阅读说明：本技术 用于乳腺癌检测及分子分型的外泌体检测装置和应用 (For breast cancer detection and the excretion body detection device and application of molecule parting ) 是由 杨延莲 李文哲 朱凌 王华艺 王琛 于 2018-05-25 设计创作，主要内容包括：本发明提供了一种用于用于乳腺癌检测及分子分型的外泌体检测装置及其应用。本发明装置包括：外泌体的提取机构；外泌体的表征机构；外泌体蛋白表达量信息测定机构和外泌体表达量信息分析机构。本发明装置在乳腺癌检测及分子分型方面的高准确度,并可指导乳腺癌患者用药。本发明中血液样本外泌体的检测可在肿瘤发展的初期便给出疾病信息,大大避免了传统病理检测的侵入性及滞后性,为乳腺癌的早筛及分子分型提供了新方案。(The present invention provides a kind of excretion body detection devices and its application for for breast cancer detection and molecule parting.Apparatus of the present invention include: the extraction mechanism of excretion body；The characterization mechanism of excretion body；Excretion body protein expression quantity information measurement mechanism and excretion body expression quantity information analysis mechanism.High accuracy of the apparatus of the present invention in terms of breast cancer detection and molecule parting, and patient with breast cancer's medication can be instructed.The detection of blood sample excretion body can just provide disease information at the initial stage of tumor development in the present invention, largely avoided the invasive and hysteresis quality of traditional pathological examination, provide new departure for the early sieve and molecule parting of breast cancer.)

1. a kind of for breast cancer detection and the excretion body detection device of molecule parting, which is characterized in that described device includes:

(1) the excretion body in cells and supernatant 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) albumen excretion body protein expression quantity information measurement mechanism: is carried out to the excretion body that the excretion body extraction mechanism is extracted The measurement of expression quantity information；

Preferably, the target protein is selected from one or more of: EpCAM albumen, HER2 albumen；

(4) excretion body expression quantity information analysis mechanism: the expression to excretion body protein expression quantity information measurement mechanism measurement Amount information is analyzed, and is carried out non-small cell lung cancer detection and is judged by stages.

2. the apparatus according to claim 1, which is characterized in that the cell is selected from one or more of: breast epithelium Cell MCF-10A, breast cancer cell MDA-MB-468, MCF-7, SK-BR-3.

3. device according to claim 1 or 2, which is characterized in that in the excretion body extraction mechanism, the excretion body Extracting method is selected from one or more of: supercentrifugation, micro-pore-film filtration method, microchannel method, preferably ultracentrifugation Method, it is highly preferred that the supercentrifugation the following steps are included:

(a) cells and supernatant is taken to be centrifuged, precipitating is abandoned in centrifugation for the first time, is taken supernatant to be centrifuged for the second time and is discarded precipitating, again Supernatant third time centrifugation is taken to discard precipitating；

Preferably, the first time centrifugal speed is 300g~1000g, preferably 800g；First time centrifugation time is 5 minutes； Second of centrifugal speed is 2000g~5000g, preferably 2000g；Second of centrifugation time is 10 minutes；The third Secondary centrifugal speed is 8000g~10000g, preferably 10000g；Third time centrifugation time is 1 hour；The centrifuging temperature is 4 ℃；

(b) supernatant obtained by membrane filtration step (a), filtered liquid are centrifuged to precipitate again to get excretion body is arrived；

Preferably, the filter sizes are 0.1~0.5 μm, preferably 0.22 μm；The centrifugal speed be 100000~ 150000g, preferably 100000g；The centrifugation time is 0.5~5 hour, preferably 2 hours；The centrifuging temperature is 4 DEG C.

4. device according to claim 3, 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 resuspended in PBS.

5. device according to any one of claim 1 to 4, which is characterized in that described in the excretion body extraction mechanism The culture medium of cells and supernatant contains 10% fetal calf serum；

Preferably, the excretion body extraction mechanism further includes following operating mechanism:

The culture medium of cells and supernatant is changed by the culture of the fetal calf serum preparation of deduction excretion body before excretion body extracts Base is cultivated；Preferably, the replacing construction is to extract first 24~48 hours.

