CN106929467B - Method and kit for separating high-purity urine exosomes - Google Patents
Method and kit for separating high-purity urine exosomes Download PDFInfo
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Abstract
The invention provides a method for separating high-purity urine exosomes, which is characterized by comprising the following steps of: step one, centrifuging a urine sample, discarding a precipitate, and collecting a supernatant; filtering the supernatant by using a filter head, and collecting filtrate; step three, putting the filtrate obtained in the step two into a dialysis bag for dialysis to obtain an exosome; step four, carrying out ultrafiltration on the exosomes obtained in the step three in an ultrafiltration tube to finally obtain concentrated urine exosomes. The invention also provides a kit for separating the high-purity urine exosomes. Compared with the conventional urine exosome separation method, the method has the advantages of low cost, simplicity in operation, high yield and purity, complete exosome structure and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method and a kit for separating high-purity urine exosomes.
Background
Exosomes (exosomes) are membrane vesicles of about 30-150nm in diameter secreted by many cells and containing various proteins and RNAs, and function to transfer information between cells. Different cells may secrete specific proteins and RNAs. The study showed that urine also contains exosomes.
One difficulty in the study of urine exosomes is the extraction and separation of exosomes from urine. The existing methods for separating exosomes mainly include the following methods:
the first method is to use different ultracentrifugation speeds (Th ry C et al curr protocol Cell Biol, 2006; 3: 1-29) or density gradient centrifugation (Tauro BJ et al methods 2012; 56: 293-.
The second method is ultrafiltration (Merchant ML et al. proteomics-Clin Appl 2010; 4: 84-96), although it is simple to operate. However, the obtained exosomes were not of high purity (Danqi Wang and Wei Sun, Proteomics, 2014; 14, 1922-.
The third method is a commercial kit, and the method can be divided into two types, one is adding an extraction reagent into a sample, and centrifuging to obtain a precipitate of exosomes (Lobb RJ et al J excell vehicles 2015; 4:27031), but the method has the defects that the purity of the extracted exosomes is not enough and the kit is expensive. Another method is to add the sample to a column (Bo AN et al.J. excell vehicles 2014; 3:23430), during the process of passing through the column, impurities such as protein in the sample enter the column, and the exosomes with larger particle sizes flow out of the column first, so that the exosomes with higher purity can be obtained more quickly. But the cost is too high to limit the application of this method.
The fourth method is to precipitate exosomes by using cheap polyethylene glycol (Rider MA et al. sci Rep 2016; 6:23978), which is similar to the principle of commercial exosome extraction kit, and has the advantages of low extraction cost and low purity of the extracted exosomes.
Therefore, a urine exosome separation method with the advantages of low cost, simple operation, high yield and purity, complete exosome structure and the like is lacked at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to establish a method for separating high-purity urine exosomes, which has the advantages of low cost, simple operation, high yield and purity, complete exosome structure and the like. In order to achieve the purpose, the invention adopts the following technical scheme, which comprises the following steps:
step one, centrifuging a urine sample, removing a precipitate, and collecting a supernatant;
step two, filtering the supernatant obtained in the step one by using a filter head, and collecting filtrate;
step three, putting the filtrate obtained in the step two into a dialysis bag for dialysis to obtain an exosome;
step four, carrying out ultrafiltration on the exosomes obtained in the step three in an ultrafiltration tube to finally obtain concentrated urine exosomes.
Further, the urine sample in step one is fresh urine, -20 ℃ frozen preserved urine and/or-80 ℃ frozen preserved urine.
Further, the time of centrifugation in the first step is 5min, and the rotation speed is 3000 rpm.
Further, the size of the filter head for filtering the supernatant in the second step is 0.22 micron.
Further, the specification of the dialysis bag used in the third step is 300kDa and/or the pore diameter of the dialysis bag is 25 nm-30 nm.
Further, in the third step, the dialysis solution is PBS or 0.9% normal saline, the dialysis time is 9 hours, and the fresh dialysis solution is replaced every 3 hours.
Further, the volume ratio of the filtrate to the dialysate used in step three was 1: 100-300. Preferably, the volume ratio of the filtrate to the dialysate used in step three is 1: 200. For example, in step three, the filtrate is 10mL, and the dialysate is 2L.
Further, the size of the ultrafiltration tube used in the fourth step is 100kDa to 200kDa, and preferably the size of the ultrafiltration tube is 100 kDa. The ultrafiltration conditions were 3000rpm for 5 min.
