Method for detecting EVs (extracellular vesicles) released by ECs (endothelial cells) and EPCs (endothelial progenitor cells) in blood

A technology of endothelial progenitor cells and endothelium, which is applied in the field of medical invention and can solve the problem of lack of kits

Inactive Publication Date: 2016-06-08
WUHAN DAFU BIOTECH CO LTD
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Problems solved by technology

The emergence of fluorescent NTA provides technical conditions for the study of EVs. However, there are no reports at home and abroad on the

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  • Method for detecting EVs (extracellular vesicles) released by ECs (endothelial cells) and EPCs (endothelial progenitor cells) in blood
  • Method for detecting EVs (extracellular vesicles) released by ECs (endothelial cells) and EPCs (endothelial progenitor cells) in blood
  • Method for detecting EVs (extracellular vesicles) released by ECs (endothelial cells) and EPCs (endothelial progenitor cells) in blood

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Embodiment Construction

[0042] The following is a specific implementation case of this project. The reagents and materials used in the subordinate applications can be obtained through market purchase channels. The pH of the PBS used in the present invention is 7.2.

[0043] The process of the present invention is as Figure 4 As shown, take 3ml of peripheral blood and put it in a 2ml sterile tube containing 3.3% sodium citrate by mass; add 6ml of filtered PBS; centrifuge after mixing, separation conditions: speed 400r / s, 35min, 4 ℃; take the top layer of liquid is plasma; take 1ml of plasma for centrifugal separation, separation conditions: speed 2000r / s, 20min, 4℃, used to remove platelets; take the supernatant for centrifugal separation, separation conditions: speed 20000r / s, 120min, 4℃, the obtained precipitate was suspended in 700μl filtered PBS as NTA to analyze the total number of cMVs; the remaining supernatant was centrifuged, and the separation condition: 169000r / s, 6h, 4℃, the obtained preci...

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Abstract

The invention relates to a method for detecting EVs (extracellular vesicles) released by ECs (endothelial cells) and EPCs (endothelial progenitor cells) in blood. EC-MVs (microvesicles), EPC-MVs, EC-EXs (exosomes) and EPC-EXs are separated from a circulatory system with two specific antibody linked immunomagnetic sand methods. The method breaks through a single specific antibody method, and the specificity and the sensitivity of extracted cells MVs and EXs are higher than those in the prior art. Besides, the cells MVs and EXs are detected with an NTS (nanoparticle tracing analysis) technique in combination of a specific antibody micro-beads method and a Q-dots method. The method can quickly, accurately and objectively analyze the levels of EC-MVs, EPC-MVs, EC-EXs and EPC-EXs in complicated samples such as blood and has advantages which a traditional analysis method does not possess. Besides, the specific and sensitive method is provided for further research of MVs and EXs serving as biomarkers of diseases.

Description

Technical field [0001] The invention relates to a method for detecting extracellular vesicles released by endothelial and endothelial progenitor cells in blood. The method has the highest sensitivity and specificity at present, and belongs to the field of medical invention. Background technique [0002] Extracellular vesicles (EVs) are small vesicles with biological activity secreted and released by cells. EVs can carry and deliver mother cell lipids, functional proteins, messenger RNAs (mRNAs) and microRNAs (miRNAs) into target cells and regulate the gene expression and function of target cells. Due to the limitation of technical level and understanding, when researchers first proposed the concept of EVs, they only regarded EVs as a "garbage bag" for cells to get rid of some useless protein waste. In the past ten years, further studies have found that the "cargo" carried by EVs has important biological significance, especially the miRNAs contained in it have been confirmed to b...

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56966G01N2333/70596G01N2333/71
Inventor 陈颜芳马晓瑭杨翼
Owner WUHAN DAFU BIOTECH CO LTD
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