CN106929467A - The method and kit of a kind of separating high-purity urine excretion body - Google Patents
The method and kit of a kind of separating high-purity urine excretion body Download PDFInfo
- Publication number
- CN106929467A CN106929467A CN201710082704.1A CN201710082704A CN106929467A CN 106929467 A CN106929467 A CN 106929467A CN 201710082704 A CN201710082704 A CN 201710082704A CN 106929467 A CN106929467 A CN 106929467A
- Authority
- CN
- China
- Prior art keywords
- excretion body
- purity
- separating high
- urine excretion
- filter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of method of separating high-purity urine excretion body, it is characterised in that comprise the following steps：Step one, urine sample centrifugation, abandon precipitation, collect supernatant；Step 2, supernatant are filtered with filter, collect filtered fluid；Step 3, the filtered fluid that step 2 is obtained is fitted into dialysis in bag filter obtains excretion body；Step 4, the excretion body that will be obtained in step 3 ultrafiltration in super filter tube finally give the urine excretion body of concentration.The present invention also provides a kind of kit of separating high-purity urine excretion body.The present invention has the advantages that low cost, simple to operate, yield and purity is high and excretion body structural integrity compared to current conventional urine excretion body separation method.
Description
Technical field
The invention belongs to biological technical field, more particularly, to the method and reagent of a kind of separating high-purity urine excretion body
Box.
Background technology
Excretion body (exosome) is that a kind of many cells secernent diameter about 30-150nm includes multiple protein
The film vesica of matter and RNA, the function with the transmission information between cell.Different cells may secrete specific albumen
Matter and RNA.Research shows also to contain excretion body in urine.
A difficult point in the research of urine excretion body is the extraction of excretion body in urine and separate.In the separation that presently, there are
The separation method of excretion body mainly has following several:
First method is using different ultracentrifugation speed (Th é ry C et al.Curr Protoc Cell
Biol,2006；3:1-29) or density gradient centrifugation (Tauro BJ et al.Methods 2012；56:293-304.),
This method is considered as the goldstandard that excretion body is separate, and the purity of the excretion body for the method separate is high, but the method needs
Equipped with expensive Ultracentrifuge and consumptive material, thereby increases and it is possible to destroy excretion body structure (Linares R et al.J
Extracell Vesicles 2015；4:29509).
Second method is ultrafiltration (Merchant ML et al.Proteomics-Clin Appl2010；4:84–
96), the method is although simple to operate.But the purity of the excretion body for obtaining it is not high (Danqi Wang and Wei Sun,
Proteomics,2014；14,1922-1932).
The third method is the kit of commercialization, and this kind of method can be divided into two classes, and a class is to add extraction agent
To in sample, centrifugation obtains precipitation (the Lobb RJ et al.J Extracell Vesicles2015 of excretion body；4:
, but the deficiency that faces of this kind of method is that the excretion body purity extracted is inadequate and kit is expensive 27031).Another kind of method
It is that sample is added to (Bo AN et al.J Extracell Vesicles2014 in pillar；3:23430), by pillar
During, the impurity such as protein in sample can be entered into pillar, and the larger excretion of particle diameter knows from experience the first stream from pillar
Go out, so can be faster obtain purity excretion body higher.But high cost limits the application of this method.
4th kind of method is that excretion body is precipitated (Rider MA et al.Sci using cheap polyethylene glycol
Rep 2016；6:23978), this kind of method and the excretion body extracts kit principle of commercialization are similar, the advantage is that and are extracted into
This is not high, has the disadvantage that the purity of the excretion body for extracting is not high.
Therefore, one is lacked at present has low cost, simple to operate, yield and purity is high and excretion body structural integrity etc. is excellent
The urine excretion body separation method of point.
The content of the invention
For defect of the prior art, the purpose of the present invention is to set up a kind of side of separating high-purity urine excretion body
Method, has the advantages that low cost, simple to operate, yield and purity is high and excretion body structural integrity.In order to achieve the above object, originally
Invention employs following technical scheme, comprises the following steps：
Step one, urine sample centrifugation, abandon precipitation, collect supernatant；
Step 2, the supernatant for obtaining step one are filtered with filter, collect filtered fluid；
Step 3, the filtered fluid that step 2 is obtained is fitted into dialysis in bag filter obtains excretion body；
Step 4, the excretion body that will be obtained in step 3 ultrafiltration in super filter tube finally give the urine excretion body of concentration.
