CN106706915A - Application of SUSD2 in preparation of kit for diagnosis and/or prognostic judgement of high-grade serous ovarian carcinoma - Google Patents
Application of SUSD2 in preparation of kit for diagnosis and/or prognostic judgement of high-grade serous ovarian carcinoma Download PDFInfo
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- CN106706915A CN106706915A CN201611255161.0A CN201611255161A CN106706915A CN 106706915 A CN106706915 A CN 106706915A CN 201611255161 A CN201611255161 A CN 201611255161A CN 106706915 A CN106706915 A CN 106706915A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Abstract
The invention discloses the application of SUSD2 as a marker for the diagnosis and/or prognostic judgement of high-grade serous ovarian carcinoma and application for detection of an SUSD2 reagent in preparation of a kit for the diagnosis and/or prognostic judgement of the high-grade serous carcinoma. In the invention, SUSD2 is used as the marker to diagnose high-grade serous ovarian carcinoma through a method of immunohistochemistry and judges the prognosis of the high-grade serous ovarian carcinoma; the SUSD2 is highly expressed in the high-grade serous ovarian carcinoma and lowly expressed in fimbriae of fallopian tube. The expression level of SUSD2 in high-grade ovarian carcinoma is related to the survival rate of patients; patients having high SUSD2 expression are poor in prognosis and low in survival rate. The SUSD2 as the marker for the diagnosis and/or prognosis of the high-grade serous ovarian carcinoma has broad application prospects and huge potential social benefits.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to SUSD2 is preparing high-level serous ovarian cancer diagnosis
And/or the application in the kit of Index for diagnosis.
Background technology
Oophoroma is gynaecology's common cancer, and case fatality rate is occupied first of gynecologic malignant tumor, is that gynemetrics's clinic faces
Ultimate challenge.Although surgical cytoreduction and platinum class Japanese yew class combined chemotherapy make 5 years survival rates of patient make moderate progress, half
Oophoroma clinical diagnosis and treatment has no substantial progress since individual century.This mainly due to lack clinical practice early diagnosis means and
The lasting therapeutic scheme of curative effect, its basic reason is the molecular mechanism of early diagnosis, generation development, prognosis to oophoroma etc.
Understanding is unclear.
High-level serous ovarian cancer (HGSOC) is ovarian cancer patients main causes of death, and proportion is larger, and extremely
Rate of dying remains high.HGSOC prognosis is very poor, and long-term surviving rate is less than 20%.Recurrence and DISTANT METASTASES IN are aftertreatment failures
Main cause.In current clinical practice, clinicopathologic stage is most important, most reliable prognostic indicator, but is still needed more
Postoperative patient is further divided into excessive risk and low-risk by accurate mark, and then takes more effective treatment method.
Tumour is the complex process of multifactor polygenes caused by abnormal, before the change of focus genetic morphology,
The change of many molecular biology may have been there is.Can be more proper with the abnormal tumor markers as oophoroma of molecular level
The heterogeneity of locality reflection tumor biological behavior, and then early diagnosis for oophoroma and Index for diagnosis provide more valuable
Instruct.
The molecular marker of the oophoroma material alterations having now been found that is concentrated mainly on nucleic acid and albumen aspect, and the former includes
Cytogenetics change, the amplification of gene, missing, mutation, fracture and merge, mRNA and miRNA etc., the latter shows as albumen table
Modification etc. changes after up to level, size, Subcellular Localization and protein translation.Because protein is the final product of gene expression,
Be also interaction between the executor of gene function, protein, it is mutually coordinated be basis that cell carries out all metabolic activities,
Therefore the change of protein level during concern ovary carcinogenesis develops, oophoroma is early diagnosed for searching and Index for diagnosis divides
Sub- mark is particularly important.
SUSD2 is the type memebrane protein of single channel 1, is often expressed in mesenchymal stem cells MSCs, blood vessel surrounding smooth myocyte, with
And many tumor cell lines.Research before shows, in many tumours (for example, liver cancer, lung cancer etc.), SUSD2 expresses low, mistake
Expression SUSD2 can suppress transfer and the propagation of tumour cell.But in breast cancer, SUSD2 is expressed compared to mammary gland benign tumor
Increase, and the transfer of breast cancer cell, induction of T cell apoptosis can be promoted after overexpression.As can be seen here, in different tumor groups
In knitting, the expression of SUSD2 occurs with tumour and the correlation of progress has larger difference.Therefore, research SUSD2 is in ovary
The change of expression quantity in carcinogenesis, evolution, early diagnosis and Index for diagnosis for oophoroma have highly important meaning
Justice.
