CN110527622A - For the excretion body detection device of glioma early detection, dynamic monitoring and Index for diagnosis and application - Google Patents
For the excretion body detection device of glioma early detection, dynamic monitoring and Index for diagnosis and application Download PDFInfo
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Abstract
The present invention provides a kind of for the excretion body detection device of glioma early detection, dynamic monitoring and Index for diagnosis and its application.Apparatus of the present invention include: excretion body extraction mechanism；Excretion body surface levies 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 glioma severity extent judge index, the marker of state of an illness real-time monitoring.
Description
Technical field
The invention belongs to field of medical examination fields, and in particular to one kind for glioma early detection, dynamic monitoring and
The excretion body detection device of Index for diagnosis and application.
Background technique
Malignant grade of gliomas is high, and treatment is difficult, poor prognosis.Glioma clinical manifestation mainly includes increased intracranial pressure table
Existing (such as headache, nausea and vomiting, personality and consciousness changing) and abnormality of nerve function (such as epilepsy, movement and/or sensory disturbance
Deng).It relies primarily on magnetic resonance imaging (MRI) and computed tomography (CT) makes preliminary imaging diagnosis to glioma.Magnetic
Resonance wave spectrogram (MRS), positron emission fault development (PET) and single photon emission tomography development (SPECT) help to identify
Tumor recurrence and radionecrosis.But due to the limitation of Imaging Technology, it is difficult to accomplish to accomplish reality over the course for the treatment of
When monitor, and final, tumor resection need to be passed through or Biopsy technique obtains tumor specimen and can just access explicitly
Pathological diagnosis.
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 glioma, 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 excretion body in utilizing, the detection of early stage, the reality of the state of an illness are carried out to glioma
When monitoring very have development prospect.
Currently, being on the one hand to establish simple and effective side in terms of the early detection for carrying out glioma using excretion body
On the one hand method is 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, provide it is a kind of for glioma early detection,
The excretion body detection device and application of dynamic monitoring and Index for diagnosis.
Before illustrating the content of present invention, it is as follows to define term used herein:
Term " EGFR " refers to: epidermal growth factor receptor, EGF-R ELISA.
Term " CXCR4 " refers to: C-X-C chemokine receptor type 4, chemokine receptors.
Term " PTTG " refers to: pituitary tumor transforming gene, pituitary tumor transforming gene.
Term " NLGN3 " refers to: neuroligin-3, nerve connection protein-3 gene.
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 a kind of for glioma early detection, dynamic monitoring
With the excretion body detection device of Index for diagnosis, described device includes:
(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: protein content, pattern, size, dense
Degree；
(3) excretion body expression quantity information measurement mechanism: the excretion body that the excretion body extraction mechanism is extracted is carried out
The measurement of expression quantity information, wherein the expression quantity information is receptor 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 glioma early detection, dynamic monitoring and Index for diagnosis 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 protein content characterization
Mechanism is BCA protein quantification mechanism；The mechanism of the morphology characterization is transmission electron microscope, preferably bion transmission electron microscope；It is described
The mechanism of size characterization is dynamic light scattering mechanism and/or nano particle trace analysis mechanism；The mechanism of 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 receptor
For EGFR and/or CXCR4；The mRNA is PTTG mRNA and/or NLGN3mRNA.
Device according to a first aspect of the present invention, wherein the mechanism of the measurement of the expressing quantity is fluidic cell machine
Structure and/or Western blot mechanism.
Preferably, 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.
Western blot mechanism measures expressing quantity the following steps are included: working as according to protein quantification as a result, taking
The excretion body for measuring 6~20 μ g albumen runs glue using the method for electrophoresis and the albumen of different molecular weight size is separated and identified；
It is incubated for corresponding antibody at corresponding molecular weight, and is detected with chemoluminescence method.
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 device described in first aspect and is producing in preparation for treating the medical treatment of tumour
Application in product；Preferably, the tumour is glioma.
The present invention relates in terms of the liquid biopsy of glioma, it is related to the albumen and gene marker field that glioma detects,
It is related to the detection field of excretion body.The main object of the present invention is to have filtered out several malignant grade of gliomas that can be used in examine
The excretion body protein marker and genetic marker of survey.Using these types of protein marker, pass through flow cytometry technique and RT-
Round pcr can judge the grade malignancy of glioma patient.By detecting several markers in circulation excretion body
Expression quantity can accomplish the screening detection of glioma early stage, and aid imaging technology prejudges grade malignancy, over the course for the treatment of
Real-time monitoring and Index for diagnosis to patient.
