CN105716928A - Separation and extraction method for cytomembrane microvesicles (MVs) and exosomes (EXs) - Google Patents
Separation and extraction method for cytomembrane microvesicles (MVs) and exosomes (EXs) Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to a separation and extraction method for cytomembrane microvesicles (MVs) and exosomes (EXs). The method is characterized by comprising the following steps that a, 3-10 ml of a peripheral blood sample is drawn and placed in an anticoagulation tube to be stored, the sample is diluted with phosphate buffer normal saline (PBS) by 2-4 times, and then centrifugation is carried out to obtain supernate plasma; b, the supernate plasma is subjected to centrifugal separation again, and sediment is MVs; c, ultra-centrifugation is carried out on the supernate plasma in the step b, and sediment is EXs. According to the method, a previous method adopting mono-specificity antibodies is broken through, and the specificity and sensibility of the extracted cell MVs and EXs are higher than those in the prior art. A specificity and sensibility direction is provided for a further study of the MVs and the EXs as biomarkers of diseases.
Description
Technical field
The present invention relates to cell membrane microcapsule bubble MVs and secrete outward the separating and extracting process of body EXs, belonging to doctor
Treat invention field.
Background technology
Extracellular vesicle (Extracellular vesicles, EVs) is a class of emiocytosis release
There is the vesicles of biologic activity.EVs portability and the lipid of transmission blast cell, functional protein
Matter, the material such as messenger RNA s (mRNAs) and Microrna s (miRNAs) enters target cell, regulation
The expression and function of genes of target cell.Due to technical merit and the restriction of understanding, researcher initially proposes
During the concept of EVs, only EVs is considered as " refuse bag " of cell, allows cell break away from some useless
Albumen rubbish.Further study show that of nearest more than ten years, " goods " entrained by EVs has weight
The biological significance wanted, the miRNAs material that especially it is comprised is it is verified that disease can be participated in
The process occurred.EVs can be divided into microvesicle (microvesicles, MVs) and secrete outward body (Exosomes,
EXs) two big class.The difference differring primarily in that generating mode of the two.MVs is to work as cell-stimulating,
The vesicles directly come off from cell membrane after damage or apoptosis, diameter is about 150nm-1000nm.EXs
It is to be discharged into extracellular, diameter by the form secreted in addition after intracellular multivesicular body and cell membrane fusion
It is about 40nm-150nm.Additionally, researcher is it is also proposed that the two biological agent played is likely to difference.
But owing to current shortage efficiently separates method, the two being separated, characteristic and function to them are done
Go out to analyze accurately and be extremely limited.
Endothelial progenitor cells (Endothelial Progenitor Cells, EPCs) and blood vessel endothelium are thin
Born of the same parents (Endothelial Cells, ECs) are the important cells directly related with vascular system, for
Maintaining stablizing of blood vessel structure and function, revascularization, tissue repair and regeneration etc. have important work
With.Having studied confirmation, circulation EPC level declines and dysfunction is high with the danger of angiopathy
Degree is relevant.Blood vessel inner skin cell function imbalance and damage are the important initiating links causing vascular lesion.
Clinical research finds, the circulation of cardiovascular disease patient (including atherosclerosis, apoplexy etc.)
The EVs (cEC-EVs) in endotheliocyte (Circulating Endothelial cells, cECs) source
Level significantly increases, and relevant to therapeutic effect and disease prognosis.In contrast, circulation endothelium
The EVs in CFU-GM (Circulating Endothelial Progenitor Cells, cEPCs) source
(cEPC-EVs) level in angiopathy patient significantly increases reduction.These researchs show
CEC-EVs and cEPC-EVs can be used for the prediction of disease as potential source biomolecule label, therapeutic effect
Assessment.Current research finds EPC-EVs, by carrying some albumen relevant to tissue regeneration, mRNAs
And miRNAs enters target cell regulator gene and expresses and cell function, thus promote vascularization, skeleton
Flesh regeneration, neuranagenesis, minimizing myocardial damage, protection acute tubular damage, minimizing injury of lung etc..
Therefore, they may play the effect of key in blood vessel injury reparation with regenerative process.,
Traditional method be difficult to sort in complex samples and detect specificity EVs, such as cEC-EVs and
cEPC-EVs.Therefore, the research and development of this respect is greatly limited.
Summary of the invention
It is an object of the invention to provide a kind of cell membrane microcapsule bubble MVs and secrete outward the separation and Extraction side of body EXs
Method, the present invention separates and MVs and EXs of purification of high-purity, is conducive to they are carried out content
Analyze and the research of function.High sensitive and high specific detection is conducive to assessing it accurately in disease
Level change under state.Further industrialization development can develop relevant test kit.
Technical scheme is as follows:
Cell membrane microcapsule bubble MVs and secrete outward the separating and extracting process of body EXs, it is characterised in that by following
Step is carried out:
A, extraction peripheral blood sample 3-10ml are placed in anticoagulant tube and preserve；Use phosphate-buffered physiology
Saline (PBS) dilutes 2-4 times, is centrifuged obtaining supernatant blood plasma；B: to supernatant blood plasma again
Secondary being centrifuged separates, and precipitate is MVs, c: the supernatant blood plasma of step b is carried out ultracentrifugation and divides
From, precipitate is EXs.
Dilute 3 times with phosphate buffered saline PBS, be centrifuged with the rotating speed of 400r/s after mixing, 35
Minute obtain supernatant blood plasma.
Described step b is: supernatant blood plasma carries out two times centrifugal separation again, and the most centrifugal turns
Speed 2000r/s, 20 minutes, preserves supernatant and carries out the most centrifugal, that second time is centrifugal rotating speed
20000r/s, 120 minutes, precipitate was MVs.
Described step c is: be centrifuged separating to being precipitated the supernatant blood plasma that thing is MVs again,
Centrifugal rotating speed 169000r/s, 6 hours, centrifuged deposit thing was EXs.
Utilize cell membrane microcapsule bubble MVs that the present invention separates and secrete outward body EXs, utilize CD105 and
CD144 as the molecular marker of ECs, utilize CD34 and KDR as the molecular marker of EPCs,
Annexin V is as the universal labelling of MVs, and CD63, as the universal labelling of EXs, utilizes two kinds not
Synantibody magnetic bead sorts cEC-MVs, cEPC-MV, cEC-EXs and cEPC-EXs, Ke Yiyong respectively
Analyze in content (comprising albumen, mRNAs and miRNA) afterwards；On the other hand, by separating
Extract MVs and EXs of blood circulation, utilize antibody magnetic bead sorting combined with fluorescent quantum dot (Q-dots)
NTA analyzes or flow cytometer showed detects cEC-MVs, cEPC-MV, cEC-EXs and cEPC-EXs water
Flat.The detection method comparison type of the present invention is analyzed the sensitivity of method and is improve tens times.
Utilize cell membrane microcapsule bubble MVs that the present invention separates and secrete outward body EXs, can quickly, accurately,
The level of EC-MVs, EPC-MVs, EC-EXs and EPC-EXs in objective analysis complex samples blood,
The present invention is that research MVs and EXs provides special and sensitive as the biomarker of disease further
Direction.
Accompanying drawing explanation
Fig. 1 a, Fig. 1 b are respectively NTA and measure CD105+MVs and CD105+EXs level；
Fig. 2 a, Fig. 2 b are respectively NTA and measure CD34+MVs and CD34+EXs level；
Fig. 3 a, Fig. 3 b respectively compare NTA and the level of streaming method survey MVs.
Fig. 4 is the cell membrane microcapsule bubble MVs that extracts of the present invention and secretes outward body EXs the technique carrying out detecting
Flow chart.
Detailed description of the invention
The following is the case study on implementation that this project is concrete, reagent used in subordinate applies and material
To be obtained by purchasing channel, market.The PH of the PBS used by the present invention is 7.2.
As shown in Figure 4, extracting 3ml peripheral blood, be loaded on 2ml containing mass concentration is 3.3% citric acid
The sterile tube of sodium；Add the PBS of 6ml filtration treatment；Centrifugation is carried out, separation condition after mixing:
Rotating speed 400r/s, 35min, 4 DEG C；Take the superiors' liquid and be blood plasma；Take 1ml blood plasma to carry out
Centrifugation, separation condition: rotating speed 2000r/s, 20min, 4 DEG C, it is used for removing platelet；
Take supernatant to be centrifuged separating, separation condition: rotating speed 20000r/s, 120min, 4 DEG C, institute
The PBS of the precipitation 700 μ l filtration treatment obtained suspends and is NTA for analyzing cMVs sum；Remaining
Supernatant is centrifuged separating, separation condition: 169000r/s, 6h, 4 DEG C, and the precipitation of gained is used
The PBS of 700 μ l filtration treatment suspends and is NTA for analyzing cEXs sum；Gained cMVs and
CEXs precipitation is separately added into the anti-human-CD105 antibody of 10 μ l biotin couplings, mixing
After hatch 24 hours at 4 DEG C；It is separately added into 10 μ l anti-biotin magnetic beads again；Reactant is put
24h in magnetic devices, with obtain after magnetic bead sorting buffer solution elution magnetic bead CD105+MVs and
CD105+EXs；The PBS being separately added into 700 μ l filtration treatment again is NTA, analyzes CD105+MVs
With the ratio shared by cMVs and cEXs in CD105+EXs；By CD105+MVs and CD105+ of gained
EXs is divided into four parts, then be separately added into the anti-human-CD144 of 10 μ l, anti-human-KDR,
Anti-human-Annexin V and anti-human-CD63 antibody 4 DEG C hatch 24 hours；Divide again
Corresponding the two of the other Qdots655 labelling with 10 μ l resist in incubated at room 90mins；Add 700 μ l
The PBS of filtration treatment is NTA.Acquired results is as shown in Fig. 1 a, Fig. 1 b, and CD105+MVs accounts for
The 24.3 of total cMVs, CD105+EXs accounts for the 21.8% of total cEXs.Pass through and different antibodies coupling
Qdots655 hatch be fluorescence NTA analyze after, the CD105+MVs coexpression Annexin of about 22%
V, the CD105+MVs coexpression CD144 of about 10%, be above KDR or CD63；The CD105+ of about 20%
EXs coexpression CD63, the CD105+EXs coexpression CD144 of about 12%, be above KDR or Annexin
V。
CMVs and cEXs sum level detection method is ibid: in cMVs and cEXs of gained precipitates
It is separately added into the anti-human-CD34 antibody of 10 μ l biotin couplings, incubates at 4 DEG C after mixing
Educate 24 hours；Add 10 μ l anti-biotin magnetic beads；Reactant is placed in 24h in magnetic devices,
CD34+MVs and CD34+EXs is obtained after magnetic bead sorting buffer solution elution magnetic bead；It is separately added into again
The PBS of 700 μ l filtration treatment is NTA, analyzes cMVs and cEXs in CD34+MVs and CD34+EXs
Shared ratio；CD34+MVs and CD34+EXs of gained is divided into four parts, then is separately added into
Anti-human-CD144, anti-human-KDR, anti-human-Annexin V of 10 μ l and
Anti-human-CD63 antibody 4 DEG C hatches 24 hours；Qdots655 with 10 μ l marks the most respectively
Corresponding the two of note resist in incubated at room 90mins；The PBS adding 700 μ l filtration treatment is NTA.
Acquired results is as shown in Fig. 2 a, Fig. 2 b, and CD34+MVs accounts for the 11.6, CD34+EXs of total cMVs
Account for the 10.8% of total cEXs.It is fluorescence NTA divides through hatching with the Qdots655 of different antibodies coupling
After analysis, the CD34+MVs coexpression Annexin V, the CD34+MVs of about 5.1% of about 10.6% is altogether
Expressing K DR, is above CD144 or CD63；The CD34+EXs coexpression CD63 of about 11.2%, about
The CD34+EXs coexpression KDR of 8.5%, is above CD144 or Annexin V.
By the MVs level of fluorescence NTA and flow cytometer showed gained, it is used for comparing both approaches detection MVs
Sensitivity.Acquired results is as shown in Fig. 3 a, Fig. 3 b, dense with the cMVs measured by NTA analysis
Degree, much higher than with the cMVs concentration measured by flow cytometer showed, illustrates the sensitivity relatively streaming side of NTA method
Method is high.
NTA detection method:
In NanoSight NS300 (Malvern Instruments, United Kingdom) outfit
405nm royal purple light Laser Diodes is for identifying MVs and EXs of Qdots655 labelling.100nm and
The polystyrene latex pearl of 200nm is for being corrected NanoSight NS300.
The sample syringe diluted is installed, squeezes into analyzing device.
Open NTA2.3 software, camera level is set to 10 detections being used for non-fluorescence sample, by phase
Machine level is heightened to 16 for the detection of fluorescent samples.
Open camera, regulate focal length, start to analyze sample.
Flow cytometer detection method:
1) by the MVs precipitation gathered and 10 μ l PE-conjugated anti-CD105or
Anti-CD34, and FITC-conjugated anti-goat CD63, or Annexin V, or
Anti-goat CD144or KDR antibody incubation.The IgG that negative control group then adds correspondence is non-specific
Property antibody.After hatching, add the PBS of 100 μ l in sample,
2) BD Accuri C6 streaming instrument (Accuri Cytometer, Ann Arbor, MI) and
CFlow Plus Analysis software is used for doing flow cytometer showed.
3) calibration microspheres (the Molecular Probes of streaming machine 200nm and 1000nm；
Eugene, OR) correction.It is analyzed record in being popped one's head in as flow cytometer showed by sample after correction is good.
Claims (4)
1. cell membrane microcapsule bubble MVs and secrete outward the separating and extracting process of body EXs, it is characterised in that by with
Lower step is carried out:
A, extraction peripheral blood sample 3-10ml are placed in anticoagulant tube and preserve；Use phosphate-buffered physiology
Saline (PBS) dilutes 2-4 times, is centrifuged obtaining supernatant blood plasma；B: to supernatant blood plasma again
Secondary being centrifuged separates, and precipitate is MVs, c: the supernatant blood plasma of step b is carried out ultracentrifugation and divides
From, precipitate is EXs.
Cell membrane microcapsule bubble MVs the most according to claim 1 and secrete outward the separation and Extraction of body EXs
Method, it is characterised in that: dilute 3 times with phosphate buffered saline PBS, after mixing, use 400r/s
Rotating speed be centrifuged, within 35 minutes, obtain supernatant blood plasma.
Cell membrane microcapsule bubble MVs the most according to claim 2 and secrete outward the separation and Extraction of body EXs
Method, it is characterised in that: described step b is: supernatant blood plasma carries out two times centrifugal separation again,
The most centrifugal rotating speed 2000r/s, 20 minutes, preserves supernatant and carries out the most centrifugal, for the second time
Centrifugal rotating speed 20000r/s, 120 minutes, precipitate was MVs.
Cell membrane microcapsule bubble MVs the most according to claim 3 and secrete outward the separation and Extraction of body EXs
Method, it is characterised in that: described step c is: to being precipitated supernatant blood plasma that thing is MVs again
Being centrifuged separating, centrifugal rotating speed 169000r/s, 6 hours, centrifuged deposit thing was EXs.
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| CN106289927A (en) * | 2016-08-28 | 2017-01-04 | 浙江省中医院 | A kind of from tumor cell supernatant, separate microcapsule bubble and the method for secreting outward body thereof |
| CN106420819A (en) * | 2016-09-30 | 2017-02-22 | 南京大学 | Application of natural nano vesicle exosomes in preparing preparation for regulating sensitivity of cells to sorafenib and C-Met signal path |
| CN106692984A (en) * | 2016-12-08 | 2017-05-24 | 武汉大学 | Tumor-targeted delivery carrier based on cell-derived micro-vacuoles, preparation method and application |
| CN107338222A (en) * | 2016-08-16 | 2017-11-10 | 上海浦美生物医药科技有限公司 | Excretion body separation method based on lipid molecule probe |
| CN107375234A (en) * | 2017-07-10 | 2017-11-24 | 武汉大学 | A kind of multifunctional carrier and preparation method and application based on cell source vesica in body fluid |
| CN108546671A (en) * | 2018-04-17 | 2018-09-18 | 马英淙 | A kind of separation method of cells in biological samples microcapsule bubble |
| CN108795852A (en) * | 2018-06-27 | 2018-11-13 | 华中科技大学 | A kind of preparation method, product and its application of people's myoblast excretion body |
| CN110073195A (en) * | 2016-11-23 | 2019-07-30 | 阿曼·沙马 | Extract the method and kit of excretion body and large biological molecule in combination |
| CN111394307A (en) * | 2020-02-17 | 2020-07-10 | 天津医科大学眼科医院 | Method for separating and purifying exosome from plasma and application |
| CN112805562A (en) * | 2018-07-31 | 2021-05-14 | 国立大学法人三重大学 | Method for producing exosome |
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Cited By (14)
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| CN107338222A (en) * | 2016-08-16 | 2017-11-10 | 上海浦美生物医药科技有限公司 | Excretion body separation method based on lipid molecule probe |
| CN106289927B (en) * | 2016-08-28 | 2019-04-16 | 浙江省中医院 | A method of separating microcapsule bubble and its excretion body from tumour cell supernatant |
| CN106289927A (en) * | 2016-08-28 | 2017-01-04 | 浙江省中医院 | A kind of from tumor cell supernatant, separate microcapsule bubble and the method for secreting outward body thereof |
| CN106420819A (en) * | 2016-09-30 | 2017-02-22 | 南京大学 | Application of natural nano vesicle exosomes in preparing preparation for regulating sensitivity of cells to sorafenib and C-Met signal path |
| CN110073195B (en) * | 2016-11-23 | 2022-03-29 | 阿曼·沙马 | Method and kit for extracting exosomes and biomacromolecules bound with exosomes |
| CN110073195A (en) * | 2016-11-23 | 2019-07-30 | 阿曼·沙马 | Extract the method and kit of excretion body and large biological molecule in combination |
| CN106692984B (en) * | 2016-12-08 | 2020-02-18 | 武汉大学 | Tumor targeted delivery carrier based on cell-derived microvesicles, and preparation method and application thereof |
| CN106692984A (en) * | 2016-12-08 | 2017-05-24 | 武汉大学 | Tumor-targeted delivery carrier based on cell-derived micro-vacuoles, preparation method and application |
| CN107375234A (en) * | 2017-07-10 | 2017-11-24 | 武汉大学 | A kind of multifunctional carrier and preparation method and application based on cell source vesica in body fluid |
| CN107375234B (en) * | 2017-07-10 | 2021-03-05 | 武汉大学 | Multifunctional carrier based on cell-derived vesicles in body fluid and preparation method and application thereof |
| CN108546671A (en) * | 2018-04-17 | 2018-09-18 | 马英淙 | A kind of separation method of cells in biological samples microcapsule bubble |
| CN108795852A (en) * | 2018-06-27 | 2018-11-13 | 华中科技大学 | A kind of preparation method, product and its application of people's myoblast excretion body |
| CN112805562A (en) * | 2018-07-31 | 2021-05-14 | 国立大学法人三重大学 | Method for producing exosome |
| CN111394307A (en) * | 2020-02-17 | 2020-07-10 | 天津医科大学眼科医院 | Method for separating and purifying exosome from plasma and application |
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