIG. 6, the expression level of the SYPL1 molecule in the plurality of colon cancer cell lines is obviously higher than that of the colonic epithelial cell line. SYPL1 is expected to be developed into a new colon cancer auxiliary diagnosis biomarker.

The preferred embodiments of the present disclosure are described in detail above with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details in the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure as long as it does not depart from the gist of the present disclosure.

Claims

1. Use of a ligand for detecting a protein marker in the manufacture of a product for use in the diagnosis of colon cancer, wherein the protein marker is a SYPL1 protein.

2. The use according to claim 1, wherein the ligand is a nucleic acid and/or a polypeptide comprising an antibody which is at least one selected from the group consisting of a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody and a polyclonal antibody.

3. The use according to claim 2, wherein the monoclonal antibody is a monoclonal antibody from abcam, cat ab184176.

4. The use according to claim 1, wherein detecting a protein marker is performed by:

2) Determining the protein expression level or the mRNA expression level of the protein marker in the sample.

5. The use of claim 4, wherein the method of step 2) is enzyme-linked immunosorbent assay method or colloidal gold test paper assay method or PCR assay method.