CN109929795A - A kind of improvement extracting method of the outer vesica of urine cellule - Google Patents
A kind of improvement extracting method of the outer vesica of urine cellule Download PDFInfo
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Abstract
The invention discloses a kind of improvement extracting methods of vesica outside urine cellule, carry out density gradient centrifugation as follows: by the sucrose solution of various concentration gradient according to concentration from successively padded down to high sequence be added test tube bottom, form the gradient liquid that concentration successively increases from top to bottom, thick vesica re-suspension liquid is slowly filled in gradient liquid top layer, is centrifuged；Wherein the sucrose solution is the 20mm Tris-HCL sucrose heavy aqueous solution of pH8.4~8.8, the thick vesica re-suspension liquid is made in the 20mm Tris-HCL aqueous solution for be resuspended in thick vesica pH8.4~8.8, this method can significantly improve extraction yield, reduce sample dosage, and it is time-consuming substantially to shorten extraction, DNA purity can be significantly improved in conjunction with differential centrifugation and ultrafiltration, clinical use is easier, more reliable, faster.
Description
Technical field
The invention belongs to field of biomedicine technology, are related to outer vesica separation and extraction technology, and in particular to a kind of urine is small
The improvement extracting method of extracellular vesica.
Background technique
With the discovery and research of the extracellular vesica of urine, urine, which has been considered as a kind of, can reflect the dynamic sample of disease.
Extracellular vesica is the nano particle of lipid membrane structure, can be divided into three classes: excretion body, microvesicle and apoptotic body.Its Forming Mechanism
It is respectively as follows: multivesicular body and plasma membrane fusion discharges excretion body；Cell membrane budding directly forms microvesicle；The capsule to fall off from dying cell
Bubble forms apoptotic body.Excretion body diameter is 40~100nm, and the diameter of microvesicle is 100~1,000nm, usually bigger than excretion body,
But lesser microvesicle is similar to excretion body size, and apoptotic body size is different (diameter 800~5,000nm), and appearance is different, but logical
It is often bigger than other kinds of extracellular vesica.During they are formed, the extracellular vesica of urine can shift various cell-specifics
Property ingredient (protein, lipid and nucleic acid) arrive target cell.As methodological study is more and more rigorous, separation, purifying and characterization side
The optimization of method has further development, and extracellular vesica has had been a great concern for treating disease, these researchs
Just promoting it was recognized that the extracellular vesica of urine serves as important medium in Pathological and physiology.
The extracting method of extracellular vesica have agent precipitate method, surpass from method, ultrafiltration, chromatographic exclusion method and density gradient from
Heart method.Agent precipitate method by it is more research confirm its extract purity it is not high, and merely using surpass from method, ultrafiltration or
Also containing more foreign protein and non-bladder complex acid in the extract that chromatographic exclusion method obtains.Meanwhile nephrotic's urine contains
Great amount of soluble protein interferes the detection of the extracellular vesicle protein of urine, and it is solvable to need to increase additional purification step removal
Property protein.Currently, surpassing from method combination density gradient centrifugation is still to extract the most efficient method of extracellular vesica, this method
The vesica of extraction contains that excretion body purity is higher, and usually the extracellular vesica that density-gradient centrifugation method extracts is called outside cellule
Vesica.However time-consuming for density gradient centrifugation, traditional density gradient centrifugation at least needs 14 hours, hinders its clinical application.
Urine is adjusted plain (also referred to as Tamm-Horsfall glucose in urine albumen (THP)) to be a kind of and is synthesized in ascending thick limb of Henle's loop
Memebrane protein is the most abundant protein of content in urine under physiological condition.In addition to interference vesicle protein detection, uromodulin can be with
The extracellular vesica of netted capture urine is formed, changes the outer vesica density of cellule, is distributed it in gradient liquid and does not concentrate, thus
Reduce the extracted amount and purity of the outer vesica of cellule.
Therefore, it in order to further adapt to the needs of clinical application and scientific research, needs to develop a kind of for urine specimen
The outer vesica of cellule improvement density gradient centrifugation method, can high yield and high-purity extraction urine medium-small cells external capsule
Bubble, and can be shortened extraction time, provide more practical, easy for the outer vesica of clinical and scientific research extraction urine cellule and rigorously may be used
The method leaned on.
