CN106399250A - Method and kit for separating exosome - Google Patents
Method and kit for separating exosome Download PDFInfo
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- CN106399250A CN106399250A CN201510469340.3A CN201510469340A CN106399250A CN 106399250 A CN106399250 A CN 106399250A CN 201510469340 A CN201510469340 A CN 201510469340A CN 106399250 A CN106399250 A CN 106399250A
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Landscapes
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Abstract
The invention discloses a method and kit for separating exosome. The invention provides a reagent. The reagent is an aqueous solution of a hydrophilic polymer or a salt solution, wherein the hydrophilic polymer is PEG, dextran sulfate, or PVP, or a composition thereof. Experimental results prove that the exosome can be successfully separated by using a plurality of hydrophilic polymers; and the yield of separated exosome can be increased by using a composition of a hydrophilic polymer and an inorganic salt.
Description
Technical field
The present invention relates to biomedicine field, more particularly, to a kind of method separating excretion body and its test kit.
Background technology
Excretion body is the vesicles (normally about 30-100 nanometer) that cell is secreted to external space of cell, separate sources excretion body
Size is variant.All types of cultured cells can secrete excretion body, and body fluids such as blood, urine, saliva and
Rich content (Kosaka et al., Silence 3 (2010) 1-7 in breast milk；Mitchell et al.,PNAS 105
(2008) 10513-10518), and excretion body can reach other cell or tissues by approach such as body fluid circulatory.Excretion body
In comprise nucleic acid, the various biomolecules such as albumen, lipid, and be probably the carrier that these materials are transported in vivo.Phase
Close research and think that excretion body may play the role of courier, it passes through, in the various signaling molecule of intercellular trafficking, to participate in
Cell-cell communication, thus regulating and controlling various physiological processes, occurs and transfer, heart disease, immune disease etc. including tumor,
Therefore excretion body has good drug delivery system potentiality to be exploited, its include or film on nucleic acid, albumen, lipid,
The materials such as enzyme are likely to become the mark of medical diagnosis on disease treatment.But the way of production to excretion body and its inclusions at present
Still insufficient with the understanding of function, therefore high-quality and efficient can obtain excretion body and its inclusions in the urgent need to a kind of
Method, to provide substantial amounts of high-quality excretion body, as further investigation excretion body function mechanism with and applied
Basis in medical diagnosis on disease or gene therapy.
The method separating excretion body at present has supercentrifugation and ultra-filtration method.Supercentrifugation mainly utilizes excretion body big
Little, density etc. is separated with the difference of surrounding material, and its shortcoming is that centrifugation apparatus are had high demands, high cost；
Need to be centrifuged repeatedly, operating procedure is complicated；Centrifugal force is big, may destroy excretion body structure, and the excretion body of therefore acquisition is received
Rate is not high.And ultra-filtration rule is to be separated from surrounding using the size characteristic of excretion body, which solve
Time-consuming for supercentrifugation, requires the defect of special installation, but due to there may be in surrounding and its size range
Other materials of identical, therefore detached excretion body purity is not high.
Content of the invention
It is an object of the present invention to provide a kind of examination separating excretion body in body fluid or cell supernatant or tissue samples
Agent.
The reagent that the present invention provides, is hydrophilic polymer or hydrophilic polymer solution.
In mentioned reagent, described hydrophilic polymer is at least one of following 3 kinds：PEG (Polyethylene Glycol), sulphuric acid Portugal
Polysaccharide and PVP (Polyvinylpyrrolidone).
In mentioned reagent, described hydrophilic polymer solution is aqueous solution, saline solution or organic solution.
In described hydrophilic polymer solution, described hydrophilic polymer concentration is 10mg/ml-300mg/ml.
In mentioned reagent, the molecular weight of described PEG is 6000-20000 dalton, the molecule of described dextran sulfate
Measure as 30000-50000 dalton, the molecular weight of described PVP is 20000-40000 dalton；
Or the molecular weight of described PEG is specially 8000 or 20000 dalton, the molecule measurer of described dextran sulfate
Body is 40000 dalton, and the molecular weight of described PVP is specially 25000 dalton or 40000 dalton.
Mentioned reagent be following 1) -3) in any one：
1) hydrophilic polymer group or hydrophilic polymer aqueous solution group；Described hydrophilic polymer group or described hydrophilic polymer
Hydrophilic polymer in aqueous solution group is following at least 2 kinds：PEG8000, PVP25000, PEG20000 and sulphuric acid Portugal
Polysaccharide 40000；
2) hydrophilic polymer saline solution, described hydrophilic polymer saline solution is made up of solute and solvent, described solute by
Hydrophilic polymer and inorganic salt composition；Described hydrophilic polymer is PEG8000 or PEG20000；
3) hydrophilic polymer or its aqueous solution, described hydrophilic polymer is PEG8000, PEG20000, dextran sulfate
40000th, PVP25000 or PVP40000.
In mentioned reagent, 1) described hydrophilic polymer group or hydrophilic polymer aqueous solution group are as follows：
PEG8000 and PVP25000；
Or PEG8000 aqueous solution and PVP25000 aqueous solution；
Or PEG20000 and dextran sulfate 40000；
Or PEG20000 aqueous solution and dextran sulfate 40000 aqueous solution；
2) described hydrophilic polymer saline solution is PEG8000 saline solution or PEG20000 saline solution；
The solute of described PEG8000 saline solution is made up of PEG8000 and NaCl；
The solute of described PEG20000 saline solution is made up of PEG20000 and NaCl.
In mentioned reagent, described body fluid is blood plasma or serum or urine or hydrothorax or spinal fluid or Cerebrospinal fluid or saliva
Or emulsion or joint fluid or seminal fluid or vaginal secretion or amniotic fluid, described cell supernatant be derived from tumor cell or inflammatory cell or
Parenchyma or Interstitial cell or epithelial cell or fibrocyte or fibroblast, described are organized as liver or lung or brain.
Described cell supernatant is derived from 3T3 cell or A549 cell or 293 cells or Hela cell.
It is a further object to provide a kind of separate excretion body in body fluid or cell supernatant or tissue samples
Test kit.
The test kit that the present invention provides, including above-mentioned reagent；
Or in above-mentioned reagent or described test kit excretion body in separating body fluid or cell supernatant or tissue samples
Application be also the scope of protection of the invention；
Or above-mentioned reagent or described test kit are in preparative separation body fluid or cell supernatant or tissue samples excretion body
In application be also the scope of protection of the invention；
In above-mentioned, described body fluid is specially blood plasma or serum or urine or hydrothorax or spinal fluid or Cerebrospinal fluid or saliva
Or emulsion or joint fluid or seminal fluid or vaginal secretion or amniotic fluid, described cell supernatant is specifically derived from tumor cell or inflammatory is thin
Born of the same parents or parenchyma or Interstitial cell or epithelial cell or fibrocyte or fibroblast, described tissue be specially liver or
Lung or brain.Described cell supernatant is specifically derived from 3T3 cell or A549 cell or 293 cells or Hela cell.
The 3rd purpose of the present invention is to provide a kind of method separating body fluid or cell supernatant or tissue samples excretion body.
The method that the present invention provides comprises the steps：By body fluid or cell supernatant or tissue samples respectively with above-mentioned examination
Agent mixes, and obtains mixed liquor, makes the hydrophilic polymer in described reagent reach working concentration in the concentration of mixed liquor, then
Described mixed liquor is stood, collects precipitation after standing, that is, obtain excretion body.
In said method,
Using described reagent and described reagent in hydrophilic polymer working concentration as follows：
Described PEG8000 aqueous solution and the mixed liquor of described PVP25000 aqueous solution；During described PEG8000 work
Concentration is 50mg/ml, and concentration during described PVP25000 work is 150mg/ml；
Or the mixed liquor of described PEG20000 aqueous solution and described dextran sulfate 40000 aqueous solution；Described PEG20000
Concentration during work is 150mg/ml, and concentration during described dextran sulfate 40000 work is 50mg/ml；
Or described PEG8000 saline solution, concentration during described PEG8000 work is 150mg/ml, and described NaCl
Concentration during work is 0.5M；
Or described PEG20000 saline solution, concentration during described PEG20000 work is 150mg/ml, and described NaCl
Concentration during work is 0.5M；
Or described PEG8000 aqueous solution, concentration during described PEG8000 work is 150mg/ml；
Or described PEG20000 aqueous solution, concentration during described PEG20000 work is 150mg/ml；
Or described dextran sulfate 40000 aqueous solution, concentration during described dextran sulfate 40000 work is 50mg/ml；
Or described PVP25000 aqueous solution, concentration during described PVP25000 work is 200mg/ml；
Or described PVP40000 aqueous solution, concentration during described PVP40000 work is 150mg/ml；
Described body fluid is specially blood plasma or serum or urine or hydrothorax or spinal fluid or Cerebrospinal fluid or saliva or emulsion
Or joint fluid or seminal fluid or vaginal secretion or amniotic fluid, the concrete tumor cell of described cell supernatant or inflammatory cell or essence thin
Born of the same parents or Interstitial cell or epithelial cell or fibrocyte or fibroblast, described tissue is specially liver or lung or brain.
