CN108841777A - The extracting method and device of extracellular vesica based on Electrostatic Absorption and its content - Google Patents
The extracting method and device of extracellular vesica based on Electrostatic Absorption and its content Download PDFInfo
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- CN108841777A CN108841777A CN201810649131.0A CN201810649131A CN108841777A CN 108841777 A CN108841777 A CN 108841777A CN 201810649131 A CN201810649131 A CN 201810649131A CN 108841777 A CN108841777 A CN 108841777A
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Abstract
The present invention provides the extracting method and device of a kind of extracellular vesica based on Electrostatic Absorption and its content, biological sample is after pretreatment, it adds in the internal extraction element equipped with anion exchange resin particles, efflux is discharged through the port of the extraction element bottom, the extraction element is cleaned with cleaning buffer solution, then elution buffer elution can be used, collect the extracellular vesica of elution, or directly extracellular vesica of the capture in resin is cracked and extracts content.Wherein, the anion exchange resin particles surface modification has at least one of following functional group：Quaternary ammonium, tertiary amine, secondary amine, primary amine.By coming into full contact with biological sample and anion exchange resin particles, the extracellular vesica for making surface negatively charged under electrostatic interaction is adsorbed on particle surface, quickly and efficiently separates to realize.The present invention separates extracellular vesica using Electrostatic Absorption, can recover over 50% extracellular vesica from a variety of biological samples in 30 minutes.
Description
Technical field
The present invention relates to extracellular vesica separation and extraction technologies, specifically, being a kind of extracellular vesica based on Electrostatic Absorption
And its extracting method and device of content.
Background technique
Cell can secrete a variety of vesicas with phospholipid bilayer structure, among these include the excretion body in endosome source
The microvesicle (Microvesicle, partial size 20-1000nm) in (Exosome, partial size 50-100nm) and plasma membrane source.Extracellular vesica quilt
It confirms to play a role in blood coagulation, intercellular signal transmitting and waste management.1 at the same time, because rich in nucleic acid, protein,
The various biomolecules such as lipid, extracellular vesica have broad application prospects in terms of the diagnosing and treating of disease.
Currently, the extraction of extracellular vesica and separation method mainly include, supercentrifugation, exclusion chromatography, immunocapture
Method, polymer sedimentation and film affinity capture method.Wherein, supercentrifugation is based on other groups in extracellular vesica and biological sample
The size and density divided are distinguished, and are separated by ultracentrifuge, the valuableness of equipment needed for this method, operating procedure
The cumbersome and rate of recovery is not high.Exclusion chromatography is the screening technique based on extracellular vesica particle size, and product purity is high, but can not
Realize the concentration to extracellular vesica.Immunocapture method is to separate by antibody with the specific binding of extracellular vesicle surface antigen
The method of extracellular vesica, this method specificity is high, but expensive, and is influenced by antigen presentation amount difference, for not
Extracellular vesica capture rate with hypotype is also not quite similar.The polymer precipitation method are to utilize polymer such as polyethylene glycol (PEG) etc.,
By the microenvironment in change body fluid, the solubility of extracellular vesica is reduced to make its sedimentation, this method is not necessarily to large scale equipment, but
It is to have a large amount of albumen to settle to influence product purity together with vesica.
Summary of the invention
The object of the present invention is to provide a kind of easy to operate and can the separation from biological sample (such as body fluid) in a short time
The method for extracting extracellular vesica.
Specifically, the present invention is generally deposited for existing extracellular vesica separation method when extracting the extracellular vesica in body fluid
The problems such as cumbersome, yield is relatively low, and sample purity is not high.In view of extracellular vesica is usually aobvious negative in physiological environment lower surface
Electric (zeta current potential is negative), using the anion exchange resin particles that surface is positively charged, through electrostatic interaction from body fluid
Fast Acquisition simultaneously separates extracellular vesica.That is, the extracellular vesica that the object of the present invention is to provide a kind of based on Electrostatic Absorption and its interior
The extracting method of inclusion.
In a first aspect, the present invention provides the extracting method of a kind of extracellular vesica based on Electrostatic Absorption and its content, it is raw
Object sample after pretreatment, adds in the internal extraction element equipped with anion exchange resin particles (filler), efflux is through institute
The port discharge for stating extraction element bottom, cleans the extraction element with cleaning buffer solution, abandons efflux, then carry out following I
Or II operation：
Operate I：Directly be added lysate into the extraction element, in the efflux of collection containing extracellular vesica and/or
Its content；
Operate II：The extraction element after cleaning is eluted with elution buffer, contains born of the same parents in the eluent of collection
Outer vesica；
Wherein, the anion exchange resin particles surface modification has at least one of following functional group：Quaternary ammonium, uncle
Amine, secondary amine, primary amine.
Resin of the present invention is selected from silica column, polystyrene resin, polyacrylic resin etc..
Preferably, the anion exchange resin particles are the silica gel particle (being commercially available) with quaternary ammonium functional group.
Preferably, the partial size of the anion exchange resin particles is 30-800 μm.
Biological sample of the present invention include but is not limited to blood plasma, serum, urine, cerebrospinal fluid, hydrothorax, ascites, saliva with
And cell culture supernatant.