Another objective of the present invention is to provide a kit for separating high-purity urine exosomes, comprising a filter head, a dialysis bag and an ultrafiltration tube; the specification of the filter head is 0.22 micron, the specification of the dialysis bag is 300kDa and/or the pore diameter of the dialysis bag is 25 nm-35 nm, and the specification of the ultrafiltration tube is 100 kDa-200 kDa. Preferably, the dialysis bag is a dialysis bag manufactured by Spectrum (spectral medicine) of America, and the specification of the dialysis bag is 300kDa, and the specification of the ultrafiltration tube is 100 kDa. And the dialyzate is PBS or 0.9% physiological saline.
Further, the present invention also includes a specification describing the method for isolating high-purity urine exosomes as described above.
Further, the method for separating high-purity urine exosomes is used according to the method for separating high-purity urine exosomes.
Compared with the prior art, the invention has the following beneficial effects:
(1) the present invention does not require expensive equipment.
(2) The method is simple and easy to operate.
(3) The consumables used in the invention are low in price, and in order to further reduce the test cost, the dialysis bag and the ultrafiltration bag
The filter tube can be reused after being cleaned.
(4) The invention can process a plurality of samples simultaneously.
(5) The exosome obtained by the invention has high purity and less impurity protein pollution.
(6) The exosome obtained by the invention has a complete structure.
As the grain diameter of the exosome is 30-150 nanometers, the aperture of the 300kDa dialysis bag adopted by the invention is converted into about 30 nanometers compared with the aperture given by a dialysis bag company (http:// plasma. com/dialysis/PoreSize. html), thereby not only ensuring that impurities smaller than 30 nanometers and heteroproteins smaller than 300kDa can be removed more quickly, but also ensuring that the exosome can not be dialyzed out. Therefore, almost all exosomes in the sample can be obtained, and the aim of obtaining high-yield exosomes is fulfilled.
Drawings
FIG. 1 is a transmission electron micrograph of urine exosomes.
FIG. 2 is the Western Blot results for urine exosomes.
Fig. 3 is a measurement result of the particle size of urine exosomes.
Detailed Description
The reagent formula of the invention is as follows:
PBS：KCl 200mg/L；NaCl 8g/L；KH2PO4 200mg/L；Na2HPO4 1.5g/L。
0.9% physiological saline: NaCl 8 g/L.
The invention is further described below with reference to the figures and examples.
Example 1
Firstly, centrifuging the freshly obtained urine at 3000rpm for 5min to obtain a precipitate, namely cells in the urine, removing the precipitate, and taking a supernatant.
And secondly, filtering the supernatant obtained in the step one through a 0.22-micron filter head to remove larger impurities.
And thirdly, taking 10mL of the filtrate obtained in the step two, and adding the filtrate into a 300kDa dialysis bag (American Spectrum company). The PBS was dialyzed for 9 hours and replaced every 3 hours for a total of 2L. The hetero-proteins and other molecules are dialyzed out, leaving the exosomes.
And fourthly, adding the exosomes in the third step into a 100kDa ultrafiltration tube, and carrying out ultrafiltration for 5min at 3000rpm so as to concentrate the volume of the exosomes.
The obtained urine exosomes are characterized by a Transmission Electron Microscope (TEM), a Western Blot and a qNano, and specific characterization results are respectively as follows:
(1) transmission Electron Microscope (TEM) photograph:
the characterization means of the exosome purity is mainly a TEM electron microscope, and if the exosome purity is insufficient, a lot of impurities exist later.
TEM pictures were taken of the urine exosomes obtained in this example, see FIG. 1. As shown in figure 1, in the visual field range, the urine exosomes are clear and complete, and only a few small white dots (namely, heteroproteins) are arranged around the urine exosomes, which indicates that the urine exosomes obtained by the method have high purity and complete exosome structures.
(2) Western Blot detection:
in Western Blot detection of urine exosomes obtained in this example, it can be known from FIG. 2 that urine exosomes express exosome marker proteins TSG101 and CD 9.
(3) qNano detection exosome particle size analysis:
in this example, a qNano picture of urine exosomes is obtained, and it can be seen from fig. 3 that the average particle size of the urine exosomes obtained by the present invention is 102nm ± 23 nm.
Example 2
Firstly, centrifuging the freshly obtained urine at 3000rpm for 5min to obtain a precipitate, namely cells in the urine, removing the precipitate, and taking a supernatant.
