CN109913417A - A method of using different cell origin excretion body hypotypes in tachysynthesis paramagnetic particle method separation cerebrospinal fluid - Google Patents
A method of using different cell origin excretion body hypotypes in tachysynthesis paramagnetic particle method separation cerebrospinal fluid Download PDFInfo
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Abstract
The present invention relates to a kind of methods using different cell origin excretion body hypotypes in tachysynthesis paramagnetic particle method separation cerebrospinal fluid, and specific steps include: that (1) pre-processes cerebrospinal fluid；(2) primary antibody that PE label is added is sorted；(3) immunomagnetic beads are added；(4) increase magnetic field, separated.Compared with prior art, the present invention precisely separates the excretion body subgroup of central nervous system difference cell origin, for targetedly to Central Nervous System Diseases and different target cell excretion body further investigations.
Description
Technical field
The present invention relates to a kind of isolation and purification methods, separate cerebrospinal fluid using tachysynthesis paramagnetic particle method more particularly, to a kind of
The method of middle difference cell origin excretion body hypotype.
Background technique
Immunomagnetic beads excretion body is can be with the specific monoclonal antibody phase that is connected with magnetic bead based on excretion body surface antigen
In conjunction in externally-applied magnetic field, the excretion body being connected by antibody with magnetic bead, which is adsorbed, to be trapped in magnetic field, and no this kind of surface is anti-
Former excretion body due to that cannot not be stopped in magnetic field in conjunction with the specific monoclonal antibody for being connected to magnetic bead without magnetism, thus
The excretion body hypotype for expressing special marker is set to separate.
Excretion body is a kind of by cell active secretion to extracellular vesica corpusculum, and diameter 30-100nm carries source
The multiple protein and RNA ingredient of cell can be used as the medium of cell-tocell exchange and substance transmitting.Neuron is to constitute nerve
The basic unit of system structure and function, astroglia are that maincenter quantity is most, undertake important metabolism and support function
Cell, microglia is the most important immunocyte of maincenter, can by it is a variety of stimulation and morbid state activate.Neuron, star
Shape spongiocyte, microglia are three kinds of main cells of cental system, can produce excretion body, be released into cerebrospinal fluid or
Blood.Excretion body is biologically active microcapsule bubble, can be absorbed by peripheral cell, carries out intercellular substance transmitting and information is handed over
Stream.Recent study finds that excretion body participates in the process of Central Nervous System Diseases, such as intracerebral inflammation in a variety of Central Nervous System Diseases pathogenic processes
Property disease, the excretion body largely discharged after microglial activation participates in progression of disease；In neurodegenerative disease, such as A Er
Zi Haimo disease, the interior excretion body containing the polymer for participating in paraprotein a- β of neuronal cell release, and participate in the propagation of a- β.
Therefore, the expression secretion situation of the excretion body of different cell origins can be used as the foundation that Central Nervous System Diseases are assessed in cerebrospinal fluid.Currently,
Cerebrospinal fluid excretion body, which separates, mainly supercentrifugation, Percoll gradient centrifugation, ultrafiltration, chromatography, and macromolecule polyalcohol is heavy
Drop method, immunomagnetic beads RNA isolation kit etc..Supercentrifugation requires sample volume big (mL-L rank), although to excretion body yield compared with
Height, but take time and effort (more than 2 hours).Common excretion body separation agent principle is the macromolecule polyalcohol precipitation method on the market,
It is short to extract excretion body time-consuming, but the disadvantage is that product excretion body purity is low, adulterates scrambled proteins and salt.Ultrafiltration can only be by diameter
Vesica less than filter sizes is separated, and it is impure to also result in product.Importantly, techniques described above can only separate
Total excretion body in cerebrospinal fluid, can not excretion body (such as the CD11b+ excretion body in microglia source) to specific subgroup
Separation also just cannot achieve the purpose using specific cells source excretion body subgroup reflection disease degree certainly.In accurate medicine
Today of continuous development, carrying out accurate parting to excretion body according to different cell origins becomes problem in the urgent need to address, this
One requirement and excretion body fluid body biopsy exploitation progress and the first step applied to clinic.Immunomagnetic ca pture can be to specific
The excretion body for expressing certain marker protein is combined, and excretion body hypotype and cell origin can be accurately distinguished after eluting.
