Kit for extracting adipose-tissue-derived exosome as well as extracting method adopting kit and applications of kit

A technology of adipose tissue and exosomes, which is applied in the direction of bone/connective tissue cells, biochemical equipment and methods, and microbial measurement/inspection, and can solve the problems of inability to quickly separate and obtain exosomes from adipose tissue

Inactive Publication Date: 2019-01-22
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem that exosomes derived from adipose tissue cannot be quickly separated from peripheral blood in the prior art, the present invention

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1 Kit for Extracting Exosomes from Adipose Tissue

[0106] Protein A magnetic beads (Pierce TM Protein A / G Magnetic Beads);

[0107] Magnetic bead washing solution: 1×PBS buffer at pH 7.4;

[0108] FABP4 antibody: IgG antibody (Abcam, Anti-FABP4 antibody (ab13979));

[0109] Cleaning solution No. 1: bovine serum albumin with a mass volume fraction of 1% is dissolved in 0.01M, pH7.4 PBS buffer;

[0110] Cleaning solution No. 2: Prepare a 0.1-0.2M glycine solution and adjust the pH to 3.0 with hydrochloric acid.

Embodiment 2

[0111] Example 2 The diagnostic kit for judging the functional state of fat

[0112] Protein A magnetic beads 2mg (Pierce TM Protein A / G Magnetic Beads);

[0113] Magnetic bead washing solution: 1×PBS buffer at pH 7.4;

[0114] FABP4 antibody: IgG antibody (Abcam, Anti-FABP4 antibody (ab13979));

[0115] Cleaning solution No. 1: 1% bovine serum albumin is dissolved in 0.01M PBS buffer (pH7.4) according to the mass volume fraction;

[0116] Cleaning solution No. 2: prepare 0.1-0.2M glycine solution, adjust to pH 3.0 with hydrochloric acid;

[0117] Neutralizing solution: prepare 0.01M Tris solution, adjust to pH 10.5 with sodium hydroxide;

[0118] Inflammation Effects Fluorescent Reporter: psiCheck2 TM As the backbone, the insert sequence is inserted into its multiple cloning site Nhe1, and the insert sequence is a 4×NFκB binding site, which is located in the Renilla luciferase coding sequence, TK promoter-firefly luciferase coding sequence in the vector upstream.

[0...

Embodiment 3

[0121] Extraction of fat-derived exosomes using the kit of Example 1

[0122] The patient, male, BMI 34, aged 48, suffered from hyperlipidemia, fatty liver and atherosclerosis. Take 3 mL of the patient's peripheral blood sample into a test tube, without adding anticoagulant, the blood coagulation reaction will be activated, and the blood will coagulate rapidly to form a jelly. The blood clot shrinks, and the pale yellow transparent liquid around it is the serum. Centrifuge at 500g for 3 minutes to take the supernatant, which is the serum to be tested.

[0123] (1) Mix protein A magnetic beads 2mg and NaN 3 Dissolve 0.1 μg in water, dilute to 200 μL to obtain a magnetic bead turbid solution, centrifuge the magnetic bead turbid solution at 1000g for 30s, discard the supernatant to obtain the first precipitate, mix the first precipitate with 1mL magnetic bead cleaning solution, and shake to 1000g Centrifuge for 1 min, discard the supernatant, and the second precipitate obtained...

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PUM

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Abstract

The invention relates to the technical field of exosome separation, and provides a kit for extracting adipose-tissue-derived exosome. The kit comprises protein A magnetic beads, a magnetic bead washing solution, an FABP4 antibody, a No.1 washing solution and a No.2 washing solution, wherein the FABP4 antibody can be combined with the adipose-tissue-derived exosome in to-be-detected serum, the protein A magnetic beads and the exosome-FABP4 antibody can have interaction, so that a protein A magnetic bead-FABP4 antibody-exosome compound is formed, the No.1 washing solution can remove the non-fatexosome ingredients in the serum, the No.2 washing solution can realize the dissociation of the interaction among the exosome, the FABP4 antibody and the protein A magnetic beads in the protein A magnetic bead-FABP4 antibody-exosome compound, and thus the adipose-tissue-derived exosome is specifically separated from the serum.

Description

technical field [0001] The invention relates to the technical field of exosome separation, in particular to a kit for extracting exosomes from adipose tissue, an extraction method and application thereof. Background technique [0002] Exosomes (exosomes) are vesicle structures secreted by cells to the outside of the cell, usually membrane-wrapped structures with a size of 30-120 nm. Exosomes can specifically wrap some proteins, lipids or nucleic acids and other substances, have biological activity, can be absorbed by recipient cells, and realize material transportation and information transmission between cells. [0003] Adipose tissue is mainly composed of a large number of clustered fat cells and a small number of macrophages, etc. It is an important energy storage organ in the human body. Adipose tissue is also an important endocrine organ in the human body, regulating important pathological processes such as insulin sensitivity, blood pressure level, endothelial functio...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12Q1/66
CPCC12N5/0653C12Q1/66
Inventor 杨国栋卢晓昭柏丹娜杨薛康袁丽君周雪莹李者龙孙汶齐石瑞静
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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