CN110527725A - Excretion body detection device and application for invasion and the judgement of Non-Invasive hypophysoma - Google Patents
Excretion body detection device and application for invasion and the judgement of Non-Invasive hypophysoma Download PDFInfo
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Abstract
The present invention provides a kind of excretion body detection device for invasion and the judgement of Non-Invasive hypophysoma, described device includes: excretion body extraction mechanism, excretion body surface sign mechanism, excretion body expression quantity information measurement mechanism and excretion body expression quantity information analysis mechanism.Apparatus of the present invention can be work perfectly well as Invasive Pituitary Adenomas and Non-Invasive judge index, state of an illness real-time monitoring, and the judgement of invasion degree and Epithelial and stromal convert marker.
Description
Technical field
The invention belongs to field of medical examination, and in particular to a kind of for the outer of aggressive and Non-Invasive hypophysoma judgement
Secrete body detection device and application.
Background technique
Hypophysoma is to occur in suprapituitary tumour, also commonly known as pituitary adenoma, is common neuroendocrine tumor
One of, account for about the 10-15% of central nerve neuroma.Most of pituitary adenoma is all benign tumour.In hypophysoma
In, a classification is pituitary tumor,nonfunctioning, wherein having percent 30 or so to show invasion.Invasive tumor develops to
Later period is difficult excision completely, causes biggish difficulty to the anaphase of patient.The diagnosis and treatment of hypophysoma mainly pass through following at present
Several iconography means judgement invasions.Head x-ray plain film, this is more original diagnostic method, according to the change of sella turcica sclerotin
The variations such as change, the calcification of saddle area determine whether tumour and antidiastole.CT scan only has diagnostic value to large-scale hypophysoma, small
Hypophysoma is easy to fail to pinpoint a disease in diagnosis.It cannot function as the main tool of diagnosis hypophysoma.MRI is checked, is the diagnosis most important tool of hypophysoma,
It can clearly illustrate the size of tumour, form, position, the relationship with surrounding structure.Even if 2~3 millimeters of tumour of diameter
It can show.But the signal of also Partial tumors is approximate with surrounding normal pituitary tissue, and the two is difficult to differentiate between, it is also necessary in conjunction with
Clinical manifestation and Endocrinological inspection are diagnosed.Imaging Method can not real-time monitoring progress, may result in nonfunctioning disease
People misses best opportunity of operation.
With the development of liquid Biopsy, the early detection of circulating tumor cell and circulating tumor cell in tumour, reality
When monitor, played a significant role in prognosis and treatment assessment, but for hypophysoma, since cell is difficult to wear
Blood-brain barrier is crossed, so circulating tumor cell is also difficult to capture in blood, and ctDNA is due to its unstability, in blood
In be easy degradation, analysis method and store method require it is all very high, it is at high price.And excretion body, a kind of size of cell secretion
Vesicles between 50nm-150nm can pass through blood-brain barrier, and abundance is very high in blood, contains in every milliliter of blood
108-1010A excretion body, in addition excretion body has one layer of phospholipid bilayer, thus it is relatively stable, it is easy analysis and saves, very greatly
The difficulty and price of analysis are reduced in degree.Therefore using circulation excretion body, the pre- of early stage is carried out to the invasion of hypophysoma
Sentence, while monitoring the progress of hypophysoma, it is extremely important.Currently, in terms of the early detection for carrying out hypophysoma using excretion body, one
Aspect is simple effective method to be established, and is on the one hand to find suitable protein marker or gene.
Summary of the invention
Therefore, the purpose of the present invention is to overcome the defects in the prior art, provides a kind of for invasion and non-invasion
Property hypophysoma judgement excretion body detection device and application.
Before illustrating the content of present invention, it is as follows to define term used herein:
Term " FOLR " refers to: folacin receptor.
Term " EpCAM " refers to: epithelial cell adhesion molecule.
Term " PTTG " refers to: pituitary tumor transforming gene, pituitary tumor transforming gene.
Term " Vimentin " refers to: vimentin.
Term " E-cadherin " refers to: the glutinous element of E- calcium.
Term " N-cadherin " refers to: the glutinous element of N- calcium.
Term " PBS " refers to: phosphate buffered saline solution.
Term " PCR " refers to: polymerase chain reaction.