Summary of the invention
It is big it is an object of the invention to solve sample dosage existing for the extracellular vesica extractive technique of existing urine, when extraction
Between long, the problems such as extraction yield is low or purity is not high, method provided by the invention can significantly improve extraction yield, reduce sample and use
Amount, and can substantially shorten extraction time-consuming, DNA purity can be significantly improved in conjunction with differential centrifugation and ultrafiltration, clinical use is simpler
Just, more reliable, faster.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
The present invention provides a kind of improvement extracting methods of vesica outside urine cellule, carry out density level bands as follows
Degree centrifugation: the sucrose solution of various concentration gradient is formed according to concentration from addition test tube bottom is successively padded down to high sequence
The gradient liquid that concentration successively increases from top to bottom slowly fills thick vesica re-suspension liquid in gradient liquid top layer, centrifugation；
Wherein the sucrose solution is the 20mm Tris-HCL sucrose heavy aqueous solution of pH8.4~8.8, the thick vesica weight
Suspension is made in the 20mm Tris-HCL aqueous solution for be resuspended in thick vesica pH8.4~8.8.
The present invention using specific pH range 20mm Tris-HCL sucrose heavy aqueous solution to thick vesica carry out density gradient from
The heart, and traditional extracting method, such as with the sucrose heavy aqueous solution pad method centrifugation of a certain concentration or with PBS sucrose heavy aqueous solution
Density gradient centrifugation etc. is compared, which can significantly improve the extraction yield of the outer vesica of urine medium-small cells, and be can be reduced sample and made
Dosage, clinic make and use more convenient.
As the preferred embodiment of the invention, the sucrose solution is that the 20mm Tris-HCL sucrose heavy water of pH8.6 is molten
Liquid, the thick vesica re-suspension liquid are made in the 20mm Tris-HCL aqueous solution for be resuspended in thick vesica pH8.6.The alkalinity item
Under part, TH albumen aggregation extent can be reduced, to reduce its capture to extracellular vesica, keep the original density of vesica, make small
Extracellular vesica concentrates on one section of specific gradient layer, vesica outside the cellule to obtain maximum production.
As the preferred embodiment of the invention, the gradient liquid in test tube top-down concentration be followed successively by 5%,
10%, 20%, 40% and 60%, which is conducive to the outer vesica of cellule in the enrichment of middle layer, is conducive to subsequent mention
Take the progress of work.
As the preferred embodiment of the invention, the centrifugation is centrifuged 5 hours at 25 DEG C.The present invention and conventional method
Difference is that traditional density gradient centrifugation needs to be centrifuged the cellule external capsule that could at least obtain certain yield for 14 hours at 4 DEG C
Bubble, and the present invention is only needed due to using 20mm Tris-HCL sucrose heavy aqueous solution in 25 DEG C of centrifugations, 5 hours energy
The outer vesica of more large-tonnage cellule is obtained, it is time-consuming to highly shortened extraction.It should be appreciated that 25 DEG C of the present invention, allow
There is certain error in the precision of instrument setting, general error is ± 2 DEG C, and the temperature in the fluctuation range is also included in the present invention
Protection in.
As the preferred embodiment of the invention, the thick vesica is that urine sample is passed through differential centrifugation at normal temperature
It extracts and obtains.The differential centrifugation is first dead with low-speed centrifugal removal cell and big cell fragment, then high speed centrifugation
Soluble protein, protein aggregate and other impurity being co-precipitated with extracellular vesica are removed, thus isolated micro-capsule
Bubble and excretion body, method is known in those skilled in the art.Usual differential centrifugation be under 4 DEG C of cryogenic conditions into
Row, and discovery when the present invention combines differential centrifugation and 20mm Tris-HCL sucrose heavy aqueous solution density gradient centrifugation,
Room temperature carries out differential centrifugation and is more advantageous to the extraction yield for improving the outer vesica of cellule.
Room temperature of the present invention refers to 18~28 DEG C.It is poor in 18~28 DEG C of progress to be referred at normal temperature using differential centrifugation
Speed centrifugation.