Further, the urine sample in step one is the urine and/or -80 DEG C that freshly voided urine, -20 DEG C of freezen protectives are crossed
The urine that freezen protective is crossed.
Further, the time being centrifuged in step one is 5min, and rotating speed is 3000rpm.
Further, the specification of the filter of filtering supernatant is 0.22 micron in step 2.
Further, the specification of the bag filter used in step 3 be 300kDa and/or bag filter aperture be 25nm~
30nm。
Further, dialyzate is PBS or 0.9% physiological saline in step 3, and dialysis time 9 hours and every 3 hours are more
Change once fresh dialyzate.
Further, filtered fluid and the use volume ratio of dialyzate are 1 in step 3:100-300.Preferably, step 3
Middle filtered fluid is 1 with the use volume ratio of dialyzate:200.Such as, filtered fluid is 10mL in step 3, and dialyzate is 2L.
Further, the specification of the super filter tube used in step 4 is 100kDa~200kDa, it is preferable that the rule of super filter tube
Lattice are 100kDa.Ultra-filtration conditions are 3000rpm, 5min.
It is a further object of the present invention to provide a kind of kit of separating high-purity urine excretion body, including filter, dialysis
Bag and super filter tube；The specification of filter is 0.22 micron, the specification of bag filter be 300kDa and/or bag filter aperture be 25nm~
35nm, the specification of super filter tube is 100kDa~200kDa.Preferably, bag filter is the life of U.S. Spectrum (spectrum medical science) company
The bag filter of product, the specification of bag filter is 300kDa, and the specification of super filter tube is 100kDa.Dialyzate, dialyzate be PBS or
0.9% physiological saline.
Further, separating high-purity urine excretion body as described above is also recite on specification including specification
Method.
Further, the method for separating high-purity urine excretion body is used as described above.
Compared with prior art, the present invention has following beneficial effect：
(1) equipment that the present invention need not be expensive.
(2) the inventive method is simple, easily operated.
(3) present invention consumptive material used is cheap, and in order to further reduce testing expenses, bag filter and super
Chimney filter can be recycled after cleaning.
(4) present invention can simultaneously process multiple samples.
(5) the excretion body purity that the present invention is obtained is high, and foreign protein pollution is few.
(6) the excretion body structural integrity that the present invention is obtained.
Because the particle diameter of excretion body is that, from 30-150 nanometers, the bag filter control bag filter of the 300kDa that the present invention is used is public
Department (http://spectrumlabs.com/dialysis/PoreSize.html) institute be scaled 30 rans to aperture, this
Sample both can guarantee that the impurity less than 30 nanometers and the foreign protein less than 300kDa can faster be got rid of, and excretion body is can guarantee that again not
Can be dialyzed away.Therefore all of excretion body in sample can be almost obtained, the purpose of the excretion body of acquisition high yield is realized.
Brief description of the drawings
Fig. 1 is the transmission electron microscope photo of urine excretion body.
Fig. 2 is the Western Blot results of urine excretion body.
Fig. 3 is the particle diameter measurements of urine excretion body.
Specific embodiment
Agent prescription of the invention：
PBS：KCl 200mg/L；NaCl 8g/L；KH2PO4200mg/L；Na2HPO4 1.5g/L。
0.9% physiological saline：NaCl 8g/L.
The present invention is described further with reference to the accompanying drawings and examples.
Embodiment 1
First, by the urine of fresh acquisition, 3000rpm is centrifuged, 5min, precipitation is the cell in urine, removal precipitation takes
Supernatant.
2nd, the supernatant that will be obtained in step one, is filtered by 0.22 micron of filter, is used to remove larger impurity.
3rd, the bag filter (Spectrum companies of the U.S.) that the filtered fluid 10mL obtained in step 2 is added to 300kDa is taken
In.Dialysed 9 hours in PBS, change a PBS within every 3 hours, PBS consumptions amount to 2L.Foreign protein and other molecules are dialysed
Go out, leave excretion body.
4th, by the excretion body in step 3, add in the super filter tube of 100kDa, 3000rpm ultrafiltration 5min is used to concentrate outward
Secrete body volume.
The present invention is entered using transmission electron microscope (TEM), Western Blot, qNano to the urine excretion body for obtaining
Row is characterized, and embodiments result is as follows respectively：
(1) transmission electron microscope (TEM) photo：
The characterization method of excretion body purity is mainly TEM Electronic Speculum, many impurity after the meeting if excretion body purity is inadequate.
