CN104678099B - Applications of the COX 2 in diagnosis, the diagnostic reagent of detection canine tumor is prepared - Google Patents

Abstract

The invention discloses the application of COX-2 in the preparation of diagnostic reagents for diagnosing and detecting canine tumors: the present invention finds through experimental research that COX-2 is effective in canine tumors (breast tumors, skin and soft tissue tumors, perianal tumors, testicular tumors, The positive expression rate in vaginal and uterine tumors) was 76.1%, while the positive expression rate in normal tissues was only 20%. Therefore, COX‑2 can be used as a marker for canine tumors. COX can be detected by immunohistochemistry ‑2 expression, the results can be used as one of the basis for the clinical diagnosis of canine tumors. The invention also discloses a kit for detecting COX-2 (including 3% H ₂ O ₂ solution, citrate buffer, PBS buffer, mouse COX-2 monoclonal antibody, goat anti-mouse IgG antibody, DAB Chromogenic solution and distilled water) and detection method (streptavidin-peroxidase staining).

Description

Application of COX-2 in preparation of diagnostic reagents for diagnosing and detecting canine tumors

technical field

The invention relates to the application of COX-2 as a tumor marker in the preparation of diagnostic reagents for diagnosing and detecting canine tumors.

Background technique

Cyclo-oxygen-ase (COX) is an essential enzyme for the synthesis of prostaglandins (PGs), and it is also a key rate-limiting enzyme in the initial step of PGs synthesis. Also known as epoxide hydrase. COX has two isoenzymes COX-1 and COX-2. The former is a structural type called element enzyme or housekeeping enzyme, which mainly exists in blood vessels, stomach, kidney and other tissues. Scholars at home and abroad generally believe that the PG produced by it participates in the normal physiological process and protective functions of the body, such as maintaining the integrity of the gastrointestinal mucosa The latter is inducible, which is an inducible enzyme that is rapidly produced by stimulation, that is, its production is induced by various damaging chemical, physical and biological factors, and then catalyzes the synthesis of PGs to participate in the inflammatory response.

At present, a large number of animal experiments, epidemiology, cytology experiments and other studies have confirmed that COX-2 plays a role in various human visceral tumors, including gastric cancer, esophageal cancer, colorectal cancer, lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer, etc. There is overexpression in cancer, etc., so the relationship between COX-2 and tumors has also received widespread attention. At present, its carcinogenic mechanisms are mainly considered to be: stimulating cell proliferation, inhibiting cell apoptosis, inhibiting the body's immune response, promoting tumor angiogenesis, participating in Metabolism of carcinogens and increase the invasiveness and metastasis of tumor cells.

There is no domestic research on the relationship between COX-2 and canine tumors.

Contents of the invention

In view of the above-mentioned prior art, the present invention finds through experimental research that there is a certain correlation between COX-2 and the occurrence and development of canine tumors, and the detection of the expression of COX-2 by immunohistochemistry can be used as one of the basis for clinical diagnosis .

The present invention is achieved through the following technical solutions:

The present invention finds through experimental research that the positive expression rate of COX-2 in canine tumors (mammary gland tumors, skin and soft tissue tumors, perianal tumors, testicular tumors, vagina and uterine tumors) is 76.1%, while the positive expression rate in normal tissues is 76.1%. The expression rate is only 20%. Therefore, COX-2 can be used as a marker for canine tumors. The expression of COX-2 can be detected by immunohistochemical methods, and the results can be used as one of the basis for clinical diagnosis of canine tumors. one. Although COX-2 is expressed in a variety of tumors and cannot achieve absolute specificity, it is well known to those skilled in the art that the non-absolute uniqueness of the specificity of clinical detection indicators cannot negate the detection indicators as one of the means for diagnosing related diseases Feasibility, for example, doctors often combine several detection indicators with clinical manifestations to make a final diagnosis. Therefore, COX-2 is feasible as a tumor marker; although COX-2 has non-exclusive specificity, it is sufficient to use COX-2 as a tumor marker in combination with other detection indicators and clinical manifestations for the detection of canine tumors (this It is also a common way in clinical practice).

