CN107315090B - Asparagine endopeptidase combines the purposes of Integrin α_5 and integrin β_1 as diagnostic reagent - Google Patents
Asparagine endopeptidase combines the purposes of Integrin α_5 and integrin β_1 as 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
- 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
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- 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)
Abstract
The present invention provides the purposes of a kind of asparagine endopeptidase joint Integrin α_5 and integrin β_1 in preparation diagnosis ovarian epithelial carcinoma or the reagent for diagnosing ovarian epithelial carcinoma outcome.The present invention, which identifies AEP by immunofluorescence common location, to form complex in people epithelial ovarian cancer cell SKOV3 and human mesothelial cells HPMC with Integrin α_5 and β 1.Meanwhile the expression quantity of Elisa technology detection discovery epithelial ovarian carcinoma patients serum and asparagine endopeptidase (AEP) and Integrin α_5 and β 1 in the excretion body in ascites source is high compared with the expression quantity in the excretion body in benign ovarian tumor patients serum source and the excretion body in liver cirrhosis ascites source.It follows that asparagine endopeptidase (AEP) and Integrin α_5 and β 1 and ovarian epithelial carcinoma and prognosis are closely related.
Description
Technical field
The invention belongs to fields of biomedicine, are 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 in the reagent of preparation diagnosis ovarian epithelial carcinoma outcome
Purposes.
Background technique
Ovarian epithelial carcinoma (Epithelial ovarian cancer, EOC) is one of gynaecology's common cancer, extremely
The rate of dying occupies gynecological tumor first place, and nearly 70% patient has been FIGO III IV phase by stages when making a definite diagnosis, and is mainly shown as abdomen
Film is planted and is shifted extensively.Peritonaeum has the feature of chronic " peritonitis ".Ovarian epithelial carcinoma prognosis is poor, how effectively to mention
The prognosis of high epithelial ovarian carcinoma patients is current problem to be solved.
Asparagine endopeptidase (Aasparaginylendopeptidase, AEP), also known as legumain
It (legumain), is the member of cysteine proteinase C13 family.The people AEP assignment of genes gene mapping is in 14q32.1, by 13 intrones
And 14 exons are constituted, and are had well-conserved.There are three types of forms by AEP: unactivated AEP, activation AEP and completely activation AEP.
The stability of the activation of AEP and conformation intensified by sugar in microenvironment, pH value and oxidation-reduction potential etc. are influenced.AEP is just
It is less in ordinary person's body tissue to express or do not express, however, thin in many solid tumor tissues, lymthoma and tumour correlation macrophage
Expression is increased in born of the same parents (Tumor-associated macrophages, TAM), the occurrence and development and disease of AEP and malignant tumour
The prognosis of people is closely related.AEP is existed in tumor microenvironment, and processing can be carried out to antigen protein and is offered, matrix is activated
Metalloproteinases, the release etc. of modulate tumor associated macrophages and the modulating vascular new life factor.
Correlative study of the AEP in ovarian epithelial carcinoma is actually rare, predominantly stays in functional experiment stage, mechanism at present
Study it is indefinite, because disease incidence and the death rate are all very high in female tumor for ovarian epithelial carcinoma, early diagnose 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 sub- single by α (α subunit, 120-170 kDa) and 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 adherency of system.Integrin is the important medium mutually exchanged between mediated cell and cell, cell and extracellular matrix.Many cancers are thin
Growth, hyperplasia, adherency and the transfer etc. of the adjustable cell of the integrin of cellular expression.Integrin α_5 can mutually be tied with 1 subunit of β
It closes.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
Report that AEP can form compound with the integrin of tumor cell surface, integrin can promote the activation of AEP, to increase
Its strong biological function.Therefore, furtheing investigate Integrin α_5 and β 1 helps to explore the new mechanism of EOC peritonaeum transfer.
Excretion body, diameter, can be in biologies such as intercellular trafficking DNA, RNA, lipid and protein between 30-100nm
Credit.It is a variety of that excretion body can be realized tumour cell and endothelial cell, mesothelial cell, immunocyte in tumor microenvironment etc.
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 companion of progressive stage EOC
It is formed with ascites.Therefore, we are from the excretion body in EOC patients serum and ascites source for asparagine endopeptidase (AEP)
And Integrin α_5 and β 1 are detected, and therefrom explore the correlation of itself and EOC patient's prognosis.
Summary of the invention
The purpose of the present invention is to provide a kind of asparagine endopeptidase (AEP) joint Integrin α_5s and integrin β_1 to make
Purposes in the reagent of standby diagnosis ovarian epithelial carcinoma or diagnosis ovarian epithelial carcinoma outcome, this purposes are wanted
It solves to diagnose the reagent of ovarian epithelial carcinoma prognosis and the technical problem that method is ineffective in the prior art.
The present invention provides a kind of asparagine endopeptidase (AEP) joint Integrin α_5s and integrin β_1 in preparation diagnosis
Purposes in the reagent of skin oophoroma or diagnosis ovarian epithelial carcinoma outcome.
The present invention obtained by lot of experiments, joint-detection asparagine endopeptidase (AEP) and Integrin α_5 and β 1
It is closely related with the prognosis of ovarian epithelial carcinoma.Specifically, the present invention by immunofluorescence common location identify AEP can with it is whole
It closes element α 5 and β 1 and forms complex in people epithelial ovarian cancer cell SKOV3 and human mesothelial cells HPMC.Meanwhile Elisa
Asparagine endopeptidase (AEP) and whole in technology detection discovery epithelial ovarian carcinoma patients serum and the excretion body in ascites source
Close element α 5 and β 1 expression quantity compared with benign ovarian tumor patients serum source excretion body and liver cirrhosis ascites source outside
The expression quantity secreted in body is high.
The present invention is compared with prior art, and technological progress is positive and effective.The present invention provides one kind to be used for
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 detection epithelial ovarian carcinoma patients tumor tissues in and peripheral blood, ascites source excretion body in asparagus fern acyl
The expression quantity of amine endopeptidase and Integrin α_5 and β 1 assesses patient's prognosis.
Detailed description of the invention
Fig. 1 is to confirm that AEP can be in epithelial ovarian cancer cell and Peritoneal Mesothelial Cells by immunofluorescence common location method
Compound is formed with Integrin α_5 and β 1.
Identification-CD63 the Identification of the antibodies of Fig. 2 excretion body
Fig. 3 is that the expression of the AEP and Integrin α_5 and β 1 in the excretion body in good malignant ovarian tumor patients serum source are poor
It is different.
Fig. 4 is that the expression of the AEP and Integrin α_5 and β 1 in the excretion body in good malignant ovarian tumor patient ascites source 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.
Specific embodiment:
Illustrate research of the present invention referring to specific embodiment.Field technology scientific research personnel is it is understood that the implementation
Example is merely to illustrate the present invention, does not limit the scope of the invention in any way.
Experiment reagent used in following embodiment and material are as follows:
RPMI-1640 culture solution (HyClone)
Dulbecco`s modified Eagle medium (high sugar, HyClone)
Fetal calf serum (Fetal bovine serum, Gibco-BRL)
Hank ' s equilibrium liquid (Gibco-BRL)
Anti-Integrin α 5, Integrin β 1(#4749, Integrin antibody sampler Kit,
CST)
AEP antibody anti-legumain(AF2199, R&D)
Lowlenthal serum is purchased from Wuhan doctor moral
Alexa Fluor 488-conjugated anti-rabbit antibody(Abcam)
Cy3-conjugated anti-goat antibody(Abcam)
DAPI is purchased from Santa Cruz Biotechnology company
Exosomes isolation kit(EXOQ5A-1, SBI, USA)
RIPA buffer (RIPA:PMSF=100:1, KeyGENBioTECH)
Embodiment 1:
Immunofluorescence common location method identifies that AEP can be in epithelial ovarian cancer cell and Peritoneal Mesothelial Cells and Integrin α_5
Compound is formed with β 1.
It is compound whether the present embodiment can form AEP in cell and Integrin α_5 and β 1 by immunofluorescence common location method
Object is identified.
Specific implementation method are as follows:
1. the acquisition and cell culture of sample: epithelial ovarian carcinoma patients peripheral blood, ascites and tumor tissues sample obtain
It is obtained in January, 2015 to 65 patients of the first maternity and infant health institute, in May, 2017 Shanghai City.The patient registered in research obtains
Informed consent form, and ratify by Shanghai City Ethics Committee, the first maternity and infant health institute.
