CN109609458A - A kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body - Google Patents
A kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body Download PDFInfo
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Abstract
The present invention provides a kind of methods of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body, after the method is concentrated by low-speed centrifugal and ultra-filtration centrifuge tube, excretion body is separated in conjunction with polymer sedimentation, the desk centrifuge and cheap ultra-filtration centrifuge tube for only needing laboratory common, operating method is simple and easy, and does not influence excretion body bioactivity.The present invention also efficiently solve nerve cell compared with other cells secretion the excretion scale of construction it is few, the problem of separation and concentration hardly possible.Simultaneously, since the liquid containing excretion body is by effectively concentration, the polymeric reagent used greatly reduces, compared to the prior art, separation purity of the invention and more efficient, strong operability can reduce and separate excretion body cost, have good scientific application promotional value and application prospect.
Description
Technical field
The invention belongs to biomedicine technical field, be related to it is a kind of low speed centrifuge and ultra-filtration centrifuge tube are combined it is outer
Body separation method is secreted, in particular to a kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body.
Background technique
Small vesica of the excretion body as a kind of cell active secretion to extracellular diameter between 30~100nm, is cell
Between the important medium that communicates participate in biological cell in the disease including cancer, infectious diseases and neurodegenerative disease
Between signal transmitting.Clinically, excretion body can play biomarker, and pharmaceutical carrier explains disease mechanism and display
The effects of intervening novel targets.The excretion body of nerve cell secretion is the carrier exchanged between nerve cell, in central nervous system disease
Important regulating and controlling effect is played in the generation of disease, development and injury repair.And nerve cell is due to splitting ability and metabolic cycles
Far below tumour cell, the secretory volume of body secretion is far below tumour cell and other cells of body.Therefore how to be enriched with and mention
Taking nerve cell excretion body is also a major challenge.
Filter centrifugation is the ultrafiltration membrane centrifuge separation excretion body using different retentions relative molecular weight (MWCO).Retention phase pair
Molecular weight refers to the relative molecular mass of maximum molecule in the molecule that can pass freely through certain pored wood material.Excretion body is a capsule
Shape corpusculum, relative molecular weight are greater than general protein, therefore select a certain size MWCO film that excretion body can be made big with other
Molecular substance separation.It is this easy to operate, it is only necessary to which that low speed centrifuge achieves that low-speed centrifugal often refers to that revolving speed is 8000r/min
Hereinafter, relative centrifugal force is 10 000 × g centrifugation below.Mainly for separating of cell, cell fragment and culture based draff etc.
Particulate matter.Therefore this step is applied time saving in extracting excretion body method, is not influenced excretion body activity, be can be used as and further mention
Pure use.Although ultrafiltration membrane centrifugation can achieve in weak solution micro constitutent recycling, ultrafiltration membrane in the prior art from
The heart also has certain limitation, for the content of solution separation, can only generally be concentrated to 10~50% concentration.
The separation most common method of excretion body mainly has at present: ultracentrifugation method, neat polymer precipitation method etc..Generally
It says, centrifuge speed is known as ultracentrifugation 30 000r/min's or more.Ultracentrifugation method needs instrument special and expensive,
Only the buying expenses of Ultracentrifuge reach 100,000 yuans or more at present, and separate often since protein mixes
It is ruptured with excretion body, it is not high to isolate excretion body purity, cannot get popularization and application.The current existing procucts of the neat polymer precipitation method
It releases, but amount of reagent is big, it is expensive to affect further experiment.Moreover, nerve cell due to splitting ability decline, compared with
The excretion scale of construction of tumor cell secretion is few, so that separation and concentration is more difficult.Existing most of laboratory is overcome to lack thus super
Supercentrifuge, only conventional speeds centrifuge, and funds are in short supply cannot make a big purchase polymer precipitation method kit etc. in large quantities no
Foot, exploitation separation excretion body strong operability, purity is high, economical and practical method, is especially badly in need of.
Summary of the invention
The shortcomings that it is an object of the invention to overcome excretion body isolation technics in the prior art and deficiency, provide one kind and are based on
The method that low speed ultrafiltration is centrifuged conjugated polymer sedimentation separation excretion body, excretion body through low-speed centrifugal and ultra-filtration centrifuge tube into
After row concentration, separated in conjunction with polymer sedimentation.The method can be applied to cell excretion body, especially refreshing
It separates and purifies through cell excretion body.The desk centrifuge and cheap ultrafiltration centrifugation that this method only needs laboratory common
Pipe, simultaneously as the liquid of the invention containing excretion body by concentration, so that the dosage of polymeric reagent greatly reduces, passes through
Repeatedly compared with previous methods, separation purity is higher, strong operability, can reduce the features such as separation excretion body cost for purification.
The purpose of the invention is achieved by the following technical solution:
A method of excretion body is separated based on low speed ultrafiltration centrifugation conjugated polymer sedimentation, comprising the following steps:
(1) culture supernatant is collected after 48~72h of neuronal cell cultures, obtains the first supernatant after centrifugation removal suspension cell
Liquid, centrifugation removal cell fragment, obtains the second supernatant again；
(2) the second supernatant is filtered by 0.22 μm of filter membrane, removes bacterium and residual cell device, obtains excretion body
Preliminary filtrate；
(3) the preliminary filtrate of excretion body in step (2) is subjected to low speed ultrafiltration centrifugation, obtains concentrate 1, it is molten that PBS is added
Low speed ultrafiltration is centrifuged again for liquid washing, and collection cannot be concentrate 2 by filter membrane product；
(4) concentrate 2 in step (3) is mixed with polyethylene glycol (PEG) solution, 4 DEG C of overnight incubations are reacted
Liquid；
(5) the reaction solution centrifuging and taking of step (4) is precipitated, precipitating is centrifuged again, gained precipitating is the excretion
Body.
The low-speed centrifugal refers to that revolving speed is 8000r/min hereinafter, relative centrifugal force is 10 000 × g centrifugation below.
Super filter tube containing ultrafiltration membrane is centrifuged by the referring to for low speed ultrafiltration centrifugation in centrifuge.
Nerve cell described in step (1) preferably includes mouse cortex nerve cell and hippocampal neurons, further
Preferably nerve cell vigor is high, the well-grown mouse cortex nerve cell of nerve fibre and/or hippocampal neurons.
The temperature of culture described in step (1) is preferably 36~38 DEG C, and more preferably 37 DEG C；The environment of the culture is excellent
It is selected as gas concentration lwevel 4%~6%, more preferable gas concentration lwevel is 5%.
Culture medium used in culture described in step (1) preferably contains nerve growth factor culture medium, further preferably
For the Neurobasal culture medium containing 2%B27 of Gibico company.
The speed of centrifugation described in step (1) is preferably 200~350g, further preferably 200g；Centrifuging temperature is excellent
4 DEG C are selected as, centrifugation time is preferably 10min.
The condition being centrifuged again described in step (1) is 2000~2500g, 4 DEG C, is centrifuged 20min.
Filter membrane described in step (2) is cellulose mixture filter membrane.
Low speed ultrafiltration centrifugation can be repeated several times the washing of addition PBS solution described in step (3) again, and preferably repeatedly 3
It is secondary.
The centrifugation of low speed ultrafiltration described in step (3) is preferably enterprising in the ultrafiltration membrane of 30000Da NMWL ultra-filtration centrifuge tube
Row.
The centrifugation of low speed ultrafiltration described in step (3), low speed ultrafiltration centrifugation again condition be preferably 3500~4000g, 4
DEG C, 30min.
The final volume of concentrate 2 described in step (3) is preferably 200~500 μ L.
PBS described in step (3) is preferably Phosphate Buffered Saline (1X).
Polyethylene glycol described in step (4) is preferably PEG6000 (molecular weight 6000)；The polyethylene glycol (PEG)
Solution is preferably the polyglycol solution that concentration is 16% (w/v).
Preferably 1:1 is mixed concentrate 2 described in step (4) by volume with polyglycol solution.
The condition of centrifugation in step (5) is preferably 1500g, 4 DEG C, is centrifuged 30min；The condition being centrifuged again is preferably
1500g, is centrifuged 10min by 4 DEG C.
The excretion body is preferably disposed at -80 DEG C and saves.
In the method, every time after centrifugation, filtering or low speed ultrafiltration centrifugation, liquid is transferred to new container, such as
In centrifuge tube etc., then carry out next step operation.
The present invention first carries out ultrafiltration centrifugation, since the size of ultra-filtration centrifuge tube filter sizes makes protein and RNA etc. big
Quantity of fluid can pass through, and a small amount of liquid containing excretion body remains in outside filter membrane, and first excretion body is concentrated；It is heavy to reuse
The reagent of shallow lake method only needs 1/the tens of the simple precipitation method；The use of reagent is greatly reduced, and does not have to mating ultrahigh speed
Centrifuge；Operating method is simple and easy.
The present invention has the following advantages and effects with respect to the prior art:
1. the present invention provides a kind of enrichment and the methods of purifying nerve cell excretion body.Efficiently solve nerve cell base
This is not divided, few compared with other cells secretion excretion scale of construction, and separation and concentration difficulty is apparently higher than the problem of the excretion body of other cells.
2. innovative precipitate in conjunction with ultra-filtration centrifuge tube and polymer of the invention extracts excretion body.The method is simply high
Effect, has carried out effective concentration to excretion body, has not needed additional expensive equipment, and do not influenced excretion body bioactivity, is to extract
A kind of effective new method of excretion body.
3. prior step of the present invention filters other albumen using ultra-filtration centrifuge tube and other macromoleculars, excretion body group concentrate on
Outside film, overcomes prior art background impurities to interfere, multiple purifying excretion body can be played the role of.
4. the added PEG amount of the obtained liquid after being concentrated by ultrafiltration of the invention is significantly compared with the neat polymer precipitation method
It reduces, can obviously reduce reagent use cost, it is economical and practical.
5. separative efficiency of the present invention is high, purification excretion body allows subsequent further analysis, identification and survey including genome
Sequence etc. has higher scientific application and popularization value.
Detailed description of the invention
Fig. 1 is the Electron microscopic findings figure for the product that embodiment 1 obtains；Wherein, the scale of A is 200nm；The scale of B
For 50nm, the scale of C is 100nm.
Fig. 2 is the immunoblot results figure that embodiment 2 detects CD63 and HSP70 albumen.
Fig. 3 is the particle diameter distribution result figure for the excretion body that embodiment 1 obtains.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The extraction and purification of 1 nerve cell excretion body of embodiment
1. extracting newborn mice primary neural cell (including cortical neurogenic cell and hippocampal neurons): using ethanol disinfection
In 24 hours newborn behind mouse head (balb/c mouse is purchased from Traditional Chinese Medicine University Of Guangzhou), frost anesthesia carries out under the conditions of 4 DEG C
PBS phosphate buffer isolates cortex and hippocampus, removes meninx and blood vessel.Then it is discarded supernatant after static 2min, to tissue
It is interior be added 0.125% trypsase (Gibico company), digest 10min after terminate digestion, be resuspended cell, 1000r/min from
Liquid, bed board are discarded supernatant after heart 5min.Promote neuronal maturation with complete medium DMEM/F12 (being purchased from Gibico company), more
Changing the nerve cell growth factor culture medium that contains without serum composition, (Neurobasal+2%B27 of Gibico company is cultivated
Base) on, at 37 DEG C, 5%CO2It is cultivated 48~72 hours under environment, collects culture supernatant in centrifuge tube 1, remove suspension cell
(200g, is centrifuged 10min by 4 DEG C) afterwards is collected supernatant liquid removal cell fragment (2000g, is centrifuged 20min by 4 DEG C), is transferred to new
Centrifuge tube 2.
2. by the liquid of centrifuge tube 2 in above-mentioned steps 1, by 0.22 μm of filter to further filtering, removal bacterium and
Residual cell device is transferred to centrifuge tube 3, obtains the preliminary filtrate of nerve cell excretion body.
3. by the preliminary filtrate 15mL of nerve cell excretion body in the step 2 be added to special 30000Da NMWL ultrafiltration from
On the filter (ultrafiltration membrane) of heart pipe (being purchased from Millipore company), concentration centrifugation
(4000g, 4 DEG C, 30min), obtains obtaining concentrate 1 on filter.
4. 15mL PBS cleaning solution (pH 7.35~7.45) is added on step 3 filter (4000g, 4 DEG C,
30min) further cleaning, is repeated 3 times, about 200~500 μ L of concentrate 2 is obtained on filter.
5. the sample slot using pipette filter from the step 4 collects concentrate 2, it is transferred to 1.5mL EP pipe.
6. being by volume the ratio of 1:1 by the PEG6000 solution that concentrate 2 in the step 5 and concentration are 16% (w/v)
Example, is mixed by inversion, and 4 DEG C of overnight incubations obtain reaction solution.
7. second day, by reaction solution in the step 6,4 DEG C of 1500g, is centrifuged 30min, abandons supernatant；Stay precipitating again
4 DEG C of 1500g, it is centrifuged 10min, carefully siphons away remaining supernatant, precipitating is excretion body.The excretion body, which is placed at -80 DEG C, to be protected
It deposits.
The identification of 2 excretion body of embodiment
1. morphologic observation
Morphologic observation is carried out to the excretion body that embodiment 1 obtains by transmission electron microscope.
(1) after the excretion body that embodiment 1 obtains being mixed well using PBS；
(2) on the load sample copper mesh for adding 20 μ L to diameter 2mm, 5min is stood；
(3) copper mesh surrounding liquid is gently blotted using filter paper, and copper mesh is then tipped upside down on into 20g/L phosphotungstic acid (pH6.8) liquid
In drop, negative staining 10min at room temperature；
(4) copper mesh is dried under incandescent lamp, is placed in searching 40~100nm of diameter capsule balloon-shaped structure under transmission electron microscope and is taken pictures,
As a result as shown in Figure 1, the scale of A is 200nm, B, C scheme to amplify compared with A, and scale is respectively 50nm, 100nm.
2. labelled protein detects
PROTEIN C D63 in the excretion body and primary neural cell that embodiment 1 obtains is detected by protein immunoblotting,
The case where HSP70.
(1) following two experimental groups are set:
Groups of cells (Cell): culture medium in the primary neural cell of culture 72 hours is sucked out, with pre-cooling 1 × PBS (pH
7.35~7.45) it cleans 2 times, 60 μ L RIPA cell pyrolysis liquids (the green skies, product number P0013B) of every hole addition, in ice face
Crack 30min.12000rpm/ ware, 4 DEG C of centrifugation 15min, takes supernatant, for use.By BCA quantification kit (be purchased from the green skies,
Article No. P0010) carry out protein quantification.
Excretion body group (Exosome): excretion 1 × PBS of body 1mL (pH 7.35 that the reserved purification of embodiment 1 obtains is taken out
~7.45) it is resuspended, obtains excretion weight suspension, RIPA lysate (being purchased from the green skies, article No. P0013B) is added by 1:1, sufficiently
Cracking, for use.Protein quantification is carried out by BCA quantification kit (being purchased from the green skies, article No. P0010).
(2) detecting step
1. measuring groups of cells and excretion body group respectively using BCA protein quantification kit (being purchased from the green skies, article No. P0010)
Excretion body labelled protein content.
2. sample protein concentration is measured according to BCA method, so that loading quantity of protein sample (50 μ g/10 μ L).
3. installing glue frame and glass plate, 10% separation gel encapsulating is prepared, after gelling to be separated is solid, it is dense illustratively to prepare 5%
Contracting glue and encapsulating are inserted into glue comb to glass plate upper limb, wait gelling solid.
4. 10 μ L protein samples are added in each swimming lane, 1 μ L marker is added in side swimming lane.First with 80V electrophoresis
30min or so continues to separate after sample enters separation gel using voltage 120V, electrophoresis 60min or so, until bromophenol blue extremely
Bottom end.
5. taking out gel after electrophoresis, cutting gel according to corresponding molecular weight marker marker, from bottom to top successively
For foam-rubber cushion, filter paper, Kynoar (PVDF) film, gel, the sequence placement for thickening filter paper, sponge are thickeied, prepares " Sanming City
Control ", 90min is turned with constant current 260mA electricity in the transfering buffering liquid of pre-cooling.
6. pvdf membrane is put into the culture dish of 5% skim milk, 1h is closed under room temperature；
7. TBST cleaning film, 3 times, each 5min.
8. the diluted CD63 of TBST (being purchased from abcam, article No. ab216130) is added on film, HSP70 (is purchased from Cell
Signal technology, article No. #4872), 4 DEG C of overnight incubations, with appropriate TBST rinsing 3 times after next day rewarming 1h, every time
5min；
9. the diluted secondary antibody of TBST (HRP marks goat anti-rabbit antibody, purchased from all creation biology) is added on film, incubate at room temperature
It is rinsed on shaking table 3 times after 60min with appropriate TBST, each 5min.
10. chemiluminescence and development: the ECL luminescent solution of existing preparation being added drop-wise on film, is carried out with 4.7 instrument of ALLIANCE
Fluorescence signal development and acquisition image.
As a result it as shown in Fig. 2, detecting the expression of the peculiar PROTEIN C D63 in product excretion body surface face through the above method, uses
HSP70 is as same protein content reference.The specific mark object CD63 expression quantity of excretion body group is apparently higher than cell protein.
