CN107271672A - Applications of the excretion body p ERK in diagnosis of colorectal carcinoma product is prepared - Google Patents
Applications of the excretion body p ERK in diagnosis of colorectal carcinoma product is prepared Download PDFInfo
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- CN107271672A CN107271672A CN201710476829.2A CN201710476829A CN107271672A CN 107271672 A CN107271672 A CN 107271672A CN 201710476829 A CN201710476829 A CN 201710476829A CN 107271672 A CN107271672 A CN 107271672A
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- erk
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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
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- 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
- G01N33/57446—Specifically defined cancers of stomach or intestine
Abstract
The invention discloses applications of the p ERK in excretion body in diagnosis colorectal cancer.The study result show that, be free of in the excretion body in the nutrient solution of normal noncancerous cells and contain more p ERK in the excretion body in p ERK, the nutrient solution of colorectal cancer cell, therefore p ERK in excretion body can be regard as the molecular marker for diagnosing colorectal cancer.The achievement in research of the present invention provides a kind of colorectal cancer non-invasive diagnosis method with wide application prospect for clinic.
Description
Technical field
The invention belongs to biomedical sector, it is related to the diagnosis of cancer, and in particular to excretion body p-ERK is in diagnosis Colon and rectum
Application in cancer.
Background technology
Tumor tissue biopsy frequently as diagnosing tumor goldstandard, but tissue Biopsy have certain limitation.It is main
Show as:Tumour has heterogeneity;Some patients are not suitable for doing tissue biopsy;Organize treatment of the hysteresis quality of biopsy to patient
It is unfavorable.Therefore diagnosis and detection technique for cancer has higher requirement.The appearance of liquid Biopsy, solves above-mentioned ask
Topic, also shortens the Diagnostic Time of cancer.As a branch of in-vitro diagnosis, liquid biopsy be exactly by detect blood or
Some of the body fluid such as urine bioactive molecule, so as to make diagnosis to diseases such as cancers.It is advantageous that can be invaded by non-
The harm of entering property sampling reduction biopsy;Because it has the features such as sensitiveness is high, detection is quick, and the early stage of tumour is conducive to send out
It is existing.Liquid biopsy mainly includes three kinds of detection methods:The detection of dissociative DNA, the detection of circulating tumor cell, cell excretion body
Detection.
Excretion body is that active secretion is homogeneous to extracellular size after intracellular many vesica bodies are merged with cytoplasma membrane, directly
Footpath is 30-100nm lipid bilayer Rotating fields vesica.Excretion body can be de- by different type cell (including tumour cell)
Fall release, in most of body fluid such as peripheral blood, urine, saliva, ascites, amniotic fluid, milk, cerebrospinal fluid, joint fluid, bronchovesicular
It can detect in the body fluid such as irrigating solution.Containing many bioactive substances in excretion body, such as protein, mRNA, microRNA,
DNA fragmentation etc., these bioactive substances can mutually be transmitted in iuntercellular, and then adjust the physiological function of cell.Tumour cell
The excretion body in source can be by number of ways modulate tumor environment, and then promotes the progress of tumour.
The content of the invention
It is an object of the invention to provide a kind of molecular marker early diagnosed available for colorectal cancer.Present invention research
It was found that, contain p-ERK in colorectal cancer cell nutrient solution in excretion body, and it is outer in the cell culture fluid of normal non-cancerous cells
Secrete and p-ERK is not contained in body.According to above achievement in research, applicants have discovered that a kind of side available for colorectal cancer non-invasive diagnosis
Method, by detecting that the presence or absence of excretion body p-ERK or expression quantity can be used to diagnose colorectal cancer in the preferred blood of body fluid.
According to an aspect of the present invention, Colon and rectum is being prepared the invention provides the reagent of p-ERK in detection excretion body
Application in cancer diagnostic products.
Further, p-ERK reagent includes p-ERK presence in any excretion body available for detection in the detection excretion body
Whether or the method for detection p-ERK expression quantity in the reagent that uses.
