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Method for separating exosome of stichopus japonicus body fluid sample

A technology of exosomes and japonicus, which is applied in the field of exosome separation of japonicus body fluid samples, and can solve the problems of japonicus exosomes without a clear extraction method

Active Publication Date: 2022-07-05
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for extracting and separating exosomes from japonicus body fluid samples, so as to solve the problem that there is no clear extraction method for japonicus exosomes

Method used

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  • Method for separating exosome of stichopus japonicus body fluid sample
  • Method for separating exosome of stichopus japonicus body fluid sample
  • Method for separating exosome of stichopus japonicus body fluid sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The isolation and extraction of exosomes from Apostichopus japonicus (AJ) coelomic fluid (CL) samples includes the following steps:

[0070] (1) Preparation of Apostichopus japonicus isotonic anticoagulant and Apostichopus japonicus isotonic buffer

[0071] The concentrations of each component of Apostichopus japonicus isotonic anticoagulant (pH=7.6) were 0.02M EGTA, 0.34M NaCl, 0.019MKCl, and 0.068M Tri-HCl, respectively. The concentration of each component in Apostichopus japonicus isotonic buffer (pH=7.6) was 0.001M EGTA, 0.34MNaCl, 0.01M Tri-HCl, respectively.

[0072] (2) Body cavity fluid sample extraction:

[0073] After dissecting the sea cucumber, place it in a disposable petri dish. After drawing 20 mL of the body cavity fluid sample with a disposable sterile syringe, it was filtered through a 300-mesh sieve, and the filtrate (ie, the body cavity fluid) was placed in a sterile enzyme-free centrifuge tube.

[0074] (3) Body cavity fluid sample processing:

...

Embodiment 2

[0085] The isolation and extraction of exosomes from Apostichopus japonicus polian vesicle fluid (PVL) samples includes the following steps:

[0086] (1) Preparation of Apostichopus japonicus isotonic anticoagulant and Apostichopus japonicus isotonic buffer

[0087] The concentrations of each component of Apostichopus japonicus isotonic anticoagulant (pH=7.6) were 0.02M EGTA, 0.34M NaCl, 0.019MKCl, and 0.068M Tri-HCl, respectively. The concentration of each component in Apostichopus japonicus isotonic buffer (pH=7.6) was 0.001M EGTA, 0.34MNaCl, 0.01M Tri-HCl, respectively.

[0088] (2) Extraction of the bursa body fluid sample:

[0089] After dissecting the sea cucumber, place it in a disposable petri dish. Use a disposable sterile syringe to draw a 5 mL sample of Burrell's bursa body fluid, filter it through a 300-mesh sieve, and place the filtrate (ie, Bury's bursa body fluid) in a sterile enzyme-free centrifuge tube.

[0090] (3) Polycystic bursa body fluid sample proces...

Embodiment 3

[0101] The isolation and extraction of exosomes from Apostichopus japonicus (AJ) coelomic fluid (CL) samples includes the following steps:

[0102] (1) Prepare isotonic anticoagulant A and Apostichopus japonicus isotonic buffer

[0103] The concentrations of each component of isotonic anticoagulant A (pH=7.6) were 0.02M EGTA, 0.36M NaCl, 0.01M KCl, and 0.03M Tri-HCl, respectively. The concentration of each component in A. japonicus isotonic buffer (pH=7.6) was 0.001M EGTA, 0.34M NaCl and 0.01M Tri-HCl, respectively.

[0104] (2) Body cavity fluid sample extraction:

[0105] After dissecting the sea cucumber, place it in a disposable petri dish. After drawing 20 mL of the body cavity fluid sample with a disposable sterile syringe, it was filtered through a 300-mesh sieve, and the filtrate (ie, the body cavity fluid) was placed in a sterile enzyme-free centrifuge tube.

[0106] (3) Body cavity fluid sample processing:

[0107] To the centrifuge tube containing the filtered b...

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Abstract

The invention relates to a stichopus japonicus body fluid sample exosome separation method. A body fluid sample of the stichopus japonicus is preliminarily filtered and then mixed with an isotonic anticoagulant for pretreatment, then exosomes in the stichopus japonicus are extracted and separated by utilizing a method of combining low-temperature medium-speed centrifugation, high-speed centrifugation and ultracentrifugation with multiple filtration, and the exosomes are resuspended by utilizing an isotonic buffer solution for subsequent experiments. The apostichopus japonicus exosome separation method provided by the invention is stable, effective and simple to operate, and the extracted exosome is relatively high in integrity, relatively high in content and relatively high in purity.

Description

technical field [0001] The present invention relates to the technical field of exosome extraction and separation, in particular to a method for separating exosomes from a sea cucumber body fluid sample. Background technique [0002] Exosomes (Exos) are multivesicular bodies derived from the invagination of the cytoplasmic membrane and contain a lipid bilayer membrane structure. They are a type of extracellular vesicles (EVs). In 2013, the Nobel Prize in Physiology or Medicine was awarded to three scientists who studied "the regulation mechanism of vesicle transport in cells". Exosomes have gradually become an international research hotspot. Exosomes are rich in various mRNAs, miRNAs and proteins, and can be transported between different cells. After being taken up by target cells, they affect the biological behavior of target cells and are important intercellular transport systems. The special membrane structure of exosomes can protect the internal RNAs from degradation by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B04B5/04B04B5/10B01D36/00G01N1/28
CPCB04B5/0414B04B5/10B01D36/00G01N1/28G01N1/4077
Inventor 霍达孙丽娜林承刚杨红生张立斌刘石林崔玮苏芳
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI