CN107315090A - Asparagine endopeptidase combines Integrin α_5 and integrin β_1 as the purposes of diagnostic reagent - Google Patents
Asparagine endopeptidase combines Integrin α_5 and integrin β_1 as the purposes of diagnostic reagent Download PDFInfo
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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
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
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- G—PHYSICS
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- 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
- G01N33/57488—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 involving compounds identifable in body fluids
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- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
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- G01N2333/914—Hydrolases (3)
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- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96466—Cysteine endopeptidases (3.4.22)
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Abstract
The invention provides the purposes of a kind of asparagine endopeptidase joint Integrin α_5 and integrin β_1 in preparing diagnosis ovarian epithelial carcinoma or diagnosing the reagent of ovarian epithelial carcinoma outcome.The present invention identifies AEP by immunofluorescence common location can be with Integrin α_5 and β 1 in people epithelial ovarian cancer cell SKOV3 and human mesothelial cells HPMC formation complex.Meanwhile, Elisa technology for detection finds the asparagine endopeptidase in the excretion body in epithelial ovarian carcinoma patients serum and ascites source（AEP）And the expression quantity in the excretion body originated compared with benign ovarian tumor patients serum of Integrin α_5 and β 1 expression quantity and the excretion body in liver cirrhosis ascites source is high.It follows that asparagine endopeptidase（AEP）And Integrin α_5 and β 1 and ovarian epithelial carcinoma and prognosis it is closely related.
Description
Technical field
The invention belongs to biomedical sector, it is related to asparagine endopeptidase（AEP）And Integrin α_5 and β 1, specifically
It is asparagine endopeptidase（AEP）Joint Integrin α_5 and β 1 are in the reagent for preparing diagnosis ovarian epithelial carcinoma outcome
Purposes.
Background technology
Ovarian epithelial carcinoma（Epithelial ovarian cancer, EOC）It is one of gynaecology's common cancer, extremely
It is the first that the rate of dying occupies gynecological tumor, has been FIGO III or IV phases by stages when nearly 70% patient makes a definite diagnosis, it is mainly shown as abdomen
Film plantation and transfer extensively.Peritonaeum has the feature of chronic " peritonitis ".Ovarian epithelial carcinoma prognosis is poor, how effectively to carry
The problem of prognosis of high epithelial ovarian carcinoma patients is to be solved at present.
Asparagine endopeptidase（Aasparaginylendopeptidase, AEP）, also known as legumain
（legumain）, it is the member of cysteine proteinase C13 families.The people AEP assignments of genes gene mapping are in 14q32.1, by 13 intrones
And 14 extrons are constituted, with well-conserved.AEP has three kinds of forms：Unactivated AEP, activation AEP and activate AEP completely.
AEP activation and the stability of conformation are intensified by sugar in microenvironment, pH value and oxidation-reduction potential etc. are influenceed.AEP is just
It is less in normal tissue to express or do not express, however, thin in the related macrophage of many solid tumor tissues, lymthoma and tumour
Born of the same parents（Tumor-associated macrophages,TAM）Middle expression is increased, the generation development of AEP and malignant tumour and disease
The prognosis of people is closely related.AEP is existed in tumor microenvironment, antigen protein can be processed and offered, and activates matrix
Release of the newborn factor of metalloproteinases, modulate tumor associated macrophages and modulating vascular etc..
Correlative studys of the AEP in ovarian epithelial carcinoma is actually rare, and functional experiment stage, mechanism are predominantly stayed at present
Research is indefinite, and because of ovarian epithelial carcinoma, the incidence of disease and the death rate are all very high in female tumor, early diagnoses early treatment energy
Patient's prognosis is enough improved, therefore, further investigation AEP is expected to the novel targets as treatment of ovarian cancer.
Integrin, is by α（αsubunit, 120-170 kDa）It is sub- single with β (β subunit, 90-100 kDa) two
The glycoprotein of position composition.Relative association of integrins expression is in ovarian epithelial carcinoma surface, and directing epithelial ovarian cancer cell is for extracellular machine
The adhesion of system.Integrin is mediated cell and cell, cell and the important medium mutually exchanged between extracellular matrix.Many cancers are thin
The integrin of cellular expression can adjust growth, hyperplasia, adhesion and transfer of cell etc..Integrin α_5 can mutually be tied with the subunits of β 1
Close.Epithelial ovarian cancer cell can be integrated plain α 5 for the adhesive attraction of Peritoneal Mesothelial Cells and β 1 is adjusted.There is document
The integrin formation compound that AEP can be with tumor cell surface is reported, integrin can promote AEP activation, so as to increase
Its strong biological function.Therefore, further investigation Integrin α_5 and β 1 help to explore the new mechanism of EOC peritonaeums transfer.
Excretion body, diameter, can be in biologies such as intercellular trafficking DNA, RNA, lipid and protein between 30-100nm
Credit.Excretion body can realize that tumour cell and the endothelial cell in tumor microenvironment, mesothelial cell, immunocyte etc. are a variety of
The signal communication of cell.Many documents have been reported that excretion body is largely present in the ascites of EOC patient, and the normal companions of progressive stage EOC
Formed with ascites.Therefore, for asparagine endopeptidase in the excretion body that we originate from EOC patients serums and ascites（AEP）
And Integrin α_5 and β 1 are detected, and therefrom explore its correlation with EOC patient's prognosis.
The content of the invention
It is an object of the invention to provide a kind of asparagine endopeptidase（AEP）Joint Integrin α_5 and integrin β_1 are in system
Purposes in the reagent of standby diagnosis ovarian epithelial carcinoma or diagnosis ovarian epithelial carcinoma outcome, described this purposes will
Solve the reagent and the not good technical problem of method effect of diagnosis ovarian epithelial carcinoma prognosis in the prior art.
The invention provides a kind of asparagine endopeptidase（AEP）Joint Integrin α_5 and integrin β_1 are on diagnosis is prepared
Purposes in the reagent of skin oophoroma or diagnosis ovarian epithelial carcinoma outcome.
It is of the invention to be drawn by lot of experiments, joint-detection asparagine endopeptidase（AEP）And Integrin α_5 and β 1
Prognosis with ovarian epithelial carcinoma is closely related.Specifically, the present invention by immunofluorescence common location identify AEP can with it is whole
Element α 5 and β 1 is closed in people epithelial ovarian cancer cell SKOV3 and human mesothelial cells HPMC formation complex.Meanwhile, Elisa
Technology for detection finds the asparagine endopeptidase in the excretion body in epithelial ovarian carcinoma patients serum and ascites source（AEP）And it is whole
It is outer that the excretion body and liver cirrhosis ascites that conjunction element α 5 and β 1 expression quantity is originated compared with benign ovarian tumor patients serum are originated
The expression quantity secreted in body is high.
The present invention is compared with prior art, and its technological progress is positive and effective.It is used for the invention provides one kind
The method of skin ovarian cancer diagnosis and Prognosis scoveillance-tumor markers asparagine endopeptidase（AEP）And Integrin α_5 and β 1
Close detection method.By detect in epithelial ovarian carcinoma patients tumor tissues and excretion body that peripheral blood, ascites are originated in asparagus fern acyl
The expression quantity of amine endopeptidase and Integrin α_5 and β 1 assesses patient's prognosis.
Brief description of the drawings
Fig. 1 is to confirm that AEP can be in epithelial ovarian cancer cell and Peritoneal Mesothelial Cells by immunofluorescence common location method
With Integrin α_5 and the formation compounds of β 1.
Identification-CD63 the Identification of the antibodies of Fig. 2 excretion bodies
The differential expression of AEP and Integrin α_5 and β 1 in the excretion body that Fig. 3 originates for good malignant ovarian tumor patients serum.
The expression of AEP and Integrin α_5 and β 1 in the excretion body that Fig. 4 originates for good malignant ovarian tumor patient ascites are poor
It is different.
Fig. 5 epithelial ovarian carcinoma patients tumor tissues, peritoneal tumor transfer stove, benign ovarian tumor specimens,
The differential expression of AEP and Integrin α_5 and β 1 in peritoneal tissues.
Fig. 6 AEP and Integrin α_5 and β 1 and ovarian epithelial carcinoma prognostic analysis.
Embodiment：
Illustrate research of the present invention referring to specific embodiment.Art scientific research personnel it is understood that the embodiment only
For illustrating the present invention, the scope of the present invention is not limited in any way.
The experiment reagent and material used in following embodiment is as follows：
RPMI-1640 nutrient solutions（HyClone）
Dulbecco`s modified Eagle medium （high sugar, HyClone）
Hyclone（Fetal bovine serum, Gibco-BRL）
Hank ' s equilibrium liquids（Gibco-BRL）
Anti-Integrinα5, Integrin β1（#4749, Integrin antibody sampler Kit, CST）
AEP antibody anti-legumain（AF2199, R&D）
Lowlenthal serum is purchased from Wuhan doctor's moral
Alexa Fluor 488-conjugated anti-rabbit antibody（Abcam）
Cy3-conjugated anti-goat antibody（Abcam）
DAPI is purchased from Santa Cruz Biotechnology companies
exosomes isolation kit（EXOQ5A-1, SBI, USA）
RIPA buffer solutions（RIPA: PMSF = 100:1, KeyGENBioTECH）
Embodiment 1:
Immunofluorescence common location method identification AEP can be in epithelial ovarian cancer cell and Peritoneal Mesothelial Cells and Integrin α_5 and β 1
Form compound.
Whether the present embodiment can form compound by immunofluorescence common location method to AEP in cell and Integrin α_5 and β 1
Thing is identified.
Specific implementation method is：
1. the acquisition and cell culture of sample：Epithelial ovarian carcinoma patients peripheral blood, ascites and tumor tissues sample are attained at
In January, 2015 to the first maternity and infant health institute, in May, 2017 Shanghai City 65 patients.The patient registered in research obtains informed
Letter of consent, and ratify by Ethics Committee of the first maternity and infant health institute, Shanghai City.
People epithelial ovarian cancerous cell line SKOV3 is cultivated with the culture mediums of RPMI 1640, adds 10%FBS and 1% antibiosis
Element.Human mesothelial cells HPMC is cultivated with DMEM culture mediums, adds 20%FBS and 1% antibiotic.Incubator environment is 5%
CO2, 37℃。
2. immunofluorescence staining：It is used for immunofluorescence technique by adding anti-AEP and Integrin α_5 and beta 1 antibodies and enters
Row detection.Integrin α_5 and β 1 are detected using Alexa Fluor 488-conjugated anti-rabbit antibody.Use Cy3-
The anti-goat antibodies of conjugated are detected to AEP.Photo is obtained by the laser confocal microscopes of Zeiss LSM 510.
Two rows are followed successively by above Fig. 1 photos：The nucleus that is dyed in SKOV3 and human mesothelial cells HPMC, AEP,
Integrin α_5 and common location.
Two rows are followed successively by below Fig. 1 photos：The nucleus that is dyed in SKOV3 and human mesothelial cells HPMC, AEP,
Integrin β_1 and common location.
Test result indicates that：AEP can form compound with Integrin α_5 and β 1 in SKOV3 and human mesothelial cells HPMC
Thing.
Conclusion：The present invention confirms that AEP can be in epithelial ovarian cancer cell and integrin alpha by immunofluorescence common location method
The formation compounds of 5 and β 1（Fig. 1）.
Embodiment 2:
By the present invention in that with excretion body separating kit（EXOQ5A-1, SBI, USA）Separate and collect ovarian epithelial carcinoma trouble
The excretion body in excretion body, benign ovarian tumor patients serum in person's serum and ascites, excretion body in liver cirrhosis ascites,
And CD63 Identification of the antibodies is carried out to it（Abcam, USA）（Fig. 2）.
The present embodiment is by using Integrin α 5, Integrin beta 1 antibodies（#4749, Integrin antibody
sampler Kit, CST, USA）And AEP antibody anti-legumain（AF2199, R&D, USA）To ovarian epithelial carcinoma
The AEP and integrin alpha in excretion body in patients serum and ascites, benign ovarian tumor patients serum and liver cirrhosis ascites
5 and β 1 carries out Elisa detections, observes its differential expression.
Specific implementation method is：
1. the separation of excretion body, extraction and identification：Obtain epithelial ovarian carcinoma patients serum and ascites, benign ovarian tumor are suffered from
After person's serum and liver cirrhosis ascites, excretion body is extracted in 30 minutes by 2500r ultracentrifugations.Supernatant ultracentrifugation 2 times
（1000 g × 10 min, 3000 g × 30 min）, and the reagent added in excretion body separating kit stays overnight.Then,
Excretion body is resuspended using PBS.Period, the excretion body extracted is identified using CD63.
2. the protein isolate lysate on 10% sds page, and be transferred on nitrocellulose filter.Incubate
Upper AEP and Integrin α_5 and beta 1 antibodies.Detected using HRP-linked secondary antibodies.Ultimate analysis epithelial ovarian carcinoma patients blood
AEP and Integrin α_5 and β 1 in the excretion body of clear and ascites, benign ovarian tumor patients serum and liver cirrhosis ascites source
Expression difference.
Fig. 2:Excretion body CD63 expression quantity is detected by Western-Blot.CD63 is excretion body Specific marker.
Fig. 3:Asparagine endopeptidase in epithelial ovarian carcinoma patients and benign ovarian tumor patients serum's excretion body
（AEP）And Integrin α_5 and β 1 expression quantity detection.In epithelial ovarian carcinoma patients serum excretion body, AEP expression quantity：
1.14 ± 0.08, Integrin α_5 expression quantity：2.46 ± 0.40, integrin β_1 expression quantity：3.10 ± 0.35, in benign ovarian tumor
In patients serum's excretion body, AEP expression quantity：0.14 ± 0.01, Integrin α_5 expression quantity：2.14 ± 0.32, integrin β_1 table
Up to amount：2.21 ± 0.05, there is conspicuousness compared with epithelial ovarian carcinoma patients difference（AEP：p<0.001, Integrin α_5:p=
0.025, integrin β_1：p=0.025）
Fig. 4:Asparagine endopeptidase in epithelial ovarian carcinoma patients and liver cirrhosis ascites excretion body（AEP）And integrin
α 5 and β 1 expression quantity detection.In epithelial ovarian carcinoma patients ascites excretion body, AEP expression quantity：1.93 ± 0.19, integrate
The plain expression quantity of α 5：3.03 ± 0.54, integrin β_1 expression quantity：2.35 ± 0.07, in liver cirrhosis ascites excretion body, AEP
Expression quantity：0.98 ± 0.05, Integrin α_5 expression quantity：1.53 ± 0.17, integrin β_1 expression quantity：1.46 ± 0.12, relatively on
Skin ovarian cancer patients difference has conspicuousness（AEP：p=0.025, Integrin α_5:p=0.038, integrin β_1：p=0.001）
Conclusion：From Fig. 2-4, the asparagine endopeptidase in the excretion body in epithelial ovarian carcinoma patients serum and ascites source
（AEP）And Integrin α_5 and β 1 expression quantity are compared with the excretion body and liver cirrhosis ascites that benign ovarian tumor patients serum originates
Expression quantity in the excretion body in source is high.
Embodiment 3:
Immunohistochemical method detection epithelial ovarian carcinoma patients tumor tissues, peritoneal tumor transfer stove, benign ovarian tumor are suffered from
Asparagine endopeptidase in person's tumor tissues, peritoneal tissues（AEP）And Integrin α_5 and β 1 expression.AEP and Integrin α_5 and β
1 expression and EOC patient clinicals are closely related.
The present embodiment chooses epithelial ovarian carcinoma patients tumor tissues, peritoneal tumor transfer stove, benign ovarian tumor patient
Tumor tissues, peritoneal tissues section, pass through the expression of immunohistochemical staining AEP and Integrin α_5 and β 1.
And statistical analysis is carried out to it by IRS points-scoring systems.（IRS points-scoring systems：AEP dye levels score：1,1~
25%;2,26~50%;3,51~75%;4,76~100%（PP, positive cell percentage）.Staining power（SI）Score
For:0, it is negative;1, it is weak;2, moderate;3, by force.IRS scorings are to combine the fraction that PP and SI are drawn jointly.IRS scorings point
For following groups：0-3, it is negative;4-6, weakly positive;7-9 is positive;10-12, strong positive.）
Specific implementation method is：
1. immunohistochemical staining：It is ready to after patient tissue section, is detected by using anti-AEP and Integrin α_5 and beta 1 antibodies
It is expressed（4 DEG C overnight）.Then, it is incubated 60 minutes at 37 DEG C using HRP-linked anti goat igg and anti-rabbit IgG.PBS washes 3
Secondary, 5 minutes every time, histotomy used haematoxylin redyeing and dehydration after being incubated 30 seconds using DAB.Each section random selection 5
High power field of view carries out IRS scorings.Count AEP and Integrin α_5 and β 1 expression and the correlation of epithelial ovarian carcinoma patients and
Prognostic analysis.
2. by being swollen from epithelial ovarian carcinoma patients tumor tissues, peritoneal tumor transfer stove, benign ovarian tumor patient
Tumor tissue, peritoneal tissues section, using Immunohistochemical detection method, detect asparagine endopeptidase in tissue（AEP）And it is whole
Element α 5 and β 1 expression is closed, and statistical analysis is carried out to it by IRS points-scoring systems.（Fig. 5）.In addition, this three's expression quantity and
The clinical stages of EOC patient, is closely related, in progressive stage（FIGO III and IV phases by stages）The tissue section strain of patient it is visible its
Expression is increased（FIGO I and II phases by stages：AEP 3.93 ± 2.19, Integrin α_5 2.40 ± 1.45, integrin β_1 0.87 ±
0.99；FIGO III and IV phases by stages：AEP 8.75 ± 2.88, Integrin α_5 5.25 ± 2.10, integrin β_1 3.30 ± 1.75,
Mann-Whitney U test, p<0.05）.
3. utilization of the detection of AEP and Integrin α_5 and β 1 in EOC prognosis is assessed：By the present invention in that with
SPSS.20.0 carries out EOC prognosis evaluations：Organize IRS scorings that AEP is divided to for two groups according to EOC（≤ 6 points,>6 points）, equally will be whole
Close element α 5, integrin β_1 and be divided into two groups（≤ 3 points,>3 points）, and prognostic analysis is carried out to it（Fig. 6）.
Fig. 5:A, AEP, Integrin α_5 and integrin β_1 are suffered from benign ovarian tumor patient tissue, malignant ovarian tumor respectively
Dyeing in person's tissue, benign ovarian tumor patient peritonaeum and malignant ovarian tumor patient's peritonaeum；B, IRS points-scoring system are counted
Expression differences of the AEP in above-mentioned tissue；It is poor that C, IRS points-scoring system count expression quantity of the Integrin α_5 in above-mentioned tissue
It is different；D, IRS points-scoring system count expression difference of the integrin β_1 in above-mentioned tissue；It can be seen that, fraction is higher, pernicious ovum
Nest tumor patient tissue and peritonaeum quantity are more；Fraction is lower, and benign ovarian tumor patient tissue and peritonaeum quantity are more, show
AEP, Integrin α_5 and integrin β_1 expressed in malignant ovarian tumor patient tissue and peritonaeum compared with benign ovarian patient tissue and
Peritonaeum is high.
Fig. 6:AEP, Integrin α_5 and integrin β_1 and malignant ovarian tumor patient's prognostic analysis（AEP survival analysises：p=
0.038；Integrin α_5 survival analysis：p=0.029, integrin β_1 survival analysis：p=0.048）, find AEP, Integrin α_5 and
Patient's prognosis of integrin β_1 height expression is poor compared with the patient of low expression.
Conclusion：Immunohistochemical staining is carried out for epithelial ovarian cancer tissue and finds it using IRS points-scoring systems
The expression of AEP and Integrin α_5 and β 1 is increased compared with benign ovarian tumor in tissue.Also, this three's expression quantity and patient's prognosis are close
Cut is closed.
Therefore, the detection of AEP and the expression quantity of Integrin α_5 and β 1 can turn into the prognosis that new index is used to assess EOC.
Claims (1)
1. a kind of asparagine endopeptidase joint Integrin α_5 and integrin β_1 are preparing diagnosis ovarian epithelial carcinoma or diagnosis
Purposes in the reagent of ovarian epithelial carcinoma outcome.
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|---|---|---|---|---|
| WO2009100110A1 (en) * | 2008-02-05 | 2009-08-13 | Medarex, Inc. | Alpha 5 - beta 1 antibodies and their uses |
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| WO2009100110A1 (en) * | 2008-02-05 | 2009-08-13 | Medarex, Inc. | Alpha 5 - beta 1 antibodies and their uses |
| CN101726602A (en) * | 2009-12-11 | 2010-06-09 | 南开大学 | Method for judging ovarian cancer prognosis by detecting Legumain protein |
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