The detection p-ERK presence or absence detects that the method for p-ERK expression quantity is selected from the following group:Immunohistochemistry is surveyed
Determine method, enzyme-linked immunosorbent assay, in situ hybridization, chromatography, liquid chromatography, SEC, high-efficient liquid phase color
Spectrometry, gas chromatography, mass spectrography, tandem mass spectrometry, substance assistant laser desorpted/ionization time of flight mass spectrometry method, electron spray
Ionization mass spectrometry, surface-enhanced laser desorption/ionization time of flight mass spectrometry method, quadrupole time-of-flight mass spectrometry (TOFMS), atmospheric pressure photoelectricity
From mass spectrography, fourier transform mass spectrometry, substance assistant laser desorpted/ionization-Fourier transform ion cyclotron resonance mass spectroscopy method,
Secondary ion mass spectrometry, radioimmunoassay, microexamination, the determination method based on micro-fluid chip, surface plasma
Resonance body, sequencing, Western blotting assays.
Further, p-ERK reagent includes p-ERK antibody in detection excretion body.The antibody include monoclonal antibody,
Polyclonal antibody or immune serum.
According to another aspect of the present invention, present invention also offers p-ERK in excretion body as mark exploitation and/
Or the application in design diagnosis of colorectal carcinoma product.
According to a further aspect of the invention, present invention also offers a kind of diagnostic products of colorectal cancer.The diagnosis
Product includes the reagent of p-ERK in detection excretion body.
Further, p-ERK reagent includes p-ERK presence in any excretion body available for detection in the detection excretion body
Whether or the method for detection p-ERK expression quantity in the reagent that uses.
The detection p-ERK presence or absence detects that the method for p-ERK expression quantity is selected from the following group:Immunohistochemistry is surveyed
Determine method, enzyme-linked immunosorbent assay, in situ hybridization, chromatography, liquid chromatography, SEC, high-efficient liquid phase color
Spectrometry, gas chromatography, mass spectrography, tandem mass spectrometry, substance assistant laser desorpted/ionization time of flight mass spectrometry method, electron spray
Ionization mass spectrometry, surface-enhanced laser desorption/ionization time of flight mass spectrometry method, quadrupole time-of-flight mass spectrometry (TOFMS), atmospheric pressure photoelectricity
From mass spectrography, fourier transform mass spectrometry, substance assistant laser desorpted/ionization-Fourier transform ion cyclotron resonance mass spectroscopy method,
Secondary ion mass spectrometry, radioimmunoassay, microexamination, the determination method based on micro-fluid chip, surface plasma
Resonance body, sequencing, Western blotting assays.
Further, p-ERK reagent includes p-ERK antibody in detection excretion body.The antibody include monoclonal antibody,
Polyclonal antibody or immune serum.
Further, the reagent may also include the reagent used in separation excretion body, the reagent used in cracking excretion body.
Reagent used in the separation excretion body includes the reagent used in any method that can separate excretion body.
The method of separation excretion body includes but is not limited to supercentrifugation, the precipitation method, molecular exclusion chromatography, surface protein mark
Remember affine separation method, immunomagnetic beads liquid chromatography, ultrafiltration, RNA isolation kit.
Further, diagnostic products of the invention can be diagnostic all types of products, including reagent, reagent
Box, chip, test paper.
The excretion body that the present invention is used to diagnose colorectal cancer comes from body fluid, and the body fluid includes blood, urine, saliva, abdomen
Water, amniotic fluid, milk, cerebrospinal fluid, joint fluid, BAL fluid.Preferably, it is of the invention to be used to diagnose colorectal cancer
Excretion body carrys out autoblood.
The step of diagnostic products of the present invention carry out diagnosis of colorectal carcinoma includes:
(1) excretion body is separated from body fluid;
(2) p-ERK expressions in detection excretion body;
(3) diagnosis of colorectal carcinoma is helped according to p-ERK expressing informations.
In above-mentioned steps, the body fluid includes blood, urine, saliva, ascites, amniotic fluid, milk, cerebrospinal fluid, joint fluid, branch
Bronchoalveolar lavage fluid.Preferably, body fluid is blood.
The physicochemical properties such as size, density and surface marker according to excretion body, at present separation body fluid and cell training
The method of excretion body has classical supercentrifugation, the precipitation method, molecular exclusion chromatography, surface protein to mark affine separation in nutrient solution
Method, immunomagnetic beads liquid chromatography, ultrafiltration, RNA isolation kit.
