CN111269872A - Method for separating scylla paramamosain tissue exosomes - Google Patents
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Abstract
The invention relates to a method for separating exosome of scylla paramamosain tissue, which mainly comprises S1) tissue extraction; s2) tissue digestion; s3) low-speed centrifugation; s4) low speed centrifugation is performed again; s5) high-speed centrifugation; s6) centrifuging at high speed again; s7) resuspending the pellet; s8) sucrose density gradient centrifugation; s9) ultracentrifugation; s10) ultracentrifugation again; s11) dialyzing by a dialysis bag; s12) filtering with a filter membrane; s13) ultracentrifugation; s14) resuspending the pellet to obtain the isolated exosome product extracted. The method for extracting the scylla paramamosain tissue exosomes combines the methods of differential centrifugation, sucrose density gradient centrifugation and dialysis filtration, is stable and effective, has high purity and simple operation, and does not need special equipment. The obtained exosome has high content and high purity.
Description
Technical Field
The invention relates to the technical field of exosome extraction and separation, in particular to a method for separating exosomes from scylla paramamosain tissues.
Background
Exosomes are a class of lipid vesicles with bilayer membranes in the 30-200nm range in diameter produced from the endosomal membrane filling. These vesicles form within the endosome, which are released exocytosially into the extracellular environment once the endosome fuses with the cell membrane. Exosomes, as cell substitutes secreted by host cells, are widely present in a variety of biological fluids, including plasma, saliva, synovial fluid, sputum, cerebrospinal fluid, lymph fluid, ascites, and the like, and carry a large number of diverse contents, mainly a variety of bioactive molecules, such as specific proteins, signal peptides, lipids, and nucleic acids derived from cells. Extracellular exosomes can be taken up by its neighboring cells, and the substances contained in exosomes will also perform corresponding biological functions, including immune regulation, escape from apoptosis, escape from immune surveillance, drug resistance, etc. within the cell.
Research time in the field of exosomes is short, and the number of species involved is small. Studies on vertebrates have been mainly focused and studies on invertebrates have been less, and have not been reported in crustaceans. Methods for extracting exosomes have been reported to include ultracentrifugation, kit, ultrafiltration, sucrose density gradient centrifugation, and the like, and various methods have respective advantages and disadvantages. The extraction of the capsid exosomes is currently more difficult because there are no stably culturable cell lines. Therefore, the method for obtaining extracellular exosomes by using culture medium to culture cells reported at present is not suitable for the moment. In addition, the exosome rapid extraction kit on the market mainly aims at mammals, and after the test, the subject group finds that the kit is not suitable for crustaceans such as blue crabs. In contrast, although the conventional ultracentrifugation method is simple in procedure, it was found that the simple ultracentrifugation method cannot separate exosomes having high purity when the experimental method was investigated in the subject group. Therefore, the method for preparing and applying the blue crab tissue exosome which is complete, stable, effective, high in purity and simple to operate is established, and has important practical value.
Disclosure of Invention
The invention aims to provide a method for extracting and separating scylla paramamosain tissue exosomes, and aims to solve the problems in the prior art.