And secondly, filtering the supernatant obtained in the step one through a 0.22-micron filter head to remove larger impurities.
And thirdly, collecting the filtrate obtained in the step two, and freezing and storing the filtrate at-80 ℃ for 3 days.
Fourthly, melting the urine frozen in the third step at room temperature
Fifthly, taking 10mL of the melted urine obtained in the fourth step, and adding the 10mL of the melted urine into a 300kDa dialysis bag. Dialysis was performed for 9 hours in 2L PBS, and PBS was changed every 3 hours. The hetero-proteins and other molecules are dialyzed out, leaving the exosomes.
Sixthly, adding the exosome in the step five into a 100kDa ultrafiltration tube, and carrying out ultrafiltration for 5min at 3000rpm so as to concentrate the volume of the exosome.
Example 3
Firstly, centrifuging the freshly obtained urine at 3000rpm for 5min to obtain a precipitate, namely cells in the urine, removing the precipitate, and taking a supernatant.
And secondly, filtering the supernatant obtained in the step one through a 0.22-micron filter head to remove larger impurities.
And thirdly, taking 10mL of filtrate obtained in the step two, and adding the filtrate into a 300kDa dialysis bag. Dialyzed against 2L of 0.9% saline for 9 hours, and replaced with 0.9% saline every 3 hours. The hetero-proteins and other molecules are dialyzed out, leaving the exosomes.
And fourthly, adding the exosomes in the third step into a 100kDa ultrafiltration tube, and carrying out ultrafiltration for 5min at 3000rpm so as to concentrate the volume of the exosomes.
For examples 2 and 3, the obtained exosomes were characterized using the present invention using Transmission Electron Microscopy (TEM), Western Blot, qNano. The detection results are similar and are not described herein again.
The invention utilizes a dialysis method, which can dialyze out the hybrid protein and other molecules and keep the exosome in the dialysis bag. Obtain the exosome with high yield and purity and complete structure. The method for obtaining the urine exosome has the advantages of low cost, simple operation and high exosome purity and yield.
Example 4
The kit for separating the high-purity urine exosomes in the embodiment comprises a filter head, a dialysis bag, an ultrafiltration tube and an instruction; the size of the filter head is 0.22 micron, the size of the dialysis bag is 300kDa, and the size of the ultrafiltration tube is 100 kDa. The specification describes a method for isolating high-purity urine exosomes and a formulation of PBS and/or 0.9% normal saline as described in any one of examples 1 to 3.
Example 5
The kit for separating the high-purity urine exosomes in the embodiment comprises a filter head, a dialysis bag, dialysate, an ultrafiltration tube and an instruction; the size of the filter head is 0.22 micron, the size of the dialysis bag is 300kDa, and the size of the ultrafiltration tube is 100 kDa. The dialysate was PBS or 0.9% normal saline. The specification describes a method for isolating high-purity urine exosomes as described in any of examples 1-3.
The above detailed description of the present invention is provided only for the purpose of illustrating the technical concepts and features of the present invention, and is intended to enable those skilled in the art to understand the present invention and implement the present invention, and not to limit the scope of the present invention. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (4)
1. A method for separating high purity urine exosomes, comprising the steps of:
step one, centrifuging a urine sample, removing a precipitate, and collecting a supernatant;
step two, filtering the supernatant obtained in the step one by using a filter head, and collecting filtrate;
step three, putting the filtrate obtained in the step two into a dialysis bag for dialysis to obtain an exosome;
wherein the specification of the dialysis bag used in the third step is 300kDa and/or the pore diameter of the dialysis bag is 25 nm-30 nm;
the dialysate in the third step is PBS or 0.9% normal saline, the dialysis time is 9 hours, and fresh dialysate is replaced every 3 hours;
step four, carrying out ultrafiltration on the exosomes obtained in the step three in an ultrafiltration tube to finally obtain concentrated urine exosomes.
2. The method for separating high purity urine exosomes according to claim 1, wherein the time of centrifugation in the first step is 5min, and the rotation speed is 3000 rpm.
3. The method for separating high purity urine exosomes according to claim 1, wherein the size of the filter head for filtering supernatant in the second step is 0.22 micron.
4. The method for separating high-purity urine exosomes according to claim 1, wherein the specification of the ultrafiltration tube used in the fourth step is 100-200 kDa, and the ultrafiltration condition is 3000rpm for 5 min.
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| 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 |
| 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 |
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