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Method for separating scylla paramamosain tissue exosome

A technology of Scylla simulans and exosomes is applied in the field of separation of Scylla simulans tissue exosomes, which can solve the problems of inability to isolate pure exosomes and inapplicability, and achieve the effect of simple operation and high purity.

Active Publication Date: 2020-06-12
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the currently reported method of using medium for cell culture to obtain extracellular exosomes is not applicable for the time being.
In addition, the rapid exosome extraction kits on the market are mainly aimed at mammals. After trying, the research team found that such kits are not suitable for crustaceans such as blue crabs.
The commonly used ultracentrifugation, although the steps are simple, but when the research group explored the experimental method, it was found that simple ultracentrifugation could not separate exosomes with high purity

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  • Method for separating scylla paramamosain tissue exosome
  • Method for separating scylla paramamosain tissue exosome
  • Method for separating scylla paramamosain tissue exosome

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Combining differential centrifugation, sucrose density gradient centrifugation and diafiltration to extract exosomes from the tissue of Scylla pseudocarpus, including the following steps:

[0042] S1): tissue extraction: take crab tissue, cut the tissue into pieces, and place in 2 mL of pre-cooled 0.3% trypsin solution;

[0043] S2): Tissue digestion: incubate and lyse at 37°C for 20 minutes; add 6 mL of pre-cooled 0.03% trypsin inhibitor to terminate the reaction;

[0044] S3): Low-speed centrifugation: Centrifuge the single-cell suspension of the tissue in S2) at 4°C and 800×g for 10 minutes in a low-temperature centrifuge. After the centrifugation, transfer the supernatant in the centrifuge tube to a new tube for later use, and discard the precipitate;

[0045] S4): centrifuge again at low speed: use a low-temperature centrifuge to centrifuge the supernatant obtained in S3) at 2000×g for 30 min at 4°C, transfer the supernatant in the centrifuge tube to a new tube aft...

Embodiment 2

[0058] Exosomes from the tissue of Scylla pseudocarpus were extracted by traditional simple differential centrifugation.

[0059] S1): tissue extraction: take crab tissue, cut the tissue into pieces, and place in 2 mL of pre-cooled 0.3% trypsin solution;

[0060] S2): Tissue digestion: incubate and lyse at 37°C for 20 minutes; add 6 mL of pre-cooled 0.03% trypsin inhibitor to terminate the reaction;

[0061] S3): Low-speed centrifugation: Centrifuge the single-cell suspension of the tissue in S2) at 4°C and 800×g for 10 minutes in a low-temperature centrifuge. After the centrifugation, transfer the supernatant in the centrifuge tube to a new tube for later use, and discard the precipitate;

[0062] S4): centrifuge again at low speed: use a low-temperature centrifuge to centrifuge the supernatant obtained in S3) at 2000×g for 30 min at 4°C, transfer the supernatant in the centrifuge tube to a new tube after centrifugation, and discard the precipitate;

[0063] S5): High-speed ...

Embodiment 3

[0067] Differential centrifugation and sucrose density gradient centrifugation (without diafiltration) were used to extract the exosomes from the tissue of Scylla pseudocarpus.

[0068] S1): tissue extraction: take crab tissue, cut the tissue into pieces, and place in 2 mL of pre-cooled 0.3% trypsin solution;

[0069] S2): Tissue digestion: incubate and lyse at 37°C for 20 minutes; add 6 mL of pre-cooled 0.03% trypsin inhibitor to terminate the reaction;

[0070] S3): Low-speed centrifugation: Centrifuge the single-cell suspension of the tissue in S2) at 4°C and 800×g for 10 minutes in a low-temperature centrifuge. After the centrifugation, transfer the supernatant in the centrifuge tube to a new tube for later use, and discard the precipitate;

[0071] S4): centrifuge again at low speed: use a low-temperature centrifuge to centrifuge the supernatant obtained in S3) at 2000×g for 30 min at 4°C, transfer the supernatant in the centrifuge tube to a new tube after centrifugation,...

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Abstract

The invention relates to a method for separating scylla paramamosain tissue exosome. The method mainly comprises the following steps: S1) performing tissue extraction; S2) performing tissue digestion;S3) performing low-speed centrifugation; S4) performing low-speed centrifugation again; S5) performing high-speed centrifugation; S6) performing high-speed centrifugation again; S7) resuspending andprecipitating; S8) performing sucrose density gradient centrifugation; S9) carrying out ultracentrifugation; S10) carrying out ultracentrifugation again; S11) dialyzing in a dialysis bag; S12) filtering with a filter membrane; S13) carrying out ultracentrifugation; and S14) resuspending and precipitating to obtain the extracted and separated exosome product. According to the method for extractingthe scylla paramamosain tissue exosome, the differential centrifugation method, the sucrose density gradient centrifugation method and the dialysis filtration method are combined, the method is stable, effective, high in purity and easy to operate, no special equipment is needed, and the extracted exosome is high in content and purity.

Description

technical field [0001] The invention relates to the technical field of extraction and separation of exosomes, in particular to a method for separating exosomes from tissues of Scylla pseudocavei. Background technique [0002] Exosomes are a class of lipid vesicles with a double-layer membrane with a diameter in the range of 30-200 nm produced by the membrane filling of endosomes. These vesicles form in endosomes, and once the endosomes fuse with the cell membrane, they are released exocytically into the extracellular environment. As cell substitutes secreted by host cells, exosomes are widely present in a variety of biological fluids, including plasma, saliva, synovial fluid, sputum, cerebrospinal fluid, lymph fluid, and ascites, etc., and carry a large number of biologically active molecules. Mainly a rich variety of content, such as specific proteins, signal peptides, lipids and nucleic acids derived from cells. Extracellular exosomes can be accepted by adjacent cells, a...

Claims

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Application Information

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IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2509/10
Inventor 李升康龚燚林善梦陈娇
Owner SHANTOU UNIV
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