CN107271672B - Application of the excretion body p-ERK in preparation diagnosis of colorectal carcinoma product - Google Patents
Application of the excretion body p-ERK in preparation diagnosis of colorectal carcinoma product Download PDFInfo
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- 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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- 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
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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
- G01N33/57446—Specifically defined cancers of stomach or intestine
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Abstract
The invention discloses application of the p-ERK in excretion body in diagnosis colorectal cancer.The study result show that being free of p-ERK in excretion body in the culture solution of normal noncancerous cells, more p-ERK is contained in the excretion body in the culture solution of colorectal cancer cell, therefore can be using p-ERK in excretion body as the molecular marker for diagnosing colorectal cancer.Research achievement of the invention provides a kind of colorectal cancer non-invasive diagnosis method with wide application prospect for clinic.
Description
Technical field
The invention belongs to fields of biomedicine, are related to the diagnosis of cancer, and in particular to excretion body p-ERK is in diagnosis Colon and rectum
Application in cancer.
Background technique
Tumor tissue biopsy frequently as diagnosing tumor goldstandard, but organize Biopsy have certain limitation.It is main
Show themselves in that tumour has heterogeneity；Certain patients are not suitable for doing tissue biopsy；Organize treatment of the hysteresis quality of biopsy to patient
It is unfavorable.Therefore there is higher requirement for the diagnosis of cancer and detection technique.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 pass through detection blood or
Certain bioactive molecules in the body fluid such as urine, to make diagnosis to diseases such as cancers.It is advantageous that can be invaded by non-
The sampling of entering property reduces the harm of biopsy；Since it has the characteristics that sensibility is high, detection is quick, and be conducive to the early stage hair of tumour
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 uniform to extracellular size after intracellular more vesica bodies are merged with cytoplasma membrane, directly
Diameter is the lipid bilayer structure vesica of 30-100nm.Excretion body can be de- by different type cell (including tumour cell)
Release is fallen, in most of body fluid such as peripheral blood, urine, saliva, ascites, amniotic fluid, milk, cerebrospinal fluid, joint fluid, bronchovesicular
It can be detected in the body fluid such as irrigating solution.Containing there are many bioactive substance 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 modulate tumor environment, and then the progress of promotion tumour through a variety of ways.
Summary of the invention
The purpose of the present invention is to provide a kind of molecular markers that can be used for colorectal cancer early diagnosis.Present invention research
It was found that contain p-ERK in colorectal cancer cell culture solution in excretion body, and it is outer in the cell culture fluid of normal non-cancerous cells
It secretes in body without containing p-ERK.According to the above research achievement, applicants have discovered that a kind of side that can be used for colorectal cancer non-invasive diagnosis
Method can be used to diagnosis colorectal cancer by the presence or absence of excretion body p-ERK in the detection preferred blood of body fluid or expression quantity.
According to an aspect of the present invention, the present invention provides the reagents of p-ERK in detection excretion body to prepare Colon and rectum
Application in cancer diagnostic products.
Further, the reagent of p-ERK includes that any p-ERK that can be used for detecting in excretion body exists in the detection excretion body
Whether or detection p-ERK expression quantity method used in reagent.
The detection p-ERK presence or absence or the method for detecting p-ERK expression quantity are selected from the following group: immunohistochemistry is surveyed
Determine method, enzyme-linked immunosorbent assay, in situ hybridization, chromatography, liquid chromatography, size exclusion chromatography, 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 measuring method based on micro-fluid chip, surface plasma
Resonance body, sequencing, Western blotting assays.
Further, the reagent for detecting p-ERK in excretion body includes the antibody of p-ERK.The antibody include monoclonal antibody,
Polyclonal antibody or immune serum.
According to another aspect of the present invention, the present invention also provides p-ERK in excretion body as marker exploitation and/
Or the application in design diagnosis of colorectal carcinoma product.
According to a further aspect of the invention, the present invention also provides a kind of diagnostic products of colorectal cancer.The diagnosis
Product includes the reagent for detecting p-ERK in excretion body.
