CN108225873A - A kind of composition for extracting urine excretion body and its preparation method and application - Google Patents
A kind of composition for extracting urine excretion body and its preparation method and application Download PDFInfo
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- CN108225873A CN108225873A CN201611155679.7A CN201611155679A CN108225873A CN 108225873 A CN108225873 A CN 108225873A CN 201611155679 A CN201611155679 A CN 201611155679A CN 108225873 A CN108225873 A CN 108225873A
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- excretion body
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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
- G01N1/34—Purifying; Cleaning
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Abstract
The invention discloses a kind of compositions for extracting excretion body, it is made of the component of following weight proportion：0.5 1.5 parts of polyethylene glycol；0.183 0.548 parts of sodium chloride.The composition and reagent of present invention extraction excretion body can high efficiency extraction excretion body, high income, obtained product purity is high, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to a kind of compositions for extracting urine excretion body and its preparation method and application.
Background technology
Exosome (excretion body) is the film property vesica that the diameter of living cells secretion is about 40-100nm, is naturally occurring in body
In liquid, including blood, saliva, urine and breast milk.Exosome is carried comprising cytosol albumen, is participated in Intracellular signals turn
Albumen, various metabolic enzymes, heat shock protein and the four transmembrane proteins and specific proteins led participate in the important tune of cellular activity
Control is expected to the early diagnosis marker as a variety of diseases.Exosome is as gene and drug carrier, tumor and swollen
Have become hot spot in terms of knurl biological study.
The method of the excretion body of extraction separation at present is various, including ultracentrifugation, density gradient centrifugation, immunomagnetic beads, ultrafiltration
Or the methods of kit, these methods respectively have advantage and disadvantage, supercentrifugation needs ultracentrifuge, and process is relatively time-consuming, and returns
Yield is unstable (may be related with rotor type), and purity is also under suspicion；In addition, repeated centrifugation operation is it is also possible to vesica
It damages, so as to reduce its quality.The excretion body purity that density-gradient centrifugation method obtains is higher, but complex steps, takes.It is super
Filter centrifugal process is simple and efficient, and do not influence the bioactivity of excretion body, is a kind of new method for extracting cell excretion body.Paramagnetic particle method
Have many advantages, such as that specificity is high, easy to operate, it is complete not influence excretion volume morphing, but efficiency is low, excretion body bioactivity is easy
It is influenced by pH and salinity, is unfavorable for downstream experiment, it is difficult to widely available.Kit is expensive, is not suitable for extensive detection.
It needs to be improved existing method, a kind of excretion body extracts reagent of low cost, easy to use is provided.
Invention content
The object of the present invention is to provide a kind of new excretion body extraction composition and methods.
The composition of present invention extraction excretion body, it is made of the component of following weight proportion：Polyethylene glycol 0.8-1.2
Part；0.292 part of sodium chloride.
Preferably, it is made of the component of following weight proportion：0.8 part of polyethylene glycol；0.292 part of sodium chloride.
The present invention also provides a kind of reagents for extracting excretion body, it is to be combined as active constituent with aforementioned, in addition can connect
The reagent that the auxiliary material or complementary ingredient received are prepared.
Wherein, the reagent is aqueous solution.
The present invention also provides a kind of preparation method for preparing aforementioned reagent, step is as follows：It is weighed according to aforementioned proportioning
Component adds water, mixing, you can.
The present invention also provides the purposes of aforementioned composition or reagent in excretion body is extracted.
The present invention also provides a kind of methods for extracting excretion body, include the following steps：
(1) aforementioned composition is taken, adds water, solution is made in mixing；
(2) measuring samples are taken, centrifuges, takes supernatant；
(3) solution of step (1) is added in the supernatant of step (2), mixing, reacts more than 12h, supernatant is abandoned in centrifugation, is received
Collection precipitation, you can.
In the solution that step (1) is prepared, a concentration of 0.16~0.24g/ml of polyethylene glycol.
It is described centrifugal to be centrifuged 10 minutes under the conditions of 4 DEG C, 2000g in step (2).
In step (3), the temperature of reaction is 4 DEG C；Centrifugation is to centrifuge 1h under the conditions of 4 DEG C, 3580g.