Described cell supernatant is specifically derived from 3T3 cell or A549 cell or 293 cells or Hela cell.
PEG is Polyethylene Glycol, and PVP is Polyvinylpyrrolidone, and the numeral after PEG and PVP represents its relative molecular weight.
Described cell supernatant is the supernatant of cell culture fluid.
Described collection is precipitated as being centrifuged, and the centrifugal force of centrifugation is 500g-15000g.
Described body fluid or cell supernatant or tissue samples are 7 with the volume ratio of mentioned reagent：1-1：1；
In described mentioned reagent, described hydrophilic polymer concentration is 10mg/ml-300mg/ml.
In the process, separation is obtained with excretion body to be detected further and/or to be measured before solution mixes
Sample is processed；Described detection includes detection of nucleic acids and/or Protein Detection, and described process includes cracking and/or purification.
Said method be specially following 1) -3) in any one：
1) sample to be tested, above-mentioned PEG8000 aqueous solution are mixed, obtain mixed liquor, make described PEG8000 mixed
Closing the concentration in liquid is 150mg/ml, more described mixed liquor is stood, and collects precipitation, that is, obtain excretion body after standing；
2) sample to be tested, above-mentioned PEG20000 aqueous solution are mixed, obtain mixed liquor, so that described PEG20000 is existed
Concentration in mixed liquor is 150mg/ml, more described mixed liquor is stood, and collects precipitation, that is, obtain excretion body after standing；
3) sample to be tested, above-mentioned dextran sulfate 40000 aqueous solution are mixed, obtain mixed liquor, make described sulphuric acid
Concentration in mixed liquor for the Gentran 40 000 is 50mg/ml, more described mixed liquor is stood, and collects precipitation after standing,
Obtain excretion body；
4) sample to be tested, above-mentioned PVP25000 aqueous solution are mixed, obtain mixed liquor, so that described PVP25000 is existed
Concentration in mixed liquor is 200mg/ml, more described mixed liquor is stood, and collects precipitation, that is, obtain excretion body after standing；
5) sample to be tested, above-mentioned PVP40000 aqueous solution are mixed, obtain mixed liquor, so that described PVP40000 is existed
Concentration in mixed liquor is 150mg/ml, more described mixed liquor is stood, and collects precipitation, that is, obtain excretion body after standing；
6) sample to be tested, above-mentioned PEG8000 aqueous solution and above-mentioned PVP25000 aqueous solution are mixed, obtain mixed liquor,
Described PEG8000 concentration in described mixed liquor is made to be 50mg/ml, and concentration in mixed liquor for the described PVP25000
For 150mg/ml, more described mixed liquor is stood, collect precipitation after standing, that is, obtain excretion body；
7) sample to be tested, above-mentioned PEG20000 aqueous solution and above-mentioned dextran sulfate 40000 aqueous solution are mixed, obtain
To mixed liquor, described PEG20000 concentration in described mixed liquor is made to be 150mg/ml, and described dextran sulfate 40000
Concentration in mixed liquor is 50mg/ml, more described mixed liquor is stood, and collects precipitation, that is, obtain excretion after standing
Body；
8) sample to be tested, above-mentioned PEG8000 saline solution are mixed, obtain mixed liquor, make described PEG8000 in institute
Stating in mixed liquor concentration is 150mg/ml, and concentration in mixed liquor for the described NaCl is 0.5M, then by described mixing
Liquid stands, that is, obtain excretion body；
9) sample to be tested, above-mentioned PEG20000 saline solution are mixed, obtain mixed liquor, so that described PEG20000 is existed
In described mixed liquor, concentration is 150mg/ml, and concentration in mixed liquor for the described NaCl is 0.5M, then will be described mixed
Close liquid standing, collect precipitation after standing, that is, obtain excretion body.
The condition of above-mentioned standing is 0-4 DEG C, standing 0.5-24h.
The experiment proves that, the present invention is with multiple using hydrophilic compound such as Polyethylene Glycol, glucosan, the poly- second of birdsing of the same feather flock together
Vinyl pyrrolidone (PVP) etc. all can be successfully separated excretion body, and PEG works in coordination with increasing with dextran sulfate with the use of having
The effect of effect, can improve, with hydrophilic polymer and inorganic salt cooperation, the yield separating excretion body.The method of the present invention is than existing
The RNA yield of the excretion body of some ultracentrifugation methods and the acquisition of SBI method is high, and is applied to the blood plasma in multiple sources,
As people source, mice source, rat source blood plasma；Be applied to the cell supernatant in multiple sources, such as 3T3, A549,293T,
Hela cell；It is applied to multiple body fluid, such as serum, urine, hydrothorax.The method is simple to operate, it is special to be not required to
Equipment.The present invention also provides a kind of test kit separating excretion body, and it can change excretion body in body fluid or cell culture fluid
In microenvironment so as to be easy in above-mentioned environment sedimentation；The excretion body high income obtaining；Be conducive to small volumes of liquids
Excretion health check-up in sample is surveyed；Avoid the injury to excretion body for the ultracentrifugation power simultaneously, separate the excretion body obtaining
Structural integrity, its inclusions are intact, suitably carry out separating further or the analysis of its inclusions of excretion body；Pass through
Control the use condition of the hydrophilic polymer of particular range, can farthest exclude the non-excretion body impurity in bottom phase,
Significantly improve the purity of excretion body, can effectively reduce the false positive rate in follow-up excretion body research.
Nucleic acid in available conventional nucleic acid or protein reagent box or method extraction separation excretion body or albumen, such as Trizol,
MiRNA extracts kit, DNA extraction kit etc..Agilent2200 Nucleic acid quality detecting system is automatically according to peak face
Long-pending conversion concentration.The materials such as appropriate glycogen or EDTA can be added in hydrophilic polymer solution to play raising excretion body receive
Rate and the effect of more preferable enriched nucleic acid, such as add the EDTA of final concentration of 0.1mM-20mM in PEG solution, and
/ or final concentration of 0.05mg/ml-5mg/ml glycogen.Tissue is all allowed through suitable enzymolysis processing, such as collagen
Enzyme, pancreatin, E.C. 3.4.21.64 etc..Standing can be carried out in room temperature (25 DEG C) or higher temperature, enters at low temperature such as 4 DEG C
During row, settling velocity can be faster and more abundant.Mixture time of repose can be any, within usual 24hr.Sample
Purification process can be done with reagent before mixing, way of purification can be for filtering, being centrifuged or sieve.And can be further to being derived from
The nucleic acid of excretion body, albumen, lipid etc. carry out separating.
Brief description
Fig. 1 is blood plasma excretion body and the various testing result of 3T3 cell conditioned medium excretion body.
Fig. 2 is to separate the blood plasma excretion body obtaining and the PEG20000 with 150mg/ml with the PEG8000 of 150mg/ml
Separate the rna content in the 3T3 cell conditioned medium excretion body obtaining.
Fig. 3 is that the PVP25000 of the PEG8000 and 150mg/ml of 50mg/ml coordinates the excretion body in separated plasma
Rna content and 150mg/ml PEG20000 separate outer in cell conditioned medium with 50mg/ml dextran sulfate 40000 cooperation
Secrete body rna content.
The rna content of the excretion body that Fig. 4 is obtained with PEG saline solution separated plasma.
Fig. 5 is the DNA content of the excretion body that PEG saline solution separated plasma obtains.
Fig. 6 is the rna content of the excretion body that PEG20000 saline solution separation cell conditioned medium obtains.
Fig. 7 is the DNA content of the excretion body that PEG20000 saline solution separation cell conditioned medium obtains.