The content of the invention
For above-mentioned prior art, it is an object of the invention to study tables of the SUSD2 in high-level serous ovarian cancer
Up to situation, and itself and high-level serous ovarian cancer clinicopathologic features and the relation of biological behaviour are analyzed, and then found
SUSD2 occurs in high-level serous ovarian cancer, develops, the effect played in transfer process.
To achieve the above object, the present invention is adopted the following technical scheme that：
The present invention compares expression of the SUSD2 in fimbriae tubae and high-level serous ovarian cancer first, finds SUSD2
The low expression in fimbriae tubae, the expression high in high-level serous ovarian cancer, difference has statistical significance.
The present invention also compares the progression free survival phase of high-level serous ovarian cancer patient, Overall survival, lymph and carry down
Shifting, peritonaeum transfer case, as a result find that the prognosis of SUSD2 expression patients high is poorer.
Summary result of study, the first aspect of the present invention, there is provided SUSD2 is diagnosed as high-level serous ovarian cancer
And/or the application of the mark of Index for diagnosis.
By determining the expression quantity of above-mentioned mark, high-level serous ovarian cancer diagnosis can be carried out and/or prognosis is sentenced
It is disconnected, including：The antidiastole and/or susceptibility analysis of high-level serous ovarian cancer or ovarian cancer tissue's transfer, high-level slurry
Fluidity treatment of ovarian cancer medicine, treatment method, treatment curative effect and prognosis assessment, the high-level serous ovarian cancer of correlated crowd or
Assessment of ovarian cancer tissue's transfer risk etc..
The expression quantity for determining above-mentioned mark needs to use detection reagent, kit or detection chip etc., therefore, the present invention
Detection reagent, kit and the detection chip of above-mentioned mark are further study, is examined for high-level serous ovarian cancer
Disconnected and/or prognosis.
Detecting the method for protein expression has various, and including but not limited to enzyme linked immunosorbent assay (ELISA) (ELISA) is for example competed
Combination of property ELISA, double crush syndrome, Western blotting, ELISA and Western blotting etc..According to above-mentioned detection method, can
Detecting the reagent of SUSD2 can be made the kit of ovarian cancer diagnosis and Index for diagnosis.The second aspect of the present invention, there is provided one
The detection method of SUSD2 is planted, step is as follows：
The wax stone of fimbriae tubae and high-level serous ovarian cancer is fabricated to organization chip (TMA), immuning tissue is carried out
The scanning of chemical staining, microscope and imaging device is electronic pictures, and diagosis simultaneously carries out statistical analysis.
Specific detection method is as follows：
TMA histotomies are closed through dewaxing to water, antigen retrieval, deactivating endogenous peroxydase and lowlenthal serum
Afterwards, respectively with primary antibody SUSD2 in moisture preservation box 4 DEG C of overnight incubations, after being washed through PBS after respectively with secondary antibody (biotin labeling
Sheep anti mouse/rabbit igg), HRPO enzyme label be incubated, DAB colour developings, haematoxylin redyes；Most afterwards through dehydration of alcohol and diformazan
Neutral gum mounting is used after benzene is transparent, is digital photograph using microscope and imaging device scanning, and to every SABC dye
Color section is scored.
Two parts, staining power and stained area are included to every scoring of TMA, staining power is not successively from low to high
Negative (-), weakly positive (+), positive (++), strong positive (+++), stained area is expressed as a percentage, i.e. 0%-100%.Dyeing
Area 0%-10% is designated as 0,11%-20% and is designated as 1,21%-50% and is designated as 2,51%-100% being designated as 3, final each point
SABC is scored at staining power * stained areas.
It is cut-off values that the SABC of SUSD2 scores with 6, is negative or weakly positive less than 6, is the positive more than or equal to 6
Or strong positive.
The determination methods of Index for diagnosis or risk profile are that SUSD2 protein expressions are positive, then ovarian cancer patients life cycle
It is short；SUSD2 protein expressions are negative, then ovarian cancer patients life cycle is long.
The third aspect of the present invention, there is provided the reagent of detection SUSD2 prepares high-level serous ovarian cancer diagnose and/or
Application in the kit of Index for diagnosis.
Preferably, the reagent of the detection SUSD2 is SUSD2 antibody.