6. device according to claim 5, which is characterized in that the culture medium that the fetal calf serum for deducting excretion body is prepared It is prepared by the following method:

(i) serum is added in basal medium, makes serum-concentration 30% in cell culture medium；

(ii) by under the conditions of obtained by step (i) 4 DEG C of cell culture medium, ultracentrifugation is overnight；Preferably, the centrifugal speed is 100000~150000g；

(iii) after ultracentrifugation, supernatant is collected, to be diluted to FBS dense by the culture medium containing 30% serum with basal medium Degree be 10%, be added 1% it is dual anti-；

(iv) culture medium is filtered with filter membrane, bottling is stand-by；Preferably, the filter sizes are 0.22 μm.

7. device according to any one of claim 1 to 6, 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.

8. device according to any one of claim 1 to 7, which is characterized in that the measuring means of the expressing quantity Protein marker expression quantity in single excretion body is calculated by flow cytometer and scanning electron microscope.

9. device according to claim 8, which is characterized in that it is described to protein marker expression quantity in single excretion body into Row calculates following steps:

(I) by excretion body in conjunction with microballoon, by expressing quantity on the microballoon of measured by flow cytometry combination excretion body It is worth fluorescence intensity level；

(II) the excretion body quantity combined on single microballoon is calculated by scanning electron microscope (SEM) result；

(III) ratio for passing through (I) and (II), obtains the result of expressing quantity in single excretion body.

10. device described in any one of claims 1 to 9 is in preparing the medical product for diagnosing and/or treating cancer Application；Preferably, the cancer is breast cancer；It is highly preferred that the application is diagnosis and/or the HER2 molecule point of breast cancer Type.

Technical field

The invention belongs to medical sciences, and in particular to a kind of for breast cancer detection and the excretion physical examination of molecule parting Survey device and application.

Background technique

At this stage, although there is a huge progress in the fields such as medical imaging and oncotherapy, the disease incidence of cancer and dead It is still high to die rate.Wherein, the disease incidence of breast cancer is also in the gesture of cumulative year after year, it has also become threatens the " No.1 of women's health Killer ".It is well known that epithelial cell adhesion factor (EpCAM) and human epidermal growth factor receptor-2 (HER2) albumen are mammary gland Very important two albuminoids marker in cancer.EpCAM is overexpressed in nearly all tumour, is the important albumen of lesion detection Target spot, while being considered as the bad mark of tumor prognosis.The overexpression of HER2 albumen, it is meant that degree of malignancy of tumor cell is more High, progression of disease speed faster, be easier to that transfer and recurrence occurs and prognosis is bad.In patient with breast cancer, there are about 20-30% For HER2 breast cancer patients with positive.Studies have shown that total life span of HER2 breast cancer patients with positive only has HER2 negative patient Half.Therefore, HER2 positive breast cancer is also referred to as " most dangerous breast cancer ".Specifically for HER2 positive breast cancer Molecular targeted agents ---(Herceptin) is exactly to be passed through with the HER2 molecule of cancer cell surfaces for " target spot " This link of HER2 molecule is blocked, to inhibit and kill tumour cell.The positive cream for the treatment of HER2 is recognized as by domestic and international expert The basic medication of gland cancer.Since listing in 1998, the whole world has had more than 720,000 patients because usingAnd benefit. Current research result also confirms that,The risk of recurrence of tumour can be reduced by 52% by combined chemotherapy, by the dead wind of patient Danger reduces by 33%.More there is 80% or so early-stage breast cancer patient to useAfter obtain healing.And for patients with terminal,Also life in patients can be improved, and significantly extends Overall survival.Therefore, breast cancer HER2 molecule parting is swollen Tumor treatment, has very important meaning in terms of being especially targeted medication.

Clinically, the common detection methods of tumour are pathologic finding, are contaminated the living tissue taken out from the patient Whether color observation, there is canceration by the form and structure decision of cell.When most of, pathologic finding is to judge whether it is cancer The most important foundation of disease.Immunohistochemistry inspection (IHC) and fluorescence in situ hybridization (FISH) are clinically used breast cancer The method of HER2 molecule parting, the means for being cut off or being punctured by operation take tissue to be analyzed.However, in tumour progression and controlling During treatment, heterogeneous, genetic drift and treatment means will lead in tumour to factors such as tumour bring pressure in tumor The expression quantity of HER2 albumen changes (Cardoso, Di Leo et al.2001, Yao, Lu et al.2014).It is more important , single needle aspiration biopsy also can because sample position limitation to testing result caused by great deviation.In addition to this, existing Detection method be it is invasive, bring considerable distress to patient, also having led to these methods can not be to the disease progression of patient Real-time monitoring, therefore largely affect the real-time and validity of targeted therapy.