A method for separating scylla paramamosain tissue exosomes mainly comprises the following steps:
s1) tissue extraction: taking crab tissue, cutting the tissue into pieces, and placing the pieces into precooled 0.3% trypsin liquid;
s2) tissue digestion: incubating and cracking at 37 ℃ for 20 min; adding pre-cooled 0.03% pancreatin inhibitor to stop reaction;
s3) low speed centrifugation: centrifuging the tissue single cell suspension in the S2) at low speed for 10min by using a low-temperature centrifuge so as to remove tissue and cell components, transferring supernatant in the centrifuge tube to a new tube for later use after centrifugation is finished, and removing precipitates;
s4) low speed centrifugation again: centrifuging the supernatant obtained in S3) at low speed and 4 deg.C for 30min by using low temperature centrifuge to remove dead cells, transferring the supernatant in the centrifuge tube to a new tube after centrifuging, and removing precipitate;
s5) high speed centrifugation: centrifuging the supernatant obtained in S4) at 4 deg.C for 1h with a low temperature centrifuge for eliminating cell debris, transferring the supernatant in the centrifuge tube to a new tube after centrifuging, and discarding the precipitate;
s6) high speed centrifugation again: centrifuging the supernatant obtained in S5) at 4 deg.C for 2h with a low temperature centrifuge to precipitate exosomes, and carefully sucking out the supernatant after centrifugation to retain the precipitate;
s7) resuspending the pellet: resuspending the pellet obtained in S6) with 0.95M sucrose solution;
s8) sucrose density gradient centrifugation: adding 2.0M sucrose solution, 1.3M sucrose solution and the precipitate resuspension obtained in S7) into the ultracentrifuge tube in sequence;
s9) ultracentrifugation: performing ultracentrifugation for 16h at 4 ℃ by using a low-temperature centrifuge, sucking a proper amount of middle layer solution of 1.3M and 0.95M sucrose solution, and re-suspending by using a proper amount of precooled PBS;
s10) ultracentrifugation again: carrying out ultracentrifugation on the upper layer solution obtained in the step S9) for 2h at 4 ℃ by using a low-temperature centrifuge, and carefully sucking out the supernatant after the ultracentrifugation is finished and reserving a precipitate;
s11) dialysis bag dialysis: resuspending the pellet obtained in S10) with precooled PBS, dialyzing for 3-5 h at 4 ℃ by using a dialysis bag with MW 8000-14000;
s12) membrane filtration: resuspending the dialyzed solution obtained in S11) with an appropriate amount of precooled PBS, sucking the resuspended solution with a disposable syringe, and slowly dripping the solution onto a cell filter membrane with the specification of 0.22 μm for filtration;
s13) ultracentrifugation: carrying out ultracentrifugation on the solution filtered in the step S12) for 2h at 4 ℃ by using a low-temperature centrifuge, and carefully sucking supernatant after the ultracentrifugation is finished and reserving a precipitate part;
s14) resuspending the pellet: and (3) using an appropriate amount of PBS to precipitate the heavy suspension obtained in S13), namely the extracted and separated exosome product.
The purpose of superseparation after dialysis is to obtain exosome precipitate after dialysis treatment through superseparation (the volume of liquid in the dialysis process is large, and the concentration of exosome is low).
The purpose of twice super-separation is to further remove foreign protein and achieve the effect of diluting sucrose, and is beneficial to improving the dialysis efficiency.
Further, the speed of the low-speed centrifugation in step 3) is 800 × g, the speed of the low-speed centrifugation in step 4) is 2000 × g, the speed of the high-speed centrifugation in step 5) is 10000 × g, the speed of the high-speed centrifugation in step 6) is 130000 × g, the speed of the ultracentrifugation in step 9) is 200000 × g, the speed of the ultracentrifugation in step 10) is 130000 × g, and the speed of the ultracentrifugation in step 13) is 130000 × g.
Wherein the scissors used in step S2) are aseptic dissecting scissors, and during tissue processing, the tissue is cut into pieces in a sterilized container containing a small amount of 0.3% trypsin solution.
Step S3) and step S4) are performed to sterilize the centrifuge tube used before the low-speed centrifugation.
The ultracentrifuge tubes used in steps S5), S6), S9), S10), and S13) were 14 × 89mm ultracentrifuge tubes. The solution in the ultracentrifuge tube is not less than 12mL during centrifugation.
The sucrose solution in steps S7) and S8) is prepared using ultrapure water.
Sucrose solution in steps S7), S9), S12), and S14), the PBS solution is a solution sterilized with a 0.22 μm filter.
In step S9), about 7mL of the intermediate layer solution was aspirated from the top when 1.3M and 0.95M sucrose solutions were aspirated.
In step S11), dialysis is performed with ultrapure water, and water is changed a plurality of times in the middle.
The volume of the PBS buffer used in step S14) was 100. mu.L, and the obtained exosomes were stored in a refrigerator at-80 ℃ for a long period of time.
Compared with the prior art, the method for extracting the scylla paramamosain tissue exosomes combines the methods of differential centrifugation, sucrose density gradient centrifugation and dialysis filtration, is stable and effective, has higher purity and simple operation, and does not need special equipment. The extracted exosome has high content and high purity, and can be used for a series of biological experiments.
