CN107304413A - A kind of excretion body quick separating and the kit of purifying - Google Patents
A kind of excretion body quick separating and the kit of purifying Download PDFInfo
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- CN107304413A CN107304413A CN201610259316.1A CN201610259316A CN107304413A CN 107304413 A CN107304413 A CN 107304413A CN 201610259316 A CN201610259316 A CN 201610259316A CN 107304413 A CN107304413 A CN 107304413A
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- excretion body
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- excretion
- quick separating
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- 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
- C12N5/0693—Tumour cells; Cancer cells
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- 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
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- 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
- C12N5/0634—Cells from the blood or the immune system
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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Abstract
The present invention relates to biological technical field, the kit of a kind of excretion body quick separating and purifying, including solution I, II, III and purification column are specifically disclosed, totally four parts are constituted, and the specifically used method of kit is also disclosed in addition.The kit of the present invention is reasonable in design, and processing, easy to operate and saving time and reagent cost need not be centrifuged repeatedly compared with existing Ultracentrifugation Method.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of excretion body quick separating and the kit of purifying.
Background technology
Excretion body (exosome) is the vesica corpusculum secreted by a variety of living cells, wherein a variety of containing protein and RNA etc.
Component, diameter is mostly between 30~100 nm.Under an electron microscope, it is seen that excretion body is wrapped up by phospholipid bilayer molecule,
Form is in flat or spherical corpusculum, and some are cup-shaped；Its existence form in body fluid, generally can be in sugarcane based on spherical structure
The g/ml of sucrose density gradient solution Midst density 1.13~1.19 scope is enriched with.It is now recognized that excretion body can be by all kinds
Cell secretion, the late endosomal risen in cell endocytic system.
Excretion body has very high abundance in the body fluid such as peripheral blood, urine, saliva, ascites, amniotic fluid, and different tissues come
The excretion body in source has differences in composition and function aspects, while this species diversity is adjusted by the dynamic of extracellular matrix and microenvironment
Control.Tumour source or the related excretion body of tumour are the important mechanisms that modulate tumor develops, the analysis to tumour excretion body
Can be with the early diagnosis, therapeutic evaluation and prognostic analysis of adjuvant therapy with detection.In addition, excretion body and its modification processing product are also
Can be as gene or the effective carrier of medicine, for oncotherapy.
The most frequently used excretion body means of purification is surpassed from method, separates using low-speed centrifugal, high speed centrifugation alternately
The close vesica particle of size.Surpass from method because simple to operate, the vesica quantity of acquisition is more and very popular, but process compares expense
When (more than 20 hours), and the rate of recovery is unstable, and purity is also under suspicion；In addition, repeated centrifugation operation is it is also possible to vesica
Cause damage, so as to reduce its quality.
The excretion body of this patent isolates and purifies kit and prepared by the principle of macromolecular filtering post, be it is a can be fast
Speed efficiently extracts exosome kit from serum, blood plasma, cells and supernatant, and the kit has following features：Behaviour
Make simple, can complete to extract and purify without ultracentrifugation, in 1 ~ 2 hour；Purity is high, and enriching quantity is big, from 2 ~ 4ml cells
200 ~ 400ng excretions body protein or 50-200ng excretion bodies RNA can be obtained in clear liquid；Cost is low, and stability is good, is readily transported,
It is easy to preserve；The extracting and purifying of excretion body are carried out suitable for the body fluid such as serum, urine, saliva, ponding and cell culture fluid.
The content of the invention
A kind of excretion body quick separating and the kit of purifying, including solution I, II, III and purification column, totally four part group
Into the system or quantity of every kind of composition are as follows：
| Specification | 10rnx | 40rnx |
| Solution I | 2.5 ml | 10 ml |
| Solution II | 2.5 ml | 10 ml |
| Solution III | 10 ml | 20 ml x 2 |
| Purification column | 10 | 40 |
Solution I, II, III after operation described below according to can efficiently separate excretion body, then be purified by purification column, can
To obtain the excretion body particle of high-purity.
Kit of the present invention can be used for the excretion body of the body fluid such as blood, urine, saliva, hydrops and cell culture fluid to lift
And purifying.
Kit of the present invention can be used for the excretion body of the mammalian body fluid such as people source, rat, mouse, rabbit lift with it is pure
Change.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York: Cold Spring Harbor
Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated,
Otherwise percentage and number are percentage by weight and parts by weight.
1. sample pre-treatments：To avoid the interference of foreign protein in serum, it is proposed that use serum free medium culture 48 hours
After regather cell supernatant.For the faster cell of growth rate, it is proposed that the cell supernatant after collection is trained with serum-free
Support base and carry out 1:2 dilution.Such as need to detect the cell of large-scale culture（Such as suspension cell）, it is proposed that first it is diluted to 1*
10^5 cells/ml, then sample and detected.
