CN108931651A - A kind of kallikrein 7 detection method of content in blood and its as the application on the blood serum designated object of diagnosis of pancreatic cancer - Google Patents
A kind of kallikrein 7 detection method of content in blood and its as the application on the blood serum designated object of diagnosis of pancreatic cancer Download PDFInfo
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- CN108931651A CN108931651A CN201710374234.6A CN201710374234A CN108931651A CN 108931651 A CN108931651 A CN 108931651A CN 201710374234 A CN201710374234 A CN 201710374234A CN 108931651 A CN108931651 A CN 108931651A
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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
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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
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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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
- G01N33/57492—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 localized on the membrane of tumor or cancer cells
Abstract
The present invention provides a kind of detection method of content of kallikrein 7 in blood, and collected blood sample to be measured centrifuging and taking supernatant and is specially stored in -80 DEG C of refrigerators after 4 DEG C of refrigerators stand 12 hours as required.Serum presses 1 with 1 × PBS liquid before detecting：5 are diluted packing experiment to be detected；96 hole elisa plates are distributed, 50 μ L test serum samples are added, jog is incubated for 2h；The liquid in hole is removed, addition wash buffer hole flushing 3 times places shaking table 5min after washbuffer is added every time, blotting paper is laid in desktop, pats elisa plate and removes liquid in hole completely；HRP is added in every hole, jog is incubated for；It adds TMB liquid and is incubated for 30min；100 μ l stop solution are added into every hole and terminate reaction；Jog mixes after stop solution terminate liquid is added, it is ensured that blue all becomes yellow；Measure optical density（OD value）, according to standard curve by optical density（OD value）Content value of the KLK7 in serum is converted to, the content detection of kallikrein 7 in blood can be completed.
Description
Technical field
The present invention relates to field of medicaments, particularly relate to the content inspection of kallikrein 7 (Kallikrein 7) in blood
Survey and its application in cancer of pancreas clinical diagnosis.
Background technique
Cancer of pancreas is a kind of digestive system tumor that grade malignancy is high, and case fatality rate accounts for about the 83% of annual morbidity.20 generation
It records the eighties, 5 years survival rates about 3% of cancer of pancreas, to reaching 7% at the beginning of 21 century.Since onset is hidden, pancreas has only in addition
Special anatomical structure and biological characteristics, existing DISTANT METASTASES IN when 60% Pancreas cancer patients are made a definite diagnosis, and to traditional chemicotherapy
Etc. treatment means it is insensitive, therefore poor prognosis, median survival interval only 6 to 9 months.Ductal adenocarcinoma of pancreas is most common in cancer of pancreas
Histological type, the histological types such as cystadenocarcinoma, neuroendocrine tumor, sarcoma, acinar cell carcinoma and lymthoma are clinical relatively smaller
See.It is still the current major measure for treating cancer of pancreas that operation, which is cut off, but is early diagnosed since shortage is effective, only about 10%~
20% patient is suitable for excision of performing the operation when medical, this some patients only accounts for the small part of Patients with Pancreatic Cancer sum.70%~
80% patient can not be cut off due to tumour, it is necessary to which quality of being made the life better by non-operative treatment simultaneously extends life cycle.Even if
It is the patient of tumor resection, previously research also indicates that expanding range of operation and extensive lymph node dissection not can be further improved pancreas
The survival rate of gland cancer.Therefore the early diagnosis for paying attention to cancer of pancreas, improves cancer of pancreas early stage diagnosis rate, increases Pancreas cancer patients operation
Cutting opportunity is an important link for improving cancer of pancreas survival rate.
It is solidifying that human tissue kallikrein 7 (kallikrein-related peptidase 7, KLK7) is also known as cuticula pancreas
Galactase (stratum corneumchymotryptic enzyme, SCCE), is initially one that identification is extracted from skin
Kind secretary protein.KLK7 gene is one of 15 members of kallikrein family, with other kallikrein genes in alkali
Basic sequence level has high homology, and the albumen and other members of same family of coding are also higher in amino acid levels homology,
Tertiary protein structure and function have many similitudes.The KLK7 egg of higher level can be detected in many body fluid of human body
It is white, such as：Amniotic fluid, ovarian cancer patients ascites, cerebrospinal fluid, milk, saliva, sperm, serum, synovium of joint liquid, urine, sweat etc..
