CN108414334A - A method of the separating-purifying excretion body from sperm - Google Patents
A method of the separating-purifying excretion body from sperm Download PDFInfo
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- CN108414334A CN108414334A CN201810154782.2A CN201810154782A CN108414334A CN 108414334 A CN108414334 A CN 108414334A CN 201810154782 A CN201810154782 A CN 201810154782A CN 108414334 A CN108414334 A CN 108414334A
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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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Abstract
The method of the present invention provides a kind of from sperm separating-purifying excretion body, the present invention is compared with conventional excretion body separating and extracting process, delete the slow-speed of revolution centrifugation step of 300g, 2000g, it avoids excretion body to be brought into the cell precipitation of centrifugation in next step by stickum as possible, reduces the loss of excretion body.Using two step dilution methods, reduce the loss of excretion body, improves its purity.It is handled overnight using 8%PEG6000, excretion body purity is made further to be promoted.Institute is all made of 0.1 μm of filter filtration treatment using PBS, reduces bringing into for exogenous impurity.Two step filtration methods (0.22 μm of filter filtration treatment) are taken, reduces greater particle size particle and is mixed into.It can obtain that quality is higher by this method and purity is more in line with the sperm source excretion body of requirement of experiment.Under the premise of guaranteeing both quality and quantity to sperm source excretion body, its purity is improved to greatest extent.
Description
Technical field
The method of the present invention relates to a kind of from sperm separating-purifying excretion body.
Background technology
Differential centrifugation is one of the most common technology of excretion body currently used for separation cell conditioned medium or body fluid source, this method
Including following steps：Low-speed centrifugal is to remove cell and Apoptosis fragment；High speed centrifugation is to eliminate big vesica；High speed from
The heart is to precipitate excretion body.Excretion body extraction flow from Beckman companies is as shown in Figure 1.
The viscosity of biological fluid has significant correlation with the purity of the excretion body detached, moreover, highly viscous biology sample
This needs longer ultracentrifugation step and higher centrifugal speed.But longer ultracentrifugation step is easy outside loss
The total amount of body is secreted, higher centrifugal speed is easily destroyed the normal morphology of excretion body.And conventional excretion body extraction flow then without
Method obtains the satisfactory excretion body of purity.Therefore, for the higher biological fluid of viscosity, such as sperm, existing excretion body
Separation method is not well positioned to meet related experiment requirement, and there is an urgent need to a kind of excretion bodies point that can be guaranteed the quality and energy guarantor measures at present
From method of purification.
Invention content
For the higher biological fluid of the viscosity such as sperm, sperm source excretion body when existing excretion body separation method extracts
When the total amount consume that the occurs and not high technical problem of purity, the present invention provides a kind of from sperm separating-purifying excretion body
Method, can obtain that quality is higher by this method and purity is more in line with the sperm source excretion body of requirement of experiment.
To achieve the above object, the technical solution used in the present invention：A kind of side of the separating-purifying excretion body from sperm
Method includes the following steps：
(1) semen sample is diluted using PBS；This step is preliminary dilute in order to make in sperm stoste viscous ingredients obtain
It releases, avoids excretion body to be brought into the cell precipitation of centrifugation in next step as possible, reduce the loss of excretion body.
(2) it by the semen sample after dilution in step (1), is centrifuged with the rotating speed of 4000g under the conditions of 4 DEG C, leaves and takes supernatant
Liquid abandons precipitation；To remove cell and dead cell.
(3) supernatant for obtaining step (2) is centrifuged under the conditions of 4 DEG C with the rotating speed of 12000g, leaves and takes supernatant, it is heavy to abandon
It forms sediment；To remove dead fragment.
(4) supernatant that step (3) obtains is diluted with PBS；Made by stickum attachment with reducing excretion body to the greatest extent
Its purity reduces.
(5) 0.22 μm of filter filtration treatment of supernatant after diluting step (4)；To remove larger diameter impurity
Grain.