Further, also included in the reagent：Lowlenthal serum, 0.01M citrates antigen retrieval buffers, 3%H2O2、DAB
Colour reagent and PBS solution.
The fourth aspect of the present invention, there is provided the reagent for suppressing SUSD2 protein expressions is preparing the high-level serosity ovum of improvement
Purposes in the medicine of nest cancer prognosis.
Beneficial effects of the present invention：
It is to lower that present invention research first finds that SUSD2 is expressed in fimbriae tubae, but in high-level serosity ovary
Expression in cancer is to raise, and the expression of SUSD2 is closely related with the prognosis of ovarian cancer patients.The present invention using SUSD2 as
Mark, diagnoses high-level serous ovarian cancer, and judge high-level serous ovarian cancer by immunohistochemical method
Prognosis.Expression high in the high-level serous ovarian cancers of SUSD2, and in normal fimbriae tubae low expression.SUSD2 is high-level
Expression in serous ovarian cancer is relevant with the survival rate of patient, and SUSD2 expression patient's prognosis high is bad, survival rate
It is low.The relation between expression and high-level serous ovarian cancer prognosis according to SUSD2, can diagnose to oophoroma
The medicine of high-level serous ovarian cancer can be improved with the judgement of prognosis, and exploitation, be widely used prospect.
Brief description of the drawings
Fig. 1：SUSD2 expressions slurry breast cancer survivorship curve (progression free survival phase) high-level with ovary；
Fig. 2：SUSD2 expressions slurry breast cancer survivorship curve (Overall survival) high-level with ovary；
Fig. 3：In lymphatic metastasis positive malignancies testing result；
Fig. 4：In node-negative metastasis lesion detection result.
Specific embodiment
The present invention is further illustrated in conjunction with the embodiments, it should explanation, the explanation of following embodiments merely to
The present invention is explained, its content is not defined.The experimental technique of unreceipted actual conditions in the embodiment of the present invention, is often
The test method of rule.
Embodiment 1：
1. test method：
We have collected 55 fimbriae tubaes of 2007-2013 and 306 operations of high-level serous ovarian cancer patient
Sample achieves wax stone.3 TMA organization chips are fabricated to using semi-automatic organization chip puncher, it is ensured that immunohistochemistry is big
Sample, high flux, standardization.Each wax stone includes fimbriae tubae and high-level serous ovarian cancer.
Organization chip (TMA) technology be by tens of to hundreds of or even more tissue samples it is neat be arranged in a load
The miniature array organization's piece being made on slide.Developed the color by SABC, in situ hybridization etc., height is realized on tissue sections
Flux obtains the expressing information based on morphologic genomics and proteomics.Tissue array technology is in tumor research
Middle extensive use, gene and change in protein situation, the evaluation of prognosis and molecule mark for studying tumour different stages of development
Evaluation, signal path research of will thing etc..Organization chip has the advantages that large sample, high flux, standardization, is to realize protein
The integrated key technology of molecular network and clinical information.The present embodiment uses TMA organization chips, and every chip top has
Nearly 200 points, can simultaneously carry out SABC and detect its expression.
After sample is through serial section, dimethylbenzene dewaxing, graded ethanol aquation, with super quick type two step method testing goal albumen
Expression, specific method is as follows：
(1) TMA sections are by 65 DEG C of roasting pieces, diformazan dewaxing, graded ethanol aquation；Wherein, dewaxing, the specific steps of aquation
For：
1. histotomy is placed in immersion 10 minutes in dimethylbenzene I, and 2. histotomy is placed in dimethylbenzene II immersions 10 minutes, 3.
Histotomy is placed in I immersions 10 minutes in absolute ethyl alcohol, and 4. histotomy is placed in absolute ethyl alcohol II and soaks 10 minutes, 5. organizes and cuts
Piece is placed in immersion 10 minutes in 95% ethanol, and 6. histotomy is placed in 80% ethanol and soaks 10 minutes, and 7. histotomy is placed in
In 75% ethanol soak 10 minutes, 8. histotomy be placed in distilled water soak 5 minutes.
(2) section is through 0.01M citrates antigen retrieval buffers (Citrate Buffer (pH 6.0)) antigen retrieval, 3%
H2O2Deactivating endogenous peroxydase, closing.Concretely comprise the following steps：
0.01M citrates antigen retrieval buffers are passed through into microwave stove heat 5 minutes, histotomy fire heating 15 high is put into
Second, cooling 30,15 circulations of heating-cooling, cool down at room temperature repeatedly；
Treated histotomy is washed with 1*PBS 3 times, five minutes every time.