In skin, KLK7 includes desmoglein, desmosome by mediating the extracellular binding proteins of boundary structure of cohesion between hydrolyzed cellular
Glycoprotein etc. leads to the furfur of skin.It, can both at home and abroad a large number of studies show that the invasion of KLK7 and tumour and transfer are closely bound up
The mechanism of energy is its hydrolyzed cellular epimatrix, promotes cell detachment, and tumour cell is easier to invasion and transfer whereby.Vladimir
The application organization chip Immunohistochemical Method detection such as Iakovlev includes the development process of cancer of pancreas difference (pancreas normal tissue, pancreas
Intraepithelial neoplasia (cin) and aggressive ductal adenocarcinoma of pancreas) full histotomy in KLK7 expression, to assess KLK7 to cancer of pancreas
Prognosis meaning, research find to start KLK7 abnormal expression, ductal adenocarcinoma of pancreas occur in the pancreatic intraepithelial neoplasia progress middle and later periods
Middle KLK7 expression is increased bad related to prognosis.In addition, have experiment in vitro researches show that：KLK7 hydrolyzes E cadherin,
The soluble E cadherin segment of generation, can significantly increase the ability of PANC-1 cell invasion extracellular matrix protein,
And cancer of pancreas PANC-1 cell aggregation is accordingly reduced.Prompt KLK7 may play important work in the invasion and transfer process of cancer of pancreas
With there are KLK7 to cause tumour cell to fall off on pancreatic cancer cell surface, and tumour early stage is caused to be shifted.
Up to the present, there are no a kind of effective blood serum designated objects in cancer of pancreas clinical diagnosis and treatment.Pass through immune group
Change experiment, it has been found that the expressing quantity of KLK7 is substantially raised relative to cancer beside organism in Pancreatic Adenocarcinoma, and pancreas group
Rich blood vessel nearby is knitted, therefore we guess that the KLK7 content of Pancreas cancer patients can also rise for normal person in serum
It is high.Therefore, if KLK7 can be as the blood serum designated object of cancer of pancreas auxiliary diagnosis, early diagnosis and treatment to Pancreas cancer patients
The selection of scheme will have great clinical value.
Summary of the invention
In consideration of it, the purpose of the present invention is to provide a kind of detection methods of serum marker of pancreatic cancer, and by the mark
Object and Imaging Technology and other biological markers use in conjunction, in the clinical early stage auxiliary diagnosis of Pancreas cancer patients.
Seminar of the present invention in early-stage study by Immunohistochemical Method demonstrate KLK7 in Pancreatic Adenocarcinoma compared with cancer by
Expression is significantly raised, then by that can make the increasing of pancreas cancer cell strain after a series of external related experiments discovery silencing KLK7 genes
It grows, shift and invasive ability decline, then find that KLK7 expression and Pancreas cancer patients post-operative survival rates are with related in serum
Property.Therefore, the detection method of content the present invention is to provide blood serum designated object KLK7 in human serum utilizes method of the invention
Can detecte the content of serum marker of pancreatic cancer KLK7, and it is easy to operate, easy to detect, efficiency is higher and low-cost.
To improve the noninvasive diagnosis rate of cancer of pancreas, provides for patient and treat much sooner and targetedly.
A kind of detection method of content of kallikrein 7 in blood, includes the following steps：
1) centrifuging and taking supernatant saves the blood sample to be measured of all collections after 4 DEG C of refrigerators stand 12 hours as required
In -80 DEG C of refrigerators, detection the previous day all blood serum samples press 1 with 1 × PBS liquid：It is to be measured that 5 ratios are diluted packing；
2) 96 hole elisa plates are distributed, 50 μ L test serum samples are added into each enzyme-linked hole, is placed in jog on shaking table and incubates
Educate 2h；
3) 50 μ l klk7 antibody are added into each hole and are placed in jog incubation 2h on shaking table at room temperature；
4) liquid in hole is removed, 1 × wash buffer hole flushing 3 times that 300 μ l are added (are put after buffer is added every time
Set shaking table 5min)；
5) blotting paper is laid in desktop, patting elisa plate keeps liquid removal in hole clean；
6) 100 1 × HRP of μ are added and are incubated for 30min in every hole, being placed on shaking table jog at room temperature；
7) step 4 and 5 is repeated；
8) it 100 μ l TMB liquid is added into every hole is placed on shaking table and be incubated for 30min at room temperature；
9) 100 μ l stop solution terminate liquids are added into every hole and terminate reaction；
10) terminate liquid jog is added to mix 30 seconds, it is ensured that blue all becomes yellow；
11) optical density (OD value) in each hole is measured with 450nm wavelength microplate reader.
12) optical density (OD value) is converted to by content value of the KLK7 in serum according to standard curve, according to standard curve
Optical density (OD value) is converted into content value of the KLK7 in serum, the content inspection of kallikrein 7 in blood can be completed
It surveys.
Blood serum designated object of the present invention by the kallikrein 7 during as diagnosis or auxiliary diagnosis cancer of pancreas
Upper application.
Detailed description of the invention
Fig. 1 is the expression of KLK7 albumen in pancreatic duct tissue by ductal adenocarcinoma of pancreas tissue and cancer.Figure A is pancreatic duct
The expression of KLK7 albumen in adenocarcinoma tissue；Scheme the expression that B is KLK7 albumen in pancreatic duct tissue by cancer.