But this technology is still immature at present, sorts magnetic bead currently without the excretion body hypotype that may be directly applied to cerebrospinal fluid.Hair
Bright content in view of the drawbacks of the prior art, and it is an object of the present invention to provide it is a kind of it is easy to operate quickly and cost is relatively low, can precisely separate
The main three kinds of cell origins of pivot (specific expressed microglia CD11b+, neuron Neun, astroglia GFAP)
Excretion body separation method.
Summary of the invention
Tachysynthesis is used it is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of
The method that paramagnetic particle method separates different cell origin excretion body hypotypes in cerebrospinal fluid.
The purpose of the present invention can be achieved through the following technical solutions:
A method of using different cell origin excretion body hypotypes in tachysynthesis paramagnetic particle method separation cerebrospinal fluid, including with
Lower step:
(1) cerebrospinal fluid is centrifugated, the impurity such as removal cell fragment take supernatant that excretion body sedimentation extraction agent is added
Afterwards, it mixes, is incubated overnight, after supernatant is removed in centrifugation, be resuspended, obtain CSF sample；
(2) primary antibody of PE label is added in the CSF sample that step (1) obtains, after incubation, excretion body sorting is added
Reagent, blending incubation obtain cerebrospinal fluid marker samples；
(3) the coated magnetic bead concussion of PE is mixed, the cerebrospinal fluid marker samples that step (2) obtain is added, are incubated for after mixing,
Obtain mixing sample；
(4) mixing sample is washed, blown and beaten, be placed in magnetic field, supernatant is discarded after overturning；
(5) after repeating step (4), by washing of precipitate, it is added excretion body dissociation solution, blending incubation, be washed out, blow and beat,
It is placed in magnetic field, supernatant, as the excretion body of particular source is collected after overturning.
Preferably, in step (1): the volume ratio of cerebrospinal fluid and excretion body sedimentation extraction agent is 5:1.
Preferably, in step (1): being resuspended and use PBS or distilled water, final volume is 250 μ L-1mL.
Preferably, in step (2): the primary antibody of PE label is CD11b, Neun or GFAP primary antibody of PE label.
Preferably, in step (2): after the primary antibody of PE label is added, the concentration of sample is 0.2-2 μ g/mL.
Preferably, in step (2): 25-100 μ L excretion body sorting reagent is added in every milliliter of CSF sample.
Preferably, in step (3): 100 μ L magnetic beads are added in every milliliter of cerebrospinal fluid marker samples.
Preferably, in step (4): cleaning solution ingredient is D-PBS (Du Shi phosphate buffer)+0.2% without excretion body blood
+ 1mM EDTA (ethylenediamine tetra-acetic acid) clearly.
Preferably, in step (4): sample is put into magnetic frame, magnet is picked up, it is whole to overturn, outwell supernatant.
Preferably, in step (5): volume >=100 μ L of excretion body dissociation solution are added in every milliliter of sample.
Divide the analysis of variance for human-body biological sample (urine, blood, saliva, ascites, Pleural effusions etc.) excretion body at present,
The cell origin of excretion body can not be all accurately distinguished, so the mixing sample of a variety of source excretion bodies can only be analyzed.This mixing
Sample is unable to satisfy the demand of accurate medicine, and it is even more impossible to undertake different function, play different role from being conceived to different cells
Angle is accurately studied.The present invention provides a kind of method of feasible, fast and convenient different cell origin excretion bodies separation.
The present invention is potential utility for diagnostic purposes, needs to test clinical sample, separation method of the invention can push away
The cell origin of the wide excretion body into various body fluid (urine, blood, saliva, ascites, Pleural effusions etc.) and cell culture fluid is sub-
Group's separation.The present invention operates combination cell specific antibody the outer of cell origins different in body fluid by simple conventional centrifugal
It secretes body to separate from the total excretion body mixed, and keeps biological activity.Potentially application is exactly outside to our technology
Body fluid body biopsy is secreted, finds the biomarker of extraordinary cell origin excretion body, thus for predicted treatment reaction and progression of disease
Foundation is provided.
The present invention only needs to combine the existing antibody applied to cell level by common centrifugal condition every time, so that it may
Complete the separation process of different cell origin excretion bodies.It is not necessarily to ultracentrifuge in separation process, can extract simultaneously every time more
A sample greatly simplifies operating instruction, and save the cost, the body fluid biopsy of the accurate parting and exploitation excretion body that make excretion body is significantly
It is conveniently possibly realized, the excretion body of extraction keeps itself biological activity.