TEM pictures are shot to the urine excretion body that the present embodiment is obtained, Fig. 1 is seen.As shown in figure 1, at it within sweep of the eye,
Urine excretion body complete display, the only little several small particles (i.e. foreign protein) of surrounding, this explanation is urinated by this method
Liquid excretion body purity is high, and excretion body structural integrity.
(2) Western Blot detections：
The urine excretion body Western Blot detections that the present embodiment is obtained, it is known that urine excretion body surface from Fig. 2
Up to excretion body mark albumen TSG101 and CD9.
(3) qNano detections excretion body granularmetric analysis：
The present embodiment obtains the qNano pictures of urine excretion body, the urine excretion that as can be seen from Figure 3 present invention is obtained
Body average grain diameter is 102nm ± 23nm.
Embodiment 2
First, by the urine of fresh acquisition, 3000rpm is centrifuged, 5min, precipitation is the cell in urine, removal precipitation takes
Supernatant.
2nd, the supernatant that will be obtained in step one, is filtered by 0.22 micron of filter, is used to remove larger impurity.
3rd, -80 DEG C of freezen protectives of filtered fluid obtained in collection step two 3 days.
4th, the urine room temperature that will be frozen in step 3 is melted
5th, the thawing urine 10mL obtained in step 4 is taken to be added in the bag filter of 300kDa.Dialyse 9 in 2L PBS
Hour, and change a PBS in every 3 hours.Foreign protein and other molecules are given, excretion body is left.
6th, by the excretion body in step 5, add in the super filter tube of 100kDa, 3000rpm ultrafiltration 5min is used to concentrate outward
Secrete body volume.
Embodiment 3
First, by the urine of fresh acquisition, 3000rpm is centrifuged, 5min, precipitation is the cell in urine, removal precipitation takes
Supernatant.
2nd, the supernatant that will be obtained in step one, is filtered by 0.22 micron of filter, is used to remove larger impurity.
3rd, the filtered fluid 10mL obtained in step 2 is taken to be added in the bag filter of 300kDa.In the physiology salts of 2L 0.9%
Water is dialysed 9 hours, and every 3 hours change 0.9% physiological saline.Foreign protein and other molecules are given, excretion is left
Body.
4th, by the excretion body in step 3, add in the super filter tube of 100kDa, 3000rpm ultrafiltration 5min is used to concentrate outward
Secrete body volume.
For embodiment 2 and 3, the present invention using the present invention using transmission electron microscope (TEM), Western Blot,
QNano is characterized to the excretion body for obtaining.Its testing result is similar to, and repeats no more here.
The present invention can be dialysed away foreign protein and other molecules, and excretion body is retained in using the method for dialysis
In bag filter.Obtain yield and purity is high and excretion body of structural integrity.The present invention uses with low cost, simple to operate and excretion
Body purity and yield method high obtain urine excretion body.
Embodiment 4
The kit of the separating high-purity urine excretion body in the present embodiment includes filter, bag filter, super filter tube and explanation
Book；The specification of filter is 0.22 micron, and the specification of bag filter is 300kDa, and the specification of super filter tube is 100kDa.Remember on specification
Carry the method and PBS and/or 0.9% physiological saline of separating high-purity urine excretion body as described in embodiment any one of 1-3
Formula.
Embodiment 5
The kit of the separating high-purity urine excretion body in the present embodiment includes filter, bag filter, dialyzate, super filter tube
And specification；The specification of filter is 0.22 micron, and the specification of bag filter is 300kDa, and the specification of super filter tube is 100kDa.Dialysis
Liquid is PBS or 0.9% physiological saline.Recite on specification outside the separating high-purity urine as described in embodiment any one of 1-3
The method for secreting body.
The specific embodiment of the present invention described in detail above, only for illustrating technology design of the invention and spy
Point, its object is to allowing those skilled in the art to will appreciate that present disclosure and implementing according to this, can not limit this with this
The protection domain of invention.It should be appreciated that one of ordinary skill in the art just can be with of the invention without creative work
Many modifications and variations are made in design.Therefore, all technical staff in the art are under this invention's idea in prior art
On the basis of by the available technical scheme of logical analysis, reasoning, or a limited experiment, all should be by claims institute
In the protection domain of determination.