Drawings
FIG. 1 shows the transmission electron microscope detection results of the scylla paramamosain exosomes after the differential centrifugation, the sucrose density gradient centrifugation and the dialysis filtration separation are combined in the embodiment 1;
FIG. 2 shows the results of particle size analysis of scylla paramamosain exosomes after example 1 combined differential centrifugation, sucrose density gradient centrifugation and diafiltration separation;
FIG. 3 shows the results of the detection of the marker protein of the scylla paramamosain exosomes in example 1 after the differential centrifugation, sucrose density gradient centrifugation and diafiltration separation are combined;
FIG. 4 is the results of particle size analysis of exosomes isolated from example 2 obtained by simple differential centrifugation;
FIG. 5 is the results of particle size analysis after exosome isolation obtained in example 3 combining differential centrifugation and sucrose density gradient centrifugation;
FIG. 6 shows the results of TEM examination of exosomes isolated from simple differential centrifugation as in example 2;
FIG. 7 shows the results of TEM examination of exosomes isolated from example 3 by differential centrifugation and sucrose density gradient centrifugation.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1
The method for extracting the scylla paramamosain tissue exosomes by combining differential centrifugation, sucrose density gradient centrifugation and dialysis filtration comprises the following operation steps:
s1), tissue extraction: taking crab tissues, cutting the tissues into pieces, and placing the pieces into 2mL of precooled 0.3% trypsin liquid;
s2) tissue digestion: incubating and cracking at 37 ℃ for 20 min; 6mL of precooled 0.03% pancreatin inhibitor was added to stop the reaction;
s3), low-speed centrifugation: centrifuging the tissue single cell suspension in the step S2) at 4 ℃ and 800 Xg for 10min by using a low-temperature centrifuge, transferring supernatant in a centrifugal tube to a new tube for later use after centrifugation is finished, and discarding precipitates;
s4), low-speed centrifugation again: centrifuging the supernatant obtained in S3) at 4 deg.C and 2000 Xg for 30min with a low-temperature centrifuge, transferring the supernatant to a new tube, and removing precipitate;
s5), high-speed centrifugation: centrifuging the supernatant obtained in S4) at 4 deg.C and 10000 Xg for 1h with a low temperature centrifuge, transferring the supernatant in the centrifuge tube to a new tube for use, and discarding the precipitate;
s6) high-speed centrifugation again: centrifuging the supernatant obtained in S5) at 4 deg.C and 130000 Xg for 2h with low temperature centrifuge, and carefully sucking out the supernatant and retaining precipitate;
s7) resuspending the pellet: resuspending the pellet obtained in S6) with 4.5mL of 0.95M sucrose solution;
s8) sucrose density gradient centrifugation: adding 4.5mL of 2.0M sucrose solution, 4.5mL of 1.3M sucrose solution and 4.5mL of S7) precipitate resuspension into the ultracentrifuge tube in turn;
s9) ultracentrifugation: centrifuging at 4 deg.C and 200000 Xg for 16h, sucking appropriate amount of middle layer solution of 1.3M and 0.95M sucrose solution, and resuspending with appropriate amount of precooled PBS;
s10) re-ultracentrifugation: centrifuging the supernatant obtained in S9) at 4 deg.C and 130000 Xg for 2h with a low-temperature centrifuge, and carefully sucking out the supernatant and retaining precipitate;
s11) dialysis bag dialysis: resuspending the pellet obtained in S10) with precooled PBS, dialyzing for 3-5 h at 4 ℃ by using a dialysis bag with MW 8000-14000;
s12), filtering by a filter membrane: resuspending the dialyzed solution obtained in S11) with an appropriate amount of precooled PBS, sucking the resuspended solution with a disposable syringe, and slowly dripping the solution onto a cell filter membrane with the specification of 0.22 μm for filtration;
s13) ultracentrifugation: centrifuging the solution filtered in the step S12) at 4 ℃ and 130000 Xg for 2h by using a low-temperature centrifuge, and carefully sucking the supernatant after the centrifugation is finished and reserving a precipitate;
s14) resuspending the pellet: and (3) using an appropriate amount of PBS to precipitate the heavy suspension obtained in S13), namely the extracted and separated exosome product.