People epithelial ovarian cancerous cell line SKOV3 is cultivated with 1640 culture medium of RPMI, and 10%FBS and 1% antibiosis is added
Element.Human mesothelial cells HPMC is cultivated with DMEM culture medium, and 20%FBS and 1% antibiotic is added.Incubator environment is 5%
CO2, 37℃。
2. immunofluorescence staining: by be added anti-AEP and Integrin α_5 and beta 1 antibodies be used for immunofluorescence technique into
Row detection.Integrin α_5 and β 1 are detected using Alexa Fluor 488-conjugated anti-rabbit antibody.Use Cy3-
The anti-goat antibody of conjugated detects AEP.Photo is obtained by 510 laser confocal microscope of Zeiss LSM.
Above Fig. 1 photo it is two rows of successively are as follows: the nucleus that is dyed in SKOV3 and human mesothelial cells HPMC, AEP,
Integrin α_5 and common location.
Below Fig. 1 photo it is two rows of successively are as follows: the nucleus that is dyed in SKOV3 and human mesothelial cells HPMC, AEP,
Integrin β_1 and common location.
The results showed that AEP can be formed in SKOV3 and human mesothelial cells HPMC with Integrin α_5 and β 1 it is compound
Object.
Conclusion: the present invention confirms that AEP can be in epithelial ovarian cancer cell and integrin alpha by immunofluorescence common location method
5 and β 1 forms compound (Fig. 1).
Embodiment 2:
By the present invention in that separating and collecting epithelial ovarian with excretion body separating kit (EXOQ5A-1, SBI, USA)
Cancer patients serum and the excretion body in ascites, the excretion body in benign ovarian tumor patients serum, liver cirrhosis ascites China and foreign countries
Body is secreted, and carries out CD63 Identification of the antibodies (Abcam, USA) (Fig. 2) to it.
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 detection, observes its differential expression.
Specific implementation method are as follows:
1. the separation of excretion body, extraction and identification: obtaining epithelial ovarian carcinoma patients serum and ascites, benign ovarian are swollen
After tumor patients serum and liver cirrhosis ascites, pass through 30 minutes extraction excretion bodies of 2500r ultracentrifugation.Supernatant ultracentrifugation 2
Secondary (1000 g × 10 min, 3000 g × 30 min), and the reagent being added in excretion body separating kit is stayed overnight.So
Afterwards, excretion body is resuspended using PBS.Period identifies extracted excretion body using CD63.
2. the protein isolate lysate on 10% sds page, and be transferred on nitrocellulose filter.It incubates
Upper AEP and Integrin α_5 and beta 1 antibodies.It is detected using HRP-linked secondary antibody.Ultimate analysis epithelial ovarian carcinoma patients blood
AEP and Integrin α_5 and β 1 in the clear and excretion body of ascites, benign ovarian tumor patients serum and liver cirrhosis ascites source
Expression difference.
Fig. 2: the expression quantity of excretion body CD63 is detected by Western-Blot.CD63 is excretion body Specific marker.
Fig. 3: the asparagine endopeptidase in epithelial ovarian carcinoma patients and benign ovarian tumor patients serum's excretion body
(AEP) and the detection of the expression quantity of Integrin α_5 and β 1.In epithelial ovarian carcinoma patients serum excretion body, the expression quantity of AEP:
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, the expression quantity of AEP: 0.14 ± 0.01, Integrin α_5 expression quantity: 2.14 ± 0.32, integrin β_1 table
Up to amount: 2.21 ± 0.05, compared with epithelial ovarian carcinoma patients difference have conspicuousness (AEP:p< 0.001, Integrin α_5:p=
0.025, integrin β_1:p=0.025)
Fig. 4: asparagine endopeptidase (AEP) in epithelial ovarian carcinoma patients and liver cirrhosis ascites excretion body and whole
Close the expression quantity detection of element α 5 and β 1.In epithelial ovarian carcinoma patients ascites excretion body, the expression quantity of AEP: 1.93 ± 0.19,
Integrin α_5 expression quantity: 3.03 ± 0.54, integrin β_1 expression quantity: 2.35 ± 0.07, in liver cirrhosis ascites excretion body,
The expression quantity of AEP: 0.98 ± 0.05, Integrin α_5 expression quantity: 1.53 ± 0.17, integrin β_1 expression quantity: 1.46 ± 0.12, compared with
Epithelial ovarian carcinoma patients difference have conspicuousness (AEP:p=0.025, Integrin α_5:p=0.038, integrin β_1:p=
0.001)
Conclusion: by Fig. 2-4 it is found that in asparagine in epithelial ovarian carcinoma patients serum and the excretion body in ascites source
Excretion body and liver cirrhosis patient of the expression quantity of peptase (AEP) and Integrin α_5 and β 1 compared with benign ovarian tumor patients serum source
Expression quantity in the excretion body in ascites source is high.