To achieve the above object, the solution of the present invention is as follows:
Application of the present invention is used for the detection of malignant grade of gliomas, early screening, prognosis and state of an illness real-time monitoring.
The present invention provides several markers surveyed applied to the physical examination of glioma excretion, the marker includes:
(1) EGF-R ELISA (epidermal growth factor receptor, EGFR) is thin using streaming
Born of the same parents' art detects the EGFR content in patient's circulating tumor excretion body surface face, can be judged by content its whether illness, lead to
It crosses content and judges its grade malignancy, the content of ki-67 in its tumor tissues is judged by content, its state of an illness is judged by content
Early advanced stage has high sensitivity and specificity.
(2) chemokine receptors (C-X-C chemokine receptor type 4, CXCR4), utilizes flow cytometry
The CXCR4 content in patient's circulating tumor excretion body surface face is detected, the early advanced stage of its state of an illness can be judged by content.
(3) pituitary tumor transforming gene (pituitary tumor transforming gene, PTTG), utilizes RT-
Round pcr detects PTTG mRNA content in patient's circulating tumor excretion body, and content can distinguish normal person and patient, realizes glue
The early screening of matter tumor.
(4) nerve connection protein-3 gene (neuroligin-3, NLGN3), using RT-PCR technology, detection patient is recycled
NLGN3mRNA content in tumour excretion body, content and grade malignancy are positively correlated, and content can distinguish normal person and patient, real
The early screening of existing glioma.
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 characterized by techniques.Its internal specific gene mRNA is characterized with RT-PCR technology
Expression quantity.
Step 5: postoperative tumor tissues, the ki-67 for providing ImmunohistochemistryResults Results in tumor group with hospital laboratory are compared.Step
Rapid 6: 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-PCR 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 10min, abandons precipitating.Supernatant 2000g-5000g is centrifuged 15 minutes, is discarded
Precipitating.Supernatant 8000g-10000g is centrifuged one hour, discards precipitating.
(2) with 0.2 μM of membrane filtration supernatant, filtered liquid is centrifuged with ultracentrifugation, 120000g-200000g from
The heart 6-15 hours.Supernatant is discarded, 120000-200000g is centrifuged 2 hours again.It will be centrifuged resulting precipitating, be suspended in 100 μ L-
Inside 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 it is different.
The morphology characterization of excretion body:
The excretion body extracted drop is siphoned away after standing 1min in the dedicated copper mesh of TEM, after being cleaned with ultrapure water, adds 1%
Phosphotungstic acid or uranium acetate, stand 30s after, 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. it according to protein quantification as a result, take the excretion body of equivalent 6-20 μ g albumen, is immunized with aldehyde radical microballoon or magnetization micro-
Ball or sulfonation microballoon combination 10min to 15h.
B. plus excessive glycine or bovine serum albumin solution stop experiment, and close the reaction site of microballoon.Later, add
Enter corresponding memebrane protein antibody, room temperature is incubated for 2h or more.If it is non-straight labeling antibody, then secondary antibody is being added later.
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. it according to protein quantification as a result, take the excretion body of equivalent 6-20 μ g albumen, but needs to guarantee used in each patient
It is protein content is certain.It runs glue using the method for electrophoresis the albumen of different molecular weight size is separated and 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-PCR:
(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.
Excretion body detection device for glioma early detection, dynamic monitoring and Index for diagnosis of the invention can have
But be not limited to it is following the utility model has the advantages that
EGFR albumen of the present invention, CXCR4 albumen, PTTG mRNA and NLGN 3mRNA, in serum excretion body
Content, fine can be used as glioma early screening, state of an illness real-time monitoring, the albumen mark of grade malignancy judgement and prognosis
Will object.
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 the size distributions and concentration of 1 excretion body of embodiment.
Fig. 3 shows 1 excretion body of embodiment TEM phenogram in conjunction with aldehyde radical microballoon.
Fig. 4 shows the expression quantity of 2 Healthy People of embodiment and patient EGFR on serum excretion body.
Fig. 5 shows expression quantity of the early and late patient EGFR of embodiment 2 on serum excretion body.
Fig. 6 shows the ROC curve of embodiment 2EGFR.
Fig. 7 shows the western blot result of embodiment 2.