Fig. 2 is KLK7 albumen content curve graph in serum.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this hair
Bright further description.
Embodiment one：KLK7 expression quantity in the pancreatectomy tissue of Pancreatic neoplasms patient is carried out by Immunohistochemical Method
Detection.It is collected in and goes to a doctor in Yichang Center People's Hospital's liver and gallbladder pancreas surgery and through pathology in October, 2015 in October, 2016
It is diagnosed as Pancreatic Adenocarcinoma and the cancer beside organism of 46 patients of cancer of pancreas.Specimen block excision after immediately 10% neutrality
Fixed in buffered formalin, by dehydration, transparent, paraffin embedding is made 4 μm of slabs and carries out immunohistochemical staining.Exempt from
The detection of epidemic disease histochemical staining is illustrated to be detected using EnVison two-step method by kit.
Detection method：EnVison two-step method
1) paraffin section is placed in 67 DEG C of baking ovens, is dried piece 2 hours；
2) paraffin section de-waxing, aquation, tap water rinse；
3) slice is put into the edta buffer liquid of PH9.0, Pressure method 2min；
4) 3% hydrogenperoxide steam generator is added dropwise on slice, is incubated for 10 minutes at room temperature, PBS flushing 3 times, 3 minutes every time (3 ×
3 minutes)；
5) PBS is removed, rabbit-anti people KLK7 antibody is added dropwise on slice, is incubated for 60 minutes at room temperature, PBS is rinsed 3 × 3 minutes；
6) PBS is removed, secondary antibody (HRPrabbit/mouse, Envision) is added dropwise on slice, is incubated for 40 minutes at room temperature,
PBS is rinsed 3 × 3 minutes；
7) PBS is removed, the DAB reagent colour development of Fresh is added dropwise on slice；
8) tap water rinses color development stopping, and haematoxylin is redyed, and 1% acidic alcohol differentiation, PBS returns indigo plant, de- through graded ethanol
Water, dimethylbenzene is transparent, neutral gum sealing.
Then using Image-Pro Plus V6.0 software to the resection organization research discovery KLK7 of 46 Pancreas cancer patients
Expression quantity dramatically increased compared with cancer beside organism, it was confirmed that KLK7 high expression in cancer of pancreas.By statistical analysis, relative to
Normal pancreatic tissue, the expression quantity of KLK7 increases by 20~70 times because of individual difference in cancerous tissue, averagely increases by 25 times or so.Fig. 1：
The expression of KLK7 albumen in ductal adenocarcinoma of pancreas tissue and cancer side pancreatic duct tissue.(immunohistochemical staining presentation graphics, SP
Method dyeing, × 400), KLK7 is expressed in ductal adenocarcinoma of pancreas tissue in strong positive (rufous), it is seen that KLK7 is mainly expressed in
In cytoplasm and on cell membrane (figure A)；It is hardly expressed in pancreatic duct tissue by the cancer, only there is minute quantity on cell membrane
The expression (figure B) of KLK7.
Embodiment two：KLK7 albumen is in Pancreas cancer patients, cystadenoma of pancreas patient, in Pancreatitis Patients and normal human serum
The detection of content.It is collected in the trouble gone to a doctor in Yichang Center People's Hospital's liver and gallbladder pancreas surgery 2 months in October, 2016 in 2016
Person's serum, it is Pancreas cancer patients (preoperative) serum 32, cystadenoma of pancreas patients serum 32, Pancreatitis Patients serum 32, another
Random collection 32 Healthy Volunteers of serum.Using elisa technique (Enzolifescience, Kallikrein-7 (human),
ELISA Kit, ADI-900-218-0001) content of KLK7 in serum is detected.
Article equipment prepares before detecting
1) centrifuging and taking supernatant is stored in -80 DEG C to all blood samples to be measured after 4 DEG C of refrigerators stand 12 hours as required
Refrigerator；
2) detection the previous day is added 10ml distilled water jog to 10 × wash buffer liquid and 10 × HRP and mixes preparation institute
It need to be at detection working solution.
3) detection the previous day all blood serum samples press 1 with 1 × PBS liquid：It is to be detected that 5 ratios are diluted packing.
Detection method
1) 96 hole elisa plates of distribution are rationally designed, 50 μ l test serum samples are added into each enzyme-linked hole；
2) 50 μ l klk7 antibody are added into each hole and are placed in jog incubation 2h on shaking table at room temperature；
3) liquid in hole is removed, 1 × wash buffer hole flushing 3 times that 300 μ l are added (are put after buffer is added every time
Set shaking table 5min)；
4) blotting paper is laid in desktop, patting elisa plate keeps liquid removal in hole clean；
5) 100 1 × HRP of μ are added and are incubated for 30min in every hole, being placed on shaking table jog at room temperature；
6) step 4 and 5 is repeated；
7) it 100 μ l TMB liquid is added into every hole is placed on shaking table and be incubated for 30min at room temperature；
8) 100 μ l stop solution terminate liquids are added into every hole and terminate reaction；
9) terminate liquid jog is added to mix 30 seconds, it is ensured that blue all becomes yellow；
10) optical density (OD value) in each hole is measured with 450nm wavelength microplate reader.