Compared with prior art, the invention has the following advantages:
1, the separation method of quick and convenient is provided, the excretion body for precisely separating central nervous system difference cell origin is sub-
Group, for targetedly to Central Nervous System Diseases and different target cell excretion body further investigations；
2, the excretion body purity is high obtained, is embodied in that entrained marker is single, impurity vesica is few, is conducive to studying
The better result of middle acquisition evidence rank.
Detailed description of the invention
Fig. 1 is that Western Blot detects excretion body specific marker object Alix and Tsg101；
Fig. 2 is the common transmissive Electronic Speculum effect that tachysynthesis paramagnetic particle method separates cerebrospinal fluid No microglial source excretion body
Figure；
Fig. 3 is the nanometer streaming inspection that tachysynthesis paramagnetic particle method separates cerebrospinal fluid No microglial source CD11b+ excretion body
Survey result.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
Key instrument and reagent used in embodiment: excretion body settles extraction agent (EXOTC50A-1, SBI system
Biosciences, the U.S.)；PE-anti-CD11b antibody (eBioscience, 4290075)；The coated magnetic bead sorting reagent of PE
Box (#17654, EasySep, the U.S.)；Without excretion body serum (EXOFBS50A-1, ExoPerfect, China)；Electron microscope
(phillip CM120, Japan)；Magnetic frame (EasySep, the U.S.)；Centrifuge tube (corning, the U.S.).
Tachysynthesis paramagnetic particle method separates cerebrospinal fluid No microglial source (CD11b+) excretion body subgroup, and specific steps are such as
Under:
(1) -80 DEG C of cerebrospinal fluid frozen are taken out, melts on ice, draws 2 milliliters of cerebrospinal fluid, are placed in the centrifugation of 4 DEG C of pre-coolings
Machine was centrifugated through 3000g*15 minutes, the impurity such as removal cell fragment.
(2) supernatant is transferred in 15 milliliters of new centrifuge tubes, and 400 μ L sedimenting reagents (SBIExoquick-TC) are added.
(3) it turns upside down and mixes 4-6 times, be placed in 4 DEG C of refrigerator overnight incubations.
(4) centrifuge tube is taken out, is centrifuged 1500g*30 minutes under the conditions of 4 DEG C, pipette tips carefully suck supernatant, but do not touch bottom
Precipitating.
(5) 4 DEG C of 1500g*5 minute centrifugations again, pipette tips, which are drawn, carefully thoroughly removes supernatant.
(6) 250 μ L PBS are added in centrifuge tube, pipette tips piping and druming is resuspended.
(7) the excretion body after resuspension is placed in 5 milliliters of Liu Shi pipes.
(8) the CD11b primary antibody of PE label is added in Liu Shi pipe, every milliliter of sample of final concentration of 0.5 μ g mixes, at room temperature
It is incubated for 3 minutes.
(9) excretion body sorting reagent (Easy Sep) is added in step (8) liquid, volume is 50 μ L, mixes, incubates at room temperature
It educates 3 minutes.
(10) by the coated magnetic bead of PE (EasySep), whirlpool concussion is mixed 30 seconds at room temperature.
(11) the sorting magnetic bead that volume is 200 μ L is added in step (9) liquid.
(12) piping and druming of liquid obtained by step (11) is mixed, is incubated at room temperature 3 minutes.
(13) cleaning solution is added to 2.5 milliliters into step (12) gained sample, softly blows and beats 2-3 times, cleaning solution ingredient
It is D-PBS+0.2% without excretion body serum+1mM EDTA.
(14) by the Liu Shi pipe of step (13) as in magnetic frame, room temperature is acted on 5 minutes.
(15) magnet is picked up, it is whole to overturn, outwell supernatant in Liu Shi pipe.
(16) Liu Shi pipe is removed, step (13)-(15) is repeated, washes twice.
(17) excretion body dissociation solution is added in Liu Shi pipe, volume is 50 μ L, is mixed, room temperature acts on 3 minutes.
(18) 50 μ L of cleaning solution, soft piping and druming 2-3 times is added.
(19) Liu Shi pipe is placed in magnetic frame, room temperature acts on 5 minutes.
(20) magnet is picked up, it is whole to overturn, it collects in Liu Shi pipe in supernatant to new EP pipe, the excretion body of as CD11b+
Subgroup can carry out subsequent analysis and detection.
Fig. 1 is that Western Blot detects excretion body specific marker object Alix and Tsg101, and specific band expression obtains
The product obtained is excretion body；
Fig. 2 is the common transmission electron microscope effect that tachysynthesis paramagnetic particle method separates cerebrospinal fluid No microglial source excretion body
Figure, shows typical vesica shape excretion body structure；
Fig. 3 tachysynthesis paramagnetic particle method separates the nanometer Liu Shi detection of cerebrospinal fluid No microglial source CD11b+ excretion body
As a result, excretion body particle diameter is between 50-180nm as the result is shown, meet excretion body particle diameter distribution.
Embodiment 2
The Neun antibody of PE label is purchased from Novus Biologicals (NBP1-92693PE).
Tachysynthesis paramagnetic particle method separates neuronal origin (Neun) excretion body subgroup in cerebrospinal fluid, the specific steps are as follows:
(1) -80 DEG C of cerebrospinal fluid frozen are taken out, melts on ice, draws 2 milliliters of cerebrospinal fluid, are placed in the centrifugation of 4 DEG C of pre-coolings
Machine was centrifugated through 3000g*15 minutes, the impurity such as removal cell fragment.
(2) supernatant is transferred in 15 milliliters of new centrifuge tubes, and 400 μ L sedimenting reagents (SBI Exoquick-TC) are added.
(3) it turns upside down and mixes 4-6 times, be placed in 4 DEG C of refrigerator overnight incubations.
(4) centrifuge tube is taken out, is centrifuged 1500g*30 minutes under the conditions of 4 DEG C, pipette tips carefully suck supernatant, but do not touch bottom
Precipitating.
(5) 4 DEG C of 1500g*5 minute centrifugations again, pipette tips, which are drawn, carefully thoroughly removes supernatant.
(6) 250 μ L PBS are added in centrifuge tube, pipette tips piping and druming is resuspended.
(7) the excretion body after resuspension is placed in 5 milliliters of Liu Shi pipes.
(8) the Neun primary antibody of PE label is added in Liu Shi pipe, every milliliter of sample of final concentration of 0.5 μ g is mixed, incubated at room temperature
It educates 3 minutes.
(9) excretion body sorting reagent (Easy Sep) is added in step (8) liquid, volume is 200 μ L, is mixed, at room temperature
It is incubated for 3 minutes.
(10) by the coated magnetic bead of PE (EasySep), whirlpool concussion is mixed 30 seconds at room temperature.
(11) the sorting magnetic bead that volume is 100 μ L is added in step (9) liquid.
(12) piping and druming of liquid obtained by step (11) is mixed, is incubated at room temperature 3 minutes.
(13) cleaning solution is added to 2.5 milliliters into step (12) gained sample, softly blows and beats 2-3 times, cleaning solution ingredient
It is D-PBS+0.2% without excretion body serum+1mM EDTA.
(14) by the Liu Shi pipe of step (13) as in magnetic frame, room temperature is acted on 5 minutes.
(15) magnet is picked up, it is whole to overturn, outwell supernatant in Liu Shi pipe.
(16) Liu Shi pipe is removed, step (13)-(15) is repeated, washes twice.
(17) excretion body dissociation solution is added in Liu Shi pipe, volume is 50 μ L, is mixed, room temperature acts on 3 minutes.
(18) 50 μ L of cleaning solution, soft piping and druming 2-3 times is added.
(19) Liu Shi pipe is placed in magnetic frame, room temperature acts on 5 minutes.
(20) magnet is picked up, it is whole to overturn, it collects in Liu Shi pipe in supernatant to new EP pipe, the excretion body of as Neun is sub-
Group, can carry out subsequent analysis and detection.
Embodiment 3
The GFAP of PE label is purchased from Novus Biologicals (NBP2-34401PE).
Tachysynthesis paramagnetic particle method separates cerebrospinal fluid astrocytes source (GFAP) excretion body subgroup, and specific steps are such as
Under:
(1) -80 DEG C of cerebrospinal fluid frozen are taken out, melts on ice, draws 2 milliliters of cerebrospinal fluid, are placed in the centrifugation of 4 DEG C of pre-coolings
Machine was centrifugated through 3000g*15 minutes, the impurity such as removal cell fragment.
(2) supernatant is transferred in 15 milliliters of new centrifuge tubes, and 400 μ L sedimenting reagents (SBIExoquick-TC) are added.
(3) it turns upside down and mixes 4-6 times, be placed in 4 DEG C of refrigerator overnight incubations.