Fig. 8 is to separate, from blood plasma, the excretion body precipitation size obtaining to compare.
Fig. 9 is the RNA comparing with supercentrifugation in separated plasma excretion body.
Figure 10 is the RNA comparing with SBI centrifuging in separated plasma excretion body.
Figure 11 is to compare the RNA separating in cell conditioned medium excretion body with supercentrifugation.
Figure 12 is to compare the RNA separating in cell conditioned medium excretion body with SBI centrifuging.
Figure 13 is excretion body rna content in rat plasma.
Figure 14 is excretion body rna content in 293T cell conditioned medium.
Figure 15 is excretion body rna content in hydrothorax.
Figure 16 is the excretion body rna content in liver tissues of rats.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
In following embodiments, hydrophilic polymer concentration unit is mass/volume, computing formula：Solute mass (g)/solution
Volume (mL).
Detect in following embodiments that redissolution volume during nucleic acid is 20ul if no special instructions.Embodiment center acid concentration
Detection the collection of illustrative plates of the larger data of quality is provided.
PEG6000(81255)、PEG8000(89510)、PEG10000(92897)、PEG20000(95172)
It is purchased from Sigma；PVP k-25 (molecular weight 25000, hereinafter referred to as PVP25000)；PVP k-30 (molecular weight 40000,
Hereinafter referred to as PVP40000) it is purchased from BASF；Dextran sulfate 50000 and dextran sulfate 40000 are purchased from
Amresco.3T3 (mouse embryo fibroblasts,CRL-2752TM), A549 (human lung adenocarcinoma cell,
CCL-185TM), 293T (people's renal epithelial cell, PTA-5554), Hela (human cervical carcinoma cell,CCL-2TM) cell line is ATCC product.NaCl (S7653) and EDTA (03609) is purchased from Sigma；Glycogen (Am9510)
Purchased from Life；Trizol is purchased from Invitrogen (15596-026), HiPure Circulating DNA Kits
Purchased from Magen (D3180-02).DMEM is purchased from GIBCO (C11995), Ribonuclease A and is purchased from Takara
(RNase A D202), Exo-FBSTMPurchased from SB (EXO-FBS-250A-1).CD63-Antibody-FITC is purchased from
BD (557288), CD81-Antibody-FITC are purchased from BD (551108).High Sensitivity RNA Reagents
Purchased from Agilent (5067-5580).Pancreatin is purchased from Thermofisher (15400054) and E.C. 3.4.21.64 is purchased from
YEASEN(10401ES60).Rat and mice all obtain from Guangdong Medical Lab Animal Center.
Embodiment 1, the molecular weight optimization of hydrophilic polymer used by separation excretion body
First, sample prepares
1st, blood plasma
Blood taking tube using EDTA anticoagulant gathers humanized's peripheral blood sample, stands 3 hours under the conditions of 4 DEG C, 4000 × g,
Centrifugation 10min, takes supernatant to be plasma sample.
2nd, cell supernatant
Using DMEM culture medium (including 10% FBS having removed excretion body), at 37 DEG C and 5%CO2Right under condition of culture
3T3 cell is cultivated, and after normally cultivating 2 days, collects (1000rpm is centrifuged 2 minutes) cell supernatant sample.
2nd, configure excretion body separating liquid
Using hydrophilic polymer PEG8000, PEG10000, PEG20000, dextran sulfate 50000, dextran sulfate
40000th, PVP25000 and PVP40000 be dissolved in water prepare excretion body separating liquid storing liquid as follows：
The PEG10000 of the PEG8000 aqueous solution, concentration 200mg/ml and 400mg/ml of concentration 200mg/ml and 400mg/ml
The PEG20000 aqueous solution of aqueous solution, concentration 200mg/ml and 400mg/ml, concentration 200mg/ml and 400mg/ml
Dextran sulfate 40000 aqueous solution of dextran sulfate 50000 aqueous solution, concentration 200mg/ml and 400mg/ml,
The PVP40000 of the PVP25000 aqueous solution, concentration 200mg/ml and 400mg/ml of concentration 200mg/ml and 400mg/ml
Aqueous solution.After weighing the hydrophilic polymer of above-mentioned various molecular weight of 20g and 40g, distinguished constant volume in 100ml water.
3rd, separate excretion body
1st, blood plasma separation excretion body
The detached humanized blood plasma 2ml that learns from else's experience is outer for 200mg/ml with the hydrophilic polymer concentration of above-mentioned two preparations respectively
Secrete body separating liquid mixing (i.e. blood plasma by volume：Separating liquid=3：1 ratio mixing), obtain mixed liquor, and make
The final concentration of each hydrophilic polymer is 50mg/ml.Turn upside down after mixing, put into 4 DEG C of refrigerator standing 0.5h.Quiet
Postpone, under the conditions of 4 DEG C, 12000 × g centrifugation 2min, removes supernatant, collects and is precipitated as blood plasma excretion body.
2nd, cell supernatant separation excretion body
The hydrophilic polymer concentration that 3T3 cell supernatant 20ml of collection is prepared with above-mentioned two respectively is taken to be 400mg/ml
Excretion body separating liquid mixing (i.e. blood plasma by volume：Separating liquid=3：1 ratio mixing), obtain mixed liquor,
And make the work final concentration of each hydrophilic polymer be 100mg/ml.Turn upside down after mixing, put into 4 DEG C of refrigerator standings
Overnight.Overnight take out afterwards, under the conditions of 4 DEG C, 1500 × g centrifugation 30min, takes out and outwell supernatant, collection is precipitated as
Cell excretion body.
4th, the detection of excretion body
1st, rna content detection
The blood plasma excretion body of above-mentioned three acquisitions and cell excretion body Trizol method are extracted RNA, using Agilent2200
Nucleic acid automatization electrophoresis system, carries out RNA concentration according to the instruction of High Sensitivity RNA detectable description
And quality testing, specific as follows：
The corresponding RNA sample of 1 μ L, 4 μ L sample buffer (totally 5 μ L) are taken to mix.The sample mixing
In PCR instrument, 72 DEG C of incubation 3min, place 2min on ice after incubation, of short duration centrifugation makes sample concentrate on 8
Bottom of the tube.8 unions are put in the sample cell of Agilent2200 nucleic acid automatization electrophoresis system, carries out automatization's electricity
Swimming.
The distribution of RNA fragment and concentration results are shown in Table 1-1 and Figure 1A-B, and Figure 1A is to be divided with the PEG8000 of 50mg/ml
From the rna content of the blood plasma excretion body obtaining, Figure 1B is to separate, with the PEG20000 of 100mg/ml, the 3T3 obtaining
The rna content of cell conditioned medium excretion body.Result shows：The parent of the 50mg/ml of the various molecular weight of this separation method (1)
Aqueous polymer all can isolate the excretion body in blood plasma；The hydrophilic polymer of the 100mg/ml of various molecular weight all can separate
Go out the excretion body in cell conditioned medium；(2) nucleic acid can be protected, can be to excretion body using conventional RNA extraction agent box
In RNA separated and be enriched with；(3) tiny RNA can be efficiently separated, separated RNA fragment is evenly distributed on
Between 25nt-6000nt, based on 25nt to 200nt；(4) with blood plasma as sample, different separation methods obtain
Excretion body RNA yield be followed successively by 50mg/ml PEG8000 be more than 50mg/ml PVP25000 be more than 50mg/ml
Dextran sulfate 40000, with cell conditioned medium as sample, what different separation methods obtained excretion body RNA yield according to
The dextran sulfate 40000 that the secondary PEG20000 for 100mg/ml is more than 100mg/ml is more than 100mg/ml's
PVP40000.
Table 1-1 is the optimization of molecular weight hydrophilic polymer
With the PEG8000 (separated plasma sample) of the working concentration 50mg/ml and PEG20000 of working concentration 100mg/ml
(separating cell conditioned medium 3T3 sample) does following separation detection tests.
2nd, take pictures detection
The PEG8000 of working concentration 50mg/ml is separated the blood plasma excretion body obtaining and working concentration 100mg/ml
PEG20000 separates the 3T3 cell conditioned medium excretion body obtaining and uses the water of 50ul resuspended respectively, is taken pictures with photographing unit, knot
Shown in fruit Fig. 1 C, show to obtain the excretion body precipitation from blood plasma and cell conditioned medium.