11) optical density (OD value) is converted to by content value of the KLK7 in serum according to standard curve, according to standard curve
Optical density (OD value) is converted into content value of the KLK7 in serum, the content inspection of kallikrein 7 in blood can be completed
It surveys.
According to testing requirements, strict control testing conditions carry out 3 independent detections, and testing result has repeatability,
In as shown in table 1.In addition, carrying out standard curve with the titer that kit provides while each independent detection tests progress
Parameter control test tests (the same detecting step of its process), and the results are shown in Table 2.
Table 1：KLK7 albumen content detection result in serum
Fig. 2 is KLK7 albumen content curve graph in serum, and according to standard curve, average content of the KLK7 in serum is about
For 32.5ng/ml, there is correlative study to show that the content of KLK7 is about 8ng/ml in normal human serum.In Pancreas cancer patients serum
KLK7 is higher by about 3 times compared with normal human serum, and cystadenoma of pancreas patient, Pancreatitis Patients are compared with normal people compared with without obvious area
Not.Therefore, through analysis shows that KLK7 according to the present invention as the serum mark in the support decision process of Pancreas cancer patients
Object has feasibility, for cancer of pancreas clinical diagnosis provide a kind of effective blood serum designated object and with Imaging Technology and its
Its biological markers use in conjunction is to improve the noninvasive diagnosis rate of Pancreas cancer patients.
Claims (2)
1. a kind of detection method of content of kallikrein 7 in blood, which is characterized in that include the following steps：
(1) Pancreas cancer patients serum is collected, centrifuging and taking supernatant is stored in -80 DEG C of ice after 4 DEG C of refrigerators stand 10-15 hours
Case, experiment the previous day all blood serum samples press 1 with 1 × PBS liquid：5 ratios, which are diluted, to be dispensed wait test detection；
(2) 96 hole elisa plates are distributed, test serum is added into each enzyme-linked hole respectively；
(3) KLK7 antibody is added into each hole and is placed in jog incubation on shaking table at room temperature, removes the liquid in hole, is added 1
× wash buffer hole flushing places shaking table after washbuffer is added every time；
(4) blotting paper is laid in desktop, patting elisa plate keeps liquid removal in hole clean；
(5) HRP is added and is incubated in every hole, being placed on shaking table jog at room temperature；
(6) step 1 time of step 4 and 5 is repeated；
(7) TMB liquid is added into every hole to be placed on shaking table and be incubated at room temperature；
(8) stop solution terminate liquid is added into every hole and terminates reaction；
(9) jog mixes after terminate liquid stop solution terminate liquid is added, it is ensured that blue all becomes yellow；
(10) optical density in each hole is measured with 450nm wavelength microplate reader；
(11) according to standard curve by optical density（OD value）Content value of the KLK7 in serum is converted to, kassinin kinin can be completed and release
Put the content detection of enzyme 7 in blood.
2. blood serum designated object of the kallikrein 7 described in claim 1 during as diagnosis or auxiliary diagnosis cancer of pancreas
Upper application.
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Citations (4)
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| CN106153917A (en) * | 2015-04-10 | 2016-11-23 | 中国医学科学院肿瘤医院 | Two groups of cancer of pancreas blood plasma diagnosis marker spectrums and application thereof |
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2017
- 2017-05-24 CN CN201710374234.6A patent/CN108931651A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070099209A1 (en) * | 2005-06-13 | 2007-05-03 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| US20120040861A1 (en) * | 2010-08-13 | 2012-02-16 | Somalogic, Inc. | Pancreatic Cancer Biomarkers and Uses Thereof |
| CN103415624A (en) * | 2010-08-13 | 2013-11-27 | 私募蛋白质体公司 | Pancreatic cancer biomarkers and uses thereof |
| CN104698170A (en) * | 2015-03-17 | 2015-06-10 | 任传利 | Method of taking IL-11 (interleukin) as biomarker in diagnosis and prognostic evaluation of pancreatic cancer |
| CN106153917A (en) * | 2015-04-10 | 2016-11-23 | 中国医学科学院肿瘤医院 | Two groups of cancer of pancreas blood plasma diagnosis marker spectrums and application thereof |
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| SARAH K. JOHNSON等: "Kallikrein 7 Enhances Pancreatic Cancer Cell Invasion by Shedding E-cadherin", 《CANCER》 * |
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Application publication date: 20181204 |