The present invention also provides a diagnostic kit for diagnosing and detecting canine tumors, including 3% H ₂ O ₂ solution, citrate buffer, PBS buffer, mouse COX-2 monoclonal antibody, goat anti-mouse IgG antibody, DAB chromogenic solution and distilled water. The present invention also provides a method for detecting COX-2 using the diagnostic kit, the steps are as follows:

(1) Embedding the tissue to be tested in paraffin; paraffin sections, routine dewaxing and hydration;

(2) 3% H ₂ O ₂ solution was incubated at room temperature for 10 minutes;

(3) Rinse with distilled water, soak in PBS buffer for 5 minutes

(4) Microwave heating of citrate buffer to 95-98°C for antigen repair for 10 minutes;

(5) Wash with PBS buffer, add primary antibody, and incubate at 37°C for 1 hour in a humid box;

(6) Wash with PBS buffer, add secondary antibody, and incubate in a wet box at 37°C for 0.25h;

(7) Washing with PBS buffer solution;

(8) Add freshly prepared DAB chromogenic solution dropwise, and incubate at room temperature for 20 minutes;

(9) Fully rinse with distilled water;

(10) Counterstaining, dehydration, transparency, and sealing;

(11) Negative control: PBS instead of primary antibody;

(12) Judging whether the test result is positive for COX-2 according to the staining result, the result can be used as one of the clinical diagnosis basis for diagnosing whether the patient belonging to the tissue to be tested has canine tumor. The criteria for judging that the test result is positive for COX-2 are as follows: positive staining of COX-2 is brownish-yellow granules, which are specifically located in the cytoplasm and around the nucleus; 5 fields of view are observed from each section, and the comprehensive distribution range and staining intensity are analyzed. For semi-quantitative processing, the distribution range is scored according to the following criteria: 0 points for the number of positive cells; 1 point for 1%-25%; 2 points for 26%-50%; 3 points for 51%-75%; 3 points for 76% -100% is 4 points; the staining intensity is scored according to the following criteria: basically no staining is 0 points; light staining, but obviously stronger than the negative control is 1 point; obvious staining is 2 points; strong staining is 3 points; the above Adding the two scores, 0 to 1 is negative, and 2 or more is positive.

detailed description

The present invention will be further described below in conjunction with embodiment.

Reagents, methods, etc. involved in the following examples, unless otherwise specified, are conventional reagents and methods in the prior art.

Example 1 Detection example of PAD1 as a tumor marker

Collect 46 cases of postoperative tumor tissue and 3 normal canine mammary gland, skin, testis, perianal, and uterine tissues dissected in the Animal Hospital Affiliated to Zhejiang University, remove the fatty part of the parenchymal tissue, and cut with surgical scissors and tweezers. Divide into small tissue pieces about 1cm ³ in size, and fix them easily with 10% neutral formalin. All tumors were observed by pathological tissue sections, and there were 25 cases of breast tumors, 11 cases of skin and soft tissue tumors, 4 cases of perianal tumors, 2 cases of testicular tumors, and 4 cases of vaginal and uterine tumors. Among breast tumors, there were 5 cases of compound carcinoma, 3 cases of ductal papillary carcinoma, 5 cases of solid carcinoma, 1 case of undifferentiated carcinoma, 1 case of mucinous carcinoma, 3 cases of fibrosarcoma, 1 case of osteosarcoma, 3 cases of compound adenoma, fibrosarcoma 1 case of adenoma, 1 case of mammary gland hyperplasia; 1 case of basal cell tumor, 1 case of fibroma, 1 case of perivascular cell tumor, 2 cases of sebaceous adenoma, 2 cases of sebaceous gland carcinoma, 2 cases of sebaceous gland epithelioma, 1 case of blastoma and 1 case of liposarcoma; 1 case of perianal adenoma and 3 cases of perianal adenocarcinoma among perianal tumors; 2 cases of interstitial cell tumor among testicular tumors; 1 case of fibrosarcoma among vaginal and uterine tumors, smooth There were 2 cases of fibroids and 1 case of leiomyosarcoma.

The above-mentioned tissue-embedded wax block is made into another slice, and streptavidin-peroxidase (SP) staining (this method is a mature conventional technical means in the prior art), used The main reagents are as follows:

The primary antibody is mouse COX-2 monoclonal antibody: Santa Cruz Company;

The secondary antibody is goat anti-mouse IgG antibody: Abbike company;

Phosphate buffer saline (PBS): Beijing Equation Biotechnology Co., Ltd.;

Diaminobenzidine (DAB) Chromogenic Kit: Beijing Zhongshan Biotechnology Co., Ltd.;

SP immunohistochemistry kit: Beijing Zhongshan Biotechnology Co., Ltd.

The method steps are as follows:

(1) Routinely dewax and hydrate the paraffin sections;

(2) 3% (mass percentage) H ₂ O ₂ solution was incubated at room temperature for 10 minutes;

(3) Rinse with distilled water, soak in PBS buffer (pH7.4) for 5 minutes

(4) Microwave heating of citric acid buffer solution (pH6.0) to 95-98°C for 10 minutes to repair the antigen;

(5) Wash with PBS buffer, add primary antibody, and incubate at 37°C for 1 hour in a humid box;

(6) Wash with PBS buffer, add secondary antibody, and incubate in a wet box at 37°C for 0.25h;

(7) Washing with PBS buffer solution;

(8) Add freshly prepared DAB chromogenic solution dropwise, and incubate at room temperature for 20 minutes;

(9) Fully rinse with distilled water;

(10) Counterstaining, dehydration, transparency, and sealing;

(11) Negative control: PBS instead of primary antibody.

In this experiment, normal mammary gland, skin, testis, perianal and uterine tissues of dogs were used as control experiments. Judgment of positive results: COX-2 positive staining is brownish-yellow granules, which are specifically located in the cytoplasm and perinucleus. Observe 5 fields of view from each section, conduct semi-quantitative processing based on the comprehensive distribution range and staining intensity, and score the distribution range according to the following criteria: 0 points for the number of positive cells; 1 point for 1%-25%; 1 point for 26% -50% is 2 points; 51%-75% is 3 points; 76%-100% is 4 points. Staining intensity is scored according to the following criteria: basically no staining is 0 point; light staining, but obviously stronger than the negative control is 1 point; obvious staining is 2 points; strong staining is 3 points. The above two scores are added together, 0-1 points are negative, 2 points or above are positive, of which 2 points are (+), and 3-6 points are (+++). SPSS13.0 software was used for data analysis, and the expression of COX- ² positive rate in different groups was compared by X2 test, P<0.05 was considered statistically significant.

The results showed that the positive expression of COX-2 in canine tumors was mainly located in the cytoplasm and around the nucleus of the tumor cells, and it was brownish-yellow granules. Among the 46 canine tumors, 30 cases showed strong positive expression of COX-2, 5 cases showed positive expression, and the positive expression rate was 76.1% (35/46); the positive expression rate of COX-2 in 15 normal tissues was 20% (3 /15). The expression of COX-2 in canine tumors and normal tissues was significantly different (P<0.01). Among the 46 tumors with immunohistochemical staining, 15 of the 19 benign tumors showed strong positive expression of COX-2, and the positive expression rate was 78.95%; 15 of the 27 malignant tumors showed strong positive expression of COX-2, and 5 It was positively expressed, and the positive expression rate was 74.1%. The expression of COX-2 in canine benign and malignant tumors was not statistically significant (P>0.05).

Conclusion: The above studies show that the expression level of COX-2 in canine tumors is significantly higher than that in normal tissues. tumor) diagnostic markers. In practical application, the expression of COX-2 can be detected by immunohistochemical method, and the result can be used as one of the basis for clinical diagnosis.

Claims (1)

1. The application of COX-2 in the preparation of diagnostic reagents for diagnosing and detecting canine tumors, the canine tumors being hemangiopericytoma.