CN107702957A - The method and reagent of excretion body are extracted in a kind of serum - Google Patents
The method and reagent of excretion body are extracted in a kind of serum Download PDFInfo
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- CN107702957A CN107702957A CN201710863183.3A CN201710863183A CN107702957A CN 107702957 A CN107702957 A CN 107702957A CN 201710863183 A CN201710863183 A CN 201710863183A CN 107702957 A CN107702957 A CN 107702957A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The invention discloses a kind of method and reagent that excretion body is extracted from serum.The present invention provides following reagent, polyglycol solution and cohesion amine aqueous solution.Method includes：Partial impurities are removed by centrifugation in the serum being collected into；Then supernatant is shifted and adds polyglycol solution and cohesion amine aqueous solution extracts excretion body.It is an advantage of the current invention that simple to operate, cost is low, and excretion body yield is high and purity is high.
Description
Technical field
The present invention relates to biomedical sector, particularly belongs to a kind of method and reagent for separating serum excretion body.
Background technology
Excretion body is a diameter of 30-150nm vesicles, is selectively packed and discharged by living cells.In the body of human body
In liquid, such as blood, urine and cerebrospinal fluid, rich content.Contain different types of lipid, nucleic acid and albumen in excretion body
Deng these materials can be transported to specific target cell, so as to play corresponding biological function.Therefore, excretion body is thin
Huge effect is played in Intercellular communication and physiology and pathologic process.
Application of the excretion body in disease research field in recent years has been carried out extensively.And the sample that serum is commonly used as clinical diagnosis
This source, and because of its excretion body rich content, thus the research in terms of clinical practice of serum excretion body is deeply being opened
Exhibition.It is reported that it is not yet found that obvious mark can come area among primary sclerotic cholangitis, cholangiocarcinoma and liver cancer
Point different diseases, and Ander Arbelaiz et al. research show (Ander Arbelaiz et al, Hepatology,
doi:10.1002/hep.29291 2017), the serum excretion of primary sclerotic cholangitis, cholangiocarcinoma and liver cancer patient
Body, obvious difference is there are between protein prescription face, this prompting serum excretion body can provide for the diagnosis of follow-up clinical
Certain basis.Hong-Ling Jia et al. research (Hong-Ling Jia et al, Sci.Rep.7,44706；doi:
10.1038/srep44706,2017) then find that miRNAs can be as the candidate molecules mark of diagnosis Kawasaki disease.
At present, the most frequently used extracting method of serum excretion body mainly has three kinds, one is ultracentrifugation method, the second is,
Immunomagnetic beads method, the third is RNA isolation kit, the related serum excretion body provided such as the Thermo companies in the U.S. and SBI companies
Reagent preparation.But three has clearly disadvantageous part, and ultracentrifugation method, the excretion body purity obtained is high, but
Rate is low；Immunomagnetic beads method, corresponding excretion body specific can be captured, but this technology is only that a small number of Zoomlions are public
Department is grasped, and such as U.S. SBI companies, is caused with high costs；RNA isolation kit extracts, it is well known that and the yield of excretion body is high, but
Be its common ground be excretion body purity it is too low, while excretion body is obtained, can also obtain the egg of high abundance in many serum
White matter.
The content of the invention
It is an object of the present invention to provide a kind of reagent for being used to extract serum excretion body.
Reagent provided by the present invention is polyglycol solution and cohesion amine aqueous solution.
Preferably, it is polyglycol solution with condensing being used in combination for amine aqueous solution, the working concentration of polyglycol solution
For 50-250mg/mL, the working concentration for condensing amine aqueous solution is 0.005-0.2mg/mL.
Preferably, it is the aqueous solution that polyglycol solution is polyethylene glycol, the molecular weight of polyethylene glycol is 6000-
10000 dalton.
Preferably, it is to condense salting liquid of the amine aqueous solution for cohesion amine.
The application method of reagent of the present invention, is comprised the following steps that：
Preferably, it is after serum sample is placed in into room-temperature water bath defrosting, 4 DEG C, 3000g, centrifuges 10 minutes, leave and take
Clearly.
Preferably, in the aqueous solution of the PEG20000 in the 500mg/mL that 1/5 volume is added into supernatant, mix.
Preferably, in the salting liquid for the cohesion amine that 1/10 volume (supernatant volume) is added into above-mentioned mixed liquor, mix.
By above-mentioned mixed liquor, 4 DEG C, stand, 30 minutes.
By the mixed liquor after standing, 4 DEG C, 5000g, centrifuge 10 minutes.
Gained precipitation is excretion body.
The reagent of the present invention can not only significantly improve the yield of excretion body, while can substantially reduce the Gao Feng in serum
Spend albumen.For the excretion body that this method obtains compared with Thermo kits, RNA yield is higher, meanwhile, to excretion body mark
Property albumen when being detected, the abundance of target protein is higher, and the abundance of other serum foreign proteins is also significantly less.The present invention provides
Excretion body reagent preparation, simple to operate, cost is low, and yield is high, while can ensure higher purity, it is not necessary to buys in addition
Some remove the kit of high-abundant protein from serum, reduce experimental cost.
