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Application of reagents for detecting cd105, cd144, cd34, kdr, annexin V and CD63 in preparation of reagents for detecting extracellular vesicles released by endothelial and endothelial progenitor cells in blood

A CD144, endothelial progenitor cell technology, applied in the field of medical invention, can solve the problem of no kits and other problems

Inactive Publication Date: 2018-02-27
WUHAN DAFU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 use of NTA for the detection of EVs by immunomagnetic beads combined with specific molecular surface markers, and there are no related kits.

Method used

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  • Application of reagents for detecting cd105, cd144, cd34, kdr, annexin V and CD63 in preparation of reagents for detecting extracellular vesicles released by endothelial and endothelial progenitor cells in blood
  • Application of reagents for detecting cd105, cd144, cd34, kdr, annexin V and CD63 in preparation of reagents for detecting extracellular vesicles released by endothelial and endothelial progenitor cells in blood
  • Application of reagents for detecting cd105, cd144, cd34, kdr, annexin V and CD63 in preparation of reagents for detecting extracellular vesicles released by endothelial and 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 PBS used in the present invention is 7.2.

[0043] The process of the present invention is as Figure 4 As shown, extract 3ml of peripheral blood and put it in a 2ml sterile tube containing 3.3% sodium citrate; add 6ml of filtered PBS; mix well and perform centrifugation. Separation conditions: speed 400r / min, 35min, 4 ℃; take the uppermost liquid as plasma; take 1ml of plasma for centrifugation, separation conditions: speed 2000r / min, 20min, 4℃, used to remove platelets; take supernatant for centrifugation, separation conditions: speed 20000r / Min, 120min, 4°C, the obtained precipitate was suspended in 700μl filtered PBS for NTA analysis of the total number of cMVs; the remaining supernatant was centrifuged, separation conditions: 169000r / min, 6h, 4°C, the obtained precipitate was u...

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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 present invention relates to the application of reagents for detecting CD105, CD144, CD34, KDR, Annexin V and CD63 in the preparation of reagents for detecting extracellular vesicles released by endothelial and endothelial progenitor cells in blood. The method has the highest sensitivity and Specificity belongs to the field of medical inventions. Background technique [0002] Extracellular vesicles (EVs) are a type of biologically active small vesicles secreted and released by cells. EVs can carry and transfer the lipids, functional proteins, messenger RNAs (mRNAs) and micro RNAs (miRNAs) of the mother cell into the target cells, and regulate the gene expression and function of the target cells. Due to the limitations of technical level and understanding, when researchers first proposed the concept of EVs, they only regarded EVs as "garbage bags" for cells, allowing cells to get rid of some useless protein garbage. Further research in the past te...

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Application Information

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Patent Type & Authority Patents(China)
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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