3rd, microscope inspection
The PEG8000 of working concentration 50mg/ml is separated the blood plasma excretion body obtaining and working concentration 100mg/ml
PEG20000 separate obtain 3T3 cell conditioned medium excretion body resuspended with 50ul water, then carry out 200 times dilution after, take
10ul Deca, on culture dish, is taken pictures under the microscope of 400 times of amplifications, relatively after different method for extracting
The density of the excretion body obtaining.Result as shown in figure ip, shows that the method for the present invention can separately obtain on blood plasma and cell
Excretion body in clear.
4th, RNase processes the impact detection that sample reclaims to excretion body nucleic acid
Plasma sample is divided into and processes blood plasma group (preparation method is ibid) and RNase process blood plasma group without RNase；
It is as follows that RNase processes blood plasma group processing method：Learn from else's experience separated plasma 2ml, using final concentration of 10ng/ml
The RNaseA of Takara is processed, 37 degree of water bath processing 1h, obtains RNase and processes blood plasma.
2ml RNase is processed blood plasma and 2ml and processes the blood plasma PEG8000 with concentration 200mg/ml respectively without RNase
Aqueous solution, obtains mixed liquor, makes in mixed liquor PEG8000 working concentration be 50mg/ml, separation method ibid,
Obtain RNase to process blood plasma excretion body and process blood plasma excretion body without RNase.
Use Trizol by the RNase process blood plasma excretion body of above-mentioned acquisition with without RNase process blood plasma excretion body respectively
Method extracts RNA, using Agilent2200 nucleic acid automatization electrophoresis system.The distribution of RNA fragment and concentration results are shown in Table
Shown in 1-2 and Fig. 1 E-F.
Fig. 1 E processes blood plasma excretion body rna content for RNase, and Fig. 1 F is to process blood plasma excretion body RNA without RNase
Content.
Table 1-2 RNase processes the impact to the excretion body RNA response rate
Result shows：This separation method (1) can isolate the humanized's blood processing through RNase or processing without RNase
Excretion body in slurry；(2) process through RNase or untreated excretion body in separated RNA in size distribution and
Notable difference is had no it was demonstrated that through extracting RNA actually from excretion body in concentration, rather than free RNA in blood plasma,
Also indicate that said method separate obtain be precipitated as excretion body.
5th, flow cytometer droplet measurement
The PEG8000 of working concentration 50mg/ml is separated the blood plasma excretion body obtaining and working concentration 100mg/ml
It is resuspended with 50ul water, using the Accuri C6 of BD company that PEG20000 separates the 3T3 cell conditioned medium excretion body obtaining
Flow cytometer, carries out granularmetric analyses, with normal Hela 3T3 cell aqueous suspensions, Hela 3T3 cell supernatant is
Comparison, according to Accuri C6 flow cytometer workbook instruction, carry out forward angle light scatter (forward scatter,
FSC) penetrate (side scatter, SSC) with scattered angle to analyze, count picture door inner cell number and its percentage ratio.Each
Test at least runs 10000 events.(1G is blood plasma to result such as Fig. 1 G-H, and 1H is supernatant；First is classified as grain
Footpath and granule complexity figure, second is classified as grain-size graph, and the 3rd is classified as granule complexity figure) shown in, not separated
3T3 cell excretion body in 8-12um, separated cell conditioned medium for the particle diameter in 40-100nm.Fig. 1 G shows
In detached excretion body suspension in blood plasma, excretion body is about 93.5%；Fig. 1 H shows excretion body in unsegregated cell conditioned medium
It is about 21.4%, the cell conditioned medium excretion body after separating is about 94.9%, show that this method can separate well, be enriched with outward
Secrete body.
6th, antibody labeling flow cytomery
The PEG8000 of working concentration 50mg/ml is separated the blood plasma excretion body obtaining and working concentration 100mg/ml
PEG20000 separate obtain 3T3 cell conditioned medium excretion body use BD company CD63-Antibody-FITC and
The antibody of two excretion body specific surface antigen of CD81-Antibody-FITC, the instruction according to operation instructions is to warp
Detached excretion body carries out antibody labeling.According to the instruction of Accuri C6 flow cytometer workbook, carry out FITC
The analysis of 488nm Air conduct measurement, using not separated cell conditioned medium as comparison, analyze the positive cell number of antibody
And its percentage ratio.Each test at least runs 10000 events.
(first is classified as particle diameter and granule complexity to result such as Fig. 1 I-J, and second is classified as CD63 antibody test, horizontal seat
It is designated as from left to right being followed successively by 101、102、103、104、105、106、107.2, the 3rd is classified as CD81 antibody test,
Abscissa is from left to right to be followed successively by 101、102、103、104、105、106、107.2；In Fig. 1 J, the first behavior separates
Front cell conditioned medium excretion body antibody test, the excretion body antibody test after the second behavior separation；Represent anti-on the right of vertical line
The positive is surveyed in health check-up, and the vertical line left side represents that antibody test is negative), Fig. 1 I is blood plasma excretion body streaming antibody test figure, figure
1J is 3T3 cell conditioned medium excretion body streaming antibody test figure it can be seen that the detached blood plasma of the present invention and cell conditioned medium
In excretion body all express two specific surface markers of CD63 and CD81, antibody staining is positive.Therefore,
Markers tests result shows that this method high efficiency separation has gone out the excretion body in blood plasma and cell conditioned medium.
Embodiment 2, the optimization of hydrophilic polymer concentration used by separation excretion body
First, sample prepares
1st, blood plasma：Same as Example 1；
2nd, cell supernatant：Identical with the method for embodiment 1, except for the difference that cell is A549；
2nd, configure excretion body separating liquid
Because embodiment 1 finds out hydrophilic polymer PEG8000, PEG20000, dextran sulfate 40000, PVP25000
It is the good excretion body separating liquid of effect with PVP40000, therefore, prepare excretion body separating liquid storing liquid with water dissolution as follows：
For blood plasma detached excretion body separating liquid：The PEG8000 aqueous solution of concentration 400mg/ml, concentration 400mg/ml
Dextran sulfate 40000, the PVP25000 of concentration 400mg/ml；
For cell supernatant detached excretion body separating liquid：The PEG20000 of concentration 400mg/ml, concentration 400mg/ml
Dextran sulfate 40000, the PVP40000 of concentration 400mg/ml.
3rd, separate excretion body
1st, blood plasma separation excretion body：Blood plasma is used for the mixing of blood plasma detached excretion body separating liquid from different respectively, obtains
To mixed liquor, the method according to the three of embodiment 1 separates, and obtains blood plasma separation excretion body；
Above-mentioned different as follows for blood plasma detached excretion body separating liquid and its working concentration：
Concentration 400mg/ml PEG8000 aqueous solution, concentration in mixed liquor for the PEG8000 be 50mg/ml, 150mg/ml,
200mg/ml；
Concentration 400mg/ml dextran sulfate 40000 aqueous solution, concentration in mixed liquor for the dextran sulfate 40000 is
50mg/ml、150mg/ml、200mg/ml；
Concentration 400mg/ml PVP25000 aqueous solution, concentration in mixed liquor for the PVP25000 be 50mg/ml, 150mg/ml,
200mg/ml.
2nd, cell supernatant separation excretion body：By cell supernatant respectively from different for the detached excretion of cell supernatant
Body separating liquid mixes, and obtains mixed liquor, and the method according to the three of embodiment 1 separates, and obtains cell excretion body；
Above-mentioned different as follows for cell supernatant detached excretion body separating liquid and its working concentration：
Concentration 400mg/ml PEG20000 aqueous solution, concentration in mixed liquor for the PEG20000 be 50mg/ml, 150mg/ml,
200mg/ml；
Concentration 400mg/ml dextran sulfate 40000 aqueous solution, concentration in mixed liquor for the dextran sulfate 40000 is
50mg/ml、150mg/ml、200mg/ml；
Concentration 400mg/ml PVP40000 aqueous solution, concentration in mixed liquor for the PVP40000 be 50mg/ml, 150mg/ml,
200mg/ml.
4th, the detection of excretion body
1st, micro measurement：Detection method is same as Example 1, and what result proved obtains excretion body.