Brief description of the drawings
Fig. 1 is that the Research of predicting markers testing result ratio that polyglycol solution extracts excretion body with reagent of the present invention is used alone
Compared with.TSG101 and CD63 is the significant protein molecular in excretion body surface face of the mankind, and it is molten that polyethylene glycol is used alone in swimming lane 1-4 positions
Liquid, 5-8 are to use reagent of the present invention.
Fig. 2 is excretion body mark CD63, CD9 and TSG101 inspection after the serum sample excretion body extracting of separate sources
The result of survey and the testing result of transmission electron microscope.
Fig. 3 is the content that RNA in gained serum excretion body is contrasted with Thermo RNA isolation kits.
Fig. 4 is the feelings for the situation and foreign protein detection that serum excretion body mark is detected compared with Thermo RNA isolation kits
Condition.
Embodiment
Material used, reagent, unless otherwise specified, are commercially obtained in following embodiments.
The size distribution and quantitative measurement of embodiment 1, serum excretion body
The serum sample 2mL mixed is taken, 4 DEG C, 3000g, centrifuges 10 minutes, leaves and takes supernatant.
The serum 1.6mL after centrifugation is taken, in the centrifuge tube for assigning to 8 1.5mL, every pipe 200uL, number consecutively 1,2,
3,4,5,6,7,8.
Into above-mentioned 8 centrifuge tubes, 50uL 500mg/mL PEG20000 solution is sequentially added, is mixed.To volume
Number in 5,6,7,8 centrifuge tube, to continuously add 20uL 0.2mg/mL cohesion amine aqueous solution, mix.
By above-mentioned mixed liquor, 4 DEG C, stand, 30 minutes.
By the mixed liquor after standing, 4 DEG C, 5000g, centrifuge 10 minutes, abandon supernatant.
Centrifuged pellet, as excretion body.
By excretion body be resuspended with 100uLPBS.
8 groups of samples are respectively taken into the 1000 times of dilutions of 20uL re-suspension liquids, then carry out NTA measure, the size distribution feelings of excretion body
For condition as shown in figure 1, it is that the excretion body that polyglycol solution obtains is used alone that Figure 1A, which is No. 1 sample, Figure 1B is that No. 5 samples use
The excretion body that reagent of the present invention obtains；The summarized results of 8 groups of data determinations is as shown in table 1.
Table 1
As a result show, the population that reagent extraction serum excretion body of the present invention obtains carries more than only addition polyglycol solution
The population obtained, the excretion body particle size distribution extracted is in 20-200nm.
Embodiment 2, separate sources serum sample excretion body extracting and index of correlation detection
The serum sample of 4 separate sources is taken, 25 DEG C of water-baths are thawed.4 DEG C, 3000g, centrifuge 10 minutes, leave and take supernatant.Point
200uL is not taken in four centrifuge tubes, and numbering is followed successively by a, b, c, d.50uL 500mg/mL are separately added into four centrifuge tubes
PEG20000 solution, mix；20uL 0.2mg/mL cohesion amine aqueous solution is sequentially added, is mixed.By above-mentioned mixing
Liquid, 4 DEG C, stand, 30 minutes.By the mixed liquor after standing, 4 DEG C, 5000g, centrifuge 10 minutes, abandon supernatant；Gained is heavy after centrifugation
It is excretion body to form sediment, and gained excretion body is resuspended with 100uL 1X PBS.Take 20uL re-suspension liquids to add RIPA lysates to be split
Solution and protein extraction, are then identified excretion body surface face marker protein CD63, CD9, TSG101, and qualification result is as schemed
Shown in 2A；Meanwhile respectively taking 20uL samples to carry out the measure of transmission electron microscope, the method used is phosphotungstic acid negative staining, this method master
To refer to the researcher such as Hong-Ling Jia report (Hong-Ling Jia et al, Scientific RepoRts, 7:
44706, DOI:10.1038/srep44706,2017), specifically include below scheme：Sample is subjected to 10 times of dilutions, after dilution
Take 100uL and dropping in be incubated 4 minutes on copper mesh, afterwards by with sample incubation after copper mesh with 2% 5 points of phosphotungstic acid dyeing
Clock, then copper mesh is placed on filter paper and dried, and with electron microscopic observation, testing result is as shown in Figure 2 B.
As a result show, reagent provided by the invention, be applicable to the excretion body extracting of different serum samples.