Fig. 8 shows embodiment 2EGFR content and the relation with contents of Ki-67, wherein figure a is that the ki-67 that hospital provides contains
It measures and the corresponding relationship of EGFR content on corresponding patients serum's excretion body and related coefficient calculated result；Scheming b is two of them disease
People, the flow cytometry results of EGFR expression quantity on serum excretion body, while respectively same patient's tumor tissues in corresponding diagram c
The ImmunohistochemistryResults Results of ki-67.
Fig. 9 shows expression quantity of the CXCR4 on serum excretion body in embodiment 3.
Figure 10 shows expression quantity of the PTTG mRNA on serum excretion body in embodiment 4.
Figure 11 shows expression quantity of the NLGN3mRNA on serum excretion body in embodiment 5.
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:
Glioma patient's venous blood comes from affiliated hospital, Medical University Of Chongqing；
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；
EGFR antibody, CXCR4 antibody are purchased from abcam company；
Fluorescence quantitative kit is purchased from Tiangeng biochemical technology Co., Ltd.
Reverse transcription reagent box is purchased from Tiangeng biochemical technology Co., Ltd.
All secondary antibodies are purchased from CST company.
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.
Embodiment 1
The extraction and the connection with aldehyde radical microballoon that the present embodiment is used to illustrate the method for the present invention excretion body.
(1) it takes 250 μ L glioma patient's venous blood to stand the serum that centrifugation obtains, is diluted to 10mL, 500g centrifugation with PBS
10min abandons precipitating.Supernatant 2000g is centrifuged 15 minutes, discards precipitating.Supernatant 10000g is centrifuged one hour, discards precipitating.
(2) with 0.2 μM of membrane filtration supernatant, filtered liquid is centrifuged with ultracentrifuge, and 120000g is centrifuged 12h
Hour.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 2.
(6) it takes 10g albumen in conjunction with the aldehyde radical microballoon of 10000 4 μm of sizes, i.e., according to concentration in step 4, takes 10 μ g
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 3.
Embodiment 2
The present embodiment is used to illustrate the difference of EGFR expression quantity in excretion body.
(1) 12 Healthy People, (wherein, be determined as early stage completely has 4 people to 23 preoperative patients, determines advanced stage completely
Have 12 people) and 8 postoperative patients are in the preoperative and postoperative one week extracting vein blood.
(2) using embodiment 1 method extract excretion body in conjunction with aldehyde radical microballoon after, add EGFR antibody (abcam,
Ab231, dilution ratio are as follows: volume ratio 1/200 with BSA solution dilute, 100 μ L are added after dilution) and fluorescent marker secondary antibody
(CST company, volume ratio are 1/1000 dilution, 100 μ L are added after dilution).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.
(P < 0.05) as shown in Figure 4, antibody and discovery Healthy People and postoperative there were significant differences (P < 0.0001) preoperative and postoperative
Also there is obvious difference (P < 0.05).In the preoperative in patient, EGFR advanced stage early stage by stages on, similarly show
Obvious difference, as shown in Figure 5.Illustrate that this method is effective, it can be in the case where area 0.90 under ROC curve
Patient and Healthy People are distinguished, expression quantity is 19.1% as differentiation standard, can be with 80% sensitivity and 75% specificity
Distinguish Healthy People and patient.As shown in fig. 6, this method presents fine specific and sensitivity.Therefore, using EGFR this
A protein marker can distinguish patient and Healthy People, accomplish early sieve.Middle and advanced stage can be distinguished, accomplishes that grade malignancy judges, and
And there are preferable sensitivity and specificity.The expression quantity of EGFR on excretion body can predict the actual pernicious journey of patient's tumour
Degree.As shown in figure 8, the expression quantity of EGFR has good correlation with the ki-67 expression quantity in tissue of patient.Excretion body
EGFR expression quantity is showed with the ki-67 index in tissue of patient to be obviously positively correlated.(Pearson correlation coefficient is
0.867, highly significant) ki-67 index is related with the grade malignancy of patient and proliferative capacity, so detecting in blood excretion body
EGFR content can be used to predict the content of Ki-67 in tissue, to predict grade malignancy, as shown in Figure 8.
(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 is added, (EGFR antibody, CST 4267, thinner ratio 1/1000 are diluted, dosage after dilution with 5% skimmed milk power
After 3mL), and the secondary antibody of connection HRP, thinner ratio 1/1000 is diluted with 5% skimmed milk power, and dosage is 3mL after dilution),
Slice result is obtained under chemical imaging instrument., as shown in Figure 7.