For the defect of existing lesion detection technology.Liquid biopsy based on blood is come into being, and liquid biopsy is for cancer The early sieve of disease, dynamic monitoring, direction of medication usage etc. have great significance.Circulating tumor cell (CTCs), Circulating tumor DNA (CtDNA), excretion body is the important liquid biopsy target of three classes.Wherein, excretion body is that the tumour liquid of latest find is living Object is examined, is a kind of membrane vesicle secreted by cell, diameter is present in almost all kinds of body fluid in 50-150nm. The CTCs few compared to content in blood, the quantity of excretion body is very huge, and 10 are up in every milliliter of blood⁸-10¹⁰It is a, it compares Degradable CtDNA in blood, the molecule that the membrane structure of excretion body carries its inside play a very good protection.By cell The excretion body of secretion carries the signaling molecule of its derived cell, including protein, mRNA, miRNA etc., participates in cell-cell communication, And entrained molecule is delivered to target cell.In cancer, excretion body is considered closely bound up with the progress of cancer, and cancer cell can Signal is delivered to by secretion excretion body and in downstream cellular or microenvironment, accelerates the proliferation of tumour, is shifted, recurrence etc..Cause This, the signal in excretion body usually can be used for reflecting the physiology and pathologic function state of its secretory cell, to provide potential Biomolecule marker, therefore be purified into excretion body from body fluid with good potential applicability in clinical practice, can be used as tumour and examine Disconnected and Index for diagnosis biomarker.However, nanoscale small size make excretion body isolate and purify and detection all suffers from Greatly challenge.Existing technological means concentrates on realizing the capture and detection to excretion body by micro-fluidic chip, but receives The complexity and high cost of meter level chip design limit the extensive use of the technology, it is difficult to realize that great amount of samples is analyzed.For more Application of the good realization excretion body in liquid biopsy field, developing low-cost, new technology easy to operate are very urgent.

Summary of the invention

Therefore, the purpose of the present invention is to overcome the defects in the prior art, provides one kind for breast cancer detection and divides The excretion body detection device of sub- parting and application.

To achieve the above object, the first aspect of the present invention provides a kind of for the outer of breast cancer detection and molecule parting Body detection device is secreted, described device includes:

(1) the excretion body in cells and supernatant 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 protein expression quantity information measurement mechanism: the excretion body that the excretion body extraction mechanism is extracted is carried out The measurement of expressing quantity information；

Preferably, the target protein is selected from one or more of: EpCAM albumen, HER2 albumen；

(4) excretion body expression quantity information analysis mechanism: to excretion body protein expression quantity information measurement mechanism measurement Expression quantity information is analyzed, and is carried out non-small cell lung cancer detection and is judged by stages.

Device according to a first aspect of the present invention, wherein the cell is selected from one or more of: galactophore epithelial cell MCF-10A, breast cancer cell MDA-MB-468, MCF-7, SK-BR-3.

Device according to a first aspect of the present invention, wherein in the excretion body extraction mechanism, the extraction side of the excretion body Method is selected from one or more of: supercentrifugation, micro-pore-film filtration method, microchannel method, preferably supercentrifugation, more excellent Selection of land, the supercentrifugation the following steps are included:

(a) cells and supernatant is taken to be centrifuged, precipitating is abandoned in centrifugation for the first time, and it takes supernatant to be centrifuged for the second time and discards precipitating, Supernatant third time centrifugation is taken to discard precipitating again；

Preferably, the first time centrifugal speed is 300g~1000g, preferably 800g；First time centrifugation time is 5 points Clock；Second of centrifugal speed is 2000g~5000g, preferably 2000g；Second of centrifugation time is 10 minutes；Described Centrifugal speed is 8000g~10000g, preferably 10000g three times；Third time centrifugation time is 1 hour；The centrifuging temperature It is 4 DEG C；

(b) supernatant obtained by membrane filtration step (a), filtered liquid are centrifuged to precipitate again to get excretion body is arrived；

Preferably, the filter sizes are 0.1~0.5 μm, preferably 0.22 μm；The centrifugal speed be 100000~ 150000g, preferably 100000g；The centrifugation time is 0.5~5 hour, preferably 2 hours；The centrifuging temperature is 4 DEG C.

Preferably, the extraction of the excretion body is further comprising the steps of:

(c) step (b) is centrifuged resulting precipitating, be resuspended in PBS.