Preferably, the differential centrifugation specifically: under room temperature, urine sample is centrifuged with 500g, goes to precipitate, supernatant
It with 2,000g centrifugation, goes to precipitate, supernatant goes to precipitate with 16,000g centrifugation, and supernatant is using ultracentrifuge with 200,000g
Centrifugal force 1 hour, obtain the thick vesica of sediment.
As the preferred embodiment of the invention, the supernatant obtained in the differential centrifugation process is by being concentrated by ultrafiltration.
Ultrafiltration is the method that excretion body is separated as power by the pressure difference of ultrafiltration membrane two sides, and method is those skilled in the art
Well known to member.Present invention applicant can remove a large amount of foreign proteins, guarantee the study found that differential centrifugation combination ultrafiltration
The purity of the outer vesica of cellule is improved while yield.
As the preferred embodiment of the invention, the ultrafiltration is concentrated by ultrafiltration in 100kd super filter tube, can be to greatest extent
Acquisition high-purity the outer vesica of cellule.
The present invention also provides a kind of improvement extracting method of vesica outside more specifically urine cellule, the method packets
Include following steps:
(1) differential centrifugation extracts thick vesica: under room temperature, urine sample is centrifuged with 500g, goes to precipitate, supernatant with 2,
000g centrifugation, goes to precipitate, and supernatant is added in 100kd super filter tube, supernatant is concentrated 10 times under 2,000 centrifugal force, then
It by the liquid after concentration with 16,000g centrifugation, goes to precipitate, supernatant is using ultracentrifuge with 200,000g centrifugal force 1
Hour, the thick vesica of sediment is obtained, thick vesica is resuspended in the 20mm Tris-HCL aqueous solution of PH 8.6 and obtains thick vesica weight
Suspension；
(2) density gradient centrifugation extracts the outer vesica of cellule: successively being filled to test tube bottom using tack needle injection dense
20mm Tris-HCL each 1ml of sucrose heavy aqueous solution of the pH8.6 of degree 5%, 10%, 20%, 40%, 60%, thick vesica is resuspended
Liquid is slowly added in configured good gradient liquid top layer, 25 DEG C with 110,000g centrifugal force 5 hours after, gradient liquid is from upper
It is divided into 12 layers under and, 6,7,8,9 layers for taking out the outer vesica of enrichment cellule are separately added into and surpass from pipe, use the 20mm of pH8.6
Tris-HCL aqueous solution is diluted to 8ml, and 200,000g is centrifuged 1 hour at 4 DEG C respectively, obtains sediment.
Above-mentioned improvement extracting method extracts thick vesica with differential centrifugation combination ultrafiltration, with 20mm Tris-HCL sucrose
Heavy aqueous solution carries out density gradient centrifugation, can obtain the outer vesica of cellule of maximum purity.
To sum up, compared with prior art, the device have the advantages that are as follows:
The present invention improves traditional density gradient centrifugal process, can exclude urine to the greatest extent and adjust element etc. to extraction urine
Interference caused by the outer vesica of cellule, reduces sample usage amount, significantly improves the extraction yield of the outer vesica of urine cellule, and
It is time-consuming to highly shortened extraction, is based on the improved method, differential centrifugation under room temperature (be not used in combination saltout and ultrafiltration) is obtained
The thick vesica obtained carries out the density gradient centrifugation that the present invention improves and extracts, and can obtain the outer vesica of cellule of maximum production；To normal
The thick vesica that the lower differential centrifugation combination 100kd ultrafiltration of temperature obtains carries out the density gradient centrifugation that the present invention improves and extracts, then can
Obtain the outer vesica of cellule of maximum purity.