Embodiment 3, RNA yield situations after excretion body extracts are contrasted with Thermo methods
Serum mixing sample is taken, 25 DEG C of water-baths are thawed, and centrifugation, take supernatant.Then 1mL samples are respectively taken, use Thermo respectively
RNA isolation kit and the method provided by the present invention carry out excretion body extracting, and gained precipitation is met at into the Shanghai bold and unconstrained biotechnology of uncle afterwards has
Limit company carries out Total RNAs extraction, and for RNA quantitative results as shown in figure 3,3A is Thermo methods, 3B is the method provided by the present invention.
Thermo method operating processes are as follows, and 200uL extracts reagents are added into serum sample, mix；4 DEG C, stand, be incubated
30 minutes；By the liquid after incubation, room temperature, 10000g, centrifuge 10 minutes, abandon supernatant；Gained precipitation is excretion body.
Present invention offer experiment process is as follows, and 250uL 500mg/mL PEG20000 is added into serum sample
The aqueous solution, mix；And then 20uL0.2mg/mL cohesion amine aqueous solutions are added, mix.By above-mentioned mixed liquor, 4 DEG C, stand, 30
Minute；By the mixed liquor after standing, 4 DEG C, 5000g, centrifuge 10 minutes, abandon supernatant, gained precipitation is excretion body.
As a result show, carry out the extracting experiment of excretion body with same sample, the excretion body RNA yield of Thermo methods is not as good as this
Invention provides the RNA yield that reagent place obtains excretion body.
Embodiment 4 and Thermo method contrasting detection excretion body marks and high-abundant protein from serum situation
Prepare single serum sample, after pretreatment, respectively take 200uL to provide experiment according to Thermo methods and the present invention respectively
Method carries out excretion body extracting, and gained excretion body precipitation is resuspended with 100uL PBS；Afterwards, 20uL re-suspension liquids are taken to add respectively
Isometric RIPA lysates carry out protein extraction, and with WB to excretion body marker protein CD63, CD9, TSG101 and blood
Aloof from politics and material pursuits abundance protein ALB and IgG is detected, and testing result is compiled as shown in figure 4, numbering label I is the method provided by the present invention
Number II is Thermo methods.
As a result showing, excretion body extraction agent provided by the invention, the yield of excretion body is higher, meanwhile, serum high abundance
The residual of albumen is less.
Claims (4)
1. a kind of reagent that excretion body is separated from serum, it is characterised in that polyglycol solution is combined with cohesion amine aqueous solution
Use.
2. according to the reagent described in right 1, it is characterised in that the polyglycol solution be polyethylene glycol the aqueous solution, poly- second
The molecular weight of glycol is 6000-10000 dalton, and the working concentration of Aqueous Solutions of Polyethylene Glycol is 50-250mg/mL.
3. according to the reagent described in right 1, it is characterised in that the cohesion amine aqueous solution is the salting liquid of cohesion amine, condenses amine salt
The working concentration of solution is 0.005-0.2mg/mL.
A kind of 4. method that excretion body is separated from serum, it is characterised in that：Comprise the following steps：
A. the aqueous solution of the 500mg/mL of 1/5 volume PEG20000 is added into the serum of certain volume；
B. the salting liquid of the 0.2mg/mL cohesion amine of 1/10 volume (serum sample volume) is added into above-mentioned mixed liquor.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218693A (en) * | 2019-07-01 | 2019-09-10 | 上海晟燃生物科技有限公司 | For extracting reagent combination, kit and the method for excretion body |
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| CN101498732A (en) * | 2008-02-03 | 2009-08-05 | 北京九强生物技术有限公司 | Improved prealbumin detection kit |
| US20150104801A1 (en) * | 2012-04-17 | 2015-04-16 | Life Technologies Corporation | Methods and compositions for exosome isolation |
| WO2016033695A1 (en) * | 2014-09-05 | 2016-03-10 | Exerkine Corporation | Exosome isolation |
| CN105483081A (en) * | 2015-11-13 | 2016-04-13 | 中国人民解放军第二军医大学 | MiRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome |
| US20160108368A1 (en) * | 2014-07-03 | 2016-04-21 | ReCyte Therapeutics, Inc. | Exosomes from clonal progenitor cells |
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101498732A (en) * | 2008-02-03 | 2009-08-05 | 北京九强生物技术有限公司 | Improved prealbumin detection kit |
| US20150104801A1 (en) * | 2012-04-17 | 2015-04-16 | Life Technologies Corporation | Methods and compositions for exosome isolation |
| US20160108368A1 (en) * | 2014-07-03 | 2016-04-21 | ReCyte Therapeutics, Inc. | Exosomes from clonal progenitor cells |
| WO2016033695A1 (en) * | 2014-09-05 | 2016-03-10 | Exerkine Corporation | Exosome isolation |
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| CN105483081A (en) * | 2015-11-13 | 2016-04-13 | 中国人民解放军第二军医大学 | MiRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome |
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| CN110218693A (en) * | 2019-07-01 | 2019-09-10 | 上海晟燃生物科技有限公司 | For extracting reagent combination, kit and the method for excretion body |
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Application publication date: 20180216 |