The particle diameter and concentration studies of 3 excretion body of embodiment
Nano particle tracer technique further identifies excretion body, and analysis detection purifies to obtain excretion bulk concentration and particle is straight
Diameter.Nanosight visual type nano particle analyzer, nano-scale particle does the shape of Brownian movement in direct dynamic observation sample
State, and the partial size of each particulate matter is immediately arrived at using Stroke-Einstein equation, and the actual concentration of particle can be measured
(a/milliliter).As a result as shown in figure 3, the concentration of products therefrom of the present invention, size etc. characterize, it is existing to meet excretion body surface.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body, which is characterized in that including
Following steps:
(1) culture supernatant is collected after 48~72h of neuronal cell cultures, obtains the first supernatant after centrifugation removal suspension cell,
Centrifugation removal cell fragment again, obtains the second supernatant；
(2) the second supernatant is filtered by 0.22 μm of filter membrane, removes bacterium and residual cell device, it is preliminary obtains excretion body
Filtrate；
(3) the preliminary filtrate of excretion body in step (2) is subjected to low speed ultrafiltration centrifugation, obtains concentrate 1, PBS solution is added and washes
The centrifugation of low speed ultrafiltration again is washed, collection cannot be concentrate 2 by filter membrane product；
(4) concentrate 2 in step (3) is mixed with polyglycol solution, 4 DEG C of overnight incubations obtain reaction solution；
(5) the reaction solution centrifuging and taking of step (4) is precipitated, precipitating is centrifuged again, gained precipitating is the excretion body；
The low-speed centrifugal refers to that revolving speed is 8000r/min hereinafter, relative centrifugal force is 10 000 × g centrifugation below.
2. the method according to claim 1 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
Super filter tube containing ultrafiltration membrane is centrifuged by the referring to for low speed ultrafiltration centrifugation in centrifuge.
3. the method according to claim 1 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
The centrifugation of low speed ultrafiltration described in step (3) carries out on the ultrafiltration membrane of 30000Da NMWL ultra-filtration centrifuge tube；
1:1 is mixed concentrate 2 described in step (4) by volume with polyglycol solution.
4. the method according to claim 1 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
The final volume of concentrate 2 described in step (3) is 200~500 μ L；
Polyglycol solution described in step (4) is the polyglycol solution that concentration is 16% (w/v).
5. the method according to claim 1 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
Nerve cell described in step (1) is mouse cortex nerve cell and hippocampal neurons；
The temperature of culture described in step (1) is 36~38 DEG C；
The environment of culture described in step (1) is gas concentration lwevel 4%~6%；
Culture medium used in culture described in step (1) is culture medium containing nerve growth factor；
The speed of centrifugation described in step (1) is 200~350g；
Low speed ultrafiltration centrifugation can be repeated several times the washing of addition PBS solution described in step (3) again.
6. the method according to claim 5 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
Nerve cell described in step (1) is that nerve cell vigor is high, and the well-grown mouse cortex nerve of nerve fibre is thin
Born of the same parents and hippocampal neurons；
The speed of centrifugation described in step (1) is 200g；
The washing of addition PBS solution described in step (3) number of repetition that low speed ultrafiltration is centrifuged again is 3 times.
7. the method according to claim 5 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
The temperature of culture described in step (1) is 37 DEG C；The environment of the culture is that gas concentration lwevel is 5%；
Culture medium used in culture described in step (1) is the Neurobasal culture medium containing 2%B27 of Gibico company.
8. the method according to claim 1 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
Low speed ultrafiltration described in step (3) centrifugation, again low speed ultrafiltration centrifugation condition be 3500~4000g, 4 DEG C,
30min；
The condition of centrifugation in step (5) is 1500g, 4 DEG C, is centrifuged 30min；The condition being centrifuged again is 1500g, 4 DEG C, is centrifuged
10min；
Polyethylene glycol described in step (4) is PEG6000.
9. the method according to claim 1 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
The temperature of centrifugation described in step (1) is 4 DEG C, and the time of centrifugation is 10min；
The condition being centrifuged again described in step (1) is 2000~2500g, 4 DEG C, is centrifuged 20min；
Filter membrane described in step (2) is cellulose mixture filter membrane；
PBS described in step (3) is 1 × PBS.
10. the method according to claim 1 based on low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body,
It is characterized by:
The excretion body, which is placed at -80 DEG C, to be saved；
In the method, every time after centrifugation, filtering or low speed ultrafiltration centrifugation, liquid is transferred to new container, then carry out down
Single stepping.
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| CN111961637A (en) * | 2020-07-08 | 2020-11-20 | 暨南大学 | Extracellular vesicle separation method based on combination of size exclusion chromatography and ultrafiltration |
| CN112322584A (en) * | 2020-12-09 | 2021-02-05 | 上海市第六人民医院 | Simple exosome extraction method |
| CN112941016A (en) * | 2021-04-23 | 2021-06-11 | 广州研华生物科技有限公司 | Ultrafilter and method for separating exosomes |
| CN114790439A (en) * | 2021-01-25 | 2022-07-26 | 谛邈生物科技(北京)有限公司 | Milk exosome and preparation method thereof |
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