Device according to a first aspect of the present invention, wherein in the excretion body extraction mechanism, the cells and supernatant Culture medium contains 10% fetal calf serum；

Preferably, the excretion body extraction mechanism further includes following operating mechanism:

The culture medium of cells and supernatant is changed by the fetal calf serum preparation of deduction excretion body before excretion body extracts Culture medium is cultivated；Preferably, the replacing construction is to extract first 24~48 hours.

Preferably, the culture medium that the fetal calf serum for deducting excretion body is prepared is prepared by the following method:

(i) serum is added in basal medium, makes serum-concentration 30% in cell culture medium；

(ii) by under the conditions of obtained by step (i) 4 DEG C of cell culture medium, ultracentrifugation is overnight；Preferably, the centrifugal speed For 100000~150000g；

(iii) after ultracentrifugation, supernatant is collected, is diluted to the culture medium containing 30% serum with basal medium FBS concentration be 10%, be added 1% it is dual anti-；

(iv) culture medium is filtered with filter membrane, bottling is stand-by；Preferably, the filter sizes are 0.22 μm.

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 the measuring means of the expressing quantity passes through flow cytometer Protein marker expression quantity in single excretion body is calculated with scanning electron microscope.

Preferably, described that calculating following steps are carried out to protein marker expression quantity in single excretion body:

(I) by excretion body in conjunction with microballoon, pass through expressing quantity on the microballoon of measured by flow cytometry combination excretion body Median fluorescence intensity value；

(II) the excretion body quantity combined on single microballoon is calculated by scanning electron microscope (SEM) result；

(III) ratio for passing through (I) and (II), obtains the result of expressing quantity in single excretion body.

The second aspect of the present invention provides device described in first aspect in preparation for diagnosing and/or treating cancer Application in medical product；Preferably, the cancer is breast cancer；It is highly preferred that the application be breast cancer diagnosis and/or HER2 molecule parting.

The purpose of the present invention is to provide a kind of method for carrying out the biopsy of breast cancer liquid using excretion body.This method can be right Breast cancer realizes in-vitro diagnosis and molecule parting.This method is easy to operate, at low cost, and detection process is rapid, the characteristic of non-intruding It avoids conventional pathological examination and gives patient's bring pain.In addition to this, this method is expected to carry out real-time tracing, root to the state of an illness According to the timely adjustment for the treatment of scheme of progression of the disease, thinking is instructed to realize that personalized medicine provides.

Application the invention discloses excretion body EpCAM, HER2 albumen as breast cancer diagnosis and molecule parting marker. The present invention provides by EpCAM expressing quantity in Flow cytometry excretion body as Diagnosis of Breast cancer, and detection HER2 expression quantity carries out the application of molecule parting to patient with breast cancer.Experiment of the invention, establishes in breast cancer cell line Complete cell excretion body Model, confirmed in the detection of clinical blood sample this method for diagnose with molecule parting can Row obtains higher susceptibility and specificity, and mutually confirms with ImmunohistochemistryResults Results.

The present invention provides application of the target protein expression amount in terms of breast cancer vitro detection in a kind of excretion body.

The application includes at least the diagnosis of breast cancer, HER2 molecule parting etc..

The target protein includes at least EpCAM albumen, HER2 albumen etc..

The present invention provides a kind of expression quantity using excretion body EpCAM albumen in cell line culture supernatant to establish breast cancer The method of diagnosis cell model.

The present invention provides a kind of expression quantity using excretion body HER2 albumen in cell line culture supernatant to establish breast cancer The method of molecule parting cell model.

The present invention also provides the foundation of the cell line excretion body detection model, the selection including cell line, cell training Support the extraction of excretion body and the detection of EpCAM or HER2 expressing quantity in supernatant.The invention can be verified in vitro in diagnosing tumor And the feasibility in molecule parting field.

Preferably, the cell line be galactophore epithelial cell MCF-10A, breast cancer cell MDA-MB-468, MCF-7, SK-BR-3。

Preferably, 10% fetal calf serum FBS is contained in the culture medium of the cell line.

Preferably, cells and supernatant excretion body extracts from 8-12 ware cell.Preferably, the cells and supernatant should be 24-48h is changed by the culture medium of fetal calf serum (FBS) preparation of deduction excretion body before excretion body extracts.

Specifically, the preparation of culture medium is carried out by the fetal calf serum (FBS) of deduction excretion body, comprising the following steps:

1) serum is added in basal medium, makes serum-concentration 30% in cell culture medium；

2) by under the conditions of 4 DEG C of the cell culture medium, centrifugal force 120000-150000g, ultracentrifugation is overnight；

3) after ultracentrifugation, supernatant is taken out, the culture medium containing 30% serum is diluted to serum with basal medium Concentration 10%, be added 1% it is dual anti-；

4) culture medium is filtered with 0.22 μm of filter membrane, bottling is stand-by.

Whether the expression quantity of EpCAM is patient with breast cancer in diagnosis or auxiliary diagnosis subject in detection subject's excretion body In application.

The expression quantity of HER2 is carrying out HER2 molecule parting or HER2 points of auxiliary to it in detection subject's sample excretion body Application in sub- parting

The present invention also provides the applications in diagnosing tumor and molecule parting field of the excretion body, point including serum From the extraction of excretion body and the detection of EpCAM or HER2 expressing quantity in serum.It can confirm that the invention is examined in tumour in vitro Disconnected and molecule parting field application.

Preferably, serum be Healthy Human Serum and Serum of Patients With Breast Cancer, it is isolated by new blood.

Specifically, the separation of serum the following steps are included:

1) whole blood of subject is collected using non-anticoagulant heparin tube；

2) standing after taking a blood sample makes its layering, and interior centrifugation, revolving speed 4000rpm are centrifuged 5-10min. for 24 hours

3) upper serum is collected after centrifugation.

The extraction of excretion body in cells and supernatant or serum, uses ultracentrifugal method.

Preferably, ultracentrifugation temperature is 4 DEG C.

Preferably, centrifugal force 100000-150000g.

Preferably, the ultracentrifugation time is 2-12h.

Preferably, the excretion weight after centrifugation is hanged into PBS.

Preferably, the PBS buffer solution that excretion body is resuspended has been subjected to 0.22 μm of membrane filtration.

EpCAM or HER2 expressing quantity in excretion body, is detected using specific recognition antibody.

Preferably, the antibody is the specific recognition antibody of EpCAM or HER2 albumen.

The present invention provides a kind of methods for calculating protein marker expression quantity in single excretion body, which comprises

1) intermediate value of EpCAM or HER2 expressing quantity on the microballoon for combining excretion body is obtained by flow cytometry results Fluorescence intensity level.

2) the excretion body quantity combined on single microballoon is calculated by scanning electron microscope (SEM) result.

By ratio 1) and 2), the result of EpCAM or HER2 expressing quantity in single excretion body is obtained.

Excretion body detection device for breast cancer detection and molecule parting of the invention can have but be not limited to following The utility model has the advantages that

1, the invention discloses by EpCAM expressing quantity in Flow cytometry excretion body as Diagnosis of Breast Cancer, and detection excretion body HER2 expression quantity carry out the application of molecule parting to patient with breast cancer.The present invention is in breast cancer cell line In establish complete cell excretion body Model, confirmed in the detection of clinical blood sample this method for diagnose and molecule The feasibility of parting, area under the curve (AUC) are up to 0.972, and testing result and pathological section immunohistochemical staining result phase Confirmation.Demonstrate high accuracy of this method in terms of breast cancer detection and molecule parting.Illustrate excretion body EpCAM and HER2 is expected to become the biomarker of clinical breast cancer diagnosis and molecule parting, and can instruct patient with breast cancer's medication.This hair The detection of bright middle blood sample excretion body can just provide disease information at the initial stage of tumor development, largely avoided traditional pathology inspection The invasive and hysteresis quality surveyed provides new departure for the early sieve and molecule parting of breast cancer.

2, the present invention relates to the methods of diagnosis and molecule parting that breast cancer is carried out by excretion body.Utilize mesh in excretion body The expression quantity for marking protein marker carries out in-vitro diagnosis and HER2 molecule parting to subject, and this method is expected to realize the morning of disease The real-time tracing of sieve and the state of an illness provides new method for auxiliary lesion detection and tracking therapeutic effect, while can also become prognosis The important means of evaluation instructs thinking to realize that personalized medicine provides, in the life quality for improving patient, extends life cycle Aspect has good application prospect.

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 the schematic diagram of the present invention that breast cancer diagnosis and HER2 molecule parting are carried out using excretion body.

Fig. 2 a shows the result figure of the EpCAM specific recognition antibody that flow cytometry measures and four kinds of cell combinations. Percentage is Percentage bound in figure；Fig. 2 b shows the HER2 specific recognition antibody that flow cytometry measures and four kinds of cell knots The result figure of conjunction.Percentage is Percentage bound in figure；Fig. 2 c show EpCAM specific recognition antibody that flow cytometry measures with The median fluorescence intensity value of four kinds of cell combinations makees histogram；Fig. 2 d shows the HER2 specific recognition that flow cytometry measures The median fluorescence intensity value of antibody and four kinds of cell combinations makees histogram.

Fig. 3 shows the excretion body extracted from cells and supernatant and carries out pattern and characterization of size figure.

Fig. 4 shows the morphology characterization figure of excretion body-microsphere compound of the present invention.

Fig. 5 a shows the streaming result figure of EpCAM expression quantity in the excretion body that four kinds of cell line of the present invention is extracted； Figure 5b shows that the EpCAM expression quantity histograms of the excretion body of four kinds of cells of the present invention secretion；Fig. 5 c shows the present invention The streaming result figure of HER2 expression quantity in the excretion body that four kinds of cell line is extracted；Fig. 5 d shows four kinds of the present invention thin The HER2 expression quantity histogram of the excretion body of intracrine.

Fig. 6 shows what Western blot (Western blot) detected EpCAM and HER2 protein content in excretion body Result figure

Fig. 7 a shows the excretion body shape appearance figure extracted in serum；Fig. 7 b shows in NTA detection serum and extracts The order of magnitude size distribution plot of excretion body.

Fig. 8 a is shown using flow cytometry in Healthy People group, and HER2- breast cancer patients group, HER2+ breast cancer group mentions The result figure of the EpCAM expression quantity detected in the excretion body taken, the percentage in figure are EpCAM antibody specificity combination excretion The positive rate of body；Fig. 8 b is shown by Healthy People group, patient's HER2- group, and patient HER2+ organizes EpCAM expression in serum excretion body Measure the scatter plot (* * P < 0.01, * * * P < 0.001, t inspection) drawn.

Fig. 9 a is shown using flow cytometry in Healthy People group, and HER2- breast cancer patients group, HER2+ breast cancer group mentions The result figure of the HER2 expression quantity detected in the excretion body taken, the percentage in figure are HER2 antibody specificity combination excretion body Positive rate；Fig. 9 b is shown by Healthy People group, patient's HER2- group, and patient HER2+ organizes HER2 expression quantity in serum excretion body The scatter plot (* * P < 0.01, * * * P < 0.001, t inspection) of drafting.

Figure 10 show using ROC curve to detection effect of the present invention in terms of breast cancer patients HER2 molecule parting into Row analysis.

Figure 11 shows the accuracy that molecular typing methods of the present invention are verified using Immunohistochemical Staining.

Figure 12 shows the calculation method of target protein marker expression amount in single excretion body of the present invention.

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, cell line MCF-10A, MDA-MB-468, MCF-7, SK-BR- used in following embodiment 3 are purchased from the Chinese Academy of Medical Sciences.

Unless specifically stated otherwise, the solvent of aqueous solution used is sterile ultra-pure water solution, water quality parameter in following embodiment It is 25 DEG C [email protected] resistivity 18.2M Ω .cm.

Unless specifically stated otherwise, reagent used in following embodiment is analytical reagents.

Unless specifically stated otherwise, PBS solution used in following embodiment is 1 × PBS solution.

Reagent and instrument used in the following embodiment are as follows:

Reagent:

PBS buffer solution, protein lysate, 4 μm of diameter of aldehyde radical latex beads are purchased from Thermo Fisher Scientific.

Glutaraldehyde, uranyl acetate, glycine, BSA are purchased from Sigma Aldrich.

EpCAM antibody, HER2 antibody are supported film purchased from the ultra-thin carbon of Cell Signaling Technology., are purchased from Beijing Zhongjingkeyi Technology Co.,Ltd.,China.

BCA protein quantification kit is purchased from Suo Laibao company.

Filter membrane, 0.45 μm of pvdf membrane are purchased from Millipore, Bedford, MA.

Instrument:

Transmission electron microscope is purchased from Hitachi, Japan, model Hitachi High-Tech 7700；

Nano particle trace analysis instrument is purchased from Britain's Malvern company, model NanoSight LM14 system；

Nanometer particle size potentiometric analyzer is purchased from Britain's Malvern company, model Zetasizer Nano ZS90；

Centrifuge is purchased from Beijing Lei Boer centrifuge Co., Ltd, model LD5-2A；

Flow cytometer is purchased from Life Technologies, Carlsbad, CA, modelacoustic Focusing cytometer, AppliedBiosystems.