(4) centrifuge tube is taken out, is centrifuged 1500g*30 minutes under the conditions of 4 DEG C, pipette tips carefully suck supernatant, but do not touch bottom
Precipitating.
(5) 4 DEG C of 1500g*5 minute centrifugations again, pipette tips, which are drawn, carefully thoroughly removes supernatant.
(6) 250 μ L PBS are added in centrifuge tube, pipette tips piping and druming is resuspended.
(7) the excretion body after resuspension is placed in 5 milliliters of Liu Shi pipes.
(8) the GFAP primary antibody of PE label is added in Liu Shi pipe, every milliliter of sample of final concentration of 0.5 μ g is mixed, incubated at room temperature
It educates 3 minutes.
(9) excretion body sorting reagent (Easy Sep) is added in step (8) liquid, volume is 100 μ L, is mixed, at room temperature
It is incubated for 3 minutes.
(10) by the coated magnetic bead of PE (EasySep), whirlpool concussion is mixed 30 seconds at room temperature.
(11) the sorting magnetic bead that volume is 200 μ L is added in step (9) liquid.
(12) piping and druming of liquid obtained by step (11) is mixed, is incubated at room temperature 3 minutes.
(13) cleaning solution is added to 2.5 milliliters into step (12) gained sample, softly blows and beats 2-3 times, cleaning solution ingredient
It is D-PBS+0.2% without excretion body serum+1mM EDTA.
(14) by the Liu Shi pipe of step (13) as in magnetic frame, room temperature is acted on 5 minutes.
(15) magnet is picked up, it is whole to overturn, outwell supernatant in Liu Shi pipe.
(16) Liu Shi pipe is removed, step (13)-(15) is repeated, washes twice.
(17) excretion body dissociation solution is added in Liu Shi pipe, volume is 50 μ L, is mixed, room temperature acts on 3 minutes.
(18) 50 μ L of cleaning solution, soft piping and druming 2-3 times is added.
(19) Liu Shi pipe is placed in magnetic frame, room temperature acts on 5 minutes.
(20) magnet is picked up, it is whole to overturn, it collects in Liu Shi pipe in supernatant to new EP pipe, the excretion body of as GFAP is sub-
Group, can carry out subsequent analysis and detection.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of method using different cell origin excretion body hypotypes in tachysynthesis paramagnetic particle method separation cerebrospinal fluid, feature exist
In, comprising the following steps:
(1) cerebrospinal fluid is centrifugated, after taking supernatant that excretion body sedimentation extraction agent is added, mixes, be incubated overnight, centrifugation is removed
After supernatant, it is resuspended, obtains CSF sample；
(2) primary antibody of PE label is added in the CSF sample that step (1) obtains, after incubation, excretion body sorting reagent is added,
Blending incubation obtains cerebrospinal fluid marker samples；
(3) the coated magnetic bead concussion of PE is mixed, the cerebrospinal fluid marker samples that step (2) obtain is added, is incubated for, obtains after mixing
Mixing sample；
(4) mixing sample is washed, blown and beaten, be placed in magnetic field, supernatant is discarded after overturning；
(5) washing of precipitate for obtaining step (4), is added excretion body dissociation solution, and blending incubation is washed out, blows and beats, is placed in magnetic
In, supernatant, as the excretion body of particular source are collected after overturning.
2. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (1): the volume ratio of cerebrospinal fluid and excretion body sedimentation extraction agent is 5:1.
3. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (1): it is resuspended and uses PBS or distilled water.
4. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (2): the primary antibody of PE label is CD11b, Neun or GFAP primary antibody of PE label.
5. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (2): after the primary antibody of PE label is added, the concentration of sample is 0.2-2 μ g/mL.
6. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (2): 25-100 μ L excretion body sorting reagent is added in every milliliter of CSF sample.
7. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (3): 100 μ L magnetic beads are added in every milliliter of cerebrospinal fluid marker samples.
8. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (4): cleaning solution ingredient is D-PBS+0.2% without excretion body serum+1mM EDTA.
9. according to claim 1 a kind of using different cell origin excretion bodies in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of hypotype, which is characterized in that in step (4): sample is put into magnetic frame, picks up magnet, whole to overturn, and outwells supernatant
Liquid.
10. according to claim 1 a kind of using different cell origin excretions in tachysynthesis paramagnetic particle method separation cerebrospinal fluid
The method of body hypotype, which is characterized in that in step (5): volume >=100 μ L of excretion body dissociation solution are added in every milliliter of sample.
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