Further, the reagent of p-ERK includes that any p-ERK that can be used for detecting in excretion body exists in the detection excretion body
Whether or detection p-ERK expression quantity method used in reagent.
The detection p-ERK presence or absence or the method for detecting p-ERK expression quantity are selected from the following group: immunohistochemistry is surveyed
Determine method, enzyme-linked immunosorbent assay, in situ hybridization, chromatography, liquid chromatography, size exclusion chromatography, 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 measuring method based on micro-fluid chip, surface plasma
Resonance body, sequencing, Western blotting assays.
Further, the reagent for detecting p-ERK in excretion body includes the antibody of p-ERK.The antibody include monoclonal antibody,
Polyclonal antibody or immune serum.
Further, the reagent may also include reagent used in reagent used in separation excretion body, cracking excretion body.
Reagent used in the separation excretion body includes reagent used in any method that can separate excretion body.
The method for separating 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 present invention is used to diagnose the excretion body of colorectal cancer from body fluid, and the body fluid includes blood, urine, saliva, abdomen
Water, amniotic fluid, milk, cerebrospinal fluid, joint fluid, BAL fluid.Preferably, of the invention for diagnosing colorectal cancer
Excretion body carrys out autoblood.
Diagnostic products of the invention carry out the step of diagnosis of colorectal carcinoma and include:
(1) excretion body is separated from body fluid；
(2) p-ERK expression in excretion body is detected；
(3) diagnosis of colorectal carcinoma is helped according to p-ERK expressing information.
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 separate body fluid and cell training at present
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 marker, not only surely
It is fixed, minimally invasive, be easy to detect, and it is quantitative accurate, the sensibility and specificity of medical diagnosis on disease will be greatly improved, such biomarker
Successful exploitation facilitate the auxiliary diagnosis of colorectal cancer, offer reference for the development of other diseases biomarker.
(2) present invention develops a kind of noninvasive method for diagnosing colorectal cancer, and this method is easy to operate, subtracts significantly
The light pain of patient.
Detailed description of the invention
Fig. 1 shows the electrophoretogram using p-ERK content in Western blot detection excretion body；
Fig. 2 shows the aspect graph using Electronic Speculum observation excretion body；
Fig. 3 shows the electrophoretogram using Western blot detection excretion body characteristics marker.
Specific embodiment
Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the 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.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The separation and identification of excretion body in 1 cell culture fluid of embodiment
1, cell culture
HT-29 cell and HUVECs cell are in containing 10% fetal calf serum and dual anti-(penicillin 100U/ml, streptomysin
In DMEM culture medium 100U/ml), in 37 DEG C, 5%CO2Under conditions of cultivate, cell in monolayer adherence grow, routinely change liquid,
Passage, selects the cell of logarithmic growth phase to be tested.
2, in cell culture fluid excretion body separation
Using ExoQuick-TCExosome Precipitation Solution kit, HT-29 cell culture is collected
Liquid and HUVECs cell culture fluid each 10ml, 3000 × g are centrifuged 15min (4 DEG C), remove cell and cell fragment, supernatant transfer
Into sterile chamber, 2ml ExoQuick-TC Exosome Precipitation Solution is added, overturns and shake examination
Pipe mixes solution, and 4 DEG C of refrigerated overnights (at least incubation 12h), test tube not rotate during incubation, next day, will
ExoQuick-TCExosome Precipitation Solution/ cell culture liquid mixture is centrifuged 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, supernatant is removed again, by test tube
1500 × g is centrifuged 5min (4 DEG C), the remaining ExoQuick-TC solution of rotary suction, and liquid is aspirated completely, is careful not to connect
The excretion body precipitating 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, for use.
3, the observation of excretion volume morphing
After the separation of excretion body, it is fixed on the buffer of the phosphate (including 4% paraformaldehyde, pH 7.4) of 0.01M
In, 4 DEG C are overnight.Next day, PBS are 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.