Detailed description of the invention
Fig. 1 is examination before 20mm Tris-HCL sucrose heavy aqueous solution density gradient (improved method) centrifugation in the embodiment of the present invention 1
Pipe internal component hierarchical diagram；
Fig. 2 is to try after 20mm Tris-HCL sucrose heavy aqueous solution density gradient (improved method) centrifugation in the embodiment of the present invention 1
Pipe internal component hierarchical diagram；
Fig. 3 is the outer vesica mark of each layer gradient liquid medium-small cells after Traditional Method in the embodiment of the present invention 1 and improved method centrifugation
Object distributional difference figure；
Fig. 4 is the outer vesica maximum production extraction scheme flow chart (A) of test example d cellule and examination in the embodiment of the present invention 2
Test the outer vesica maximum purity extraction scheme flow chart (B) of a h cellule；
Fig. 5 is that the cellule external capsule that test example a~h is obtained in the embodiment of the present invention 2 steeps transmission electron microscope picture；
Fig. 6 is the outer vesica marker Western Blot mirror of cellule that test example b~h is obtained in the embodiment of the present invention 2
Fixed figure (A), nano particle TRPS count results (B) and the outer vesica purity result (C) of cellule.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below.But this should not be interpreted as in the present invention
The range for stating theme is only limitted to embodiment below, all that model of the invention is belonged to based on the technology that the content of present invention is realized
It encloses.In addition to specializing, technological means used in following embodiment and operating method are well known to those skilled in the art
Conventional means and method, institute using raw material is commercial goods.
Embodiment 1
The comparison of different sucrose heavy aqueous solution density-gradient centrifugation methods
1, sucrose heavy aqueous solution configures
(1) configuration of PBS sucrose heavy aqueous solution
0.03g PBS pulvis and 1.5g sucrose are weighed in test tube, heavy water is added and is settled to 30ml, adjusts pH to 7.4, mixes
5% PBS sucrose heavy aqueous solution is obtained after dissolution.0.02g PBS pulvis and 12g sucrose are weighed in test tube, it is fixed that heavy water is added
Hold to 20ml, adjust pH to 7.4, obtains 60% PBS sucrose heavy aqueous solution after mixing dissolution.10%, 20% and 40% sucrose weight
The solution of the above two concentration of aqueous solution mixed configuration in proportion.
(2) configuration of 20mm Tris-HCL sucrose heavy aqueous solution
0.07g Tris pulvis and 1.5g sucrose are weighed in test tube, heavy water is added and is settled to 30ml, concentrated hydrochloric acid tune is added
PH to 8.6 obtains 5% 20mm Tris-HCL sucrose heavy aqueous solution after mixing dissolution.Weigh 0.05g Tris pulvis and 12g
Sucrose is added heavy water and is settled to 20ml in test tube, and concentrated hydrochloric acid is added to pH8.6, obtains 60% 20mm after mixing dissolution
Tris-HCL sucrose heavy aqueous solution.10%, 20% and 40% sucrose heavy aqueous solution is mixed in proportion with the solution of above two concentration
Close configuration.
2, differential centrifugation extracts thick vesica
By urine sample with 500g centrifugation 10 minutes, removal is precipitated, and supernatant went to precipitate with 2,000g centrifugation 20 minutes,
Supernatant after then 2,000g is centrifuged is in centrifuge Fixed Angle Rotor with 16,000g centrifugation 20 minutes.Use Beckman XL-80
Ultracentrifuge (Beckman Coulter) is by 16,000g supernatant with 200,000g centrifugal force in SW41Ti horizontal rotor
To be centrifuged 1 hour under maximum acceleration and lower deceleration, thick vesica sediment is obtained.
3, the extraction of the outer vesica of urine cellule
The precipitating (thick vesica) that differential centrifugation extracts is resuspended in PBS (Traditional Method) water of 3ml PH 7.4 respectively
(H2O) in solution or 20mm Tris-HCL (improved method) water (H of PH 8.62O) in solution, using tack needle injection successively to
Test tube bottom fills PBS (Traditional Method) the sucrose heavy aqueous solution or pH8.6 of 5%, 10%, 20%, 40%, 60% PH 7.4
Thick vesica re-suspension liquid is slowly added in using sample loading gun configured by 20mm Tris-HCL (improved method) each 1ml of sucrose heavy aqueous solution
Good gradient liquid top layer (attached drawing 1 is shown in the layering of test tube interior ingredient before improved method is centrifuged), respectively at 4 DEG C and 25 DEG C,
After being centrifuged 5 hours under 110,000g centrifugal force, dividing 12 layers from top to bottom for gradient liquid, (test tube interior ingredient divides after improved method centrifugation
Layer see attached drawing 2) take out be added surpass from pipe, use the PBS aqueous solution (Traditional Method) of PH 7.4 or the 20mm Tris-HCL of pH8.6
Aqueous solution (improved method) is diluted to 8ml, and 200,000g is centrifuged 1 hour at 4 DEG C respectively, and precipitating is detected using Western Blot
Outer vesica marker Alix and the TSG101 albumen of 12 layers of fraction medium-small cells is to determine cellule external capsule alveolar layer.It identifies and records small
Layering where extracellular vesica, the outer vesica extraction of later cellule take out these layerings containing vesica outside cellule together,
Without identifying again.