In the present invention, the preprocess method of biological sample is：After biological sample centrifugation, supernatant is collected, supernatant is straight
It connects for subsequent extracted；Alternatively, the filtrate of filtering, collection is used for subsequent extracted after supernatant is mixed with sample-loading buffer.Example
Such as, first the biological sample is mixed in proportion with sample-loading buffer, is added in extraction element and captures extracellular vesica, with cleaning
Buffer washes away impurity, with vesica outside lysate tniema and extracts content, or is eluted and collected complete with elution buffer
Extracellular vesica.
Preferably, the formula of the sample-loading buffer is：40-100mM Bis-tris propane and 0-150mM
NaCl。
The preprocess method of biological sample is specially：Biological sample is centrifuged 10-30 minutes in 1500-3000g, in collection
Clearly, by supernatant and sample-loading buffer by proper volume than mix (for example, for blood plasma, serum, ascites biological sample, inchoate aspect
Product accounts for the 25-75% of total volume after mixing, urine, cerebrospinal fluid, saliva and cell conditioned medium biological sample initial volume is accounted for mixed
The 50%-100% of total volume after conjunction), the membrane filtration in 0.45 μm or 0.8 μm of aperture is then used, gained filtrate mentions for subsequent
It takes.
In the present invention, at least containing the NaCl of 50-500mM in the cleaning buffer solution.For example, the formula of cleaning buffer solution
For：20-50mM Bis-tris propane and 50-500mM NaCl.The ionic strength of the buffer be enough will to be adsorbed on yin from
The impurity protein of sub-exchange resin particle surface elutes, to improve final product purity.
At least containing the NaCl of 500mM-1M in the elution buffer.The ionic strength of the buffer is enough to be adsorbed on
The extracellular vesica on anion exchange resin particles surface elutes.
The lysate can be commercial reagents, such as Qiazol, RIPA or Trizol.
In the specific embodiment of the present invention, the extracellular vesica for being adsorbed on anion exchange resin particles surface can
With without elution, Direct Pyrolysis and in extracellular vesica nucleic acid and albumen extract.
In the present invention, the amount ratio of the anion exchange resin particles and the biological sample is 0.1-5g：0.1-
150mL.For example, about 0.1g filler can be used for handling the plasma/serum of 0-2mL, 0-3mL cell conditioned medium, about 0.5g filler is available
In processing 0-15mL urine.
The process flow of different sample optimizations：
1. blood plasma, serum
Optimal sample handles volume 1-2mL, and 1500-3000g is centrifuged 10-30 minutes, supernatant is collected, with Bis- containing 100mM
The sample-loading buffer of tris propane and 150mM NaCl (pH=7), than mixing, then use 0.45 μm of aperture by isometric
Membrane filtration adds to gained filtrate in the 15mL centrifugal adsorbing column extraction element of the filler of SAX containing 0.1g, and 300g is centrifuged 10 points
Zhong Hou abandons efflux.The cleaning of 4mL Bis-tris containing 50mM propane and 250mM NaCl (pH=7) is added into inner tube
Buffer, 300g are centrifuged 10 minutes, replace outer tube.It is required according to subsequent experimental, alternative carries out in following operation.Operate I：
800 μ LQiazol lysates are added into pipe, are incubated for 10 minutes, 5000g is centrifuged 5 minutes, collects efflux；Operate II, Xiang Guan
The interior NaCl solution that 500 μ L 1M are added, stands 10 minutes, and 5000g is centrifuged 5 minutes, collects efflux.
2. urine
Optimal sample handles volume 10-15mL, and 1500-3000g is centrifuged 10-30 minute, collection supernatant, with 0.45 μm of aperture
Membrane filtration, gained filtrate is added in the 50mL centrifugal adsorbing column extraction element of the filler of SAX containing 0.5g, 300g centrifugation 10
After minute, efflux is abandoned.The clear of 8mL Bis-tris containing 50mM propane and 150mM NaCl (pH=7) is added into inner tube
Wash buffer, 300g are centrifuged 10 minutes, replace outer tube.It is required according to subsequent experimental, alternative carries out in following operation.Operation
I：1mLQiazol lysate is added into pipe, is incubated for 10 minutes, 5000g is centrifuged 5 minutes, collects efflux；Operate II, Xiang Guan
The interior NaCl solution that 1mL 1M is added, stands 10 minutes, and 5000g is centrifuged 5 minutes, collects efflux.
3. cell conditioned medium
Optimal sample handles volume 10-15mL, and 1500-3000g is centrifuged 10-30 minutes, supernatant is collected, with HCl or acetic acid
Adjust pH value to 6, with 0.45 μm of the membrane filtration in aperture.Gained filtrate is added to the 50mL centrifugal adsorbing column of the filler of SAX containing 0.5g
In extraction element, 300g is centrifuged after ten minutes, abandons efflux.Into inner tube be added 8mL Bis-tris containing 50mM propane with
The cleaning buffer solution of 150mM NaCl (pH=7), 300g are centrifuged 10 minutes, replace outer tube.It is required according to subsequent experimental, following
Alternative carries out in operation.Operate I：1mLQiazol lysate is added into pipe, is incubated for 10 minutes, 5000g is centrifuged 5 minutes, is received
Collect efflux；II is operated, the NaCl solution of 1mL 1M is added into pipe, stands 10 minutes, 5000g is centrifuged 5 minutes, collects outflow
Liquid.