Among them, the centrifuge tubes used in S3) and S4) were sterilized before low-speed centrifugation. The ultracentrifuge tubes used in S5), S6), S9), S10), and S13) were 14 × 89mm ultracentrifuge tubes. The solution in the ultracentrifuge tube is not less than 12mL during centrifugation. S7) and S8) were prepared using ultrapure water. S7), S9), S12), and S14), and the PBS solution is a solution sterilized with a 0.22 μm filter. S9) was extracted about 7mL of the solution from the top to the bottom, when the intermediate layer solution of 1.3M or 0.95M sucrose solution was extracted. S11), dialysis is performed with ultrapure water, and water is changed a plurality of times in the middle. S14) was used in a volume of 100. mu.L, and the obtained exosome was stored in a refrigerator at-80 ℃ for a long period of time.
Example 2
Extracting scylla paramamosain tissue exosome by adopting a traditional simple differential centrifugation method.
S1), tissue extraction: taking crab tissues, cutting the tissues into pieces, and placing the pieces into 2mL of precooled 0.3% trypsin liquid;
s2) tissue digestion: incubating and cracking at 37 ℃ for 20 min; 6mL of precooled 0.03% pancreatin inhibitor was added to stop the reaction;
s3), low-speed centrifugation: centrifuging the tissue single cell suspension in the step S2) at 4 ℃ and 800 Xg for 10min by using a low-temperature centrifuge, transferring supernatant in a centrifugal tube to a new tube for later use after centrifugation is finished, and discarding precipitates;
s4), low-speed centrifugation again: centrifuging the supernatant obtained in S3) at 4 deg.C and 2000 Xg for 30min with a low-temperature centrifuge, transferring the supernatant to a new tube, and removing precipitate;
s5), high-speed centrifugation: centrifuging the supernatant obtained in S4) at 4 deg.C and 10000 Xg for 1h with a low temperature centrifuge, transferring the supernatant in the centrifuge tube to a new tube for use, and discarding the precipitate;
s6) high-speed centrifugation again: centrifuging the supernatant obtained in S5) at 4 deg.C and 130000 Xg for 2h with low temperature centrifuge, and carefully sucking out the supernatant and retaining precipitate;
s7) resuspending the pellet: and (4) resuspending the precipitate obtained in the step S6) by using a proper amount of filter-sterilized 1XPBS solution to obtain an exosome product.
Example 3
Differential centrifugation and sucrose density gradient centrifugation (without dialysis filtration) are adopted to extract the scylla paramamosain tissue exosomes.
S1), tissue extraction: taking crab tissues, cutting the tissues into pieces, and placing the pieces into 2mL of precooled 0.3% trypsin liquid;
s2) tissue digestion: incubating and cracking at 37 ℃ for 20 min; 6mL of precooled 0.03% pancreatin inhibitor was added to stop the reaction;
s3), low-speed centrifugation: centrifuging the tissue single cell suspension in the step S2) at 4 ℃ and 800 Xg for 10min by using a low-temperature centrifuge, transferring supernatant in a centrifugal tube to a new tube for later use after centrifugation is finished, and discarding precipitates;
s4), low-speed centrifugation again: centrifuging the supernatant obtained in S3) at 4 deg.C and 2000 Xg for 30min with a low-temperature centrifuge, transferring the supernatant to a new tube, and removing precipitate;
s5), high-speed centrifugation: centrifuging the supernatant obtained in S4) at 4 deg.C and 10000 Xg for 1h with a low temperature centrifuge, transferring the supernatant in the centrifuge tube to a new tube for use, and discarding the precipitate;
s6) high-speed centrifugation again: centrifuging the supernatant obtained in S5) at 4 deg.C and 130000 Xg for 2h with low temperature centrifuge, and carefully sucking out the supernatant and retaining precipitate;
s7) resuspending the pellet: resuspending the pellet obtained in S6) with 4.5mL of 0.95M sucrose solution;
s8) sucrose density gradient centrifugation: adding 4.5mL of 2.0M sucrose solution, 4.5mL of 1.3M sucrose solution and 4.5mL of S7) precipitate resuspension into the ultracentrifuge tube in turn;
s9) ultracentrifugation: centrifuging at 4 deg.C and 200000 Xg for 16h, sucking appropriate amount of middle layer solution of 1.3M and 0.95M sucrose solution, and resuspending with appropriate amount of precooled PBS;
s10) re-ultracentrifugation: centrifuging the supernatant obtained in S9) at 4 deg.C and 130000 Xg for 2h with a low-temperature centrifuge, and carefully sucking out the supernatant and retaining precipitate;
s11) resuspending the pellet: and (3) using a proper amount of filter sterilized 1XPBS solution to precipitate the heavy suspension obtained in S10), namely the extracted and separated exosome product.