To achieve the above object, the first aspect of the present invention provides one kind and sentences for invasion and Non-Invasive hypophysoma
Disconnected excretion body detection device, described device include:
(1) the excretion body in serum excretion body extraction mechanism: is extracted by centrifugation；
(2) excretion body surface levies mechanism: the excretion body extracted to the excretion body extraction mechanism characterizes, with determination
Really excretion body has been extracted, wherein the characteristic index is selected from one or more of: pattern, size, concentration；
(3) excretion body expression quantity information measurement mechanism: the excretion body extracted to the excretion body extraction mechanism is expressed
Measure the measurement of information, wherein the expression quantity information is albumen and/or the expression quantity information of mRNA；
(4) excretion body expression quantity information analysis mechanism: the expression to excretion body expression quantity information measurement mechanism measurement
Amount information is analyzed, and invasion and the judgement of Non-Invasive hypophysoma are carried out.
Device according to a first aspect of the present invention, wherein in the excretion body extraction mechanism, the extraction packet of the excretion body
Include following steps:
(a) serum is taken to be diluted with PBS, precipitating is abandoned in centrifugation for the first time, is taken supernatant to be centrifuged for the second time and is discarded precipitating, takes again
Clear third time centrifugation discards precipitating；
Preferably, the extension rate is 10~40 times, preferably 40 times；The first time centrifugal speed be 300g~
1000g, preferably 500g；First time centrifugation time is 10 minutes；Second of centrifugal speed is 2000g~5000g, preferably
For 2000g；Second of centrifugation time is 15 minutes；The third time centrifugal speed is 8000g~10000g, preferably
10000g；Third time centrifugation time is 1 hour；
(b) supernatant obtained by membrane filtration step (a), filtered liquid centrifugation discard supernatant, are centrifuged to obtain precipitating again,
Obtain excretion body；
Preferably, the filter sizes are 0.1~0.5 μm, preferably 0.2 μm；The centrifugal speed be 120000~
200000g, preferably 120000g；The centrifugation time is 6~15 hours, preferably 12 hours；The centrifugal speed again is
120000~200000g, preferably 120000g；The centrifugation time again is 2 hours.
Preferably, the extraction of the excretion body is further comprising the steps of:
(c) step (b) is centrifuged resulting precipitating, be suspended in PBS.
Device according to a first aspect of the present invention, wherein in excretion body surface sign mechanism, the mechanism of the morphology characterization
For transmission electron microscope, preferably bion transmission electron microscope；The mechanism of the size characterization is dynamic light scattering mechanism and/or nanometer
Grain trace analysis mechanism；The mechanism of the concentration characterization is nano particle trace analysis mechanism.
Device according to a first aspect of the present invention, wherein in excretion body expression quantity information measurement mechanism, the albumen
For FOLR albumen and/or EpCAM albumen；The mRNA is PTTG mRNA.
Device according to a first aspect of the present invention, wherein the measuring means of the expressing quantity is fluidic cell mechanism
And/or Western blot mechanism.
Fluidic cell mechanism measurement expressing quantity the following steps are included:
(I) immune with aldehyde radical microballoon or magnetization according to protein quantification as a result, take the excretion body of 6~20 μ g albumen of equivalent
Microballoon or sulfonation microballoon combine, and binding time is 10 minutes to 15 hours；
(II) it increases the bovine serum albumin solution in 15mM biology glycine or greater than 10% and stops experiment, and close micro-
The reaction site of ball；Corresponding memebrane protein antibody is added, room temperature is incubated for；If it is non-straight labeling antibody, then two are being added later
It is anti-；
(III) it is monitored using the flow cytometer of corresponding channel and determines sun with the Comparative result of isotype control Ab
Property rate, determines expression quantity.
Device according to a first aspect of the present invention, wherein Western blot mechanism measures expressing quantity and includes
Following steps: according to protein quantification as a result, taking the excretion body of 6~20 μ g albumen of equivalent, glue is run to difference using the method for electrophoresis
The albumen of molecular size range is separated and is identified；Be incubated for corresponding antibody at corresponding molecular weight, and with chemoluminescence method into
Row detection.