2. operating procedure
1) 2ml cell supernatants are collected；
2) with 3,000 × g, 4 °C of centrifugation 15min, to remove residual cell or cell fragment；
3) supernatant is transferred in clean 10ml glass tubes, and as standby on ice；
4) a clean 10ml glass tube separately is taken, according to the mixed liquor of following proportions solution I, II, III；
| Culture supernatant volume | Mixed liquor cumulative volume | Solution I | Solution II | Solution III |
| 2 ml | 0.75 ml | 0.125 ml | 0.125 ml | 0.5 ml |
| 3 ml | 1.125 ml | 0.187 ml | 0.187 ml | 0.75 ml |
| 4 ml | 1.5 ml | 0.25 ml | 0.25 ml | 1 ml |
5) 5 ~ 10s is shaken, is mixed；
6) 0.75ml above-mentioned mixed liquor is added into every 2ml supernatants；
7) lid is covered, in acutely shaking 30s on oscillator, 4 DEG C are incubated 30min；Sample through processing would generally be formed 3 layers or
2 layers of solution, sop up upper strata or lower floor's solution
8) remaining sample is transferred in 1.5ml centrifuge tube, 3min is centrifuged with 5000x g, sample will continue to show 3 layers, abandon
Fall supernatant and lower floor's solution；
9) remaining sample is transferred in 0.5ml centrifuge tube, repeats the above steps 8 once；
10) centrifugation lid is opened, 10min is placed and treats that nature dries；
11) 1 × PBS of 4 times of precipitation volumes is added, precipitation is resuspended in piping and druming repeatedly, is completely dispersed it, in incubation on horizontal shaker
10min（At a high speed, one replication piping and druming can be carried out again halfway）；
12) 5min is centrifuged with 5000x g, supernatant is sucked out as in purification column（Careful operation, should not be drawn onto precipitation）, remain
It is remaining to be deposited in 4 DEG C of reservations；
13) purification column is centrifuged into 5min with 1000x g, collects obtained filtered solution；
14) solution obtained by above-mentioned 13 is the excretion body for being dissolved in PBS, can continue on for follow-up test or temporary in 4 DEG C, such as
It need to preserve for a long time please in -80 DEG C of refrigerators, it is proposed that used in 3 months.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Liquid to be checked for the present invention is not particularly limited, and can be the mammals such as people source, rat, mouse, rabbit
Body fluid, can also be said animal cell nutrient solution.
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (3)
1. a kind of excretion body quick separating and the kit of purifying, it is characterised in that can be rapidly and efficiently using the kit
Extract and purifying excretion body.
2. a kind of excretion body quick separating and the kit of purifying, it is characterised in that the kit has following characteristics：
(a) four kinds of different components are carried, are solution I, solution II, solution III and purification column respectively；
(b) extraction of excretion body can be carried out for liquid such as blood, urine, saliva, feedwater, cell culture fluids；
(c) it is simple to operate, it can complete to extract and purify without ultracentrifugation, in 1 ~ 2 hour；
(d) the excretion body purity obtained is high, and enriching quantity is big, and 200 ~ 400ng excretion bodies can be obtained from 2 ~ 4ml cell supernatants
Albumen or 50-200ng excretion bodies RNA；
(e) reagent stability is good, can be transported in normal temperature, is easy to preserve.
3. a kind of excretion body quick separating and the kit of purifying, it is characterised in that the kit can be for Ren Yuan, big
The mammalian body fluids such as mouse, mouse, rabbit and its cell culture fluid can carry out the extraction of excretion body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610259316.1A CN107304413A (en) | 2016-04-25 | 2016-04-25 | A kind of excretion body quick separating and the kit of purifying |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610259316.1A CN107304413A (en) | 2016-04-25 | 2016-04-25 | A kind of excretion body quick separating and the kit of purifying |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107304413A true CN107304413A (en) | 2017-10-31 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201610259316.1A Withdrawn CN107304413A (en) | 2016-04-25 | 2016-04-25 | A kind of excretion body quick separating and the kit of purifying |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111840646A (en) * | 2020-08-03 | 2020-10-30 | 北京诺赛启研再生医学研究院有限公司 | Stem cell composition for filling chest fat and application thereof |
| CN113046303A (en) * | 2021-04-09 | 2021-06-29 | 上海宇玫博生物科技有限公司 | Rapid extraction kit for exosome separation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468642A (en) * | 2013-09-23 | 2013-12-25 | 山西大学 | Method for separating exosome from cell culture medium |
| WO2014145940A2 (en) * | 2013-03-15 | 2014-09-18 | The Translational Genomics Research Institute | Hybridoma clones and monoclonal antibodies to cd9 |
| CN105388055A (en) * | 2015-12-11 | 2016-03-09 | 浙江省肿瘤医院 | Method for separating tumor cell derived-exosomes from urine |
-
2016
- 2016-04-25 CN CN201610259316.1A patent/CN107304413A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014145940A2 (en) * | 2013-03-15 | 2014-09-18 | The Translational Genomics Research Institute | Hybridoma clones and monoclonal antibodies to cd9 |
| CN103468642A (en) * | 2013-09-23 | 2013-12-25 | 山西大学 | Method for separating exosome from cell culture medium |
| CN105388055A (en) * | 2015-12-11 | 2016-03-09 | 浙江省肿瘤医院 | Method for separating tumor cell derived-exosomes from urine |
Non-Patent Citations (2)
| Title |
|---|
| QIAGEN公司: "exoEasy Maxi Kit Handbook", 《EXOEASY MAXI KIT HANDBOOK》 * |
| 王仁峰: "ExoQuick提取人血清外泌体方法改良及比较", 《实用医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111840646A (en) * | 2020-08-03 | 2020-10-30 | 北京诺赛启研再生医学研究院有限公司 | Stem cell composition for filling chest fat and application thereof |
| CN113046303A (en) * | 2021-04-09 | 2021-06-29 | 上海宇玫博生物科技有限公司 | Rapid extraction kit for exosome separation |
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Effective date of registration: 20200904 Address after: Room 302-304, 3 / F, building 2, No.1, Tianmu Road, Jishan street, Yuecheng District, Shaoxing City, Zhejiang Province Applicant after: Shaoxing Youming Biotechnology Co., Ltd Address before: 201203 Shanghai city Pudong New Area Cailun Road No. 1 No. 303 88 Applicant before: SHANGHAI UMIBIO SCIENCE AND TECHNOLOGY Co.,Ltd. |
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Application publication date: 20171031 |