3% hydrogen peroxide solution is added dropwise on slide, 37 degree stand 15 minutes, get rid of surplus liquid.
Washed 3 times with 1*PBS, each five minutes.
Confining liquid (Normal Goat Serum, 5%BSA solution) is added dropwise, is incubated 30 minutes at 37 degree, get rid of surplus liquid.
(3) SUSD2 primary antibody (1: 100) 50 μ L are added dropwise, are stored at room temperature 2 hours.
(4) 1*PBS buffer solutions are washed 3 times, every time 5 minutes.
(5) secondary antibody (sheep anti mouse/rabbit igg of biotin labeling) (1: 100) 50 μ L are added dropwise, are stored at room temperature 20 minutes.
(6) 1*PBS buffer solutions are washed 3 times, every time 5 minutes.
(7) the μ L of horseradish peroxidase 50 are added dropwise, are stored at room temperature 15 minutes.
(8) 1*PBS buffer solutions are washed 4 times, every time 5 minutes.
(9) DAB colour developings, grasp dye levels under the microscope, and dyeing is terminated in clear water is put into after reaching promising result,
Rinsed 10 minutes with running water afterwards.
(10) haematoxylin redyeing 5 minutes, running water is rinsed 10 minutes.
(11) 5% hydrochloride alcohols decolourize 5 seconds, and ammoniacal liquor is anti-blue 15 seconds, and running water is rinsed 10 minutes.
(12) dehydration, it is transparent, comprise the following steps that：1. histotomy is placed in immersion 3 minutes in 80% ethanol, 2. 95% second
Soaked in alcohol 3 minutes, 3. II soaks 5 minutes in absolute ethyl alcohol, and 4. absolute ethyl alcohol I immersions 5 minutes, 5. soak 10 in dimethylbenzene II
Minute, 6. dimethylbenzene I soaks 10 minutes.
(13) resin mounting, microscopy.
All of immunostained section is independently scored through 2 Pathology Doctors 's using blind.Occur in tumour cell slurry pale brown
Coloured particles are considered as positive signal.4 grades of staining power point：0, it is negative；1, weakly positive；2, it is positive；3, strong positive；Positive cell
4 grades of percentage point：0,0~10%；1,11~20%；2：21~50%；3,51-100%.The gross score of each sample is
Drawn by tumour cell staining power and the two-part product of positive tumor cell percentage, scope is 0~9.For convenience
Statistics, immunohistochemical staining is divided into low expression (i.e. the histochemical staining gross score of sample is less than 3) and expression high (i.e. by we
The histochemical staining gross score of sample is more than or equal to 3) 2 groups.
2. result of the test：
(1) expressions of the SUSD2 in fimbriae tubae and high-level serous ovarian cancer：
The results are shown in Table 1.
Table 1：Expressions of the SUSD2 in fimbriae tubae and high-level serous ovarian cancer
Control group VS tumor groups (χ2=22.725, p ＜ 0.001).
Result shows the high-level slurry breast cancer of ovary, and SUSD2 expression quantity is remarkably reinforced.
(2) relation of expression of the SUSD2 in high-level serous ovarian cancer and Clinical symptoms
The results are shown in Table 2.
Table 2：Expression of the SUSD2 in high-level serous ovarian cancer and the relation of Clinical symptoms
Progression free survival phase, 2 years (χ are less than or equal to more than 2 years VS2=15.045, p ＜ 0.001)；Overall survival, greatly
2 years (χ are less than in 2 years VS2=33.457, p ＜ 0.001))；Lymphatic metastasis, the non-transfer group (χ of transfer group VS2=4.546, p
＜ 0.05)；Peritonaeum is shifted, and transfer group VS is transfer group (χ2=10.973, p ＜ 0.005).
Result shows, in high-level serous ovarian cancer, SUSD2 expression group poor prognosis high.
(3) SUSD2 expressions are analyzed with high-level serous ovarian cancer survivorship curve
Result is shown in Fig. 1 and Fig. 2 respectively.As seen from the figure：Expressions of the SUSD2 in high-level serous ovarian cancer
Progression free survival phase and Overall survival to ovarian cancer patients is related；In expression group high (middle position progression free survival phase 15 months, in
Position Overall survival 27.55 months)；Low expression group (middle position progression free survival phase 28.08 months, middle position Overall survival 61 months).
To sum up, SUSD2 can be used to the diagnosis as high-level serous ovarian cancer and prognostic evaluation index, can especially comment
The postoperative survival condition of valency patient, for the treatment of clinician provides suggestion and refers to.
Embodiment 2：
Wax stone sample tissue is detected using the method for the embodiment of the present invention 1, microscopy result is respectively such as Fig. 3 and Fig. 4
It is shown.Wherein, the expression degree of SUSD2 is higher in the tissue stripping and slicing shown in Fig. 3, there is obvious brown yellow granule on cell membrane, is
SUSD2 detects positive findings, is high-level serous ovarian cancer by pathological examination result.
The expression degree of SUSD2 is relatively low in histotomy shown in Fig. 4, is that SUSD2 detections are cloudy without obvious brown yellow granule
Property result, by pathological examination result be node-negative metastasis.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (7)
1.SUSD2 diagnosed as high-level serous ovarian cancer and/or Index for diagnosis mark application.
2. reagent the answering in the kit of high-level serous ovarian cancer diagnosis and/or Index for diagnosis is prepared of SUSD2 is detected
With.
3. application as claimed in claim 2, it is characterised in that the high-level serous ovarian cancer diagnosis and/or prognosis are sentenced
It is disconnected to include：The antidiastole and/or susceptibility analysis of high-level serous ovarian cancer or ovarian cancer tissue's transfer, high-level slurries
Property treatment of ovarian cancer medicine, treatment method, treatment curative effect and prognosis assessment, and the high-level serous ovarian cancer of correlated crowd
Or ovarian cancer tissue shifts the assessment of risk.
4. application as claimed in claim 2, it is characterised in that the reagent of the detection SUSD2 is SUSD2 antibody.
5. application as claimed in claim 2, it is characterised in that also included in the reagent of the detection SUSD2：Lowlenthal serum,
0.01M citrates antigen retrieval buffers, 3%H2O2, DAB colour reagents and PBS solution.
6. a kind of detection method of the SUSD2 of non-diagnostic purpose, it is characterised in that step is as follows：
The wax stone of fimbriae tubae and high-level serous ovarian cancer is fabricated to tissue T MA, immunohistochemical staining is carried out, is shown
Micro mirror and imaging device scanning are electronic pictures, and diagosis simultaneously carries out statistical analysis.
7. purposes of the reagent of SUSD2 protein expressions in the medicine for improving high-level serous ovarian cancer prognosis is prepared is suppressed.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107422123A (en) * | 2017-07-26 | 2017-12-01 | 复旦大学附属中山医院 | A kind of kit for being used to diagnose OSCC |
| CN110551819A (en) * | 2019-08-23 | 2019-12-10 | 伯克利南京医学研究有限责任公司 | Application of group of ovarian cancer prognosis related genes |
| CN110646271A (en) * | 2019-09-20 | 2020-01-03 | 四川大学华西医院 | Preparation method of paraffin section of mouse or rat acute pancreatitis tissue |
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| CN104215768A (en) * | 2014-08-07 | 2014-12-17 | 北京大学 | Application of SUSD2 protein as marker |
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| CN104215768A (en) * | 2014-08-07 | 2014-12-17 | 北京大学 | Application of SUSD2 protein as marker |
Non-Patent Citations (2)
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| JN SHEETS, ET.AL.: "SUSD2 expression in high-grade serous ovarian cancer correlates with increased patient survival and defective mesothelial clearance.", 《ONCOGENESIS》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107422123A (en) * | 2017-07-26 | 2017-12-01 | 复旦大学附属中山医院 | A kind of kit for being used to diagnose OSCC |
| CN107422123B (en) * | 2017-07-26 | 2019-04-19 | 复旦大学附属中山医院 | It is a kind of for diagnosing the kit of oral squamous cell carcinoma |
| CN110551819A (en) * | 2019-08-23 | 2019-12-10 | 伯克利南京医学研究有限责任公司 | Application of group of ovarian cancer prognosis related genes |
| CN110551819B (en) * | 2019-08-23 | 2023-05-16 | 伯克利南京医学研究有限责任公司 | Application of ovarian cancer prognosis related genes |
| CN110646271A (en) * | 2019-09-20 | 2020-01-03 | 四川大学华西医院 | Preparation method of paraffin section of mouse or rat acute pancreatitis tissue |
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Application publication date: 20170524 |