The advantages of the present invention are as follows:
(1) albumen is a kind of new biomarkers in excretion body in blood, is different from traditional biological mark, not only surely
It is fixed, minimally invasive, be easy to detection, it is and quantitative accurate, the Sensitivity and Specificity of medical diagnosis on disease will be greatly improved, such biomarker
Successful exploitation contribute to the auxiliary diagnosis of colorectal cancer, be that the development of other diseases biomarker is offered reference.
(2) present invention develops a kind of noninvasive method for diagnosing colorectal cancer, and this method is easy to operate, subtract significantly
The light pain of patient.
Brief description of the drawings
Fig. 1 displays detect the electrophoretogram of p-ERK contents in excretion body using Western blot;
Fig. 2 displays utilize the aspect graph of electron microscopic observation excretion body;
Fig. 3 displays detect the electrophoretogram of excretion body characteristicses mark using Western blot.
Embodiment
The present invention is further illustrated with reference to specific embodiment, embodiments of the invention are only used for explaining the present invention,
It is not intended to limit protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The separation and identification of excretion body in the cell culture fluid of embodiment 1
1st, cell culture
HT-29 cells and HUVECs cells are in containing 10% hyclone and dual anti-(penicillin 100U/ml, streptomysin
In DMEM culture mediums 100U/ml), in 37 DEG C, 5%CO2Under conditions of cultivate, cell in monolayer adherence growth, routinely change liquid,
Passage, the cell of selection exponential phase is tested.
2nd, in cell culture fluid excretion body separation
Using ExoQuick-TCExosome Precipitation Solution kits, HT-29 cell culture is collected
Liquid and HUVECs cell culture fluids each 10ml, 3000 × g, centrifugation 15min (4 DEG C) remove cell and cell fragment, supernatant transfer
Into sterile chamber, 2ml ExoQuick-TC Exosome Precipitation Solution are added, examination is overturned and rock
Pipe mixes solution, and 4 DEG C of refrigerated overnights (being at least incubated 12h), test tube should not rotate during incubation, next day, will
ExoQuick-TCExosome Precipitation Solution/ cell culture liquid mixtures centrifuge 30min with 1500 × g
(room temperature or 4 DEG C), after centrifugation, the cream-coloured or white precipitate in test tube bottom is excretion body, again except supernatant, by test tube
1500 × g, centrifugation 5min (4 DEG C), the ExoQuick-TC solution of rotary suction residual, liquid is aspirated totally, is careful not to connect
The excretion body precipitation prepared is touched, is precipitated using 60 μ l DEPC water again suspension excretion body, the packing of 10 μ l/EP pipes is put in -80
DEG C refrigerator cold-storage, it is stand-by.
3rd, the observation of excretion volume morphing
Excretion body separation after, be fixed on 0.01M phosphate (include 4% paraformaldehyde, pH is buffer solution 7.4)
In, 4 DEG C are overnight.Next day, PBS is fixed on 1%O,sO4 30 minutes after rinsing.After distilled water flushing, by excretion body sediment
It is dehydrated in the alcohol of concentration gradient, carries out dyeing 30 minutes using containing 1% uranium acetate, be embedded in Taab 812.
After polymerization is stayed overnight at 60 DEG C, section, ultra-thin section is observed using electron microscope.
As a result:The form of excretion body under the excretion body in cell culture fluid, Electronic Speculum is isolated as shown in Fig. 2 in vesica knot
Structure.
4th, excretion body surface region feature marker detection
Using Western Blot methods detection excretion body surface region feature mark CD9.
(1) excretion body is cracked
200 μ l RIPA buffer, vortex 15s are added in the fresh excretion body precipitation prepared, are placed at room temperature
5min (is waited to be completely dissolved), adds 2*Laemmli buffer, and 95 DEG C are heated 5 minutes, before gel loading is added to, on ice
Cooling 5 minutes.RIPA buffer and Laemmli buffer formula are as shown in table 1.
The solution formula of table 1
HT-29 the and HUVECs cells of exponential phase are conventionally collected, are cracked using the above method, obtain thin
Born of the same parents' albumen.
2nd, BCA methods determine excretion body protein concentration
1. 10 μ l standard specimens and excretion body protein are drawn or cell protein is added separately in each hole of 96 orifice plates;
2. 200 μ l BCA working solutions are added per hole, mixing 30s is rocked, covers various kinds sample wells;
3. standard detection:30min is incubated at 37 DEG C or room temperature places 2~16h;
4. room temperature, ELIASA 562nm wavelength detectings absorbance (OD) are cooled to;
5. standard curve is drawn according to absorbance and calculates the concentration of excretion body protein or cell protein.