Testing
Performing morphology detection on the extracted exosomes by using a transmission electron microscope:
20 mu.L of the exosome suspension obtained in example 1-3 was respectively dropped on a copper mesh, and after standing at room temperature for 10min, the remaining solution on the copper mesh was removed by suction with filter paper, and the solution was stained with 2% phosphotungstic acid for 1-3 min, and the staining solution was removed by suction with filter paper. And dropping a drop of ultrapure water on a copper net, sucking the ultrapure water by using filter paper, standing the ultrapure water at room temperature for drying, and observing the dried ultrapure water by using a projection electron microscope to obtain a double-layer membrane vesicle structure.
As can be seen from fig. 1, the scylla paramamosain exosomes obtained in example 1 have substantially the same appearance as exosomes in higher model organisms, and both have a typical cup-stand-like structure.
As can be seen from FIG. 2, the size of the scylla paramamosain exosome isolated in example 1 is mainly concentrated between 100-200nm by Nanosight detection, and is basically consistent with that of other species.
As can be seen in fig. 3, in example 1, both the exosome marker proteins CD9 and TSG101 were detected in isolated scylla paramamosain exosomes, but the non-marker protein Calnexin was not detected, which indicates that the purity of the isolated exosomes meets the requirement.
As can be seen from fig. 4, the scylla paramamosain exosomes isolated in example 2 were detected by Nanosight, and the size of the scylla paramamosain exosomes was between 27-524nm, and the vesicles larger than 200nm occupied a larger proportion, and probably were other large vesicles than exosomes, indicating that the purity of the isolated exosomes was very low.
As can be seen in FIG. 5, the size of the scylla paramamosain exosomes isolated in example 3 is mainly concentrated between 100-200nm by Nanosight detection, but a small portion of 200-545nm vesicles are also detected in the extracted exosome sample, indicating that the purity of the isolated exosomes is insufficient.
As can be seen from fig. 6, in example 2, a large number of vesicles with different sizes are found by morphological observation under a transmission electron microscope, and the background value is high, so that the vesicle morphology is difficult to distinguish.
Fig. 7 shows that, in the morphological observation of example 3 under a transmission electron microscope, the scylla paramamosain exosomes obtained in example 3 have substantially the same appearance as exosomes in higher model organisms, and both have typical saucer-shaped structures, but have high background values, form a large number of crystals, and influence the morphological observation of vesicles.
From the above examples 1-3, it can be seen that the method for extracting scylla paramamosain tissue exosomes of the present invention combines the methods of differential centrifugation, sucrose density gradient centrifugation and diafiltration, and is stable and effective, high in purity and simple in operation, and does not need special equipment. The extracted exosome has high content and high purity, and can be used for a series of biological experiments.
In addition, in the experiment, two times of low-speed centrifugation and two times of high-speed centrifugation in example 1 are changed into one time of low-speed centrifugation for 40min and one time of high-speed centrifugation for 3h, but because the sample has certain viscosity, only two times of centrifugation easily cause that the exosome is wrapped by large granular cell debris or a large amount of protein and is centrifuged along with the centrifugation, so that a large amount of loss is caused.