4 DEG C of density gradient centrifugations of PBS (Traditional Method), 25 DEG C of density gradient centrifugations of PBS (Traditional Method), 20mm Tris-HCL
Each cellule that 25 DEG C of density gradient centrifugation of (improved method) 4 DEG C of density gradient centrifugations and 20mm Tris-HCL (improved method) obtains
The distributional difference result (see attached drawing 3) of outer vesica acceptance of the bid will object shows using improved method saccharose gradient liquid of the invention at 25 DEG C
Lower ultracentrifugation can obtain vesica distribution layer outside the cellule more concentrated than other three kinds of schemes, focus primarily upon the 6th, 7,8,9
Layer.
Embodiment 2
The comparison of different differential centrifugation scheme combination Tris-HCL (improved method) sucrose heavy aqueous solution density gradient centrifugations
1, the extraction of the outer vesica of urine cellule
Test example a:30% sucrose cushions method
Thick vesica is obtained according to differential centrifugation described in embodiment 1 at normal temperature, thick vesica is resuspended in 6ml 20mm
Tris-HCL water (H2O) in solution (PH 8.6), using tack needle injection successively to test tube bottom fill thick vesica re-suspension liquid,
30% and 60% 20mm Tris-HCL sucrose heavy aqueous solution (PH 8.6) each 1ml, 25 DEG C are centrifuged under 110,000g centrifugal force
After 5 hours, the sucrose heavy aqueous solution layer addition for taking out 1ml 30% surpasses from pipe, uses 20mm Tris-HCL aqueous solution (PH
8.6) it is diluted to 8ml, 200,000g is centrifuged 1 hour at 4 DEG C, is precipitated as the sample rich in extracellular vesica.
Test example b: differential centrifugation combines and saltouts and improve density-gradient centrifugation method under room temperature
(1) differential centrifugation, which combines to saltout, extracts thick vesica
Thick vesica is extracted according to differential centrifugation described in embodiment 1 at normal temperature, difference is after 2,000g is centrifuged,
Taking supernatant that sodium chloride is added makes its concentration 0.58M, is placed at room temperature for 2 hours, 2,000g centrifugations 20 minutes, after obtaining supernatant
The centrifugation of 16,000g is carried out again.
(2) extraction of the outer vesica of urine cellule
The thick vesica that step (1) obtains is extracted into the outer vesica of urine cellule by the following method: thick vesica is resuspended in
20mm Tris-HCL (improved method) water (H of 3ml PH 8.62O) in solution, using tack needle injection successively to test tube bottom
20mm Tris-HCL (improved method) each 1ml of sucrose heavy aqueous solution of the pH8.6 of filling 5%, 10%, 20%, 40%, 60%, will
Thick vesica re-suspension liquid is slowly added in configured good gradient liquid top layer (see attached drawing 1) using sample loading gun, 110 at 25 DEG C,
After being centrifuged 5 hours under 000g centrifugal force, vesica enriched layer outside cellule is taken out into addition and is surpassed from pipe, the 20mm of pH8.6 is used
Tris-HCL aqueous solution is diluted to 8ml, and 200,000g is centrifuged 1 hour at 4 DEG C respectively, obtains sediment.
Differential centrifugation combines improvement density-gradient centrifugation method at c:4 DEG C of test example
Thick vesica is extracted according to differential centrifugation described in embodiment 1 at 4 DEG C, and according to described in test example b step (2)
Method extracts the outer vesica of cellule.
