Methods for extracting exosomes and exosome proteins

An extraction method and exosome technology, applied in the biological field, can solve the problems of inability to meet the requirements of specific extraction and analysis, interference of exosome analysis, separation of exosomes, etc., and achieve high recovery rate and fast extraction speed. , the effect of high specificity extraction

CN110343664BActive Publication Date: 2021-12-24ACADEMY OF MILITARY MEDICAL SCI
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2021-12-24

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Abstract

The invention discloses a method for extracting exosomes and exosome proteins from human body fluid or cell culture fluid. The method for extracting exosomes and exosome proteins provided by the present invention includes: adding extraction materials to the liquid to be extracted containing exosomes to extract exosomes; the extraction materials are metal oxides or modified with the metal oxides The functional material; the metal oxide can double coordinate with the phosphate ion; the liquid to be extracted is the body fluid from the human body, the dilution of the body fluid or the culture product of cells. Experiments have proved that the exosome extraction method of the present invention can achieve efficient extraction of exosomes in human body fluids or cell culture fluids, the extraction time is short, the obtained exosomes are of high purity, and complete exosomes can be obtained by eluting with an eluent. Exosomes, and the extraction materials used can be recycled and reused; in addition, it can be found that the obtained exosome proteins have many types and are more closely related to the function of exosomes.
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Description

technical field

[0001] The invention relates to a method for extracting exosomes and exosome proteins from human body fluid or cell culture fluid in the field of biotechnology. Background technique

[0002] Exosomes are a type of vesicle-like structure secreted by mammalian cells through the process of "endocytosis-efflux", with a diameter of about 30-150nm. Exosomes exist not only in cell culture fluid, but also in body fluids such as blood, urine, amniotic fluid, breast milk, semen, saliva, and pleural effusion. Their components include lipid bilayers and cell-derived proteins and RNAs. , DNA and other substances. Almost all cells can produce exosomes, and exosomes from different cell sources contain not only common components related to cell formation, structure, and material transport, but also specific molecules related to the biological functions of the source cells. Exosomes, as an important way of intercellular communication and delivery of a large number of biolog...

Examples

Embodiment 1

[0066] Embodiment 1, exosome extraction

[0067] 1. Extraction of exosomes from human serum and LC-MS / MS analysis of their proteins

[0068] Step 1: Serum sample pretreatment

[0069] Take 100 μL of human serum (with the informed consent of the person), add DMEM buffer solution to the serum at a volume ratio of 1:1 for dilution to obtain a serum dilution; perform gradient density centrifugation on the serum dilution to remove dead cells and / or cell residues, and centrifuge The conditions are: 300g centrifugation for 10 minutes, 2000g centrifugation for 10 minutes, 10000g centrifugation for 30 minutes, take the supernatant, filter the supernatant through a 0.2μm filter membrane, and collect the filtrate, which is the pretreated serum sample.

[0070] Step 2: Extraction of exosomes from serum samples

[0071] Weigh 5mg TiO 2 , respectively rinsed twice with pure water and DMEM buffer to obtain the cleaned TiO 2 ; to the cleaned TiO 2 Add 200 μL of the pretreated serum sampl...