Embodiment 3
The present embodiment is used to illustrate the difference of CXCR4 expression quantity in serum excretion body.
(1) early stage patient 4, end-stage patients 12, preoperative extracting vein blood.
(2) using embodiment 1 method extract excretion body in conjunction with aldehyde radical microballoon after, add CXCR4 antibody (abcam,
Ab167, dilution ratio are volume ratio 1/100, with BSA solution dilute, 100 μ L are added after dilution) and fluorescent marker secondary antibody
(CST, dilution ratio are that volume ratio is 1/1000, and 100 μ L are added after dilution).It is detected using BD streaming instrument, with nonreactive
10% setting-out of body negative control, obtains relative fluorescence expression quantity.
(3) Prism software is utilized, above data is analyzed.
There were significant differences up to measurer for the serum excretion body surface of CXCR4.As shown in figure 9, expression of the CXCR4 in serum excretion body
Measure the grade malignancy it is also predicted that patient.
Embodiment 4
The present embodiment is for illustrating that the expression quantity of PTTG mRNA in excretion body can carry out early sieve.
(1) 23 patients and 12 Healthy Peoples are carried out preoperative taking blood.
(2) 500 μ LTrizol reagents, cracking 10 is added in the 50 μ L excretion liquid suspensions extracted using the method for embodiment 1
Minute.Chloroform extraction is used later, leaves and takes water phase, is extracted mRNA therein with 250 μ L isopropanols and ethyl alcohol respectively and is extracted
mRNA.MRNA is washed with straight alcohol.After outwelling ethyl alcohol and drying, 10 μ L water is added to obtain pure mRNA.
(3) reverse transcription reagent box reverse transcription (QuantScript RT Kit Cat#KR103-04, Tiangeng) is utilized.
(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 10, it is found that Healthy People and patients serum
PTTG mRNA expression in excretion body has significant difference, may be used as the early screening of glioma.
Embodiment 5
The present embodiment is for illustrating that the expression quantity of NLGN3mRNA in excretion body can carry out early sieve.
(1) 19 patients and 9 Healthy Peoples are carried out preoperative taking blood.
(2) the 50 μ L excretion liquid suspensions extracted using the method using embodiment 1 that the method for embodiment 1 is extracted, are added
Enter 500 μ L Trizol reagents, cracks 10 minutes.Later use chloroform extraction, leave and take water phase, respectively with 250 μ L isopropanols with
Ethyl alcohol extracts mRNA therein.MRNA is washed with straight alcohol.After outwelling ethyl alcohol and drying, 10 μ L water is added to obtain pure mRNA.(3) sharp
With reverse transcription reagent box reverse transcription (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.
(6) as shown in figure 11, the NLGN3mRNA expression in Healthy People and patients serum's excretion body has significant difference, can
For use as the early screening of glue tumor.
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 glioma early detection, dynamic monitoring and Index for diagnosis, which is characterized in that institute
Stating device includes:
(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: protein content, 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 receptor 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 glioma early detection, dynamic monitoring and Index for diagnosis 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 protein content characterization is BCA protein quantification mechanism；The mechanism of the 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 concentration
The mechanism of 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 receptor is EGFR and/or CXCR4；The mRNA is PTTG mRNA and/or NLGN3 mRNA.
6. device according to any one of claim 1 to 5, which is characterized in that the machine of the measurement of the expressing quantity
Structure is 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 preparing the application in the medical product for treating tumour；It is excellent
Selection of land, the tumour are glioma.
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| CN107557441A (en) * | 2017-10-27 | 2018-01-09 | 中南大学湘雅医院 | Glioma diagnosis marker Circ2:23823258|23823569 and application |
| US20180104187A1 (en) * | 2016-10-19 | 2018-04-19 | Northwestern University | Extracellular vesicle-based diagnostics and engineered exosomes for targeted therapeutics against cancer |
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| US20180104187A1 (en) * | 2016-10-19 | 2018-04-19 | Northwestern University | Extracellular vesicle-based diagnostics and engineered exosomes for targeted therapeutics against cancer |
| CN107557441A (en) * | 2017-10-27 | 2018-01-09 | 中南大学湘雅医院 | Glioma diagnosis marker Circ2:23823258|23823569 and application |
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