CN116396923A - Extraction method of mouse testis tissue exosome - Google Patents
Extraction method of mouse testis tissue exosome Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
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- C—CHEMISTRY; METALLURGY
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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 invention discloses a method for extracting mouse testis tissue exosomes, which comprises the following steps: (1) Fresh mouse testis tissue is taken and washed 3 times by a PBS solution which is precooled at 4 ℃ and contains 1% of green-streptomycin, and the tissue is sheared on ice; (2) Placing the sheared tissues in a complete culture medium without exosomes for culture; (3) collecting the culture supernatant; (4) removing tissue fragments from the supernatant by centrifugation; (5) Centrifuging the supernatant to remove cell debris in the supernatant; (6) Taking the centrifuged supernatant, and filtering and sterilizing the supernatant by a 0.22 mu m filter; (7) centrifuging the filtrate to obtain exosome precipitate; (8) suspending the precipitate in pre-chilled PBS for washing; (9) Centrifuging the exosome suspension to obtain purified exosome; (10) The exosome pellet was resuspended in pre-chilled PBS to give an exosome suspension. The method can efficiently extract high-concentration and high-purity exosomes from the supernatant of the testis tissue culture of the mice.
Description
[ field of technology ]
The invention belongs to the technical field of biology, and particularly relates to a method for extracting mouse testis tissue exosomes.
[ background Art ]
Exosomes (Exosomes) are tiny vesicles with lipid bilayer membranes secreted by most cells in the body, approximately 30-150nm in diameter. Exosomes, which are membrane vesicles commonly found in body fluids, can participate in signal communication and substance exchange between cells, mediate physiological and pathological processes, and may play an important role in testes. Exosome inclusion has a wide variety of components such as miRNA, protein, mRNA, lncRNA, etc. The exosomes can specifically recognize target cells and transfer these components into receptor cells, and regulate the biological activity of the receptor cells through the components such as miRNAs carried by the exosomes, and participate in physiological or pathological processes such as cell differentiation, cell migration, immune response, tumor invasion and the like.
As exosome studies have increased, so has related studies of exosomes derived from various cell sources. However, at present, the extraction method of exosomes at home and abroad is mainly focused on cells, and the extraction method of exosomes derived from testis tissue needs to be further discussed.
[ invention ]
Aiming at the problem that the research on the extraction method of the testicular tissue source exosomes is relatively less in the prior art, the invention provides the extraction method of the mouse testicular tissue exosomes, which can extract and purify the exosomes to the greatest extent.
The aim of the invention is achieved by the following technical scheme:
the extraction method of the mouse testis tissue exosome comprises the following steps:
1) Taking a fresh mouse testis tissue sample, washing the fresh mouse testis tissue sample with a pre-cooled PBS solution containing 1% (v/v) of green-streptomycin for 3 times, and cutting the tissue on ice;
2) Placing the sheared tissue in a complete culture medium with 1% (v/v) of green-streptomycin without exosomes for culture;
3) Collecting culture supernatant;
4) Removing tissue fragments from the supernatant by a first centrifugation;
5) Taking supernatant obtained by the first centrifugation, performing the second centrifugation, and removing cell fragments in the supernatant by the second centrifugation;
6) Taking supernatant obtained by the second centrifugation, and filtering and sterilizing by a 0.22 mu m filter;
7) Centrifuging the filtrate obtained in the previous step for the third time to obtain exosome precipitate;
8) Suspending the exosome precipitate obtained in the previous step in precooled PBS for washing to obtain exosome suspension;
9) Performing fourth centrifugation on the exosome suspension obtained in the previous step to obtain a purified exosome;
and (3) resuspending the exosome precipitate in precooled PBS to obtain exosome suspension, and carrying out exosome WB, electron microscopy and particle size identification.
In the invention, the following components are added:
culturing the strain in a complete medium without exosomes of 1% (v/v) of the green-streptomycin, wherein the culture conditions are as follows: 37 ℃,5% CO 2 Culturing for 4h.
The first centrifugation in the step 4) is carried out under the following conditions: centrifuge at 1000g for 5min at 4 ℃.
The second centrifugation in the step 5) is carried out under the following centrifugation conditions: centrifuge at 10000g for 10min at 4 ℃.
The third centrifugation in the step 7) is carried out under the following centrifugation conditions: centrifuge at 100000g for 70min at 4 ℃.
Precooling as described in step 8), preferably at 4 ℃.
The fourth centrifugation in the step 9) is carried out under the following centrifugation conditions: centrifuge at 100000g for 70min at 4 ℃.
Compared with the prior art, the invention has the following advantages:
the extraction method of the mouse testis tissue exosome has strong operability, and truly solves the problems that the exosome extraction from tissue sources is difficult, the exosome extraction reagent is expensive, time-consuming, the product purity is low and the like. Therefore, the method of the invention greatly improves the extraction quality and extraction efficiency of exosomes.
[ description of the drawings ]
FIG. 1 is a sediment chart of exosomes extracted by the method for extracting exosomes from testis tissue of a mouse according to the embodiment of the invention;
FIG. 2 is a diagram showing the WB results of a method for extracting mouse testis tissue exosomes according to the example of the present invention;
FIG. 3 is an exosome electron microscope image extracted by the method for extracting the exosome of the testis tissue of the mouse according to the embodiment of the invention;
fig. 4 is a graph showing the particle size of exosomes extracted by the method for extracting exosomes from mouse testis tissue according to the embodiment of the present invention.
[ detailed description ] of the invention
The following describes the invention in more detail with reference to examples. The following examples are intended to illustrate the invention and are not intended to be limiting. The experimental examples were conducted in accordance with the conventional methods without specifying the specific conditions.
Example 1:
the extraction method of the mouse testis tissue exosome comprises the following specific extraction steps:
(1) Taking a fresh mouse testis tissue sample, washing the fresh mouse testis tissue sample with a pre-cooled PBS solution containing 1% (v/v) of green-streptomycin for 3 times, and cutting the tissue on ice;
(2) Mouse testis tissue supernatant acquisition: placing the sheared tissue in 1% (v/v) blue-streptomycin exosome-free complete medium for culture, placing 3ml culture solution into each 1g tissue, placing at 37deg.C, 5% CO 2 Culturing in an incubator for 4 hours;
(3) Collecting culture supernatant after 4 hours, centrifuging for 5 minutes at 4 ℃ and 1000g, transferring the supernatant into another centrifuge tube, and removing tissue fragments;
(4) Taking the supernatant, performing secondary centrifugation at 4 ℃ and 10000g for 10 minutes, transferring the supernatant into another centrifuge tube, and removing cell debris;
(5) Filtering and sterilizing the supernatant obtained by the second centrifugation with a 0.22 μm filter, and collecting the filtrate into a super-high speed centrifuge tube;
(6) Centrifuging the filtrate for a third time at 4deg.C and 100000g for 70min, and removing supernatant to obtain exosome precipitate;
(7) Adding precooled PBS into a super-high speed centrifuge tube, blowing exosome sediment by using a 1000ul gun head, re-suspending the exosome in PBS solution, and washing the exosome;
(8) Centrifuging the exosome suspension for the fourth time at 4deg.C and 100000g for 70min, wherein yellowish precipitate appears at the bottom of the ultra-high speed centrifuge tube to obtain purified exosome (figure 1);
(9) Blowing 1ml of precooled PBS (phosphate buffer solution) to precipitate exosomes to obtain 1ml of exosome heavy suspension, wherein CD9 and CD63 are common markers of the exosomes, and the WB result (figure 2) and an exosome electron microscope image (figure 3) extracted by the extraction method show that the exosomes are successfully extracted;
(10) The diameter of the membrane vesicle exosomes secreted by living cells was about 30 to 150nm, and it can be seen from Table 1 that the average particle diameter of exosomes was 86nm and the average concentration was 1.64×10 10 particle/mL, the grain size diagram (figure 4) of exosomes extracted by the extraction method of the invention shows that most of exosomes have grain sizes in the range of 60-100 nm, which indicates that the exosomes extracted by the extraction method of the invention have higher purity and concentration.
FIG. 1 is a precipitation chart of exosomes extracted by the method for extracting exosomes from mouse testis tissue according to the present example;
FIG. 2 is a diagram showing the WB results of a method for extracting mouse testis tissue exosomes according to the present example;
fig. 3 is an exosome electron microscope image extracted by the method for extracting the exosome of the testis tissue of the mouse according to the embodiment;
fig. 4 is a graph showing the particle size of exosomes extracted by the method for extracting exosomes from mouse testis tissue according to the present example.
Table 1:
sample name average particle size (nm) concentration (Particles/mL)
Mice 86.0.1.64×10 10
Example 2:
the extraction method of the mouse testis tissue exosome comprises the following steps:
1) Taking a fresh mouse testis tissue sample, washing the fresh mouse testis tissue sample with a pre-cooled PBS solution containing 1% (v/v) of green-streptomycin for 3 times, and cutting the tissue on ice;
2) The sheared tissues are placed in a complete medium without exosomes for 1% (v/v) of green-streptomycin for culture under the following conditions: 37 ℃,5% CO 2 Culturing for 4h;
3) Collecting culture supernatant;
4) Tissue fragments in the supernatant were removed by a first centrifugation under the following conditions: centrifuging at 4deg.C at 1000g for 5min;
5) Taking supernatant obtained by the first centrifugation, performing the second centrifugation, removing cell fragments in the supernatant by the second centrifugation, and centrifuging at 4 ℃ for 10min at a rotating speed of 10000 g;
6) Taking supernatant obtained by the second centrifugation, and filtering and sterilizing by a 0.22 mu m filter;
7) Centrifuging the filtrate obtained in the previous step for the third time to obtain exosome precipitate, and centrifuging at 100000g at 4deg.C for 70min;
8) Suspending the exosome precipitate obtained in the previous step in precooled PBS for washing to obtain exosome suspension;
9) And (3) carrying out fourth centrifugation on the exosome suspension obtained in the step to obtain a purified exosome, wherein the centrifugation conditions are as follows: centrifuging at 100000g at 4deg.C for 70min;
and (3) resuspending the exosome precipitate in precooled PBS to obtain exosome suspension, and carrying out exosome WB, electron microscopy and particle size identification.
Comparative example 1:
the existing extraction method of mouse testis tissue exosomes adopts an ultrafiltration method, and comprises the following steps:
(1) Grinding testis tissue of a mouse, diluting the tissue with a trehalose solution with 3-5 times of volume, and shaking and mixing for 10min.1000g, centrifuging at 25 ℃ for 10min, and collecting a supernatant;
(2) Filtering the supernatant with a 0.8um filter head, and then filtering with a 0.22nm filter head;
(3) Adding into a ultrafiltration tube, centrifuging for 10-20min, concentrating to 100ul, and collecting concentrated solution as exosome working solution.
Comparative example 2:
the existing exosome extraction method adopts size exclusion chromatography, and comprises the following steps:
(1) Grinding testis tissue of a ground mouse, adding 3-5 times of a volume of a pbs buffer solution, uniformly mixing, 1000g, centrifuging at 4 ℃ for 10min, and collecting a supernatant;
(2) Centrifuging the supernatant twice at 10000g for 10min at 4deg.C, collecting supernatant, and filtering with 0.22nm filter head;
(3) The filtrate flows through the chromatographic column, and substances with the size larger than the pore diameter of the gel particles cannot enter the pore diameter, and the exosome working solution is eluted.
Results and summary:
1. from a comparison of example 1 and comparative example 1, it can be seen that the method of comparative example 1 needs to be carried out in combination with centrifugation, but the greatest problem of comparative example 1 is that the retentate on one side of the filter membrane is continuously accumulated during the filtration process, so that the filtration efficiency is lower and lower, and in addition, cholesterol close to the size of the exosomes is also retained, thus affecting the purity of the product.
2. By comparing example 1 with comparative example 2, it can be seen that the eluent of the method of comparative example 2 dilutes the exosomes, resulting in lower concentrations; furthermore, the method of comparative example 2 is a widely accepted technique for separating exosomes in body fluids, is not used for extraction of tissue exosomes, and is time-consuming and unsuitable for large-scale sample processing.
The invention has been described in further detail in connection with the specific embodiments, but the invention is not limited thereto. Variations or substitutions of technical features of the present technical solution will be considered to be within the scope of the present invention by those of ordinary skill in the art to which the present invention pertains without departing from the inventive concept.
Claims (6)
1. The extraction method of the mouse testis tissue exosome is characterized by comprising the following steps: the method comprises the following steps:
1) Taking a fresh mouse testis tissue sample, washing the fresh mouse testis tissue sample with a pre-cooled PBS solution containing 1% v/v of green-streptomycin at 4 ℃ for 3 times, and cutting the tissue on ice;
2) Placing the sheared tissues in a complete culture medium without exosomes of 1% v/v green-streptomycin for culture;
3) Collecting culture supernatant;
4) Removing tissue fragments from the supernatant by a first centrifugation;
5) Taking supernatant obtained by the first centrifugation, performing the second centrifugation, and removing cell fragments in the supernatant by the second centrifugation;
6) Taking supernatant obtained by the second centrifugation, and filtering and sterilizing by a 0.22 mu m filter;
7) Centrifuging the filtrate obtained in the previous step for the third time to obtain exosome precipitate;
8) Suspending the exosome precipitate obtained in the previous step in precooled PBS for washing to obtain exosome suspension;
9) Performing fourth centrifugation on the exosome suspension obtained in the previous step to obtain a purified exosome;
and (3) resuspending the exosome precipitate in precooled PBS to obtain exosome suspension, and carrying out exosome WB, electron microscopy and particle size identification.
2. The method for extracting mouse testis tissue exosomes of claim 1, wherein: culturing the strain in a complete culture medium without exosomes of 1% v/v green-streptomycin, wherein the culture conditions are as follows: 37 ℃,5% CO 2 Culturing for 4h.
3. The method for extracting mouse testis tissue exosomes of claim 1, wherein: the first centrifugation in the step 4) is carried out under the following conditions: centrifuge at 1000g for 5min at 4 ℃.
4. The method for extracting mouse testis tissue exosomes of claim 1, wherein: the second centrifugation in the step 5) is carried out under the following centrifugation conditions: centrifuge at 10000g for 10min at 4 ℃.
5. The method for extracting mouse testis tissue exosomes of claim 1, wherein: the third centrifugation in the step 7) is carried out under the following centrifugation conditions: centrifuge at 100000g for 70min at 4 ℃.
6. The method for extracting mouse testis tissue exosomes of claim 1, wherein: the fourth centrifugation in the step 9) is carried out under the following centrifugation conditions: centrifuge at 100000g for 70min at 4 ℃.
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