Method and reagent of extracting exosome from urine

A technology of exosomes and reagents, applied in the field of biomedicine, can solve the problems of low yield of exosomes, difficulty in obtaining analysis experiments, high cost, etc., and achieve the effect of high purity, high yield and low cost

Inactive Publication Date: 2017-12-29
WAYEN BIOTECHNOLGIES SHANGHAI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A typical shortcoming of the ultra-high-speed centrifugation method is that the yield of exosomes is too low, and it is difficult to obtain a sufficient amount for subsequent analysis experiments; while the kit method, such as those provided by Thermo and SBI in the United States Reagents for the preparation of related urine exosomes are too expensive

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  • Method and reagent of extracting exosome from urine
  • Method and reagent of extracting exosome from urine
  • Method and reagent of extracting exosome from urine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Comparing the effects of different molecular weight polyethylene glycols on the extraction rate of urine exosomes

[0023] 1) Take the frozen urine sample, thaw in a water bath at 37°C; centrifuge at 3000g at 4°C for 10 minutes, take the supernatant, and divide it into 4 centrifuge tubes, each 20mL, numbered as I, II, III, IV; The sodium chloride solution of polyethylene glycol with a molecular weight of 2000 was added into the No. I tube, and mixed; the sodium chloride solution of polyethylene glycol with a molecular weight of 4000 was added into the No. II tube, and mixed; 6000 sodium chloride solution of polyethylene glycol, and mix well; add sodium chloride solution of polyethylene glycol with a molecular weight of 8000 to No. Afterwards, centrifuge at 5000g at 4°C for 60 minutes; discard the supernatant, and resuspend the obtained pellet with 100uL PBS.

[0024] 2) Take 20uL of the obtained exosome resuspension for nanoparticle tracking analysis (NTA) m...

Embodiment 2

[0028] Example 2, the effect of TCEP on the extraction of urine exosomes

[0029] 1) Sample preparation: Take the frozen urine sample, thaw it in a water bath at 37°C; centrifuge at 3000g for 10 minutes at 4°C, take the supernatant, and divide it into 4 centrifuge tubes, each 20mL, numbered 1, 2, 3 , 4;

[0030] 2) Reagent configuration: prepare a sodium chloride solution of polyethylene glycol 4000 with a concentration of 400 mg / mL and a sodium chloride solution of TCEP with a concentration of 50 mg / mL.

[0031] 3) Extraction of urine exosomes: Add only 5mL polyethylene glycol 4000 sodium chloride solution to tube No. 1 and tube 2 and mix well; Diol 4000 and TCEP with a volume ratio of 5:1 mixed reagent 5mL, mix well; put the above mixed solution, 4 ℃, stand still, 12h; then 4 ℃, 5000g, centrifuge 60 minutes; discard the supernatant, the obtained precipitate Resuspend with 100uL PBS.

[0032] 4) Determination of urinary exosome surface marker protein, particle size, and pa...

Embodiment 3

[0036] Example 3, using the reagent of the present invention to extract urine exosomes from different sources

[0037] 1) Take 5 cases of frozen urine samples from different sources, thaw in a water bath at 37°C; centrifuge at 3000g at 4°C for 10 minutes, take the supernatant, put them in 5 centrifuge tubes, each 20mL, numbered a, b , c, d, e; then add 5 mL of the mixed solution of the reagent polyethylene glycol of the present invention and TCEP (refer to Example 2 for the configuration method), and mix well; put the above mixed solution at 4°C and let it stand for 12h; then 4 Centrifuge at 5000g for 60 minutes; discard the supernatant, and resuspend the resulting pellet with 100uL PBS.

[0038] 2) Take 20uL of the obtained exosome resuspension and add an equal volume of RIPA lysate for lysis and protein extraction, and detect the exosome markers TSG101, CD63, and CD9 by WB. The test results are as follows: image 3 As shown in A.

[0039] 3) Take 12uL of the obtained exoso...

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Abstract

The invention discloses a method and a reagent of separating an exosome from urine. The reagent is a mixed solution of polyethylene glycol and tri-(2-carboxyethyl) phosphine (TCEP). The experimental method sequentially comprises the steps of centrifuging the urine, mixing the reagent according to a certain proportion, adding the mixed reagent into the urine according to a certain proportion, performing mixing, standing overnight at 4 DEG C, after centrifugation the next day, discarding supernatant, and obtaining a sediment, namely the exosome. The method and the regent have the advantages that the exosome in the urine is high in yield and purity, the operation is simple, and the cost is low.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method and a reagent for isolating urine exosomes. Background technique [0002] Exosomes are encapsulated cystic vesicles with bilayer lipid molecules with a diameter of 30-200nm, which are selectively packaged and released by living cells, and are abundant in human body fluids. Exosomes contain different types of lipids, nucleic acids, and proteins, which can be transported with body fluids to specific target organs and related target cells, thereby exerting their corresponding biological functions. Related studies have also shown that exosomes play a huge role in intercellular communication as well as physiological and pathological processes. [0003] Pisitkun T et al. (Pisitkun T et al, Proc Natl AcadSci US A 2004; 101:13368–73) Obtained exosomes in urine for the first time by ultracentrifugation and identified disease-associated proteins in exosomes . Later, Takaya Shimura et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N23/22
CPCG01N23/2202G01N2223/07
Inventor 张庆华宋凯王晓楠
Owner WAYEN BIOTECHNOLGIES SHANGHAI INC
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