4. cerebrospinal fluid
Optimal sample handles volume 2-4mL, and 1500-3000g is centrifuged 10-30 minute, collection supernatant, with 0.45 μm of aperture
Membrane filtration adds to gained filtrate in the 15mL centrifugal adsorbing column extraction element of the filler of SAX containing 0.1g, and 300g is centrifuged 10 points
Zhong Hou abandons efflux.The cleaning of 4mL Bis-tris containing 50mM propane and 150mM NaCl (pH=7) is added into inner tube
Buffer, 300g are centrifuged 10 minutes, replace outer tube.It is required according to subsequent experimental, alternative carries out in following operation.Operate I：
800 μ LQiazol lysates are added into pipe, are incubated for 10 minutes, 5000g is centrifuged 5 minutes, collects efflux；Operate II, Xiang Guan
The interior NaCl solution that 500 μ L 1M are added, stands 10 minutes, and 5000g is centrifuged 5 minutes, collects efflux.
5. hydrothorax, ascites
Optimal sample handles volume 10-15mL, and 1500-3000g is centrifuged 10-30 minutes, with Bis-tris containing 100mM
Propane and 150mM NaCl (pH=7) sample-loading buffer by isometric than mixing, will after the membrane filtration in 0.8 μm of aperture
Gained filtrate adds in the 50mL centrifugal adsorbing column extraction element of the filler of SAX containing 0.5g, and 300g is centrifuged after ten minutes, and abandoned stream goes out
Liquid.The cleaning buffer solution of 8mL Bis-tris containing 50mM propane and 150mM NaCl (pH=7), 300g are added into inner tube
Outer tube is replaced in centrifugation 10 minutes.It is required according to subsequent experimental, alternative carries out in following operation.Operate I：It is added into pipe
1mLQiazol lysate is incubated for 10 minutes, and 5000g is centrifuged 5 minutes, collects efflux；II is operated, 1mL 1M is added into pipe
NaCl solution, stand 10 minutes, 5000g be centrifuged 5 minutes, collect efflux.
6. saliva
Optimal sample handles volume 1-3mL, and 1500-3000g is centrifuged 10-30 minutes, supernatant is collected, with Bis- containing 100mM
Tris propane and 150mM NaCl (pH=7) sample-loading buffer, than mixing, then use the filter in 0.45 μm of aperture by isometric
Film filtering, gained filtrate is added in the 15mL centrifugal adsorbing column extraction element of the filler of SAX containing 0.1g, and 300g is centrifuged 10 minutes
Afterwards, efflux is abandoned.The cleaning that 4mL Bis-tris containing 50mM propane and 150mM NaCl (pH=7) is added into inner tube is slow
Fliud flushing, 300g are centrifuged 10 minutes, replace outer tube.It is required according to subsequent experimental, alternative carries out in following operation.Operate I：To
800 μ LQiazol lysates are added in pipe, are incubated for 10 minutes, 5000g is centrifuged 5 minutes, collects efflux；II is operated, into pipe
The NaCl solution of 500 μ L 1M is added, stands 10 minutes, 5000g is centrifuged 5 minutes, collects efflux.
Second aspect, the present invention provide the extraction element of a kind of extracellular vesica based on Electrostatic Absorption and its content, institute
Extraction element is stated in centrifugal adsorbing column (Fig. 1 a), liquid transfer gun head (Fig. 1 b), solid-phase extraction column (Fig. 1 c), syringe (Fig. 1 d)
At least one.
Wherein, anion exchange resin particles are filled between the upper and lower sieve plate of the centrifugal adsorbing column inner tube；
Inside the liquid transfer gun head be arranged in parallel with two pieces of sieve plates on the direction of pipette tips axis oriented normal, between two pieces of sieve plates
Filled with anion exchange resin particles；
Anion exchange resin particles are filled between the upper and lower sieve plate of the solid-phase extraction column；
Two pieces of sieve plates are arranged in parallel on the direction vertical with piston shaft inside the syringe, are filled between two pieces of sieve plates
There are anion exchange resin particles.
Wherein, the anion exchange resin particles surface modification has at least one of following functional group：Quaternary ammonium, uncle
Amine, secondary amine, primary amine.
Preferably, the aperture of the sieve plate is 10 μm.
By above-mentioned technical proposal, the present invention at least has following advantages and beneficial effect：
(1) easy to operate.This method utilizes the electrostatic interaction between extracellular vesica and anion exchange resin particles,
To realize separation to the extracellular vesica in biological sample, without large scale equipment, whole can be completed in 15~30 minutes
Operating procedure extracts extracellular vesica time-consuming about 3~8 hours using ultracentrifuge, (such as using PEG sedimentation in contrast
The ExoQuick kit of SBI) extract extracellular vesica needed for time view sample type between 1~12 hour.
(2) efficiency of pcr product, purity is high.When prior art extracts extracellular vesica from body fluid, all it is inevitably present miscellaneous
The pollution of matter albumen.Although density-gradient centrifugation method can obtain the extracellular vesica of higher degree, cumbersome, equipment is high
It is expensive to be unfavorable for promoting.And including ultracentrifugation, the extracellular vesica including the methods of PEG sedimentation and aqueous two-phase extraction extracts skill
Art cannot be effectively removed protein impurities.The method of the present invention is after capturing extracellular vesica, by introducing high ionic strength
NaCl solution can thoroughly elute impurity protein, to improve product purity.It is extracellular according to gained by taking plasma sample as an example
Vesica nucleic acid and total protein are assessed, the yield and purity of extracellular vesica obtained by the method for the present invention about exceed the speed limit from
10 times of heart method and 4 times or so, be 2 times and 50 times of PEG sedimentation or so, is 3.5 times and 1.5 times of exoEasy method or so.
(3) this method can effectively be compatible with downstream nucleic acid and protein analysis.After cleaned removal protein impurities, absorption
Extracellular vesica in anion exchange resin particles can with Direct Pyrolysis and in it nucleic acid and protein analyze, because
It almost can be ignored for the buffer volume in exchanger resin stratum granulosum at this time, cracking process and subsequent analysis are not cleaned
The influence of buffer composition.
(4) present invention separates extracellular vesica using Electrostatic Absorption, can be in 30 minutes from a variety of biological samples
In recover over 50% extracellular vesica.
Detailed description of the invention
Fig. 1 be the present invention is based on the extracellular vesica of Electrostatic Absorption and its extraction element of content, centrifugal adsorbing column (a),
Liquid transfer gun head (b), solid-phase extraction column (c), syringe (d)；Wherein, 1- sieve plate, 2- biological sample, 3- anion exchange resin
Grain, 4- inner tube, 5- outer tube, 6- pipette tips, 7-SPE column, 8- injection needle tube.
Fig. 2 is the ratio that remaining fEVs accounts for total amount fEVs in the embodiment of the present invention 1.
Fig. 3 is adsorption effect of the anion exchange resin particles to fEVs of different assembling forms in the embodiment of the present invention 2.
Fig. 4 is influence of the conductivity to EVs capture effect in the embodiment of the present invention 3.
Fig. 5 is influence of the pH value to EVs capture effect in the embodiment of the present invention 4.
Fig. 6 is the transmission electron microscope photo that EVs is separated in the embodiment of the present invention 5.
Fig. 7 is the Western blot experimental result of blood plasma EVs in the embodiment of the present invention 5.
Fig. 8 is the relationship of anionic exchange medium dosage and EVs RNA total amount in the embodiment of the present invention 6.
Fig. 9 is influence of the anion exchange resin particles dosage to house-keeping gene RT-PCR result in the embodiment of the present invention 6.
Figure 10 is influence of the flow velocity to human normal plasma EVs capture effect in the embodiment of the present invention 7.
Figure 11 is EVs RNA total amount obtained by the blood plasma of different initial volumes in the embodiment of the present invention 8.
Figure 12 is the GAPDH RT-PCR result of EVs RNA obtained by the blood plasma of different initial volumes in the embodiment of the present invention 8.
Figure 13 is the relationship of anionic exchange medium dosage and EVs RNA total amount in the embodiment of the present invention 9.
Figure 14 is anion exchange resin particles dosage in the embodiment of the present invention 9 to the shadow of house-keeping gene RT-PCR result
It rings.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Filler PS-SAX used in the following embodiment, article No. 201*7 (717) are limited purchased from Hangzhou swimming continent water process science and technology
Company, C8-SAX (article No. C8SAX-100) and SAX (article No. SAX-2-100) are purchased from Shenzhen comma Bioisystech Co., Ltd.
Suction-operated of 1 anion exchange resin particles of embodiment to extracellular vesica
1, reagent and consumptive material prepare
The anion exchange resin particles used include the granules of polystyrene (PS-SAX) with quaternary ammonium functional group, band C8 and
The silica gel particle (C8-SAX) of quaternary ammonium functional group and silica gel particle (SAX) with quaternary ammonium functional group, partial size is between 30-800 μm.Carefully
After birth green fluorescence probe DIO.Commercial human serum.With DI water dilution pH7.4 PBS liquid, be configured to respectively conductivity 2mS/cm,
The buffer of 4mS/cm and 10mS/cm.
2, the extracellular vesica (fEVs) of green fluorescence label is prepared
1300g is centrifuged 15 minutes after commercial human plasma is thawed, and removes cytoplasm remains.Later by supernatant be transferred to newly from
In heart pipe, after 3000g is centrifuged 15 minutes, take supernatant and PBS liquid with volume ratio 1:9 mixing, and 13000g is centrifuged 30 points at 4 DEG C
Clock.Supernatant is dispensed into and is centrifuged 1 hour from (every pipe corresponds to 2mL blood plasma) in pipe, 100,000g surpassing for 20mL, supernatant is abandoned, it will
The precipitating that 20mL surpasses from pipe tube bottom is resuspended in 1mL PBS liquid, is continued 100,000g and is centrifuged 1 hour, will be deposited in the born of the same parents of tube bottom
Outer vesica is resuspended in the PBS liquid of 100 μ L (corresponding 2mL blood plasma) containing 5%DIO dyestuff, is incubated for 2 hours at room temperature.It is saturating overnight
Analysis, removes remaining DIO dyestuff.
3, anion exchange resin particles adsorb fEVs
By the extracellular vesica of the fluorescent marker of same volume (fEVs) suspension be added to conductivity 2mS/cm, 4mS/cm and
It is mixed in the buffer of 10mS/cm and is made into A, B and C working solution.The fluorescence intensity of analytical unit volume tri- kinds of working solutions of A, B and C
IA、IBAnd IC.Then tri- kinds of working solutions of A, B, C are divided into several equal portions, PS-SAX, C8-SAX of 0.1g is added thereto respectively
Or SAX.4 DEG C of slight oscillatories are stayed overnight, and supernatant is drawn after low-speed centrifugal.The fluorescence intensity of analytical unit volume supernatant A ', B ' and C '
IA’、IB’And IC’。
Supernatant remains the work fEVs ratio of original shared by fEVs, yield=Ix’/Ix× 100%, x indicate A, B or C, as a result such as
Shown in Fig. 2, the silica gel particle (SAX) with quaternary ammonium functional group has preferable fEVs capture ability, and the shadow changed by conductivity
Sound is smaller.
Adsorption effect of the different device form of embodiment 2 to fEVs
1, reagent and consumptive material prepare
The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is situated between partial size
In 30-800 μm.The extracellular vesica (fEVs) of green fluorescence label, preparation method is the same as experimental example 1.The centrifugal adsorbing column used is
The centrifugation void column of outer pipe volume 15mL.It the use of liquid-transfering gun pipette tips is 1mL standard pipette tips.It the use of syringe is that 1mL is disposably injected
Device.The SPE void column (12mL) for the use of solid-phase extraction column (SPE column) being internal diameter 13mm.Sieve plate used is 10 unless otherwise noted
The hydrophobic sieve plate in μm aperture.Specific assembling mode is as shown in Figure 1.
2, absorption of the anion exchange resin particles of different assembling forms to fEVs
FEVs is added in the PBS buffer solution of pH 7.4, and the fluorescence intensity I of analytical unit volume fEVs solution0.It takes
1mL fEVs solution is added in device as the sample individually tested.Cooperate corresponding sample processing method (for example, from
Heart adsorption column is blown and beaten using 200-5000g low-speed centrifugal, liquid-transfering gun pipette tips using liquid-transfering gun, and SPE column is loaded using negative pressure,
Such as gauge pressure -30KPa~-5KPa) come into full contact with fEVs solution with solid phase.Collect efflux, and analytical unit volume efflux
Fluorescence intensity I1.The ratio that the adsorbed fEVs of anion exchange resin particles accounts for original solution fEVs is yield=(I0-
I1)/I0× 100%.Experimental result is as shown in figure 3, four kinds of device forms can adsorb fEVs.
Influence of 3 conductivity of embodiment to EVs capture effect
1, reagent and consumptive material prepare
The centrifugal adsorbing column used is the centrifugation void column of outer pipe volume 15mL, and selected sieve plate is the hydrophobic sieve in 10 μm of aperture
Plate.The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is 30-800 μm.It is used
Biological sample, the 22rv1 cell conditioned medium of no excretion body fetal calf serum culture medium culture are stand-by after 0.45 μm of membrane filtration.Make
The conductivity that cell conditioned medium is adjusted with DI water and NaCl, between 4-80mS/cm.EVs nucleic acid extraction used kit used is
miRNeasy Mini Kit(Qiagen).Gained RNA is analyzed with Agilent Agilent RNA 6000Pico Kit.
2, it captures the EVs in cell conditioned medium and extracts RNA
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is 0.1g.By 3mL cell conditioned medium be added to from
In heart pipe inner tube, 300g is centrifuged after ten minutes, abandons efflux.3mL Bis-tris containing 50mM propane+ is added into inner tube
The cleaning buffer solution of 150mM NaCl, 300g are centrifuged 10 minutes, replace outer tube.To inner tube be added Qiazol lysate, 5000g from
It the heart 5 minutes, collects efflux and extracts RNA according to kit operating process.With Agilent RNA 6000Pico Kit reagent
Box analyzes extracted RNA total amount, as a result as shown in Figure 4.The conductivity of optimization is 13.31mS/cm.
Influence of the embodiment 4pH value to EVs capture effect
1, reagent and consumptive material prepare
The centrifugal adsorbing column used is the centrifugation void column of outer pipe volume 15mL, and selected sieve plate is the hydrophobic sieve in 10 μm of aperture
Plate.The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is 30-800 μm.It is used
Biological sample, for the 22rv1 cell conditioned medium of no excretion body fetal calf serum culture medium culture.Use 100mM HCl or NaOH solution
It is between 5~8 to adjust the pH value of cell conditioned medium.EVs nucleic acid extraction used kit used is miRNeasy Mini Kit
(Qiagen).Gained RNA is analyzed with Agilent Agilent RNA 6000Pico Kit.
2, it captures the EVs in cell conditioned medium and extracts RNA
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is 0.1g.By 3mL cell conditioned medium be added to from
In heart pipe inner tube, 300g is centrifuged after ten minutes, abandons efflux.3mL 50mM Bis-tris propane+ is added into inner tube
The cleaning buffer solution of 150mM NaCl, 300g are centrifuged 10 minutes, replace outer tube.To inner tube be added Qiazol lysate, 5000g from
It the heart 5 minutes, collects efflux and extracts RNA according to kit operating process.With Agilent RNA 6000Pico Kit reagent
Box analyzes extracted RNA total amount, as a result as shown in Figure 5.The result shows that as pH=6, EVs yield highest.
The capture and characterization of 5 human normal plasma EVs of embodiment
1, reagent and consumptive material prepare
The centrifugal adsorbing column used is the centrifugation void column of outer pipe volume 15mL, and selected sieve plate is the hydrophobic sieve in 10 μm of aperture
Plate.The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is 30-800 μm.By quotient
Employment blood plasma unfreezing, 3000g takes supernatant after being centrifuged 15 minutes, with 100mM Bis-tris propane+150mM NaCl solution
By volume 1:1 mixing, it is stand-by then to obtain supernatant A with 0.45 μm of membrane filtration.Cleaning buffer solution used is 50mM Bis-tris
Propane+150mM NaCl, EVs elution buffer used are 1M NaCl solution.
2, blood plasma EVs is captured
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is 0.1g.1mL supernatant A is added in centrifuge tube
Pipe, 300g are centrifuged after ten minutes, abandon efflux.4mL cleaning buffer solution is added into inner tube, 300g is centrifuged 10 minutes, and replacement is outer
Pipe.EVs elution buffer is added into inner tube, 5000g centrifugation elutes 5 minutes.
3, the transmission electron microscope detection of EVs is eluted
The 10 μ l EVs solution eluted is added drop-wise on copper mesh and is incubated at room temperature 10 minutes, is cleaned with DI water, then uses acetic acid
Uranyl negative staining 1 minute.It can loading shooting electromicroscopic photograph after drying.Isolated EVs electromicroscopic photograph is as shown in fig. 6, can
See and both contain the microcapsule bubble (MVs) that partial size is greater than 200nm, also contains the lesser excretion body (exosome) of partial size.
4, the Western blot detection of EVs is eluted
By 10 μ l EVs suspensions and 3.3 4 × sample-loading buffers of μ l (BiofurawTM protein sample buffer,
Shanghai Tian Neng Science and Technology Ltd.) mix after 95 DEG C of water-baths boil 5min, after centrifugation be added pre-prepared colloid (BiofurawTM Precast
Gel, Shanghai Tian Neng Science and Technology Ltd.), electrophoresis 1 hour under 100V constant pressure.It usesVE-180PRE fast-turn construction system
Albumen is gone on the pvdf membrane after methyl alcohol process.After being closed 4 hours with 5% skimmed milk power, be incubated for it is 1 anti-(anti-CD63 and
Anti-calnexin it) and with the Tween-20PBST containing 0.1% elutes.Be incubated for it is 2 anti-, and with the PBST containing 0.1%Tween-20
Elution.Finally, substrate is added dropwise and is imaged.As a result as shown in Figure 7.The result shows that extracellular vesica positive protein matter marker CD63
Band is high-visible, and extracellular vesica negative proteins matter marker calnexin is without bands visible.
Influence of 6 amount of filler of embodiment to human normal plasma EVs contact conditions
1, reagent and consumptive material prepare
The centrifugal adsorbing column used is the centrifugation void column of outer pipe volume 15mL, and selected sieve plate is the hydrophobic sieve in 10 μm of aperture
Plate.The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is 30-800 μm.By quotient
Employment blood plasma unfreezing, 3000g takes supernatant after being centrifuged 15 minutes, with 100mM Bis-tris propane+150mM NaCl solution
By volume 1:1 mixing, it is stand-by then to obtain supernatant A with 0.45 μm of membrane filtration.Cleaning buffer solution used is 50mM Bis-tris
Propane+150mM NaCl, EVs elution buffer used are 1M NaCl solution.
2, blood plasma EVs is captured
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is respectively 0.05g, 0.1g, 0.2g and 0.4g.
Centrifuge tube inner tube is added in 1mL supernatant A, 300g is centrifuged after ten minutes, abandons efflux.4mL cleaning buffer solution is added into inner tube,
300g is centrifuged 10 minutes, replaces outer tube.Qiazol lysate is added to inner tube, 5000g is centrifuged 5 minutes, collects efflux.
3, RNA macroanalysis
Using the Standard Operating Procedure of miRNeasy Mini Kit kit, the RNA of EVs is extracted, and uses Agilent
RNA 6000Pico Kit kit analyzes RNA total amount, as a result as shown in Figure 8.
4、RT-PCR
The RNA sample of 1-5ng is taken to carry out subsequent house-keeping gene detection, the specific detection method is as follows：
(1) excretion body total serum IgE reverse transcription
The reverse transcription reagent box for using Beijing health to produce for century Bioisystech Co., Ltd carries out reverse transcription, and (article No. is
CW2582M), concrete operations process is as follows：
1. preparing reverse transcription reaction system on ice according to below table：
2. mixing, of short duration centrifugation makes the solution on tube wall be collected into tube bottom.
It is incubated for 30 minutes 3. cDNA synthetic reaction condition is 42 DEG C, 85 DEG C are incubated for 5 minutes.
4. after reaction, adding ddH2O dilutes 1 times, and of short duration centrifugation is placed on ice, then carries out fluorescent quantitative PCR,
It such as needs to save for a long time, -20 DEG C can be placed in.
(2) reference gene primer and PCR amplification system
1. reference gene primer
It is total to have selected 2 reference genes, respectively RPL37A and GAPDH.The amplimer and probe sequence of each gene
It arranges as follows：
Primer and probe sequence (the SEQ ID NO of 1 two house-keeping genes of table:1-6)
2. PCR amplification system
QPCR detection reaction system provided by the invention is 20 μ L：2 × PCR Master Mix 10 μ L, Forward
Primer, Reverse Primer and Probe concentration are respectively 0.2 μM, 4 μ L of reverse transcription product, mend ddH2O to 20 μ L.The present invention
In PCR Master Mix used purchased from health be century Biotechnology Co., Ltd, article No. CW0932.
The PCR response procedures are：95 DEG C of initial denaturation 10min；95 DEG C of 15s, 60 DEG C of 30s, 15 circulations；95 DEG C of 15s, 60
DEG C 30s collects fluorescence, 35 circulations.
(3) testing result
Comprehensive Agilent 2100 (Fig. 8) and RT-PCR result (Fig. 9), the media particle dosage of optimization are 0.1g.Wherein,
Total RNA content that Agilent 2100 is extracted from isolated EVs as the result is shown (comprising miRNA, lncRNA and
Total serum IgE including circRNA), the copy number quantity of the house-keeping gene RT-PCR mRNA as the result is shown.
Influence of 7 flow velocity of embodiment to human normal plasma EVs capture effect
1, reagent and consumptive material prepare
The centrifugal adsorbing column used is the centrifugation void column of outer pipe volume 15mL, and selected sieve plate is the hydrophobic sieve in 10 μm of aperture
Plate.The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is 30-800 μm.By quotient
Employment blood plasma unfreezing, 3000g takes supernatant after being centrifuged 15 minutes, with 100mM Bis-tris propane+150mM NaCl solution
By volume 1:1 mixing, it is stand-by then to obtain supernatant A with 0.45 μm of membrane filtration.Cleaning buffer solution used is 50mM Bis-tris
propane+150mM NaCl。
2, the sample flow under different centrifugal force
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is respectively 0.1g.4mL supernatant A is added and is centrifuged
Pipe inner tube, respectively to detect trickle after 100g, 200g, 300g and 500g centrifugal force 30 seconds, 1 minute and 2 minutes
Product.The result shows that flow rate of liquid is gradually risen by 0.4cm/s to 4cm/s as centrifugal rotational speed gradually rises.
3, blood plasma EVs is captured
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is respectively 0.1g.1mL supernatant A is added and is centrifuged
Pipe inner tube abandons efflux respectively after ten minutes with 100g, 200g, 300g and 500g centrifugal force.4mL is added into inner tube
Cleaning buffer solution, 300g are centrifuged 10 minutes, replace outer tube.Qiazol lysate is added to inner tube, 5000g is centrifuged 5 minutes, is collected
Efflux.
4, RNA macroanalysis
Using the Standard Operating Procedure of miRNeasy Mini Kit kit, the RNA of EVs is extracted, and uses Agilent
RNA 6000Pico Kit kit analyzes RNA total amount, and RNA total amount result is as schemed under different centrifugal force/flow conditions
Shown in 10.The result shows that the centrifugal rotational speed of optimization is 300g.
The influence that 8 blood plasma initial volume of embodiment captures EVs
1, reagent and consumptive material prepare
The centrifugal adsorbing column used is the centrifugation void column of outer pipe volume 15mL, and selected sieve plate is the hydrophobic sieve in 10 μm of aperture
Plate.The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is 30-800 μm.By quotient
Employment blood plasma unfreezing, 3000g takes supernatant after being centrifuged 15 minutes, with 100mM Bis-tris propane+150mM NaCl solution
By volume 1:1 mixing, it is stand-by then to obtain supernatant A with 0.45 μm of membrane filtration.Cleaning buffer solution used is 50mM Bis-tris
propane+150mM NaCl。
2, blood plasma EVs is captured
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is respectively 0.1g.Respectively by 0.1mL, 0.2mL,
Centrifuge tube inner tube is added in 0.5mL, 1mL and 2mL supernatant A, and 300g is centrifuged after ten minutes, abandons efflux.It is clear that 4mL is added into inner tube
Wash buffer, 300g are centrifuged 10 minutes, replace outer tube.Qiazol lysate is added to inner tube, 5000g is centrifuged 5 minutes, collects stream
Liquid out.
3, RNA macroanalysis
Using the Standard Operating Procedure of miRNeasy Mini Kit kit, the RNA of EVs is extracted, and uses Agilent
RNA 6000Pico Kit kit analyzes RNA total amount, and RNA total amount result is as shown in figure 11 under different condition.As a result
Show to increase to 2mL by 0.1mL with blood plasma initial volume, gained RNA total amount gradually rises.
4, RT-PCR detects EVs mRNA
Extraction carries out reverse transcription reaction after obtaining EVs RNA, carries out the special base genetic test of GAPDH, specific detection method
See embodiment 6, as a result as shown in figure 12.
Comprehensive Agilent 2100 is with RT-PCR's as a result, the 15mL centrifugal adsorbing column of 0.1g filler assembling can be used for extracting
Volume is no less than the blood plasma EVs of 2mL.
Influence of 9 amount of filler of embodiment to Healthy People urine EVs capture effect
1, reagent and consumptive material prepare
The centrifugal adsorbing column used is the centrifugation void column of outer pipe volume 50mL, and selected sieve plate is the hydrophobic sieve in 20 μm of aperture
Plate.The anion exchange resin particles used are the silica gel particle (SAX) with quaternary ammonium functional group, and partial size is 30-800 μm.Acquisition
Healthy People middle section urina sanguinis is no less than 50mL, by 0.45 μm of membrane filtration of supernatant after 3000g centrifugation, freezes stand-by.EVs core used
It is miRNeasy Mini Kit (Qiagen) that acid, which extracts used kit,.Gained RNA Agilent Agilent RNA
6000Pico Kit is analyzed.
2, the EVs in urine is captured
Mode as shown in Figure 1a assembles centrifuge tube, and added SAX amount of filler is 0.25g, 0.5g, 1g and 2g.15mL is urinated
Liquid is added in centrifuge tube inner tube, and 300g is centrifuged after ten minutes, abandons efflux.50mM Bis-tris is added into inner tube
The cleaning buffer solution of propane+150mM NaCl, 300g are centrifuged 10 minutes, replace outer tube.Qiazol cracking is added to inner tube
Liquid, 5000g are centrifuged 5 minutes, collect efflux.
3, RNA is extracted
RNA is extracted according to miRNeasy Mini Kit kit operating process.With Agilent RNA 6000Pico Kit
Kit analyzes extracted RNA total amount, as a result as shown in figure 13.
4, RT-PCR detects EVs mRNA
Extraction carries out reverse transcription reaction after obtaining EVs RNA, carries out the special base genetic test of GAPDH, specific detection method
See embodiment 6, as a result as shown in figure 14.The result shows that the amount of filler of optimization is 0.5g.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Bibliography
1.Hill,A.F.,Exosomes and Microvesicles.2016.
2.(a)Sokolova,V.；Ludwig,A.K.；Hornung,S.；Rotan,O.；Horn,P.A.；Epple,M.；
Giebel,B.,Characterisation of exosomes derived from human cells by
nanoparticle tracking analysis and scanning electron microscopy.Colloids and
surfaces.B,Biointerfaces 2011,87(1),146-50；(b)Hood,J.L.；San,R.S.；Wickline,
S.A.,Exosomes released by melanoma cells prepare sentinel lymph nodes for
tumor metastasis.Cancer research 2011,71(11),3792-801.
3.Bosma,J.；Wesselingh,J.,pH dependence of ion‐exchange equilibrium of
proteins.AIChE journal 1998,44(11),2399-2409.
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Claims (10)
1. the extracting method of the extracellular vesica based on Electrostatic Absorption and its content, which is characterized in that biological sample is preprocessed
Afterwards, it adds in the internal extraction element equipped with anion exchange resin particles, port of the efflux through the extraction element bottom
Discharge cleans the extraction element with cleaning buffer solution, abandons efflux, then carries out following I or II operation：
Operate I：Lysate is directly added into the extraction element, containing in extracellular vesica and/or its in the efflux of collection
Inclusion；
Operate II：The extraction element after cleaning is eluted with elution buffer, and extracellular capsule is contained in the eluent of collection
Bubble；
Wherein, the anion exchange resin particles surface modification has at least one of following functional group：It is quaternary ammonium, tertiary amine, secondary
Amine, primary amine.
2. the method according to claim 1, wherein the resin is selected from silica column, polystyrene resin, gathers
Acrylic resin.
3. according to the method described in claim 2, it is characterized in that, the anion exchange resin particles are band quaternary ammonium functional group
Silica gel particle.
4. the method according to claim 1, wherein the partial size of the anion exchange resin particles is 30-800
μm。
5. the method according to claim 1, wherein the biological sample includes blood plasma, serum, urine, brain ridge
Liquid, hydrothorax, ascites, saliva and cell culture supernatant.
6. the method according to claim 1, wherein the preprocess method of biological sample is：By the biological sample
After product centrifugation, supernatant is collected, supernatant is directly used in subsequent extracted；Alternatively, being filtered after supernatant is mixed with sample-loading buffer, receive
The filtrate of collection is used for subsequent extracted.
7. according to the method described in claim 6, it is characterized in that, the preprocess method of biological sample is specially：Biological sample
It is centrifuged 10-30 minutes in 1500-3000g, collects supernatant, supernatant is mixed with sample-loading buffer by proper volume ratio, is then used
The membrane filtration that 0.45 μm or 0.8 μm of aperture, gained filtrate are used for subsequent extracted.
8. the method according to claim 1, wherein at least containing 50-500mM's in the cleaning buffer solution
NaCl；At least containing the NaCl of 500mM-1M in the elution buffer.
9. method according to claim 1-8, which is characterized in that the anion exchange resin particles with it is described
The amount ratio of biological sample is 0.1-5g：0.1-150mL.
10. the extraction element of the extracellular vesica based on Electrostatic Absorption and its content, which is characterized in that the extraction element is selected from
At least one of centrifugal adsorbing column, liquid transfer gun head, solid-phase extraction column, syringe；
Wherein, anion exchange resin particles are filled between the upper and lower sieve plate of the centrifugal adsorbing column inner tube；
Two pieces of sieve plates are arranged in parallel with on the direction of pipette tips axis oriented normal inside the liquid transfer gun head, are filled between two pieces of sieve plates
There are anion exchange resin particles；
Anion exchange resin particles are filled between the upper and lower sieve plate of the solid-phase extraction column；
Two pieces of sieve plates are arranged in parallel with inside the syringe on the direction vertical with piston shaft, are filled with yin between two pieces of sieve plates
Ion-exchange resin particles；
Wherein, the anion exchange resin particles surface modification has at least one of following functional group：It is quaternary ammonium, tertiary amine, secondary
Amine, primary amine.
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| CN109825472A (en) * | 2019-03-01 | 2019-05-31 | 易春 | A kind of extracting method and kit of extracellular vesica |
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| CN112322583A (en) * | 2020-09-28 | 2021-02-05 | 上海思路迪生物医学科技有限公司 | Blood coagulation-based extracellular vesicle capture technology |
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