2nd, rna content detection
The various blood plasma excretion bodies of above-mentioned three acquisitions and various cell excretion body Trizol method are extracted RNA, uses
Agilent2200 nucleic acid automatization electrophoresis system,
The distribution of RNA fragment and concentration results are shown in Table 2 and Fig. 2, and wherein, Fig. 2A is that the PEG8000 of 150mg/ml separates
The rna content of the blood plasma excretion body obtaining, Fig. 2 B is that the PEG20000 of 150mg/ml separates on the 3T3 cell obtaining
The rna content of clear excretion body, result shows：The hydrophilic polymer of the various concentration of this separation method (1) all can be isolated
Excretion body in serum and cell conditioned medium；(2) with blood plasma as sample, the excretion body RNA that different separation methods obtains
The PEG8000 that yield is followed successively by 150mg/ml is poly- more than the sulphuric acid Portugal that the PVP25000 of 200mg/ml is more than 50mg/ml
Sugar 40000；With cell conditioned medium as sample, the excretion body RNA yield that different separation methods obtains is followed successively by 150mg/ml
PEG20000 be more than 50mg/ml dextran sulfate 40000 be more than 150mg/ml PVP40000.
The optimization of table 2 hydrophilic polymer concentration
In above-mentioned table, PEG8000 is used for blood plasma detached excretion body, and it is detached that PEG20000 is used for cell supernatant
Excretion body；PVP25000 is used for blood plasma detached excretion body, and PVP40000 is used for cell supernatant detached excretion body,
Dextran sulfate 40000 is used for separating excretion body from blood plasma and cell conditioned medium.
Embodiment 3, hydrophilic polymer use cooperatively and separate excretion body
First, sample prepares
1st, blood plasma：Same as Example 1；
2nd, cell supernatant：Identical with the method for embodiment 1, except for the difference that cell is Hela；
2nd, configure excretion body separating liquid
Finding out the optimal hydrophilic polymer of separated plasma excretion body due to embodiment 2 is PEG8000 and PVP25000,
Separating the optimal hydrophilic polymer of cell supernatant excretion body is PEG20000 and dextran sulfate 40000, therefore, can
With by 2 kinds of hydrophilic polymer mixed preparing excretion body separating liquid storing liquids, as follows：
For blood plasma detached excretion body separating liquid：The PEG8000 aqueous solution of concentration 300mg/ml, concentration 300mg/ml
PVP25000；
For cell supernatant detached excretion body separating liquid：The PEG20000 of concentration 300mg/ml, concentration 300mg/ml
Dextran sulfate 40000.
Process plasma sample with PEG8000 and/or PVP25000；
Process cell conditioned medium sample with PEG20000 and/or dextran sulfate 40000.
3rd, separate excretion body
1st, blood plasma separation excretion body：Blood plasma is used for the mixing of blood plasma detached excretion body separating liquid from different respectively, obtains
To mixed liquor, the method according to the three of embodiment 1 separates, and obtains detached excretion body from blood plasma；
Above-mentioned different as follows for blood plasma detached excretion body separating liquid and its working concentration：
The PEG8000 aqueous solution of concentration 300mg/ml, concentration in mixed liquor for the PEG8000 is 50mg/ml；
The PVP25000 aqueous solution of concentration 300mg/ml, concentration in mixed liquor for the PVP25000 is 150mg/ml；
The PEG8000 aqueous solution of concentration 300mg/ml and the PVP25000 aqueous solution of concentration 300mg/ml, PEG8000
Concentration in mixed liquor is 50mg/ml, and concentration in mixed liquor for the PVP25000 is 150mg/ml.
2nd, cell supernatant separation excretion body：Blood plasma is separated for cell supernatant detached excretion body from different respectively
Liquid mixes, and obtains mixed liquor, and the method according to the three of embodiment 1 separates, and obtains cell supernatant separation excretion body；
Above-mentioned different as follows for cell supernatant detached excretion body separating liquid and its working concentration：
The PEG20000 aqueous solution of concentration 300mg/ml, concentration in mixed liquor for the PEG20000 is 150mg/ml；
Dextran sulfate 40000 aqueous solution of concentration 300mg/ml, concentration in mixed liquor for the dextran sulfate 40000
For 50mg/ml；
Dextran sulfate 40000 aqueous solution of the PEG20000 aqueous solution of concentration 300mg/ml and concentration 300mg/ml,
Concentration in mixed liquor for the PEG20000 is 150mg/ml, and concentration in mixed liquor for the dextran sulfate 40000 is
50mg/ml.
4th, the detection of excretion body
1st, micro measurement：Detection method is same as Example 1, and what result proved obtains excretion body.
2nd, rna content detection
The various blood plasma excretion bodies of above-mentioned three acquisitions and various cell excretion body Trizol method are extracted RNA, uses
Agilent2200 nucleic acid automatization electrophoresis system,
The distribution of RNA fragment and concentration results are shown in Table 3 and Fig. 3, and Fig. 3 A is PEG8000 and 150mg/ml of 50mg/ml
PVP25000 coordinate separated plasma in excretion body rna content, Fig. 3 B be 150mg/ml PEG20000 with
50mg/ml dextran sulfate 40000 cooperation separates the excretion body rna content in cell conditioned medium, by the concentration of RNA
Represent the quantity of excretion body, result shows：The excretion body separating liquid being used cooperatively using 2 kinds of hydrophilic polymers is separated,
Better (being embodied by the concentration of RNA) than being used alone hydrophilic polymer separation excretion body effect, wherein for blood plasma
Good excretion body separating liquid is PEG8000 and PVP25000 aqueous solution, and concentration during PEG8000 work is 50mg/ml,
Concentration during PVP25000 work is 150mg/ml, is PEG20000 for the optimal excretion body separating liquid of cell supernatant
With dextran sulfate 40000 aqueous solution, and PEG20000 work when concentration be 150mg/ml, dextran sulfate 40000
Concentration during work is 50mg/ml.
The using cooperatively of the hydrophilic coordination compound of table 3
Embodiment 4, hydrophilic polymer are used cooperatively with inorganic salt and separate excretion body
A, hydrophilic polymer and inorganic salt use cooperatively separated plasma excretion body
First, sample preparation
1st, blood plasma：Same as Example 1；
2nd, configure excretion body separating liquid storing liquid
Multiple salt concentration gradient PEG8000 saline solution：PEG8000, the NaCl of different content and water are mixed, obtains
The concentration of PEG8000 saline solution, wherein PEG8000 is that the concentration of 450mg/ml, NaCl is respectively 300mM, 1.5M
With 3M.
3rd, separate excretion body
2ml blood plasma is mixed with multiple salt concentration gradient PEG8000 saline solution respectively, obtains mixed liquor, make mixed liquor
The concentration of middle PEG8000 and NaCl is as follows respectively：
PEG8000 concentration is 150mg/ml, and NaCl concentration is 100mM；
PEG8000 concentration is 150mg/ml, and NaCl concentration is 0.5M；
PEG8000 concentration is 150mg/ml, and NaCl concentration is 1M；
Method according to the three of embodiment 1 separates, and obtains different salinity detached blood plasma excretion body.
By the 2ml blood plasma PEG8000 aqueous solution with 450mg/ml respectively, obtain mixed liquor, make in mixed liquor
The concentration of PEG8000 is 150mg/ml, and the method according to the three of embodiment 1 separates, and obtains being not added with what salt separation obtained
Blood plasma excretion body.
4th, the detection of excretion body
1st, micro measurement：Detection method is same as Example 1, and what result proved obtains excretion body.
2nd, rna content detection
The excretion body Trizol method obtaining that separates from blood plasma of above-mentioned three acquisitions is extracted RNA, uses
Agilent2200 nucleic acid automatization electrophoresis system.The distribution of RNA fragment and concentration results are shown in Table 4 and Fig. 4 (PEG8000
Concentration is 150mg/ml, and NaCl concentration is 0.5M), result shows：Salt ion in this separation method (1) separating liquid
Concentration is the excretion body that all can isolate during 0-2M in blood plasma；(2) during the final concentration of 1M of salt ion, the excretion obtaining
Body RNA concentration and yield highest.Therefore, the excretion body separating liquid being used cooperatively using hydrophilic polymer and inorganic salt is divided
From better (being embodied by the concentration of RNA) than being used alone hydrophilic polymer separation excretion body effect, outside separated plasma
Secreting body optimal separation liquid is PEG8000 saline solution, and PEG8000 working concentration is 150mg/ml, NaCl working concentration
For 0.5M.