The present invention is using the method for particular composition and reagent extraction excretion body, high income, the product purity being prepared
Also it is higher while easy to operate, ultracentrifugation is not needed to, application easy to spread is of low cost, can be developed into diagnostic kit,
Diagnose and treat for various diseases provides basis.
The present invention is described in further details below by specific embodiment, but is not the limit to the present invention
System, the above according to the present invention, according to the ordinary technical knowledge and customary means of this field, not departing from, the present invention is above-mentioned
Under the premise of basic fundamental thought, the modification, replacement or change of other diversified forms can also be made.
Description of the drawings
The Electronic Speculum qualification result of Fig. 1 excretion bodies
The Electronic Speculum qualification result of Fig. 2 excretion bodies
The Electronic Speculum qualification result of Fig. 3 excretion bodies
The western blot testing results of Fig. 4 excretion bodies
Specific embodiment
The preparation of 1 excretion body extracts reagent of the present invention of embodiment
Taking polyethylene glycol 0.8g；Sodium chloride 0.292g adds distilled water, mixing, constant volume to 5ml, you can.
The preparation of 2 excretion body extracts reagent of the present invention of embodiment
Taking polyethylene glycol 1.0g；Sodium chloride 0.292g adds distilled water, mixing, constant volume to 5ml, you can.
The preparation of 3 excretion body extracts reagent of the present invention of embodiment
Taking polyethylene glycol 1.2g；Sodium chloride 0.292g adds distilled water, mixing, constant volume to 5ml.
The extraction separation method of 4 excretion body of the present invention of embodiment
1st, experimental method
A. 0.8 part of polyethylene glycol, 0.292 part of sodium chloride add in distilled water mixing, and constant volume to 5ml is prepared into preparation.
B. freshly voided urine is collected, 4 DEG C of 2000g are centrifuged 10 minutes, -80 DEG C of refrigerators of supernatant are taken to freeze spare.
C. the solution prepared by bis- step of a, b mixes, and overturns mixing repeatedly, 4 DEG C of refrigerator cold-storages overnight (or place 12h with
On).
D.4 DEG C 3580g centrifugation 1h, abandon supernatant, collect precipitation.
2nd, it detects
The Electronic Speculum identification of excretion body and western blot detections.
The Electronic Speculum identification of 2.1 excretion bodies
2.1.1 method
A. 10 μ l of pipette samples are added dropwise in precipitating 1min on copper mesh；
B. filter paper sucks supernatant liquid；
C. uranium acetate 10 μ l are drawn to be added dropwise in precipitating 1min on copper mesh；
D. filter paper sucks supernatant liquid；
E. several minutes of air drying, upper machine；
F.80kv-120kv it is imaged.
2.1.2 as a result, below figure 1- Fig. 3, the saucer shape vesica of 40-100 μm of diameter is seen under mirror.
The western blot detections of 2.2 excretion bodies
2.2.1 method
A collects protein sample (Protein sample preparation)
A, it plus in 200 μ L SDS buffer solutions to excretion body particle, is vortexed 15 seconds；
B, it is placed at room temperature for 5min (reach complete cracking).
B electrophoresis (Electrophoresis)
A, PAGE gel is prepared；
B, sample treatment
The SDS-PAGE albumen sample-loading buffers concentrated in right amount are added in the protein sample of collection.100 DEG C or boiling water bath
Heating 3-5 minutes, with abundant albuminate.
C, loading and electrophoresis
After being cooled to room temperature, protein sample is directly loaded in SDS-PAGE glue well.One glue is examined
Mas bright blue dyes, another glue transferring film, carries out subsequent experimental.
C transferring films (Transfer)
D closes (Blocking)
To contain the Tris buffer salines+0.05%Tween (TBS-T) of 5% dry milk closings 1h.
E primary antibodies are incubated (Primary antibody incubation)
With excretion body specific antibody (CD63 and CD9), with 1:1000 dilutions (5% dry milk in TBS-T), 4 DEG C of incubations
Blot overnight.It is rinsed 3 times with TBS-T.
F secondary antibodies are incubated (Secondary antibody inucubation)
With goat-anti rabbit HRP antibody, with 1:20,000 dilutions (5% dry milk in TBS-T) are incubated at room temperature 1h.With TBS-
T is rinsed 3 times.
G-protein detects (Detection of proteins)
Trace is incubated with chemiluminescent substrate, develops or is shown on other vision facilities on film.
2.2.2 result such as Fig. 4, the visible CD63 bands at 43kDa, the visible CD9 bands at 26KDa.Internal reference albumen β-
Actin is in 42kDa or so appearance.
2.2.4 yield and purity
By detection, the yield of the method for the present invention is 2.42 ± 0.48 particles/μ l Urine specimens；Gained particle is through low concentration
The purity of product that polyethylene glycol washs or short time ultracentrifugation is prepared is higher than 1.0 × 1010Particle number/μ g albumen.
To sum up, the present composition and reagent can be with high efficiency extraction excretion body, high income, the product purities being prepared
Height, and of low cost, simple and convenient extraction, application prospect are good.
Claims (10)
1. a kind of composition for extracting excretion body, it is characterised in that：It is made of the component of following weight proportion：Polyethylene glycol
0.8-1.2 parts；0.292 part of sodium chloride.
2. pharmaceutical composition according to claim 1, it is characterised in that：It is made of the component of following weight proportion：
0.8 part of polyethylene glycol；0.292 part of sodium chloride.
3. a kind of reagent for extracting excretion body, it is characterised in that：It is to combine as active constituent, add described in claims 1 or 2
The reagent that upper acceptable auxiliary material or complementary ingredient are prepared.
4. reagent according to claim 3, it is characterised in that：The reagent is aqueous solution.
5. a kind of preparation method for preparing the reagent described in claim 3 or 4, it is characterised in that：Step is as follows：It will according to right
1 or 2 proportionings is asked to weigh component, add water, mixing, you can.
6. purposes of the reagent described in composition or claim 3 or 4 in excretion body is extracted described in claims 1 or 2.
A kind of 7. method for extracting excretion body, it is characterised in that：Include the following steps：
(1) composition described in claims 1 or 22 is taken, adds water, solution is made in mixing；
(2) measuring samples are taken, centrifuges, takes supernatant；
(3) solution of step (1) is added in the supernatant of step (2), mixing, reacts more than 12h, supernatant is abandoned in centrifugation, and it is heavy to collect
It forms sediment, you can.
8. according to the method described in claim 7, it is characterized in that：In the solution that step (1) is prepared, polyethylene glycol it is dense
It spends for 0.16~0.24g/ml.
9. according to the method described in claim 7, it is characterized in that：It is described centrifugal in 4 DEG C, 2000g conditions in step (2)
Lower centrifugation 10 minutes.
10. according to the method described in claim 7, it is characterized in that：In step (3), the temperature of reaction is 4 DEG C；Centrifugation is 4
DEG C, centrifuge 1h under the conditions of 3580g.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611155679.7A CN108225873A (en) | 2016-12-14 | 2016-12-14 | A kind of composition for extracting urine excretion body and its preparation method and application |
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| CN201611155679.7A CN108225873A (en) | 2016-12-14 | 2016-12-14 | A kind of composition for extracting urine excretion body and its preparation method and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108148809A (en) * | 2017-09-18 | 2018-06-12 | 兰州大学 | A kind of method that excretion body is detached in the supernatant from tumour cell |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103717284A (en) * | 2011-06-08 | 2014-04-09 | 新加坡科技研究局 | Purification of biological products by constrained cohydration chromatography |
| CN105087546A (en) * | 2015-08-18 | 2015-11-25 | 张灏 | Exosome extraction kit and application thereof in liquid biopsy of diseases including tumors |
-
2016
- 2016-12-14 CN CN201611155679.7A patent/CN108225873A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103717284A (en) * | 2011-06-08 | 2014-04-09 | 新加坡科技研究局 | Purification of biological products by constrained cohydration chromatography |
| CN105087546A (en) * | 2015-08-18 | 2015-11-25 | 张灏 | Exosome extraction kit and application thereof in liquid biopsy of diseases including tumors |
Non-Patent Citations (1)
| Title |
|---|
| WWWJJJ1328: "（"PEGwj2016.3.30血清外囊泡（外泌体）提取方案"", 《百度文库：HTTPS://WENKU.BAIDU.COM/VIEW/F38F8E389EC3D5BBFC0A748D.HTML》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108148809A (en) * | 2017-09-18 | 2018-06-12 | 兰州大学 | A kind of method that excretion body is detached in the supernatant from tumour cell |
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