Claims (10)
1. a kind of method of separating high-purity urine excretion body, it is characterised in that comprise the following steps：
Step one, urine sample centrifugation, abandon precipitation, collect supernatant；
Step 2, the supernatant for obtaining step one are filtered with filter, collect filtered fluid；
Step 3, the filtered fluid that step 2 is obtained is fitted into dialysis in bag filter obtains excretion body；
Step 4, the excretion body that will be obtained in step 3 ultrafiltration in super filter tube finally give the urine excretion body of concentration.
2. the method for separating high-purity urine excretion body according to claim 1, it is characterised in that in the step one from
The time of the heart is 5min, and rotating speed is 3000rpm.
3. the method for separating high-purity urine excretion body according to claim 1, it is characterised in that mistake in the step 2
The specification for filtering the filter of supernatant is 0.22 micron.
4. the method for separating high-purity urine excretion body according to claim 1, it is characterised in that used in the step 3
To bag filter specification for the aperture of 300kDa and/or the bag filter be 25nm~30nm.
5. the method for separating high-purity urine excretion body according to claim 1, it is characterised in that in the step 3 thoroughly
Analysis liquid is PBS or 0.9% physiological saline, and once fresh dialyzate is changed in dialysis time 9 hours and every 3 hours.
6. the method for separating high-purity urine excretion body according to claim 1, it is characterised in that used in the step 4
The specification of the super filter tube for arriving is 100kDa~200kDa, and ultra-filtration conditions are 3000rpm, 5min.
7. a kind of kit of separating high-purity urine excretion body, it is characterised in that including filter, bag filter and super filter tube；Institute
The specification for stating filter is 0.22 micron, the specification of the bag filter be 300kDa and/or the bag filter aperture be 25nm~
35nm, the specification of the super filter tube is 100kDa~200kDa.
8. the kit of separating high-purity urine excretion body according to claim 7, it is characterised in that also including dialysis
Liquid, the dialyzate is PBS or 0.9% physiological saline.
9. the kit of separating high-purity urine excretion body according to claim 7, it is characterised in that also including explanation
Book, the method that the separating high-purity urine excretion body as described in claim any one of 1-6 is recite on the specification.
10. the kit of separating high-purity urine excretion body according to claim 7, it is characterised in that will according to right
The method of the separating high-purity urine excretion body described in any one of 1-6 is asked to use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710082704.1A CN106929467B (en) | 2017-02-16 | 2017-02-16 | Method and kit for separating high-purity urine exosomes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710082704.1A CN106929467B (en) | 2017-02-16 | 2017-02-16 | Method and kit for separating high-purity urine exosomes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106929467A true CN106929467A (en) | 2017-07-07 |
| CN106929467B CN106929467B (en) | 2021-05-18 |
Family
ID=59423180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710082704.1A Active CN106929467B (en) | 2017-02-16 | 2017-02-16 | Method and kit for separating high-purity urine exosomes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106929467B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439625A (en) * | 2018-10-31 | 2019-03-08 | 上海交通大学 | A kind of preparation method of the controllable scale of NK cell exosomes |
| CN110283776A (en) * | 2019-06-26 | 2019-09-27 | 上海交通大学医学院附属第九人民医院 | A kind of separation method of extracellular vesica |
| CN111269872A (en) * | 2020-01-21 | 2020-06-12 | 汕头大学 | Method for separating scylla paramamosain tissue exosomes |
| CN115109742A (en) * | 2022-05-22 | 2022-09-27 | 上海交通大学 | Clinical grade high-purity exosome separation and purification kit in blood or urine |
| CN118006550A (en) * | 2024-04-07 | 2024-05-10 | 四川天府亨特生命科技有限公司 | Method for extracting exosomes by using cell culture medium |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103900890A (en) * | 2014-03-07 | 2014-07-02 | 南方医科大学第三附属医院 | Method for extracting urinary micro vesicle using nanofilm concentration |
