CN108931650A - A kind of kallikrein 7 activity test method 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 activity test method in blood and its as the application on the blood serum designated object of diagnosis of pancreatic cancer Download PDFInfo
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- CN108931650A CN108931650A CN201710373623.7A CN201710373623A CN108931650A CN 108931650 A CN108931650 A CN 108931650A CN 201710373623 A CN201710373623 A CN 201710373623A CN 108931650 A CN108931650 A CN 108931650A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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
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Abstract
The present invention provides a kind of activity test method 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.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, is added wash buffer hole flushing 3 times, shaking table 5min is placed after washbuffer is added every time, blotting paper is laid in desktop, patting elisa plate keeps liquid removal in hole clean；KLK7 FRET fluorogenic substrate McaRPKPVENvalWRK (Dnp) NH is added230min is incubated in every hole, being placed in jog on shaking table at 37 DEG C；It is detected at excitation wavelength 320nm, wavelength of transmitted light 405nm in fluorescence microplate reader.
Description
Technical field
The present invention relates to field of medicaments, particularly relate to the activity 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 detection
Method and Imaging Technology and other biological markers use in conjunction, the clinical early stage auxiliary diagnosis for Pancreas cancer patients
In.
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, proliferation, transfer and the invasive ability of pancreatic cancer cell can be made to decline after then finding silencing KLK7 gene, so
There is correlation with Pancreas cancer patients post-operative survival rates by a series of KLK7 level in external related experiments discovery serum afterwards.Cause
This, the present invention is to provide detection method of content of the marker KLK7 in human serum, can detecte using method of the invention
The content of serum marker of pancreatic cancer KLK7, and it is easy to operate, easy to detect, low-cost.To improve the non-of cancer of pancreas
Invasive diagnostic rate is provided for patient and is treated much sooner and targetedly.
A kind of activity test method 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 μ L KLK7FRET fluorogenic substrate McaRPKPVENvalWRK (Dnp) NH are added2In in every hole, make its eventually it is dense
Degree is 10 μM, and jog on shaking table is placed at 37 DEG C and is incubated for 30min；
7) it is detected at excitation wavelength 320nm, wavelength of transmitted light 405nm in fluorescence microplate reader.
Blood serum designated object of the present invention by the kallikrein 7 during as diagnosis or auxiliary diagnosis cancer of pancreas
On 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.
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.
Example one：KLK7 expression quantity in the pancreatectomy tissue of Pancreatic neoplasms patient is examined by Immunohistochemical Method
It surveys.It is collected in and goes to a doctor in Yichang Center People's Hospital's liver and gallbladder pancreas surgery and examined through pathology in October, 2015 in October, 2016
Pancreatic Adenocarcinoma and the cancer beside organism broken as 46 patients of cancer of pancreas.Neutrality after specimen block's excision immediately 10% is slow
It rushes in formalin and fixes, by dehydration, transparent, paraffin embedding is made 4 μm of slabs and carries out immunohistochemical stainings.It is immune
Histochemical staining detection 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 is rinsed 3 times, 3 minutes (3X3 every time
Minute)；
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.
Example two：KLK7 albumen is in Pancreas cancer patients, cystadenoma of pancreas patient, living in Pancreatitis Patients and normal human serum
The detection of property.It is collected in what 2 months in October, 2016 in 2016 went to a doctor in No.1 Clinical Medical College, Sanxia Univ.'s liver and gallbladder pancreas surgery
Patients serum, 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 FRET technology (Enzolifescience, Kallikrein-7
(human),ELISAKit,ADI-900-218-0001；McaRPKPVENvalWRK (Dnp) NH2, R&D systems, 2624-
SE) activity 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 checked after 4 DEG C of refrigerators stand 12 hours as required
Refrigerator；
2) 270ml distilled water was added into 10 × wash buffer liquid and prepares 1 × washbuffer in normal detection the previous day
Temperature saves；
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.
Experimental method
1) 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；
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 μ L KLK7FRET fluorogenic substrate McaRPKPVENvalWRK (Dnp) NH are added2In in every hole, make its eventually it is dense
Degree is 10 μM, and jog on shaking table is placed at 37 DEG C and is incubated for 30min；
6) it is detected at excitation wavelength 320nm, wavelength of transmitted light 405nm in fluorescence microplate reader.
Testing result has repeatability, wherein as shown in table 1.
Table 1：KLK7 albumen Activity determination result in serum
Through analysis shows that KLK7 compared with normal human serum is higher by about 280% in Pancreas cancer patients serum, and cystadenoma of pancreas is suffered from
Person, Pancreatitis Patients are compared with normal people compared with no significant difference.Therefore KLK7 according to the present invention is as Pancreas cancer patients
Blood serum designated object in support decision process is feasible.A kind of promising tumor marker and image are provided for the clinical diagnosis of cancer of pancreas
Technology and other biological markers use in conjunction are to improve the noninvasive diagnosis of Pancreas cancer patients.
Claims (2)
1. a kind of activity test method of marker in blood, which is characterized in that the marker is human tissue kallikrein
7, include 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 10-15 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, test serum sample is added into each enzyme-linked hole, jog on shaking table is placed in and is incubated for；
(3) klk7 antibody is added into each hole and is placed in jog incubation on shaking table at room temperature；
(4) liquid in hole is removed, 1 × wash buffer hole flushing is added, places shaking table 5min after buffer is added every time；
(5) blotting paper is laid in desktop, patting elisa plate keeps liquid removal in hole clean；
(6) KLK7 FRET fluorogenic substrate McaRPKPVENvalWRK (Dnp) NH is added2In in every hole, keep its final concentration of, then
Jog on shaking table is placed at 37 DEG C to be incubated for；
(7) it is detected at excitation wavelength 320nm, wavelength of transmitted light 405nm in fluorescence microplate reader, mark can be completed
The Activity determination of will object 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 (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2468651A1 (en) * | 2003-06-13 | 2004-12-13 | Eleftherios P. Diamandis | Detection of neurodegenerative diseases |
| WO2005075667A1 (en) * | 2004-02-03 | 2005-08-18 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with kallikrein 7 (klk7) |
| CN106198980A (en) * | 2010-08-13 | 2016-12-07 | 私募蛋白质体公司 | Cancer of pancreas biomarker and application thereof |
-
2017
- 2017-05-24 CN CN201710373623.7A patent/CN108931650A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2468651A1 (en) * | 2003-06-13 | 2004-12-13 | Eleftherios P. Diamandis | Detection of neurodegenerative diseases |
| WO2005075667A1 (en) * | 2004-02-03 | 2005-08-18 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with kallikrein 7 (klk7) |
| CN106198980A (en) * | 2010-08-13 | 2016-12-07 | 私募蛋白质体公司 | Cancer of pancreas biomarker and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ENZO LIFE SCIENCES INC.: "《Kallikrein-7 (human) ELISA Kit》", 9 August 2016 * |
| ISRAEL S. FERNANDEZ等: "Crystal Structure of Huma Epidermal Kallikrein 7 （hK7） Synthesized Directly in its Native State in E.coli: Insights into the Atomic Basis of its Inhibition by LEKT1 Domain 6(LD6)", 《JOURNAL OF MOLECULAR BIOLOGY》 * |
| SARAH K. JOHNSON等: "Kallikrein 7 Enhances Pancreatic Cancer Cell Invasion by Shedding E-cadherin", 《CANCER》 * |
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Application publication date: 20181204 |