Claims (5)
1. A method for separating exosomes of scylla paramamosain tissues is characterized by mainly comprising the following steps:
s1) tissue extraction: taking crab tissue, cutting the tissue into pieces, and placing the pieces into precooled 0.3% trypsin liquid;
s2) tissue digestion: incubating and cracking at 37 ℃ for 20 min; adding pre-cooled 0.03% pancreatin inhibitor to stop reaction;
s3) low speed centrifugation: carrying out low-speed centrifugation on the tissue single-cell suspension in the step S2) for 10min by using a low-temperature centrifuge, transferring supernatant in a centrifugal tube to a new tube for later use after the centrifugation is finished, and discarding precipitates;
s4) low speed centrifugation again: centrifuging the supernatant obtained in S3) at low speed and 4 ℃ for 30min by using a low-temperature centrifuge, transferring the supernatant in the centrifuge tube to a new tube for later use after the centrifugation is finished, and discarding the precipitate;
s5) high speed centrifugation: centrifuging the supernatant obtained in S4) at 4 deg.C for 1h at high speed by using low temperature centrifuge, transferring the supernatant in the centrifuge tube to a new tube for use, and discarding the precipitate;
s6) high speed centrifugation again: centrifuging the supernatant obtained in S5) at 4 deg.C for 2h with a low temperature centrifuge, and carefully sucking out the supernatant and retaining precipitate;
s7) resuspending the pellet: resuspending the pellet obtained in S6) with 0.95M sucrose solution;
s8) sucrose density gradient centrifugation: adding 2.0M sucrose solution, 1.3M sucrose solution and the precipitate resuspension obtained in S7) into the ultracentrifuge tube in sequence;
s9) ultracentrifugation: performing ultracentrifugation for 16h at 4 ℃ by using a low-temperature centrifuge, sucking a proper amount of middle layer solution of 1.3M and 0.95M sucrose solution, and re-suspending by using a proper amount of precooled PBS;
s10) ultracentrifugation again: carrying out ultracentrifugation on the upper layer solution obtained in the step S9) for 2h at 4 ℃ by using a low-temperature centrifuge, and carefully sucking out the supernatant after the ultracentrifugation is finished and reserving a precipitate;
s11) dialysis bag dialysis: resuspending the pellet obtained in S10) with precooled PBS, dialyzing for 3-5 h at 4 ℃ by using a dialysis bag with MW 8000-14000;
s12) membrane filtration: resuspending the dialyzed solution obtained in S11) with an appropriate amount of precooled PBS, sucking the resuspended solution with a disposable syringe, and slowly dripping the solution onto a cell filter membrane with the specification of 0.22 μm for filtration;
s13) ultracentrifugation: carrying out ultracentrifugation on the solution filtered in the step S12) for 2h at 4 ℃ by using a low-temperature centrifuge, and carefully sucking supernatant after the ultracentrifugation is finished and reserving a precipitate part;
s14) resuspending the pellet: and (3) using an appropriate amount of PBS to precipitate the heavy suspension obtained in S13), namely the extracted and separated exosome product.
2. The method for separating the scylla paramamosain tissue exosomes according to claim 1, wherein the speed of the low-speed centrifugation in step 3) is 800 × g, the speed of the low-speed centrifugation in step 4) is 2000 × g, the speed of the high-speed centrifugation in step 5) is 10000 × g, the speed of the high-speed centrifugation in step 6) is 130000 × g, the speed of the ultracentrifugation in step 9) is 200000 × g, the speed of the ultracentrifugation in step 10) is 130000 × g, and the speed of the ultracentrifugation in step 13) is 130000 × g.
3. The method for separating the scylla paramamosain tissue exosomes according to claim 1, wherein in the step S2), the tissue is cut into pieces in a container which is sterilized and contains 0.3% trypsin solution during the tissue treatment.
4. The method for extracting and separating scylla paramamosain tissue exosomes according to claim 1, wherein the PBS solution and the sucrose solutions in step S7), step S9), step S12) and step S14) are all solutions sterilized by using a 0.22 μm filter membrane.
5. The method for extracting and separating scylla paramamosain tissue exosomes according to claim 1, wherein the middle layer solution of 1.3M and 0.95M sucrose solutions is extracted from top to bottom in step S9).
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