Embodiment 3:
It is swollen that immunohistochemical method detects epithelial ovarian carcinoma patients tumor tissues, peritoneal tumor transfer stove, benign ovarian
The expression of asparagine endopeptidase (AEP) and Integrin α_5 and β 1 in tumor specimens, peritoneal tissues.AEP and integrin alpha
The expression of 5 and β 1 and EOC patient clinical 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 slice, pass through the expression of immunohistochemical staining AEP and Integrin α_5 and β 1.
And it is for statistical analysis to its by IRS points-scoring system.(IRS points-scoring system: the scoring of AEP dye levels: 1,1~
25%;2,26~50%;3,51~75%;4,76~100%(PP, positive cell percentage).Staining power (SI) score
Are as follows: 0, it is negative;1, it is weak;2, moderate;3, by force.IRS scoring is the score obtained jointly in conjunction with PP and SI.IRS scoring point
For following groups: 0-3, it is negative;4-6, weakly positive;7-9 is positive;10-12, strong positive.)
Specific implementation method are as follows:
1. immunohistochemical staining: after getting out patient tissue slice, by using anti-AEP and Integrin α_5 and beta 1 antibodies
Detect its expression (4 DEG C overnight).Then, it is incubated for 60 minutes using HRP-linked anti goat igg and anti-rabbit IgG at 37 DEG C.PBS
It washes 3 times, 5 minutes every time, histotomy used haematoxylin redyeing and dehydration after being incubated for 30 seconds using DAB.Each slice random selection 5
A high power field of view carries out IRS scoring.Count the expression and the correlation of epithelial ovarian carcinoma patients of AEP and Integrin α_5 and β 1
And prognostic analysis.
2. by selecting epithelial ovarian carcinoma patients tumor tissues, peritoneal tumor transfer stove, benign ovarian tumor patient swollen
Tumor tissue, peritoneal tissues slice detect in tissue asparagine endopeptidase (AEP) and whole using Immunohistochemical detection method
The expression of element α 5 and β 1 are closed, and for statistical analysis to its by IRS points-scoring system.(Fig. 5).In addition, this three's expression quantity and
The clinical stages of EOC patient, is closely related, the tissue section strain of progressive stage (FIGO by stages III and IV phase) patient it is visible its
Expression increase (FIGO I and II phase 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 phase 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 assessment EOC prognosis: by the present invention in that with
SPSS.20.0 carries out EOC prognosis evaluation: organizing IRS scoring to be divided to AEP according to EOC is two groups (≤6 points, > 6 points), equally will be whole
Close element α 5, integrin β_1 is divided into two groups (≤3 points, > 3 points), and carries out prognostic analysis (Fig. 6) to it.
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 statistics
Expression difference 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 score is higher, pernicious ovum
Nest tumor patient tissue and peritonaeum quantity are more；Score is lower, and benign ovarian tumor patient tissue and peritonaeum quantity are more, shows
AEP, Integrin α_5 and integrin β_1 express 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 prognostic analysis (AEP survival analysis: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
The highly expressed patient's prognosis of integrin β_1 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 system
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 phase is closed.
Therefore, the detection of 1 expression quantity of AEP and Integrin α_5 and β can become the prognosis that new index is used to assess EOC.
Claims (1)
1. the asparagine endopeptidase joint Integrin α_5 and integrin β_1 in excretion body source diagnose ovarian epithelial carcinoma in preparation
Or the purposes in the reagent of diagnosis ovarian epithelial carcinoma outcome.
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| 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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Non-Patent Citations (4)
| Title |
|---|
| Legumain:A Biomarker for Diagnosis and Prognosis of Human Ovarian Cancer;Lina Wang等;《Journal of Cellular Biochemistry》;20120322;第113卷;第2679-2686页 * |
| Lewis y 抗原和整合素α、β1，αv、β3在上皮性卵巢癌中的表达及其耐药和预后的关系;朱连成等;《现代肿瘤医学》;20160731;第24卷(第13期);第2132-2133页 * |
| The expression of asparaginyl endopeptidase promotes growth potential in epithelial;Qinyi等;《Cancer Biology & Therapy》;20170315;第18卷(第4期);第222-228页 * |
| 整合素α5β1、FN及CD44V6在卵巢上皮性肿瘤中的表达及其意义;常瑞霞等;《肿瘤研究与临床》;20071231(第3期);摘要 * |
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