3rd, 20 μ g excretions body proteins and cell protein is taken to carry out polyacrylamide gel electrophoresis, transferring film, with 2% ox respectively
Seralbumin (BSA) is closed 1 hour, is added primary antibody (4 DEG C of incubator overnights), TBST and is washed that film, secondary antibody are incubated 45 minutes, TBST washes
Film, development is exposed with the low background chemiluminescence instruments of Western blot.
4th, result
As a result as shown in figure 3, detecting the markers characteristic CD9 of excretion body table in the excretion body protein of separation
Reach, show the success of excretion body separation and Extraction.
P-ERK differential expression in the excretion body of embodiment 2
Step:Take the excretion body protein row polyacrylamide gel electrophoresis after being quantified in 20 μ g steps 1, transferring film, with 2%
Bovine serum albumin(BSA) (BSA) is closed 1 hour, is added primary antibody (4 DEG C of incubator overnights), TBST and is washed film, secondary antibody incubation 45 minutes, TBST
Film is washed, development is exposed with the low background chemiluminescence instruments of Western blot.
As a result:The Western blot results for taking the μ g of excretion body protein 20 of HT-29 cells, HUVECs cells to carry out respectively
As shown in figure 1, not containing p-ERK in HUVECs cell excretion bodies, and contain more p-ERK in HT-29 cell excretion bodies.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improve can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
Claims (10)
1. detect application of the reagent of p-ERK in excretion body in diagnosis of colorectal carcinoma product is prepared.
2. application according to claim 1, it is characterised in that the reagent includes detection p-ERK presence or absence or detection
The reagent used in the method for p-ERK expression quantity.
3. application according to claim 2, it is characterised in that the detection p-ERK presence or absence or detection p-ERK expression
The method of amount is selected from the following group:Immunohistochemical assay, enzyme-linked immunosorbent assay, in situ hybridization, chromatography, liquid phase
Chromatography, SEC, high performance liquid chromatography, gas chromatography, mass spectrography, tandem mass spectrometry, Matrix-assisted swash
Photodesorption/ionization time of flight mass spectrometry method, electrospray ionization mass spectrometry, surface-enhanced laser desorption/ionization time of flight mass spectrometry
Method, quadrupole time-of-flight mass spectrometry (TOFMS), atmospheric pressure photoionization mass spectrography, fourier transform mass spectrometry, substance assistant laser desorpted/electricity
From-Fourier transform ion cyclotron resonance mass spectroscopy method, secondary ion mass spectrometry, radioimmunoassay, microexamination, base
In the determination method of micro-fluid chip, surface plasma body resonant vibration, sequencing, Western blotting assays.
4. the application according to any one of claim 1-3, it is characterised in that the reagent includes p-ERK antibody.
5. application according to claim 4, it is characterised in that the antibody include monoclonal antibody, polyclonal antibody or
Immune serum.
6. the application according to any one of claim 1-3, it is characterised in that the diagnostic products include kit, core
Piece, test paper.
7. the application according to any one of claim 1-3, it is characterised in that excretion body comes from body fluid, the body fluid choosing
From with the following group:Blood, urine, saliva, ascites, amniotic fluid, milk, cerebrospinal fluid, joint fluid, BAL fluid.
8. application according to claim 7, it is characterised in that the excretion body carrys out autoblood.
9. applications of the p-ERK as mark in developing and/or designing diagnosis of colorectal carcinoma product in excretion body.
10. application according to claim 9, it is characterised in that excretion body comes from body fluid, the body fluid is selected from the following group:
Blood, urine, saliva, ascites, amniotic fluid, milk, cerebrospinal fluid, joint fluid, BAL fluid.
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Cited By (2)
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CN113194820A (en) * | 2019-03-04 | 2021-07-30 | 艾索波特株式会社 | Method and system for providing cancer diagnosis information using liquid biopsy based on artificial intelligence by using exosome |
CN113980903A (en) * | 2021-11-04 | 2022-01-28 | 中南大学湘雅医院 | Colorectal cancer cell exosome and separation and extraction method and application thereof |
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