Device according to a first aspect of the present invention, wherein in excretion body expression quantity information measurement mechanism, the mRNA
Expression quantity information measurement the following steps are included:
(i) excretion body is taken, Trizol lysate is added to crack；
(ii) add chloroform extraction, extract mRNA therein with the isopropanol and ethyl alcohol of ice；
(iii) it is dissolved with water, and quantitative with RNA quantitative instrument；
It (iv) is cDNA by rna transcription；
(v) real-time quantitative fluorescence PCR is carried out using SYBR Green I chimeric fluorescent method, obtains mrna expression amount.
The second aspect of the present invention provides first aspect device and produces in preparation for diagnosing and/or treating the medical treatment of tumour
Application in product；Preferably, the tumour is hypophysoma.
The present invention relates in terms of the liquid biopsy of hypophysoma, it is related to the aggressive judgement field of hypophysoma, is related to hypophysoma
The albumen of detection and gene marker field, are related to the detection field of excretion body.The main object of the present invention be filtered out it is several
A excretion body protein marker and genetic marker that can be used in hypophysoma invasion detection.Using these types of protein marker,
By flow cytometry technique and RT-qPCR technology, can the invasion degree to hypophysoma patient judge.It is several by detecting
A marker circulation excretion body in expression quantity, can accomplish hypophysoma invasion and Non-Invasive judgement, auxiliary at
Degree prejudges as a possibility that technology is to invasion, real-time monitoring over the course for the treatment of and the Index for diagnosis to patient.
In addition, it has also been found that, the Epithelial and stromal conversion conversion of tumour can pass through albumen in excretion body and gene mark
The differential expression of will object observes.
The application is used for the detection of hypophysoma grade malignancy, early screening, prognosis and state of an illness real-time monitoring.
The present invention provides several markers surveyed applied to the physical examination of hypophysoma excretion, the marker includes:
(1) folacin receptor (FOLR) detects circulating tumor excretion body surface face FOLR content using flow cytometry,
Its content and invasion are negatively correlated, convert with EMT negatively correlated.
(2) epithelial cell adhesion molecule (EpCAM), using flow cytometry to the EpCAM in circulating tumor excretion body surface face
Content is detected, and content and invasion are negatively correlated, is converted with EMT negatively correlated.
(3) pituitary tumor transforming gene (pituitary tumor transforming gene, PTTG), utilizes RT-
QPCR technology detects PTTG mRNA content in excretion body, and content and invasion are positively correlated.
(4) vimentin (Vimentin) detects vimentin mRNA content in excretion body using RT-qPCR technology,
Content and invasion are positively correlated, and are converted with EMT negatively correlated.
(5) N- calcium is glutinous plain (N-cadherin), detects vimentin mRNA content in excretion body using RT-qPCR technology,
Its content and invasion are positively correlated, and are converted with EMT negatively correlated.
(6) E- cadherins (E-cadherin), using flow cytometry to the E- in circulating tumor excretion body surface face
Cadherin content is detected, and content and invasion are negatively correlated, is converted with EMT negatively correlated.
Specifically, which comprises
Step 1: extracting patient or Healthy People is preoperative or postoperative one week venous blood, stand to be centrifuged and obtain serum.Serum is straight
It connects extraction excretion body or is stored in -80 DEG C of refrigerators.
Step 2: by continuous centrifugal, extracting the excretion body in serum.
Step 3: the pattern of characterization excretion body, size and concentration.Excretion body has been extracted really to determine.
Step 4: taking the excretion body of a certain range concentration, the expression of its particular surface albumen is characterized with Flow Cytometry
Amount.With the expression quantity of its surface specific protein of western blot characterized by techniques.Its internal specific base with RT-qPCR characterized by techniques
Because of the expression quantity of mRNA.
Step 5: patient information is corresponding with gained expression quantity information.
The method of marker in above-mentioned detection excretion body, including following technology: excretion body extractive technique, excretion bodily form looks table
Sign technology (mainly including scanning transmission microscope, scanning electron microscope, nano particle trace analysis technology), the total egg of excretion body
White content detection technology (BCA albuminimetry), excretion body protein assay technology (flow cytometry and Western blot
Technology) and excretion body mRNA content characterization technique (RT-qPCR technology).
Specifically, the above-mentioned detection device for giving excretion body is based on following steps:
Excretion body extracts:
(1) serum for obtaining people's venous blood now takes or deposits in -80 degrees Celsius.After low temperature thawing, 250 μ L- are taken
1mL is diluted to 10mL with PBS, and 300g-1000g is centrifuged 10 minutes, abandons precipitating.Supernatant 2000g-5000g is centrifuged 15 minutes, is abandoned
Fall precipitating.Supernatant 8000g-10000g is centrifuged one hour, discards precipitating.
(2) with 0.2 μM of membrane filtration supernatant, filtered liquid is centrifuged with ultracentrifuge, 120000g-200000g
Centrifugation 6-15 hours.Supernatant is discarded, 120000-200000g is centrifuged 2 hours again.It will be centrifuged resulting precipitating, be suspended in 100 μ
Inside L-500 μ L PBS.
The protein content of excretion body characterizes:
The albumen of the excretion body extracted is quantified using BCA albuminimetry.Specific steps and dosage are according to examination
The difference of agent box and distinguish.
The morphology characterization of excretion body:
The excretion body extracted drop is siphoned away after standing 1 minute in the dedicated copper mesh of TEM, after being cleaned with ultrapure water, adds 1%
Phosphotungstic acid or uranium acetate, stand 30s after, siphon away, dry.It is characterized with bion transmission electron microscope.
The size and concentration of excretion body characterize:
A certain amount of excretion liquid suspension is taken, after being diluted to 1mL with PBS, characterizes size, or benefit using dynamic light scattering technique
Size and excretion bulk concentration are analyzed with nano particle trace analysis technology.
Excretion body protein expression quantity determination techniques:
(1) flow cytometry.A. micro- with aldehyde radicalization according to protein quantification as a result, take the excretion body of equivalent 6-20 μ g albumen
Ball or magnetization immune microsphere or sulfonation microballoon combine, and binding time is to arrive 15h in 10 minutes.
B. it increases the bovine serum albumin solution in 15mM biology glycine or greater than 10% and stops experiment, and close microballoon
Reaction site.Later, corresponding memebrane protein antibody is added, room temperature is incubated for 2h or more.If it is non-straight labeling antibody, then later
Add secondary antibody.
C. it is monitored and is determined with the Comparative result of isotype control Ab positive using the flow cytometer of corresponding channel
Rate determines expression quantity.
(2) Western blot technology: a. as a result, take the excretion body of equivalent 6-20 μ g albumen, but is needed according to protein quantification
Guarantee to be protein content used in each patient to be certain.Glue is run to the albumen of different molecular weight size using the method for electrophoresis
It is separated and is identified.Later, it is incubated for corresponding antibody at corresponding molecular weight, and is detected with chemoluminescence method.
Excretion body mRNA content characterization technique RT-qPCR:RT-qPCR, embodiment 4 are to the RT-qPCR of PTTG mRNA 5
Involved in the RT-qPCR of vimentin and n-cad is detected.
(1) 50 μ L excretion bodies are taken, Trizol lysate is added to crack.
(2) add chloroform extraction, extract mRNA therein with the isopropanol and ethyl alcohol of ice.
(3) it is dissolved with the water of 10 μ L, and quantitative with RNA quantitative instrument.
It (4) is cDNA by rna transcription.
(5) Real Time PCR is carried out using SYBR Green I chimeric fluorescent method, obtains mrna expression amount.
The excretion body detection device for invasion and the judgement of Non-Invasive hypophysoma of the invention can have but unlimited
In following the utility model has the advantages that
The FOLR albumen that the method for the present invention uses, EpCAM albumen, content of the PTTG mRNA in excretion body can be fine
Ground turns as Invasive Pituitary Adenomas and Non-Invasive judge index, state of an illness real-time monitoring, the judgement of invasion degree and Epithelial and stromal
Change marker.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows 1 excretion body TEM morphology characterization figure of embodiment.
Fig. 2 shows 1 excretion body of embodiment TEM phenograms in conjunction with aldehyde radical microballoon.
Fig. 3 shows the size distribution of 1 excretion body of embodiment.
Fig. 4 shows 2 folacin receptor of embodiment in the expression quantity of circulating tumor excretion body.
Fig. 5 shows embodiment 3EpCAM in the expression quantity of circulating tumor excretion body.
Fig. 6 shows the ROC curve of 2 folacin receptor of embodiment.
Fig. 7 shows the ROC curve of embodiment 3EpCAM receptor.
Fig. 8 shows the western blot result of embodiment 2 and 3.
Fig. 9 shows the PTTG mrna expression amount of 4 circulating tumor excretion body of embodiment.
Figure 10 shows the Epithelial and stromal conversion information expressed in 5 excretion body of embodiment.
Specific embodiment
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for specifically describing in more detail.
This part carries out general description to the material and test method that arrive used in present invention test.Although being
It realizes many materials used in the object of the invention and operating method is it is known in the art that still the present invention still uses up herein
It may detailed description.It will be apparent to those skilled in the art that within a context, if not specified, material therefor of the present invention and behaviour
It is well known in the art as method.
Unless specifically stated otherwise, the solvent of aqueous solution used is sterile ultra-pure water solution in following embodiment.
Unless specifically stated otherwise, reagent used in following embodiment is analytical reagents.
Reagent and instrument used in the following embodiment are as follows:
Reagent:
Hypophysoma venous patient blood derives from Tiantan Hospital's neurosurgery and clinical laboratory.；
PBS, Thermo fisher. uranium acetate is derived from, Bellingwell company is derived from.
Ethyl alcohol, chloroform are purchased from Chinese medicines group.
Trizol reagent is purchased from Life technologies company.
Filter membrane is purchased from Millex-GP.
Carbon film supports copper mesh, is purchased from middle mirror tech；
BCA kit is purchased from Suo Laibao company；
BCA kit is purchased from Suo Laibao company；
FOLR antibody, E-cadherin antibody are purchased from abcam company；
EpCAM antibody is purchased from CST company；
All secondary antibodies are purchased from CST company.
Fluorescence quantitative kit is purchased from Tiangeng biochemical technology Co., Ltd.；
Reverse transcription reagent box is purchased from Tiangeng biochemical technology Co., Ltd.
SDS lysis buffer is purchased from Thermo fisher；Bis-tris GEL is purchased from Thermo fisher company,
Aldehyde radical microballoon is purchased from Life technologies company.
Instrument:
Ultracentrifuge is purchased from Bake Mann, model Optima XPN；
TEM is purchased from Hitachi, Ltd., model HITACHI 7700；
Nano particle tracing instrument is purchased from Malvern company, model NanoSight LM14；
BD streaming instrument is purchased from BD company, model C 6；
Chemical imaging instrument is purchased from Bio-rad company, model ChemiDoc MP chemiluminescence imaging system.
Embodiment 1
The extraction and the connection with aldehyde radical microballoon that the present embodiment is used to illustrate excretion body of the present invention.
(1) it takes 250 μ L hypophysoma venous patient blood to stand the serum that centrifugation obtains, is diluted to 10mL, 500g centrifugation with PBS
10 minutes, abandon precipitating.Supernatant 2000g is centrifuged 15 minutes, discards precipitating.Supernatant 10000g is centrifuged 1 hour, discards precipitating.
(2) with 0.2 μM of membrane filtration supernatant, filtered liquid is centrifuged with ultracentrifuge, and 120000g centrifugation 12 is small
When.Supernatant is discarded, 120000g is centrifuged 2 hours again.It will be centrifuged resulting precipitating, be suspended in inside 100 μ L PBS.
(3) hanging drop obtained by 10 μ L steps (2) is taken to support on copper mesh in 400 mesh carbon films, after washing twice, with 1%
Uranium acetate negative staining, 80Kv TEM shooting, finds successfully to be extracted excretion body.Morphology characterization is as shown in Figure 1.
(4) BCA test is carried out according to the kit specification of Suo Laibao company, measures concentration in 1mg/mL albumen or so.
(5) shown after taking suspension obtained by 100 μ L steps (2) that 1500 μ LPBS is added to dilute with the nano particle of Malvern company
The detection of track instrument, obtains size distribution and concentration, as shown in Figure 3.
(6) take 10g albumen in conjunction with the aldehyde radical microballoon of 10000 4 μm of sizes.I.e. according to concentration in step 4,10 μ g are taken
Measure volume excretion liquid suspension, the microsphere suspension liquid with 10000 microsphere volumes, in 4 degrees Celsius of shaking tables be incubated for 12h with
On, effect is combined with TEM detection, as shown in Figure 2.
Embodiment 2
The present embodiment is used to illustrate the difference of FOLR expression quantity in excretion body.
(1) 10 aggressive patient and 10 preoperative extracting vein bloods of Non-Invasive patient.
(2) using embodiment 1 method extract excretion body in conjunction with aldehyde radical microballoon after, add FOLR antibody (abcam,
Ab3361, volume ratio be 1/200 with BSA solution dilute, 100 μ L are added after dilution) and fluorescent marker secondary antibody CST, 4410S,
Volume ratio 1/1000 dilutes, and 100 μ L are added after dilution.It is detected using BD streaming instrument, with no antibody negative controls
10% setting-out, obtains relative fluorescence expression quantity.
(3) Prism software is utilized, above data is analyzed.
Invasion and Non-Invasive notable difference on the FOLR expression quantity of circulating tumor excretion body.(p=as shown in Figure 4
0.0006 < 0.001, * * *), illustrate that this method is effective, invasion can be distinguished in the case where area 0.94 under ROC curve
Property and Non-Invasive.Under the experiment condition, it is believed that folacin receptor expression quantity is 28.5% to be used as differentiation standard, can be with
90% sensitivity and 90% specificity distinguish aggressive patient and Non-Invasive patient, as shown in Figure 6.
(4) this method presents specificity and sensitivity well.It therefore, can be with using this protein marker of FOLR
Invasion and Non-Invasive are distinguished, accomplishes to judge in advance.
(5) the excretion body for taking 10 μ g albumen cracks (purchase of Thermo Fisher company) with SDS lysate, is cooled to room
Protein sample is loaded to Bis-tris glue (purchased from Thermo fisher company) well by Wen Hou, under the voltage of 90mV into
Row electrophoresis.Primary antibody is added, (FOLR antibody, model are same as above, thinner ratio 1/1000, are diluted with 5% skimmed milk power, dosage after dilution
After 3mL), and the secondary antibody of connection HRP, CST, 7076S, thinner ratio 1/1000 are diluted with 5% skimmed milk power, after dilution
Dosage is 3mL), slice result is obtained under chemical imaging instrument.As a result, as shown in Figure 8.
Embodiment 3
The present embodiment is used to illustrate the difference of EpCAM expression quantity in excretion body.
(1) 10 aggressive patient and 10 preoperative extracting vein bloods of Non-Invasive patient.
(2) using embodiment 1 method extract excretion body in conjunction with aldehyde radical microballoon after, add EpCAM antibody (EpCAM,
CST, #2929,1/200 with BSA solution dilute) and fluorescent marker secondary antibody.It is detected using BD streaming instrument, with no antibody
10% setting-out of negative control, obtains relative fluorescence expression quantity.
(3) Prism software is utilized, above data is analyzed.
Invasion and Non-Invasive notable difference on the EpCAM expression quantity of circulating tumor excretion body.(p=as shown in Figure 5
0.0014 < 0.01, * *), illustrate that this method is effective, invasion can be distinguished in the case where area 0.88 under ROC curve
With Non-Invasive.As shown in fig. 7, this method presents fine specific and sensitivity.Under the experiment condition, it is believed that
EpCAM expression quantity is 66.5% to be used as differentiation standard, can be distinguished with 80% sensitivity and 90% specificity aggressive patient and
Non-Invasive patient.
(4) the excretion body for taking 10 μ g albumen cracks (purchase of Thermo Fisher company) with SDS lysate, is cooled to room
Protein sample is loaded to Bis-tris glue (purchased from Thermo fisher company) well by Wen Hou, under the voltage of 90mV into
Row electrophoresis.Primary antibody (CST, #2929, thinner ratio 1/1000 are diluted with 5% skimmed milk power, and dosage is 3mL after dilution) is being added,
And after the secondary antibody of connection HRP, CST, 7076S, thinner ratio 1/1000 are diluted with 5% skimmed milk power, and dosage is after dilution
3mL), slice result is obtained under ultraviolet.
Embodiment 4
The present embodiment is used to illustrate to carry out early sieve using the expression quantity of PTTG mRNA in RT-qPCR technology excretion body.
(1) 7 patients and 6 Healthy Peoples are carried out preoperative taking blood.
(2) 500 μ L Trizol reagents, cracking is added in the 50 μ L excretion liquid suspensions extracted using the method for embodiment 1
10 minutes.Chloroform extraction is used later, leaves and takes water phase, extracts mRNA therein with 250 μ L isopropanols and ethyl alcohol respectively.With pure
Ethanol washing mRNA.After outwelling ethyl alcohol and drying, 10 μ L water is added to obtain pure mRNA.(3) reverse transcription reagent box reverse transcription is utilized
(QuantScript RT Kit Cat#KR103-04, Tiangeng).
(4) using fluorescence quantitative kit, (Tiangeng, Supernal Premix Plus Kit (Cat#FP205-02) are carried out
Relative quantification does internal reference with GAPDH gene.
(5) Prism software is utilized, above data is analyzed.As shown in figure 9, outside discovery Healthy People and patients serum
The PTTG mRNA expression secreted in body has significant difference, may be used as the invasion and Non-Invasive screening of hypophysoma.
Embodiment 5
The Epithelial and stromal conversion information that the present embodiment is used to illustrate to express in excretion body.
(1) 6 aggressive patients and 6 Non-Invasive patients are carried out preoperative taking blood.
(2) 500 μ L Trizol reagents, cracking is added in the 50 μ L excretion liquid suspensions extracted using the method for embodiment 1
10 minutes.Chloroform extraction is used later, leaves and takes water phase, extracts mRNA therein with 250 μ L isopropanols and ethyl alcohol respectively.With pure
Ethanol washing mRNA.After outwelling ethyl alcohol and drying, 10 μ L water is added to obtain pure mRNA, 10 μ g excretion bodies are used for flow cytometry.
(3), reverse transcription reagent box reverse transcription (QuantScript RT Kit Cat#KR103-04, Tiangeng) is utilized.
(4) RT-qPCR fluorescence quantitative kit (Tiangeng, Supernal Premix Plus Kit (Cat#FP205- are utilized
02), internal reference is done with GAPDH gene.Detect relative amount of the vimentin mRNA in patients serum's excretion body to GAPDH, N-
Relative amount of the cadherin mRNA to GAPDH.
(5) the E-cadherin protein content in streaming method detection patients serum's excretion body, EpCAM protein content are adopted
After extracting excretion body in conjunction with aldehyde radical microballoon with the method for embodiment 1, add EpCAM antibody (EpCAM, CST, #2929, according to
Volume ratio 1/200 is diluted with BSA solution and E-cadherin antibody, abcam, ab1416, molten with BSA according to volume ratio 1/200
Liquid dilution, respectively 100 μ L of each additions) and fluorescent marker secondary antibody (CST, #4410, according to volume ratio 1/1000 use BSA solution it is dilute
It releases, 100 μ L is added after dilution).It is detected using BD streaming instrument, with 10% setting-out of no antibody negative controls, obtains phase
To luciferase expression amount.
(6) Prism software is utilized, above data is analyzed.
(7) as shown in Figure 10, higher Vimentin and N-cadherin content is shown in aggressive patient, and less
EpCAM and E-cadherin content.And this has corresponded to the Epithelial and stromal conversion of tumour.
Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention
Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right
It is required that range comprising the equivalent replacement of each factor.
Claims (10)
1. a kind of excretion body detection device for invasion and the judgement of Non-Invasive hypophysoma, which is characterized in that described device
Include:
(1) the excretion body in serum excretion body extraction mechanism: is extracted by centrifugation；
(2) excretion body surface levies mechanism: the excretion body extracted to the excretion body extraction mechanism characterizes, certain to determine
Excretion body is extracted, wherein the characteristic index is selected from one or more of: pattern, size, concentration；
(3) expression quantity letter excretion body expression quantity information measurement mechanism: is carried out to the excretion body that the excretion body extraction mechanism is extracted
The measurement of breath, wherein the expression quantity information is albumen and/or the expression quantity information of mRNA；
(4) excretion body expression quantity information analysis mechanism: the expression quantity of excretion body expression quantity information measurement mechanism measurement is believed
Breath is analyzed, and invasion and the judgement of Non-Invasive hypophysoma are carried out.
2. the apparatus according to claim 1, which is characterized in that in the excretion body extraction mechanism, the excretion body is mentioned
Take the following steps are included:
(a) serum is taken to be diluted with PBS, precipitating is abandoned in centrifugation for the first time, is taken supernatant second to be centrifuged and is discarded precipitating, takes supernatant the again
Centrifugation discards precipitating three times；
Preferably, the extension rate is 10~40 times, preferably 40 times；The first time centrifugal speed is 300g~1000g,
Preferably 500g；First time centrifugation time is 10 minutes；Second of centrifugal speed is 2000g~5000g, preferably
2000g；Second of centrifugation time is 15 minutes；The third time centrifugal speed is 8000g~10000g, preferably 10000g；
Third time centrifugation time is 1 hour；
(b) with supernatant obtained by membrane filtration step (a), filtered liquid centrifugation discards supernatant, be centrifuged again precipitating to get
To excretion body；
Preferably, the filter sizes are 0.1~0.5 μm, preferably 0.2 μm；The centrifugal speed be 120000~
200000g, preferably 120000g；The centrifugation time is 6~15 hours, preferably 12 hours；The centrifugal speed again is
120000~200000g, preferably 120000g；The centrifugation time again is 2 hours.
3. the apparatus of claim 2, which is characterized in that the extraction of the excretion body is further comprising the steps of:
(c) step (b) is centrifuged resulting precipitating, be suspended in PBS.
4. device according to any one of claim 1 to 3, which is characterized in that described in excretion body surface sign mechanism
The mechanism of morphology characterization is transmission electron microscope, preferably bion transmission electron microscope；The mechanism of the size characterization is dynamic light scattering
Mechanism and/or nano particle trace analysis mechanism；The mechanism of the concentration characterization is nano particle trace analysis mechanism.
5. device according to any one of claim 1 to 4, which is characterized in that the excretion body expression quantity information measurement
In mechanism, the albumen is FOLR albumen and/or EpCAM albumen；The mRNA is PTTG mRNA.
6. device according to any one of claim 1 to 5, which is characterized in that the measuring means of the expressing quantity
For fluidic cell mechanism and/or Western blot mechanism.
7. device according to claim 6, which is characterized in that fluidic cell mechanism measurement expressing quantity include with
Lower step:
(I) according to protein quantification as a result, taking the excretion body of 6~20 μ g albumen of equivalent, in conjunction with microballoon, binding time is 10 minutes
By 15 hours；
(II) it increases the bovine serum albumin solution in 15mM biology glycine or greater than 10% and stops experiment, and close microballoon
Reaction site；Corresponding memebrane protein antibody is added, room temperature is incubated for；If it is non-straight labeling antibody, then secondary antibody is being added later；
(III) it is monitored and is determined with the Comparative result of isotype control Ab positive using the flow cytometer of corresponding channel
Rate determines expression quantity.
8. device according to claim 6, which is characterized in that Western blot mechanism measures expressing quantity packet
It includes following steps: according to protein quantification as a result, taking the excretion body of 6~20 μ g albumen of equivalent, running glue to not using the method for electrophoresis
Albumen with molecular size range is separated and is identified；It is incubated for corresponding antibody at corresponding molecular weight, and uses chemoluminescence method
It is detected.
9. device according to any one of claim 1 to 8, which is characterized in that the excretion body expression quantity information measurement
In mechanism, the mrna expression amount information measurement the following steps are included:
(i) excretion body is taken, Trizol lysate is added to crack；
(ii) add chloroform extraction, extract mRNA therein with isopropanol and ethyl alcohol；
(iii) it is dissolved with water, and quantitative with RNA quantitative instrument；
It (iv) is cDNA by rna transcription；
(v) real-time quantitative fluorescence PCR is carried out using SYBR Green I chimeric fluorescent method, obtains mrna expression amount.
10. device described in any one of claims 1 to 9 is in preparing the medical product for diagnosing and/or treating tumour
Application；Preferably, the tumour is hypophysoma.
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| CA2875418A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
| CN106366196A (en) * | 2016-08-31 | 2017-02-01 | 上海美吉生物医药科技有限公司 | EpCAM antibody immunomagnetic bead and preparation method thereof |
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| CA2875418A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
| CN106366196A (en) * | 2016-08-31 | 2017-02-01 | 上海美吉生物医药科技有限公司 | EpCAM antibody immunomagnetic bead and preparation method thereof |
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