Test example d: differential centrifugation combines improvement density-gradient centrifugation method under room temperature (see attached drawing 4A)
Thick vesica is extracted according to differential centrifugation described in embodiment 1 at normal temperature, and according to test example b step (2) institute
It states method and extracts the outer vesica of cellule.
Differential centrifugation combination 30kd ultrafiltration and improvement density-gradient centrifugation method at e:4 DEG C of test example
(1) differential centrifugation combination ultrafiltration extracts thick vesica
Thick vesica is extracted according to differential centrifugation described in embodiment 1 at 4 DEG C, difference is after 2,000g is centrifuged,
Take supernatant be added 30kd super filter tube in, under 2,000g centrifugal force by supernatant be concentrated 10 times, then carry out 16,000g from
The heart.
(2) extraction of the outer vesica of urine cellule
The outer vesica of cellule is extracted according to test example b step (2) the method.
Test example f: differential centrifugation combination 30kd ultrafiltration and improvement density-gradient centrifugation method under room temperature
(1) differential centrifugation combination ultrafiltration extracts thick vesica
Thick vesica is extracted according to differential centrifugation described in embodiment 1 at normal temperature, difference is after 2,000g is centrifuged,
Take supernatant be added 30kd super filter tube in, under 2,000g centrifugal force by supernatant be concentrated 10 times, then carry out 16,000g from
The heart.
(2) extraction of the outer vesica of urine cellule
The outer vesica of cellule is extracted according to test example b step (2) the method.
Differential centrifugation combination 100kd ultrafiltration and improvement density-gradient centrifugation method at g:4 DEG C of test example
(1) differential centrifugation combination ultrafiltration extracts thick vesica
Thick vesica is extracted according to differential centrifugation described in embodiment 1 at 4 DEG C, difference is after 2,000g is centrifuged,
It takes supernatant to be added in the super filter tube of 100kd, supernatant is concentrated 10 times under 2,000g centrifugal force, then carry out 16,000g's
Centrifugation.
(2) extraction of the outer vesica of urine cellule
The outer vesica of cellule is extracted according to test example b step (2) the method.
Test example h: differential centrifugation combination 100kd ultrafiltration and improvement density-gradient centrifugation method under room temperature (see attached drawing 4B)
(1) differential centrifugation combination ultrafiltration extracts thick vesica
Thick vesica is extracted according to differential centrifugation described in embodiment 1 at normal temperature, difference is after 2,000g is centrifuged,
It takes supernatant to be added in the super filter tube of 100kd, supernatant is concentrated 10 times under 2,000g centrifugal force, then carry out 16,000g's
Centrifugation.
(2) extraction of the outer vesica of urine cellule
The outer vesica of cellule is extracted according to test example b step (2) the method.
The content of the test and operating condition of above-mentioned test example a~h see the table below.
2, the inspection of the outer vesica of urine cellule
The outer vesica (see attached drawing 5) of cellule that every group of test example obtains, transmission electron microscope picture are observed using transmission electron microscope (TEM)
Middle filled arrows show TH albumen, and hollow arrow shows the extracellular vesica with the adherency of TH albumen.Result of study shows Tris-HCL not
Gradient of continuous density is centrifuged (test example b~h), and than the centrifugation of 30%Tris sucrose cushions is used alone, (test example a) can be removed more
TH albumen.
Vesica total protein content outside the cellule that every group of test example obtains, Western Blot mirror are detected using coomassie method
(see attached drawing 6A), more every group of excretion body marker protein difference, adjustable resistance pulse sense (TRPS) and count every group of nanometer fixed figure
Grain quantity variance, the nano particle number as a result extracted with every milliliter of urine volume calculate (see attached drawing 6B).With the cellule extracted
The outer vesica purity of the nano particle number and total protein concentration ratio calculation cellule of outer vesica sample (see attached drawing 6C).The result shows that
TEM transmission electron microscope results (see attached drawing 5) and Western Blot qualification result (see attached drawing 6A) display, b and d are in room temperature for test example
Lower differential centrifugation without combining ultrafiltration, can extract more extracellular vesicas and vesica marker Alix, TSG101 and
Syntenin-1, TRPS nano particle count results (see attached drawing 6B) show test example d at normal temperature differential centrifugation without combine
The maximum amount of nano particle number can be obtained in the case where ultrafiltration and salting out method, therefore, test example d is that the outer vesica maximum of cellule produces
It measures extraction scheme (its flow chart is shown in attached drawing 4A)；With nano particle number/total protein concentration calculated purity (see attached drawing 6C) as a result, it has been found that,
Differential centrifugation combination 100kd ultrafiltration can obtain the outer vesica of cellule of maximum purity to test example h at normal temperature, be cellule external capsule
Steep maximum purity extraction scheme (its flow chart is shown in attached drawing 4B).
Claims (8)
1. a kind of improvement extracting method of the outer vesica of urine cellule, it is characterised in that carry out as follows density gradient from
The heart: the sucrose solution of various concentration gradient is formed from addition test tube bottom is successively padded down to high sequence from upper according to concentration
And the gradient liquid that lower concentration successively increases, thick vesica re-suspension liquid is slowly filled in gradient liquid top layer, is centrifuged；
Wherein the sucrose solution is the 20mm Tris-HCL sucrose heavy aqueous solution of pH8.4~8.8, the thick vesica re-suspension liquid
It is to be made in the 20mm Tris-HCL aqueous solution for be resuspended in thick vesica pH8.4~8.8.
2. the improvement extracting method of the outer vesica of urine cellule according to claim 1, it is characterised in that the sucrose is molten
Liquid is the 20mm Tris-HCL sucrose heavy aqueous solution of pH8.6, and the thick vesica re-suspension liquid is that thick vesica is resuspended in pH8.6
It is made in 20mm Tris-HCL aqueous solution.
3. the improvement extracting method of the outer vesica of urine cellule according to claim 1, it is characterised in that the gradient liquid
Top-down concentration is followed successively by 5%, 10%, 20%, 40% and 60% in test tube.
4. the improvement extracting method of the outer vesica of urine cellule according to claim 3, it is characterised in that the centrifugation is
It is centrifuged 5 hours at 25 DEG C.
5. the improvement extracting method of the outer vesica of urine cellule described in any one of -4 according to claim 1, it is characterised in that
The thick vesica is to extract urine sample by differential centrifugation at normal temperature to obtain.
6. the improvement extracting method of the outer vesica of urine cellule according to claim 5, it is characterised in that the differential from
The supernatant obtained during the heart is by being concentrated by ultrafiltration.
7. the improvement extracting method of the outer vesica of urine cellule according to claim 6, it is characterised in that the ultrafiltration is
It is concentrated by ultrafiltration in 100kd super filter tube.
8. the improvement extracting method of the outer vesica of urine cellule according to claim 7, it is characterised in that the method packet
Include following steps:
(1) differential centrifugation extracts thick vesica: under room temperature, urine sample being centrifuged with 500g, goes to precipitate, supernatant is with 2,000g
Centrifugation, goes to precipitate, and supernatant is added in 100kd super filter tube, and supernatant is concentrated 10 times under 2,000 centrifugal force, then will be dense
Liquid after contracting goes to precipitate with 16,000g centrifugation, and supernatant uses ultracentrifuge with 200,000g centrifugal force 1 hour,
The thick vesica of sediment is obtained, thick vesica is resuspended in the 20mm Tris-HCL aqueous solution of PH8.6 and obtains thick vesica re-suspension liquid；
(2) density gradient centrifugation extracts the outer vesica of cellule: successively filling concentration to test tube bottom using tack needle injection
5%, 20mm Tris-HCL each 1ml of sucrose heavy aqueous solution of 10%, 20%, 40%, 60% pH8.6, by thick vesica re-suspension liquid
Slowly be added in configured good gradient liquid top layer, 25 DEG C with 110,000g centrifugal force 5 hours after, gradient liquid from upper and
Under be divided into several layers, take out the layer that the outer vesica of cellule is enriched with, be separately added into and surpass from pipe, use the 20mm Tris-HCL of pH8.6
Aqueous solution is diluted to 8ml, and 200,000g is centrifuged 1 hour at 4 DEG C respectively, obtains sediment.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111398008A (en) * | 2020-03-11 | 2020-07-10 | 易春 | Method for quickly and quantitatively concentrating extracellular vesicle sample |
| 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 |
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