At 60 DEG C after polymerization overnight, slice, ultra-thin section is observed using electron microscope.
As a result: isolating the excretion body in cell culture fluid, the form of excretion body is as shown in Fig. 2, be in vesica knot under Electronic Speculum
Structure.
4, excretion body surface region feature marker detection
Excretion body surface region feature marker CD9 is detected using Western Blot method.
(1) excretion body is cracked
200 μ l RIPA buffer are added in the fresh excretion body precipitating prepared, vortex 15s is placed at room temperature
5min (wait be completely dissolved), be added 2*Laemmli buffer, 95 DEG C heat 5 minutes, before being added to gel loading, on ice
It is 5 minutes cooling.The formula of RIPA buffer and Laemmli buffer are as shown in table 1.
1 solution formula of table
HT-29 the and HUVECs cell for conventionally collecting logarithmic growth phase, is cracked using the above method, is obtained thin
Born of the same parents' albumen.
2, BCA method measures excretion body protein concentration
1. drawing 10 μ l standard specimens and excretion body protein or cell protein being added separately in each hole of 96 orifice plates；
2. the BCA working solution of 200 μ l is added in every hole, rocks and mix 30s, cover various kinds sample wells；
3. standard detection: in 37 DEG C of incubation 30min or being placed at room temperature for 2~16h；
4. being cooled to room temperature, microplate reader 562nm wavelength detecting absorbance (OD)；
5. drawing standard curve according to absorbance value and calculating the concentration of excretion body protein or cell protein.
3,20 μ g excretion 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, primary antibody (4 DEG C shaking tables stay overnight) is added, TBST washes film, secondary antibody is incubated for 45 minutes, TBST washes
Film is exposed development with the low background chemiluminescence instrument of Western blot.
4, result
As a result as shown in figure 3, detecting the table of the markers characteristic CD9 of excretion body in isolated excretion body protein
It reaches, shows the success of excretion body separation and Extraction.
The differential expression of p-ERK in 2 excretion body of embodiment
Step: the excretion body protein row polyacrylamide gel electrophoresis after quantifying in 20 μ g steps 1, transferring film, with 2% are taken
Bovine serum albumin(BSA) (BSA) is closed 1 hour, primary antibody (4 DEG C of shaking tables are stayed overnight) is added, TBST washes film, secondary antibody incubation 45 minutes, TBST
Film is washed, is exposed development with the low background chemiluminescence instrument of Western blot.
As a result: the Western blot result for taking the 20 μ g of excretion body protein of HT-29 cell, HUVECs cell to carry out respectively
As shown in Figure 1, not containing p-ERK in HUVECs cell excretion body, and contain more p-ERK in HT-29 cell excretion body.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (10)
1. detecting reagent the answering in preparation diagnosis of colorectal carcinoma product of p-ERK in the excretion body in colorectal cancer cell source
With, which is characterized in that the reagent includes reagent used in separation excretion body, and/or reagent used in cracking excretion body.
2. application according to claim 1, which is characterized in that the reagent includes detection p-ERK presence or absence or detection
Reagent used in the method for p-ERK expression quantity.
3. application according to claim 2, which is characterized 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, size exclusion chromatography, 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 measuring method of micro-fluid chip, surface plasma body resonant vibration, sequencing, Western blotting assays.
4. application according to any one of claim 1-3, which is characterized in that the reagent includes the antibody of p-ERK.
5. application according to claim 4, which is characterized in that the antibody include monoclonal antibody, polyclonal antibody or
Immune serum.
6. application according to any one of claim 1-3, which is characterized in that the diagnostic products include kit, core
Piece, test paper.
7. application according to any one of claim 1-3, which is characterized 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, which is characterized in that the excretion body carrys out autoblood.
9. p-ERK is developing and/or is designing diagnosis of colorectal carcinoma production as marker in the excretion body in colorectal cancer cell source
Application in product.
10. application according to claim 9, which is characterized in that excretion body comes from body fluid, and 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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