(6) supernatant that step (5) obtains is sufficiently mixed with PEG6000, makes the final concentration of 8wt% of PEG6000, then 4
DEG C overnight；
After (7) 4 DEG C are stayed overnight, the mixture that step (6) is obtained is centrifuged under the conditions of 4 DEG C with the rotating speed of 12000g, is left and taken
Precipitation, abandons supernatant；To concentrate, purify sperm source excretion body.
(8) precipitation that step (7) obtains is resuspended with PBS, is centrifuged with the rotating speed of 100000g under the conditions of 4 DEG C；Centrifugation knot
Shu Hou abandons supernatant, leaves and takes precipitation, to remove big vesica；Precipitation is resuspended with PBS again, is placed under the conditions of 4 DEG C；
(9) re-suspension liquid for obtaining step (8) is centrifuged under the conditions of 4 DEG C with the rotating speed of 100000g；After centrifugation, abandon
Supernatant leaves and takes precipitation, which is the sperm source excretion body obtained by separating-purifying.
Preferably, the volume ratio of PBS and semen sample is 3 in the step (1):1.
Preferably, centrifugation time is 15 minutes in the step (2).
Preferably, centrifugation time is 30 minutes in the step (3).
Preferably, the volume ratio of PBS and supernatant is 9 in the step (4):1.
Preferably, PEG6000 is containing the water-soluble of 160g/L PEG6000 and 58.44g/L NaCl in the step (6)
Liquid.
Preferably, centrifugation time is 30 minutes in the step (7).
Preferably, centrifugation time is 70 minutes in the step (8), and standing time is 1 hour.
Preferably, centrifugation time is 70 minutes in the step (9).
The present invention provides a kind of excretion bodies using above method separating-purifying.
The beneficial effects of the present invention are：The present invention compared with conventional excretion body separating and extracting process,
1, the slow-speed of revolution centrifugation step for deleting 300g, 2000g, avoid as possible excretion body by stickum bring into next step from
In the cell precipitation of the heart, the loss of excretion body is reduced.
2, using two step dilution methods, reduce the loss of excretion body, improve its purity.
3, it is handled overnight using 8%PEG6000, excretion body purity is made further to be promoted.
4, institute is all made of 0.1 μm of filter filtration treatment using PBS, reduces bringing into for exogenous impurity.
5, two step filtration methods (0.22 μm of filter filtration treatment) are taken, reduces greater particle size particle and is mixed into.
In conclusion using the method for the present invention, under the premise of guaranteeing both quality and quantity to sperm source excretion body, to greatest extent
Improve its purity.
Description of the drawings
Fig. 1 is the excretion body extraction flow from Beckman companies；
Fig. 2 is the flow chart of present invention method of separating-purifying excretion body from sperm；
Fig. 3 is that refining excretion body ultracentrifugation compares；
Fig. 4 is that refining excretion body protein concentration compares；
Fig. 5 is that refining excretion body Electronic Speculum compares.
Specific implementation mode
The method of the present invention is compared with conventional excretion body separating and extracting process, the method for still continuing to use differential centrifugation, but is directed to
The higher characteristic of semen liquefied duration, has carried out a series of improvement, as shown in Fig. 2, being broadly divided into 9 steps：
1st step：Semen sample is with volume ratio for 1:3 ratio is diluted with PBS (0.1 μm of filter filtration treatment).This step is
In order to make viscous ingredients in sperm stoste obtain preliminarily diluted, excretion body is avoided to be brought into the cell precipitation of centrifugation in next step as possible
In, reduce the loss of excretion body.Through overtesting, when the volume ratio of semen sample and PBS are more than 1:When 3, diluted effect is not achieved
Fruit；When the volume ratio of semen sample and PBS are less than 1:When 3, it is be easy to cause the waste of sperm, follow-up work amount in addition can be caused acute
Increase.
2nd step：Semen sample after dilution is centrifuged 15 minutes with the rotating speed of 4000g under the conditions of 4 DEG C, is left and taken on sample
Clear liquid abandons precipitation, to remove cell and dead cell.
3rd step：Sample supernatant is centrifuged 30 minutes under the conditions of 4 DEG C with the rotating speed of 12000g, sample supernatant is left and taken,
Precipitation is abandoned, to remove dead fragment.
4th step：By sample supernatant again with volume ratio for 1:9 ratio is further with PBS (0.1 μm of filter filtration treatment)
Dilution is made the reduction of its purity by stickum attachment to reduce excretion body to the greatest extent.Through overtesting, find when sample supernatant with
The volume ratio of PBS is more than 1:It is too dense when 9, diluted effect is not achieved；When the volume ratio of semen sample and PBS are less than 1:9
When, follow-up work amount can be caused to increase severely, unsuitable large-scale application is universal.It was found that the volume ratio of sample supernatant and PBS are
1：9 dilution proportions, effect is best, and most suitable, because can be loaded with 1 pipe 50ml centrifuge tubes after dilution, greatly reduces work
It measures.
5th step：By 0.22 μm of filter filtration treatment of the sample supernatant after dilution, to remove larger diameter impurity
Grain.
6th step：By filtered sample supernatant and 2 × PEG6000 (by 16g PEG6000,5.844g NaCl are molten
In 100mL ddH2O is prepared) it is 1 by volume：1 ratio is sufficiently mixed, and makes the final concentration of 8wt% of PEG6000, then 4 DEG C of mistakes
Night.
7th step：After 4 DEG C is stayed overnight, mixed liquor is centrifuged 30 minutes under the conditions of 4 DEG C with the rotating speed of 12000g, precipitation is left and taken,
Supernatant is abandoned, to concentrate, purify sperm source excretion body.
8th step：Will precipitation with PBS (0.1 μm of filter filtration treatment) be resuspended, under the conditions of 4 DEG C with
The rotating speed of 100000g centrifuges 70 minutes.After centrifugation, supernatant is abandoned, leaves and takes precipitation, to remove big vesica.Again will
Precipitation is resuspended with PBS (0.1 μm of filter filtration treatment), is placed 1 hour under the conditions of 4 DEG C.
9th step：It is centrifuged 70 minutes with the rotating speed of 100000g under the conditions of 4 DEG C.After centrifugation, supernatant is abandoned, it is heavy to leave and take
It forms sediment, precipitation is the sperm source excretion body obtained by separating-purifying.
Excretion body is extracted from sperm using conventional excretion body separation method and the method for the present invention, data are by same individual
Obtained by sperm stoste with a same volume.By comparing conventional excretion body separation method and the method for the present invention in sperm source
Excretion body surpasses measures excretion body total protein concentration (as schemed from audio-visual picture (as shown in Figure 3), gained excretion body grain size, BCA methods after the fast heart
Shown in 4) and excretion body Electronic Speculum (as shown in Figure 5), strong evidence the method for the present invention is practical, significant effect.It can from Fig. 3
Go out, sperm source excretion body surpasses from intuitively, the more conventional processing of its color is slightly deep after purification processes, and excretion body is prompted to obtain after the fast heart
Taken amount is more.It is further verified by BCA methods, detects the amount of excretion body total protein under two kinds of different disposals, it can from Fig. 4
Go out, the more conventional processing of gained excretion body total protein concentration is more after purification processes.Gained excretion body is carried out most finally by Electronic Speculum
Intuitive detection, as can be seen from Figure 5 after purification processes, the more conventional processing purity of gained excretion body is obviously improved, the excretion bodily form
State rule is neat, clear-cut as it can be seen that having no apparent impurity adherency.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of method of the separating-purifying excretion body from sperm, which is characterized in that include the following steps：
(1) semen sample is diluted using PBS；
(2) it by the semen sample after dilution in step (1), is centrifuged with the rotating speed of 4000g under the conditions of 4 DEG C, leaves and takes supernatant, abandon
Precipitation；
(3) supernatant for obtaining step (2) is centrifuged under the conditions of 4 DEG C with the rotating speed of 12000g, is left and taken supernatant, is abandoned precipitation；
(4) supernatant that step (3) obtains is diluted with PBS；
(5) 0.22 μm of filter filtration treatment of supernatant after diluting step (4)；
(6) supernatant that step (5) obtains is sufficiently mixed with PEG6000, makes the final concentration of 8wt% of PEG6000, then 4 DEG C of mistakes
Night；
After (7) 4 DEG C are stayed overnight, the mixture that step (6) is obtained is centrifuged under the conditions of 4 DEG C with the rotating speed of 12000g, leaves and takes precipitation,
Abandon supernatant；
(8) precipitation that step (7) obtains is resuspended with PBS, is centrifuged with the rotating speed of 100000g under the conditions of 4 DEG C；Centrifugation terminates
Afterwards, supernatant is abandoned, precipitation is left and taken；Precipitation is resuspended with PBS again, is placed under the conditions of 4 DEG C；
(9) re-suspension liquid for obtaining step (8) is centrifuged 70 minutes under the conditions of 4 DEG C with the rotating speed of 100000g；After centrifugation,
Supernatant is abandoned, precipitation is left and taken, which is the sperm source excretion body obtained by separating-purifying.
2. according to the method described in claim 1, it is characterized in that, the volume ratio of PBS and semen sample is in the step (1)
3:1。
3. according to the method described in claim 1, it is characterized in that, centrifugation time is 15 minutes in the step (2).
4. according to the method described in claim 1, it is characterized in that, centrifugation time is 30 minutes in the step (3).
5. according to the method described in claim 1, it is characterized in that, the volume ratio of PBS and supernatant is 9 in the step (4):
1。
6. according to the method described in claim 1, it is characterized in that, PEG6000 is containing 160g/L in the step (6)
The aqueous solution of PEG6000 and 58.44g/L NaCl.
7. according to the method described in claim 1, it is characterized in that, centrifugation time is 30 minutes in the step (7).
8. according to the method described in claim 1, it is characterized in that, centrifugation time is 70 minutes in the step (8), when placement
Between be 1 hour.
9. according to the method described in claim 1, it is characterized in that, centrifugation time is 70 minutes in the step (9).
10. a kind of excretion body using such as claim 1-9 either method separating-purifyings.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810154782.2A CN108414334A (en) | 2018-02-23 | 2018-02-23 | A method of the separating-purifying excretion body from sperm |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810154782.2A CN108414334A (en) | 2018-02-23 | 2018-02-23 | A method of the separating-purifying excretion body from sperm |
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| CN108414334A true CN108414334A (en) | 2018-08-17 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110003008A1 (en) * | 2008-02-22 | 2011-01-06 | Agency For Science, Technology And Research (A*Star) | Mesenchymal stem cell particles |
| CN105209881A (en) * | 2013-03-13 | 2015-12-30 | 迈阿密大学 | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| CN105779586A (en) * | 2015-12-28 | 2016-07-20 | 四川农业大学 | Method for separating exosomes from animal plasma and for detecting purity |
| CN106544317A (en) * | 2016-11-07 | 2017-03-29 | 四川农业大学 | A kind of method that spermatid and excretion body are isolated and purified from pig semen |
| US20170296626A1 (en) * | 2014-09-05 | 2017-10-19 | Exerkine Corporation | Exosome isolation |
-
2018
- 2018-02-23 CN CN201810154782.2A patent/CN108414334A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110003008A1 (en) * | 2008-02-22 | 2011-01-06 | Agency For Science, Technology And Research (A*Star) | Mesenchymal stem cell particles |
| CN105209881A (en) * | 2013-03-13 | 2015-12-30 | 迈阿密大学 | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| US20170296626A1 (en) * | 2014-09-05 | 2017-10-19 | Exerkine Corporation | Exosome isolation |
| CN105779586A (en) * | 2015-12-28 | 2016-07-20 | 四川农业大学 | Method for separating exosomes from animal plasma and for detecting purity |
| CN106544317A (en) * | 2016-11-07 | 2017-03-29 | 四川农业大学 | A kind of method that spermatid and excretion body are isolated and purified from pig semen |
Non-Patent Citations (1)
| Title |
|---|
| 杨诚 等: "基于PEG6000 富集精液来源外泌体的提取及鉴定", 《南方医科大学学报》, vol. 36, no. 11, pages 1531 - 1535 * |
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