CN104678099B - Applications of the COX 2 in diagnosis, the diagnostic reagent of detection canine tumor is prepared - Google Patents
Applications of the COX 2 in diagnosis, the diagnostic reagent of detection canine tumor is prepared Download PDFInfo
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- CN104678099B CN104678099B CN201510094627.2A CN201510094627A CN104678099B CN 104678099 B CN104678099 B CN 104678099B CN 201510094627 A CN201510094627 A CN 201510094627A CN 104678099 B CN104678099 B CN 104678099B
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- cox
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
Abstract
The invention discloses applications of the COX 2 in diagnosis, the diagnostic reagent of detection canine tumor is prepared：The present invention pass through experimental studies have found that, COX 2 is in canine tumor (tumor of breast, skin and soft tissue neoplasm, crissum tumour, orchioncus, vagina and cervix tumor) in positive expression rate be 76.1%, and positive expression rate in the normal tissue is only 20%, therefore, COX 2 can be applied as the mark of canine tumor, detect the expression of COX 2 with immunohistochemical method, its result can as whether one of the foundation of clinical diagnosis with canine tumor.The invention also discloses kit (including the 3%H for detecting COX 22O2Solution, citrate buffer solution, PBS, the monoclonal antibodies of mouse COX 2, goat anti-mouse IgG antibody, DAB nitrite ions and distilled water) and detection method (dyeing of streptomysin avidin peroxidase).
Description
Technical field
Application the present invention relates to COX-2 as tumor markers in diagnosis, the diagnostic reagent of detection canine tumor is prepared.
Background technology
Epoxidase (cyclo-oxygen-ase, COX), enzyme necessary to being prostaglandin (PGs) synthesis is also that PGs is closed
Into the key rate-limiting enzyme in initial step.Also known as epoxide hydratase.COX has two kinds of isodynamic enzymes COX-1 and COX-2.Before
Person is referred to as key element enzyme or house keeping enzyme for structural type, is primarily present in the tissue such as blood vessel, stomach, kidney, and domestic and foreign scholars are generally recognized
For the PG that it is produced participates in body normal physiological processes and defencive function, gastrointestinal mucosa integrality, regulation blood platelet work(are such as maintained
Energy and renal hemodynamic；The latter is induction type, is through stimulating the rapid induced enzyme for producing, i.e., by various damaging chemistry, physics and life
The thing factor induces it to produce, and then is catalyzed PGs synthesis participation inflammatory reactions.
It is various in the mankind that the research of the aspect such as a large amount of zoopery, epidemiology, cytologic experiments at present has proven to COX-2
Internal tumor, the middle presence such as including stomach cancer, the cancer of the esophagus, colorectal cancer, lung cancer, prostate cancer, cancer of pancreas, carcinoma of urinary bladder, breast cancer
Overexpression, therefore COX-2 has also obtained common concern with the relation of tumour, it is now recognized that its carcinogenic mechanism mainly has：Stimulate thin
Born of the same parents propagation, suppress Apoptosis, suppress organism immune response, promote tumor angiogenesis, participate in carcinogen metabolism and
Increase invasion and transfer ability of tumour cell etc..
COX-2 is domestic without research at present with the relation of canine tumor.
The content of the invention
For above-mentioned prior art, the present invention pass through experimental studies have found that, the generation of COX-2 and canine tumor, development are present
Certain correlation, with immunohistochemical method detect COX-2 expression can as the foundation of clinical diagnosis it
One.
The present invention is achieved by the following technical solutions：
The present invention pass through experimental studies have found that, COX-2 canine tumor (tumor of breast, skin and soft tissue neoplasm, crissum swell
Knurl, orchioncus, vagina and cervix tumor) in positive expression rate be 76.1%, and positive expression rate in the normal tissue
Only 20%, therefore, COX-2 can be applied as the mark of canine tumor, and COX-2 is detected with immunohistochemical method
Expression, its result can as whether one of the foundation of clinical diagnosis with canine tumor.Although COX-2 is various swollen
In knurl express, do not reach absolute specificity, but, known in one of ordinary skill in the art, clinical detection index it is specific
It is non-it is absolutely unique can not negate Testing index as diagnosis relevant disease one of means feasibility, such as doctor often will
Final diagnosis is made in several Testing index combination clinical manifestations.Therefore, COX-2 has feasibility as tumor markers；Although
COX-2 has not exclusive specificity, but combines other Testing index and clinical manifestation also foot using COX-2 as tumor markers
For detection canine tumor (this mode is also normal method at present clinically).
Present invention also offers a kind of diagnostic kit for diagnosing, detecting canine tumor, including 3%H2O2Solution, lemon
Acid buffer, PBS, mouse COX-2 monoclonal antibodies, goat anti-mouse IgG antibody, DAB nitrite ions and distilled water.This
Invention additionally provides the method that COX-2 is detected using the diagnostic kit, and step is as follows：
(1) tissue to be detected is carried out into FFPE；Paraffin section, conventional dewaxing, aquation；
(2) 3%H2O2Solution is incubated at room temperature 10 minutes；
(3) distilled water flushing, PBS soaks 5 minutes
(4) citrate buffer solution heating using microwave repairs antigen 1 0min to 95~98 DEG C；
(5) PBS washing, plus primary antibody, is incubated 1h by 37 DEG C in wet box；
(6) PBS washing, plus secondary antibody, is incubated 0.25h by 37 DEG C in wet box；
(7) PBS washing；
(8) Fresh DAB nitrite ions are added dropwise, 20min is incubated at room temperature；
(9) distilled water is fully rinsed；
(10) redye, be dehydrated, transparent, mounting；
(11) negative control：PBS replaces primary antibody；
(12) judge whether testing result is COX-2 positive according to coloration result, the result can be to be detected as diagnosing
Whether patient belonging to tissue suffers from one of foundation of clinical diagnosis of canine tumor.Judge that testing result is COX-2 positive standard
For：COX-2 positive stainings are brown yellow granule, and specificity is located in cytoplasm and nucleus week；Seen respectively from every section
5 visuals field are examined, comprehensive distribution and staining power carry out sxemiquantitative treatment, and distribution presses following scale：It is positive thin
Born of the same parents' number is designated as 0 point for 0；1%-25% is 1 point；26%-50% is 2 points；51%-75% is 3 points；76%-100% is 4 points；Dye
Intensity of colour presses following scale：Substantially not colored is 0 point；Coloring is light, but person is 1 point to be significantly stronger than negative control；Coloring is bright
Aobvious person is 2 points；Coloring powerhouse is 3 points；Above two scoring is added, and 0~1 point is feminine gender, and 2 points or more persons are the positive.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
Involved reagent, method etc. in following embodiments, unless otherwise noted, are conventional examination of the prior art
Agent, method.
The PAD1 of embodiment 1 as tumor markers detection example
Collect postoperative tumor tissues 46 and dissected in the attached pets hospital of Zhejiang University dog normal breast, skin,
Each 3 parts of testis, crissum, uterine tissue, the fats portion in removal parenchymal tissue, is cut with surgical scissors and tweezers, point
Into size about 1cm3The small tissue blocks of left and right, are easily fixed with 10% neutral formalin.All tumours are through pathologic group
Sections observation is knitted, it is tumor of breast 25, skin and soft tissue neoplasm 11,4, crissum tumour, orchioncus 2 to clarify a diagnosis
Example, vagina and cervix tumor 4.Cancer 5, conduit papillary carcinoma 3, parenchyma type cancer 5, undifferentiated carcinoma are combined in tumor of breast
1, mucinous carcinoma 1, fibrosarcoma 3, osteosarcoma 1, compound adenoma 3, adenofibroma 1, the proliferation of mammary gland 1；Skin
Basal cell tumor 1 in skin and soft tissue neoplasm, fibroma 1, hemanyiopericytoma 1, steatadenoma 2, carcinoma of sebaceous glands 2
Example, sebaceous epithelioma 2, Trichoblastoma 1, embryonal-cell lipoma 1；Crissum adenoma 1 in crissum tumour, crissum gland cancer 3
Example；Orchioncus interstitial cells knurl 2；Fibrosarcoma 1 in vagina and cervix tumor, liomyoma 2, leiomyosarcoma
1.
The wax stone of above-mentioned organization embedding is done into another section, streptomysin avidin-peroxidase (SP) is carried out
Dyeing (the method is the conventional technical means of existing maturation in the prior art), used main agents are as follows：
Primary antibody is mouse COX-2 monoclonal antibodies：Santa Cruz companies；
Secondary antibody is goat anti-mouse IgG antibody：Abbike companies；
Phosphate buffer (PBS)：Beijing equation bio tech ltd；
Diaminobenzidine (DAB) colour reagent box：Beijing Zhong Shan Bioisystech Co., Ltd；
SP immunohistochemical kits：Beijing Zhong Shan Bioisystech Co., Ltd.
Method and step is as follows：
(1) by the dewaxing of paraffin section routine, aquation；
(2) 3% (mass percent) H2O2Solution is incubated at room temperature 10 minutes；
(3) distilled water flushing, PBS (pH7.4) soaks 5 minutes
(4) citrate buffer solution (pH6.0) heating using microwave repairs antigen 1 0min to 95~98 DEG C；
(5) PBS washing, plus primary antibody, is incubated 1h by 37 DEG C in wet box；
(6) PBS washing, plus secondary antibody, is incubated 0.25h by 37 DEG C in wet box；
(7) PBS washing；
(8) Fresh DAB nitrite ions are added dropwise, 20min is incubated at room temperature；
(9) distilled water is fully rinsed；
(10) redye, be dehydrated, transparent, mounting；
(11) negative control：PBS replaces primary antibody.
The normal breast, skin, testis, crissum, uterine tissue of this experimental dog are used as check experiment.Positive findings is sentenced
It is fixed：COX-2 positive stainings are brown yellow granule, and specificity is located in cytoplasm and nucleus week.Seen respectively from every section
5 visuals field are examined, comprehensive distribution and staining power carry out sxemiquantitative treatment, and distribution presses following scale：It is positive thin
Born of the same parents' number is designated as 0 point for 0；1%-25% is 1 point；26%-50% is 2 points；51%-75% is 3 points；76%-100% is 4 points.Dye
Intensity of colour presses following scale：Substantially not colored is 0 point；Coloring is light, but person is 1 point to be significantly stronger than negative control；Coloring is bright
Aobvious person is 2 points；Coloring powerhouse is 3 points.Above two scoring is added, and 0~1 point is feminine gender, and 2 points or more persons are the positive, wherein 2
It is divided into (+), 3~6 points is (+++).Data analysis, the expression ratio of difference group COX-2 positive rates are carried out using SPSS13.0 softwares
Relatively use X2Inspection, P ＜ 0.05 are statistically significant.
Result shows：Positive expressions of the COX-2 in canine tumor is predominantly located in the endochylema of tumour cell and around karyon,
It is brown yellow granule.30 COX-2 are expressed in strong positive in 46 canine tumors, 5 positive expressions, and positive expression rate is
76.1% (35/46)；The positive expression rate of COX-2 is 20% (3/15) in 15 parts of normal structures.COX-2 is in canine tumor and normally
Structural differential expression is statistically significant (P ＜ 0.01).In 46 tumours for carrying out immunohistochemical staining, 19 benign swollen
15 COX-2 are expressed in strong positive in knurl, and positive expression rate is 78.95%；15 COX-2 are in strong positive in 27 malignant tumours
Expression, 5 positive expressions, positive expression rate is 74.1%.COX-2 is dog is good, the expression in malignant tumour is anticipated without statistics
Adopted (P ＞ 0.05).
Conclusion：Research above shows that expressions of the COX-2 in canine tumor is more obvious than normal structure to be increased, therefore,
COX-2 can (tumor of breast, skin and soft tissue neoplasm, crissum tumour, orchioncus, vagina and uterus be swollen as canine tumor
Knurl) diagnostic marker.It is the expression of detectable COX-2 with immunohistochemical method during practical application, its result can
As clinical diagnosis according to one of pick.
Claims (1)
- Applications of the 1.COX-2 in diagnosis, the diagnostic reagent of detection canine tumor is prepared, the canine tumor is hemanyiopericytoma.
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