The RNA response rate of excretion body in table 4 blood plasma
3rd, DNA content detection
PEG8000 saline solution (NaCl working concentration 0.5M) is separated the excretion body of the blood plasma obtaining, according to Magen
The instruction of HiPure Circulating DNA Kits description carry out the extracting of DNA, using Agilent2200 core
Sour automatization electrophoresis system carries out DNA detection.
DNA fragmentation distribution and concentration results are shown in Table 5, and (PEG8000 concentration is 150mg/ml, and NaCl concentration is with Fig. 5
0.5M), result shows：This separation method (1) can protect nucleic acid, can be right using conventional DNA extraction agent box
DNA in excretion body is separated and is enriched with；(2) DNA can be efficiently separated, separated DNA fragmentation distribution is divided equally
Cloth is in 100nt-1500nt.
The DNA response rate of excretion body in table 5 blood plasma
B, hydrophilic polymer are used cooperatively with inorganic salt and separate cell supernatant excretion body
First, sample preparation
1st, cell supernatant：Same as Example 1, only replace with 293T cell；
2nd, configure excretion body separating liquid
Prepare excretion body separating liquid storing liquid：
Multiple salt concentration gradient PEG20000 saline solution：PEG20000, different amounts of NaCl and water are mixed, obtains
The concentration of PEG20000 saline solution, wherein PEG20000 be 450mg/ml, NaCl concentration be 300mM, 1.5M with
3M.
3rd, separate excretion body
20ml 293T cell supernatant is mixed with multiple salt concentration gradient PEG20000 saline solution respectively, is mixed
Liquid, makes the concentration of PEG20000 and NaCl in mixed liquor as follows respectively：
PEG20000 concentration is 150mg/ml, and NaCl concentration is 100mM；
PEG20000 concentration is 150mg/ml, and NaCl concentration is 0.5M；
PEG20000 concentration is 150mg/ml, and NaCl concentration is 1M；
Method according to the three of embodiment 1 separates, and obtains different salinity detached cell supernatant excretion body.
By the 20ml cell supernatant PEG20000 aqueous solution with 450mg/ml respectively, obtain mixed liquor, make to mix
The concentration closing PEG20000 in liquid is 150mg/ml, obtains being not added with salt detached cell supernatant excretion body.
4th, the detection of excretion body
1st, micro measurement：Detection method is same as Example 1, and what result proved obtains excretion body
2nd, rna content detection
The cell supernatant excretion body Trizol method of above-mentioned three acquisitions is extracted RNA, using Agilent2200 nucleic acid certainly
Dynamicization electrophoresis system.
The distribution of RNA fragment and concentration results are shown in Table 6, and (PEG20000 concentration is 150mg/ml, and NaCl concentration is with Fig. 6
0.5M), result shows：In this separation method (1) separating liquid, salt ionic concentration is all to isolate blood plasma during 0-2M
In excretion body；(2) during the final concentration of 1M of salt ion, the excretion body RNA concentration obtaining and yield highest.Therefore,
The excretion body separating liquid being used cooperatively using hydrophilic polymer and inorganic salt is separated, and separates than being used alone hydrophilic polymer
Excretion body effect good (being embodied by the concentration of RNA), separating cell supernatant excretion body optimal separation liquid is PEG20000
Saline solution, and PEG20000 working concentration is 150mg/ml, NaCl working concentration is 0.5M.
The excretion body RNA response rate in table 6 cell conditioned medium
3rd, DNA content detection
By PEG20000 saline solution (NaCl working concentration 0.5M), from cell conditioned medium, detached excretion body extracts DNA,
Using Agilent2200 nucleic acid automatization electrophoresis system.
DNA fragmentation distribution and concentration results are shown in Table 7, and (PEG20000 concentration is 150mg/ml, NaCl concentration with Fig. 7
For 0.5M), result shows：This separation method (1) can protect nucleic acid, using conventional DNA extraction agent box is
DNA in excretion body can be separated and be enriched with；(2) DNA can be efficiently separated, separated DNA fragmentation distribution
It is distributed in 100nt-1500nt.
The DNA response rate of excretion body in table 7 cell conditioned medium
Embodiment 5, hydrophilic polymer use cooperatively, with inorganic salt, the application separating excretion body
The following examples are all used cooperatively with inorganic salt using hydrophilic polymer and separate excretion body：
A, compare hydrophilic polymer and inorganic salt and use cooperatively (Ribo), ultracentrifugation method and SBI method and separate blood
Slurry excretion body
First, sample preparation
1st, blood plasma：Same as Example 1；
2nd, configure excretion body separating liquid
Prepare excretion body separating liquid storing liquid：
PEG8000 saline solution：PEG8000, NaCl and water are mixed, obtains PEG8000 saline solution, wherein PEG8000
Concentration be 450mg/ml, NaCl concentration be 1.5M.
3rd, separate excretion body
Using following three kinds of method separated plasma excretion bodies：
1st, hydrophilic polymer of the present invention is used cooperatively with inorganic salt and separates (abbreviation Ribo method)
2ml blood plasma is mixed with PEG8000 saline solution, obtains mixed liquor, make PEG8000 working concentration in mixed liquor
For 150mg/ml, NaCl working concentration is 0.5M, and the method according to the three of embodiment 1 separates, and obtains hydrophilic polymeric
Thing uses cooperatively the detached excretion body from blood plasma with inorganic salt.
2nd, SBI method
2ml blood plasma is used the Exosome extraction agent ExoQuick serum exosome precipitation of SBI
Solution and ExoQuick TC is operated, and obtains SBI method detached blood plasma excretion body.
4th, the detection of excretion body
1st, take pictures detection
Method in above-mentioned three 1 and 2 separation is obtained excretion body precipitation uses the water of 50ul resuspended respectively, is taken pictures with photographing unit,
The amount of the excretion body relatively obtaining after different method for extracting.
Result is as shown in Figure 8 it can be seen that the method for the present invention is bigger than SBI reagent separates the excretion body precipitation obtaining
Little big, that is, its excretion body yield is higher.
2nd, rna content detection
The excretion body Trizol method that above-mentioned three various separation methods are obtained extracts RNA, using Agilent2200 nucleic acid
Automatization's electrophoresis system.
The distribution of RNA fragment and concentration results are as follows：
(1) compare with ultracentrifugation method
The distribution of RNA fragment and concentration results are shown in Table 8 and Fig. 9 (Ribo), and result shows：This method energy high efficiente callback blood
RNA in slurry excretion body, its response rate is more than 9.5 times of conventional ultracentrifugation method.
In table 8 blood plasma, the Ribo method separation excretion body RNA response rate is high
(2) compare with SBI method
The distribution of RNA fragment and concentration results are shown in Table 9 and Figure 10 (Ribo), and result shows：This method energy high efficiente callback
RNA in blood plasma excretion body, its response rate is 2.4 times of SBI method.
In table 9 blood plasma, the Ribo method separation excretion body RNA response rate is high
Therefore, using the method for the present invention, better than SBI method separation excretion body effect (being embodied by the concentration of RNA).
3rd, Small RNA high-flux sequence detection
The excretion body that above-mentioned various separation methods are obtained extracts RNA, using Illumina companySmall
RNA Sample Prep Kit test kit carries out the library construction of small RNA, mainly comprises the following steps 3'adaptor even
Connect；5'adaptor connects；First chain cDNA synthesis；PCR expands；Clip size selects, and with Agilent 2200
TapeStation carries out library quality inspection.By the library of quality inspection, mixed according to pooling ratio, pooling
Concentration afterwards is 2.1nM；Using Illumina'sRapid SBS Kit-HS (50Cycles) and
Rapid SR Cluster Kit reagent, the instruction according to HiSeq 2500User Guide is operated the computer, and
Operation standard sequencing program；Sequencing program is run and is finished, and carries out bioinformatic analysis to the data obtained.
If table 10 is with shown in table 11, the inventive method (1) separates the miRNA of excretion body in the blood plasma obtaining to result
Number is approximately 1.54 times of SBI method；(2) ratio in tiny RNA for the miRNA of excretion body in the blood plasma that separation obtains
1.67 times of about SBI method of example ratio.
The number of miRNA is detected in table 10 blood plasma excretion body
SBI-1 and SBI-2 in above-mentioned table 10 is 2 repetitions, Ribo-1 and Ribo-2 in above-mentioned table 10 is 2
Secondary repetition.
Tiny RNA distribution in table 11 blood plasma
SBI-1 and SBI-2 in above-mentioned table 11 is 2 repetitions, Ribo-1 and Ribo-2 in above-mentioned table 10 is
2 repetitions.
B, compare that aqueous polymer and inorganic salt use cooperatively (Ribo) and SBI method separates excretion body in cell conditioned medium
First, sample preparation
Cell supernatant：Same as Example 1, different is only that cell is changed to Hela cell.
2nd, configure excretion body separating liquid
Prepare excretion body separating liquid storing liquid：
PEG20000 saline solution：PEG20000, NaCl and water are mixed, obtains PEG20000 saline solution, wherein
The concentration of PEG20000 is the concentration of 450mg/ml, NaCl is 1.5M.
3rd, separate excretion body
Adopt separated plasma excretion body with the following method：
1st, hydrophilic polymer of the present invention is used cooperatively with inorganic salt and separates (abbreviation Ribo method)
20ml Hela cell supernatant is mixed with PEG20000 saline solution, obtains mixed liquor, make in mixed liquor
PEG20000 working concentration is 150mg/ml, and NaCl working concentration is 0.5M, and the method according to the three of embodiment 1 is divided
From obtaining hydrophilic polymer and inorganic salt and use cooperatively detached cell supernatant excretion body.
2nd, ultracentrifugation method：
20ml Hela cell supernatant is carried out centrifugally operated (Thery et al., Cum with reference to ultracentrifugal method
Protoc.Cell.Biol.,Chapter 3,Unit 3:22 (2006)), obtain ultracentrifugation detached thin
Born of the same parents' supernatant excretion body.
3rd, SBI method
20ml Hela cell supernatant is used the Exosome extraction agent ExoQuick serum exosome of SBI
Precipitation solution and ExoQuick TC is operated, and obtains outside the detached cell supernatant of SBI method
Secrete body.
4th, the detection of excretion body
Rna content detects
The excretion body Trizol method that above-mentioned three various separation methods are obtained extracts RNA, using Agilent2200 nucleic acid
Automatization's electrophoresis system.
The distribution of RNA fragment and concentration results are as follows：
(1) compare with ultracentrifugation method
The distribution of RNA fragment and concentration results are shown in Table 12 and Figure 11 (Ribo), and result shows：This method energy high efficiente callback
RNA in cell conditioned medium excretion body, its response rate is more than 2.1 times of conventional ultracentrifugation method.
In table 12 cell conditioned medium, the Ribo method separation excretion body RNA response rate is high
(2) compare with SBI method
The distribution of RNA fragment and concentration results are shown in Figure 12 (Ribo) and table 13, and result shows：This method energy high efficiente callback
RNA in cell conditioned medium excretion body, its response rate is more than 1.7 times of SBI method.
In table 13 cell conditioned medium, the Ribo method separation excretion body RNA response rate is high
The nucleic acid of excretion body in C, separation separate sources blood plasma
First, sample preparation
Blood plasma：Blood taking tube using EDTA anticoagulant gathers humanized's peripheral blood sample, big mouse peripheral blood sample and little
Mouse peripheral blood sample, stands 3 hours under the conditions of 4 DEG C, and 4000 × g is centrifuged 10min, takes supernatant to be people
Source blood plasma, rat plasma and mice plasma.
2nd, configure excretion body separating liquid
Prepare excretion body separating liquid storing liquid：
PEG8000 saline solution：PEG8000, NaCl and water are mixed, obtains PEG8000 saline solution, wherein PEG8000
Concentration be 450mg/ml, NaCl concentration be 1.5M.
3rd, separate excretion body
The blood plasma of 2ml separate sources is mixed with PEG8000 saline solution, obtains mixed liquor, make PEG8000 in mixed liquor
Working concentration is 150mg/ml, and NaCl working concentration is 0.5M, and the method according to the three of embodiment 1 separates, and obtains
Human plasma excretion body, rat plasma excretion body and mice plasma excretion body.
4th, the detection of excretion body
Rna content detects
The human plasma excretion body of above-mentioned three acquisitions, rat plasma excretion body and mice plasma excretion body are used Trizol respectively
Method extracts RNA, using Agilent2200 nucleic acid automatization electrophoresis system.
The distribution of RNA fragment and concentration results are shown in Table 14 and Figure 13 (Ribo), and result shows：This separation method (1)
The excretion body in people, rat and little mouse blood plasma all can be isolated；(2) people, rat separate with little mouse blood plasma
Fragment distribution and RNA concentration be slightly different.
The RNA response rate of excretion body in table 14 separate sources blood plasma
D, separation separate sources cell conditioned medium excretion body
First, sample preparation
Cell supernatant：Same as Example 1, different is only cell is 3T3, A549,293T, Hela.
2nd, configure excretion body separating liquid
Prepare excretion body separating liquid storing liquid：
PEG20000 saline solution：PEG20000, NaCl and water are mixed, obtains PEG20000 saline solution, wherein
The concentration of PEG20000 is the concentration of 450mg/ml, NaCl is 1.5M.
3rd, separate excretion body
Respectively by 20ml 3T3 cell supernatant, A549 cell supernatant, 293T cell supernatant, Hela cell
Clear liquid is mixed with PEG20000 saline solution, obtains mixed liquor, makes PEG20000 working concentration in mixed liquor be 150mg/ml,
NaCl working concentration be 0.5M, according to embodiment 1 three method separate, obtain 3T3 cell supernatant excretion body,
A549 cell supernatant excretion body, 293T cell supernatant excretion body, Hela cell excretion body.
4th, the detection of excretion body
Rna content detects
By above-mentioned 3T3 cell supernatant excretion body, A549 cell supernatant excretion body, 293T cell supernatant excretion body,
Hela cell excretion body extracts RNA with Trizol method, using Agilent2200 nucleic acid automatization electrophoresis system.
The distribution of RNA fragment and concentration results are shown in Table 15 and Figure 14 (293T cell), and result shows：This separation method
(1) the excretion body in 3T3, A549,293T and Hela cell supernatant can all be isolated；(2)3T3、A549、
The fragment distribution of 293T and Hela cell supernatant detached excretion body RNA and RNA concentration are slightly different.
Table 15 is the RNA response rate of excretion body in separate sources cell conditioned medium
E, the multiple body fluid excretion bodies of separation
First, sample preparation
1st, serum separates：Gather humanized's peripheral blood sample using the blood taking tube being not added with anticoagulant, stand under the conditions of 4 DEG C
4 hours it is seen that clot separates out.4000 × g, centrifugation 10min, it is seen that faint yellow serum, take supernatant to be also blood
Clearly.2000 × g centrifugation 10min can be reused, at utmost to ensure Serology Quality after taking out supernatant.
2nd, urine separation：Collect humanized's urine, 4000 × g, be centrifuged 15min, remove cell and precipitated impurities,
Take supernatant, obtain urine supernatant.
3rd, hydrothorax separates：Collect humanized hydrothorax, 4000 × g, be centrifuged 15min, remove cell and
Precipitated impurities, take supernatant, obtain hydrothorax supernatant.
2nd, configure excretion body separating liquid
Prepare excretion body separating liquid storing liquid：
PEG8000 saline solution：PEG8000, NaCl and water are mixed, obtains PEG8000 saline solution, wherein PEG8000
Concentration be 450mg/ml, NaCl concentration be 1.5M.
PEG20000 saline solution：PEG20000, NaCl and water are mixed, obtains PEG20000 saline solution, wherein
The concentration of PEG20000 is the concentration of 450mg/ml, NaCl is 1.5M.
PEG8000 saline solution separation serum.
PEG20000 saline solution separated urine supernatant and hydrothorax supernatant.
3rd, separate excretion body
1st, 2ml serum is mixed with PEG8000 saline solution, obtain mixed liquor, make PEG8000 work in mixed liquor dense
Spend for 150mg/ml, NaCl working concentration is 0.5M, the method according to the three of embodiment 1 separates, and obtains outside serum
Secrete body.
2nd, 20ml urine supernatant is mixed with PEG20000 saline solution, obtain mixed liquor, make PEG20000 in mixed liquor
Working concentration is 150mg/ml, and NaCl working concentration is 0.5M, and the method according to the three of embodiment 1 separates, and obtains
Urine supernatant excretion body.
3rd, 20ml hydrothorax supernatant is mixed with PEG20000 saline solution, obtain mixed liquor, make in mixed liquor
PEG20000 working concentration is 150mg/ml, and NaCl working concentration is 0.5M, and the method according to the three of embodiment 1 is divided
From obtaining hydrothorax supernatant excretion body.
4th, the detection of excretion body
Rna content detects
The serum excretion body of above-mentioned three acquisitions, urine supernatant excretion body and hydrothorax supernatant excretion body are used respectively
Trizol method extracts RNA, using Agilent2200 nucleic acid automatization electrophoresis system.
The distribution of RNA fragment and concentration results are shown in Table 16 and Figure 15 (hydrothorax), and result shows：This separation method
(1) people source serum, urine, the excretion body in hydrothorax can all be isolated；(2) body fluid of separate sources separates
Excretion body RNA concentration be slightly different.
The RNA response rate of excretion body in table 16 separate sources body fluid
The RNA of excretion body in F chorista sample
First, sample preparation
Rat and mouse liver are as the tissue samples in Mus source.
2nd, configure excretion body separating liquid
Prepare excretion body separating liquid storing liquid：
PEG20000 saline solution：PEG20000, NaCl and water are mixed, obtains PEG20000 saline solution, wherein
The concentration of PEG20000 is the concentration of 450mg/ml, NaCl is 1.5M.
3rd, separate excretion body
20mg liver tissues of rats and murine liver tissue are shredded after homogenate, respectively with 0.35% pancreatin, protease
37 degree of K (20mg/ml) digests 0.5 hour, takes supernatant through 1200rpm centrifugation 2min after digestion, then with PEG20000
Saline solution mixes, and obtains mixed liquor, makes the final concentration of 150mg/ml of PEG20000, the final concentration of 0.5M of NaCl；
Method according to the three of embodiment 1 separates, and obtains tissue-derived excretion body.
4th, excretion body rna content detection
The tissue samples excretion body of above-mentioned three acquisitions is extracted RNA with Trizol method, using Agilent2200 nucleic acid certainly
Dynamicization electrophoresis system.The distribution of RNA fragment and concentration results are shown in Table 17 and Figure 16 (rats'liver), and result shows：This point
Excretion body from method energy chorista sample.
The rna content of excretion body in table 17 tissue samples
Claims (10)
1. a kind of reagent separating excretion body in body fluid or cell supernatant or tissue samples, is hydrophilic
Polymer or hydrophilic polymer solution.
2. reagent according to claim 1 it is characterised in that：Described hydrophilic polymer be as
At least one of lower 3 kinds：PEG, dextran sulfate and PVP.
3. reagent according to claim 1 or claim 2 it is characterised in that：Described hydrophilic polymer is molten
Liquid is aqueous solution, saline solution or organic solution；
In described hydrophilic polymer solution, described hydrophilic polymer concentration is 10mg/ml-300mg/ml.
4. according to claim 2 reagent it is characterised in that：The molecular weight of described PEG is
6000-20000 dalton, the molecular weight of described dextran sulfate is 30000-50000 dalton, institute
The molecular weight stating PVP is 20000-40000 dalton；
Or the molecular weight of described PEG is specially 8000 or 20000 dalton, described dextran sulfate
Molecular weight be specially 40000 dalton, the molecular weight of described PVP be specially 25000 dalton or
40000 dalton.
5. according to described reagent arbitrary in claim 1-4 it is characterised in that：Described reagent be as
Lower 1）-3）In any one：
1）Hydrophilic polymer group or hydrophilic polymer aqueous solution group；Described hydrophilic polymer group or described
Hydrophilic polymer in hydrophilic polymer aqueous solution group is following at least 2 kinds：PEG8000、PVP25000、
PEG20000 and dextran sulfate 40000；
2）Hydrophilic polymer saline solution, described hydrophilic polymer saline solution is made up of solute and solvent,
Described solute is made up of hydrophilic polymer and inorganic salt；Described hydrophilic polymer be PEG8000 or
PEG20000；
3）Hydrophilic polymer or its aqueous solution, described hydrophilic polymer be PEG8000, PEG20000,
Dextran sulfate 40000, PVP25000 or PVP40000.
6. reagent according to claim 5 it is characterised in that：
1）Described hydrophilic polymer group or hydrophilic polymer aqueous solution group are as follows：
PEG8000 and PVP25000；
Or PEG8000 aqueous solution and PVP25000 aqueous solution；
Or PEG20000 and dextran sulfate 40000；
Or PEG20000 aqueous solution and dextran sulfate 40000 aqueous solution；
2）Described hydrophilic polymer saline solution is PEG8000 saline solution or PEG20000 saline solution；
The solute of described PEG8000 saline solution is made up of PEG8000 and NaCl；
The solute of described PEG20000 saline solution is made up of PEG20000 and NaCl.
7. according to described reagent arbitrary in claim 1-6 it is characterised in that：Described body fluid is blood
Slurry or serum or urine or hydrothorax or spinal fluid or Cerebrospinal fluid or saliva or emulsion or joint fluid or
Seminal fluid or vaginal secretion or amniotic fluid, described cell supernatant is derived from tumor cell or inflammatory cell or essence is thin
Born of the same parents or Interstitial cell or epithelial cell or fibrocyte or fibroblast, described be organized as liver or lung or
Brain.
8. a kind of test kit separating excretion body in body fluid or cell supernatant or tissue samples, including
Arbitrary described reagent in claim 1-7；
Or arbitrary described reagent or described test kit are separating body fluid or cell in claim 1-7
Application in excretion body in supernatant or tissue samples；
Or in claim 1-7 arbitrary described reagent or described test kit in preparative separation body fluid or
Application in cell supernatant or tissue samples excretion body；
Described body fluid be specially blood plasma or serum or urine or hydrothorax or spinal fluid or Cerebrospinal fluid or
Saliva or emulsion or joint fluid or seminal fluid or vaginal secretion or amniotic fluid, concrete being derived from of described cell supernatant is swollen
Oncocyte or inflammatory cell or parenchyma or Interstitial cell or epithelial cell or fibrocyte or one-tenth fibre
Dimension cell, described tissue is specially liver or lung or brain.
9. a kind of method separating body fluid or cell supernatant or tissue samples excretion body, including as follows
Step：By body fluid or cell supernatant or tissue samples any one described examination with claim 1-8 respectively
Agent mixes, and obtains mixed liquor, makes the hydrophilic polymer in described reagent reach work in the concentration of mixed liquor
Make concentration, more described mixed liquor is stood, collect precipitation after standing, that is, obtain excretion body.
10. method according to claim 9 it is characterised in that：
Using described reagent and described reagent in hydrophilic polymer working concentration as follows：
Described PEG8000 aqueous solution and the mixed liquor of described PVP25000 aqueous solution；Described PEG8000
Concentration during work is 50mg/ml, and concentration during described PVP25000 work is 150mg/ml；
Or the mixed liquor of described PEG20000 aqueous solution and described dextran sulfate 40000 aqueous solution；
Concentration during described PEG20000 work is 150mg/ml, and described dextran sulfate 40000 works
When concentration be 50mg/ml；
Or described PEG8000 saline solution, concentration during described PEG8000 work is 150mg/ml, and
Concentration during described NaCl work is 0.5M；
Or described PEG20000 saline solution, concentration during described PEG20000 work is 150mg/ml,
And concentration during described NaCl work is 0.5M；
Or described PEG8000 aqueous solution, concentration during described PEG8000 work is 150mg/ml；
Or described PEG20000 aqueous solution, concentration during described PEG20000 work is 150mg/ml；
Or described dextran sulfate 40000 aqueous solution, dense during described dextran sulfate 40000 work
Spend for 50mg/ml；
Or described PVP25000 aqueous solution, concentration during described PVP25000 work is 200mg/ml；
Or described PVP40000 aqueous solution, concentration during described PVP40000 work is 150mg/ml；
Described body fluid be specially blood plasma or serum or urine or hydrothorax or spinal fluid or Cerebrospinal fluid or
Saliva or emulsion or joint fluid or seminal fluid or vaginal secretion or amniotic fluid, the concrete tumor of described cell supernatant is thin
Born of the same parents or inflammatory cell or parenchyma or Interstitial cell or epithelial cell or fibrocyte or one-tenth fiber finer
Born of the same parents, described tissue is specially liver or lung or brain.
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