| CN105505854A (en) * | 2016-01-14 | 2016-04-20 | 上海市第六人民医院 | Acquisition method for exosomes derived from human urinary cells and application |
| CN106383227A (en) * | 2008-10-06 | 2017-02-08 | 莫尔豪斯医学院 | Detection of HIV-related proteins in urine |
-
2017
- 2017-02-16 CN CN201710082704.1A patent/CN106929467B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106383227A (en) * | 2008-10-06 | 2017-02-08 | 莫尔豪斯医学院 | Detection of HIV-related proteins in urine |
| CN103900890A (en) * | 2014-03-07 | 2014-07-02 | 南方医科大学第三附属医院 | Method for extracting urinary micro vesicle using nanofilm concentration |
| CN105505854A (en) * | 2016-01-14 | 2016-04-20 | 上海市第六人民医院 | Acquisition method for exosomes derived from human urinary cells and application |
Non-Patent Citations (4)
| Title |
|---|
| RICHARD J. LOBB等: "Optimized exosome isolation protocol for cell culture supernatant and human plasma", 《JOURNAL OF EXTRACELLULAR VESICLES》 * |
| 林韩翡等: "健康成人24h尿液细胞外囊泡定量及大小分布", 《J SOUTH MED UNIV》 * |
| 王晓良: "《应用分子药理学》", 30 September 2015, 中国协和医科大学出版社 * |
| 胡国文 等: "旋转超滤：一种提取细胞外泌体的新方法", 《第二军医大学学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439625A (en) * | 2018-10-31 | 2019-03-08 | 上海交通大学 | A kind of preparation method of the controllable scale of NK cell exosomes |
| CN110283776A (en) * | 2019-06-26 | 2019-09-27 | 上海交通大学医学院附属第九人民医院 | A kind of separation method of extracellular vesica |
| CN111269872A (en) * | 2020-01-21 | 2020-06-12 | 汕头大学 | Method for separating scylla paramamosain tissue exosomes |
| CN111269872B (en) * | 2020-01-21 | 2021-10-29 | 汕头大学 | Method for separating scylla paramamosain tissue exosomes |
| CN115109742A (en) * | 2022-05-22 | 2022-09-27 | 上海交通大学 | Clinical grade high-purity exosome separation and purification kit in blood or urine |
| CN118006550A (en) * | 2024-04-07 | 2024-05-10 | 四川天府亨特生命科技有限公司 | Method for extracting exosomes by using cell culture medium |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106929467B (en) | 2021-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106929467A (en) | The method and kit of a kind of separating high-purity urine excretion body | |
| Leberman | The isolation of plant viruses by means of “simple” coacervates | |
| CN103479682B (en) | Preparation method for plant source active component nano-scale membrane type vesicle | |
| Jirgensons et al. | A short textbook of colloid chemistry | |
| CN108865971A (en) | A kind of separation method and its separator of excretion body | |
| Siakotos et al. | Procedures for the isolation of two distinct lipopigments from human brain: lipofuscin and ceroid | |
| Peyret | A protocol for the gentle purification of virus-like particles produced in plants | |
| CN110540961B (en) | Annexin V-FITC exosome capture affinity magnetic bead, preparation method thereof and method for extracting exosome by using same | |
| CN112899218A (en) | Dual tangential flow filtration system for exosome extraction, and preparation method and application of exosome | |
| CN107523536A (en) | A kind of extracting method of tissue-derived excretion body and application | |
| CN102746412A (en) | Method for extracting momordica charantiap polysaccharide | |
| CN110283776A (en) | A kind of separation method of extracellular vesica | |
| CN114591905A (en) | Method for preparing apoptosis vesicle from human red blood cell and application | |
| CN109439625A (en) | A kind of preparation method of the controllable scale of NK cell exosomes | |
| CN103900890A (en) | Method for extracting urinary micro vesicle using nanofilm concentration | |
| CN107338222A (en) | Excretion body separation method based on lipid molecule probe | |
| CN104984739B (en) | A kind of preparation method and applications of gelatin affinity chromatography medium | |
| CN115109742A (en) | Clinical grade high-purity exosome separation and purification kit in blood or urine | |
| CN114397388A (en) | Urine exosome extraction kit based on combination of PEG precipitation method and SEC column method and application | |
| Prince et al. | Virus-like particles in human plasma and serum: Role of platelet lysosomes | |
| Van der Geest | A method for the purification of polyhedra | |
| CN115873786A (en) | Large-scale extraction method of exosomes in fresh milk | |
| CN112574950A (en) | Method for extracting cell exosomes | |
| US4977091A (en) | Method for preparing phosphatidylinositol from vegetable matter | |
| CN107866561B (en) | Extract solution from aloe separates single size In4The method of Ni nanosphere |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |