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Vesicle and application thereof

A technology of vesicles and markers, applied in the field of biomedicine, can solve the problems of short treatment period, easy production of autoantibodies, and high treatment cost

Pending Publication Date: 2021-07-20
EV CELL BIOTECH GUANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For a long time, for the treatment of hemophilia, injection of exogenous blood coagulation factors has been the main clinical intervention. However, this method has various problems such as high treatment cost, short treatment period, and easy production of autoantibodies, so it cannot be practical and effective. treatment

Method used

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  • Vesicle and application thereof
  • Vesicle and application thereof
  • Vesicle and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] The isolation culture of embodiment 1 MSCs

[0125] Excess CO was used according to the guidelines of the Animal Ethics Committee 2 Sacrifice the mice, remove the tibia and femur under aseptic conditions, peel off the muscles and connective tissue attached to them, further separate the metaphysis, expose the bone marrow cavity, and extract the volume fraction of 10% with a 10mL sterile syringe. The bone marrow cavity was washed repeatedly with PBS of fetal bovine serum, filtered through a cell strainer with a pore size of 70 μm, centrifuged at 500 g for 5 min, the supernatant was removed, and the cell pellet at the bottom was collected, resuspended in PBS, centrifuged at 500 g for 5 min again, and the final cell pellet was collected. Then, the cells were sorted by flow cytometry, and BMMSCs were sorted out by using CD34- and CD90+ as sorting criteria. Finally, the cells were resuspended in Dex(-) medium and inoculated in a 10cm diameter cell culture dish at 37°C, 5% CO...

Embodiment 2

[0132] Example 2 Obtaining of inducible vesicles

[0133] The MSCs (bone marrow-derived MSCs, BMMSCs) cultured to the second generation in Example 1 were continued to be cultured with the medium (Dex(+) culture fluid) in Example 1 until the cells were confluent 80%-90%, and then used Rinse twice with PBS, add serum-free medium (α-MEM medium) containing 500nM STS to induce apoptosis, incubate at 37°C for 24h, and collect cell supernatant for isolation and extraction of IEVs.

[0134] Separation and extraction of IEVs from the collected culture supernatant, the operation process is as follows: figure 2 As shown, the specific steps include: after centrifuging at 800g for 10 minutes, collecting the supernatant; then centrifuging at 2000g for 10 minutes, then collecting the supernatant; then centrifuging at 16000g for 30 minutes, removing the supernatant, and resuspending IEVs in sterile PBS; After centrifugation at 16000g for 30 minutes, the supernatant was removed and IEVs were...

Embodiment 3

[0138] The analysis of embodiment 3 IEVs

[0139] 1. Quantification of IEVs and analysis of membrane proteins

[0140] Utilize flow cytometry to carry out quantitative analysis to the IEVs that embodiment 2 obtains, measurement time point is the 1st, the 4th, the 8th, the 16th and the 24th h, the result shows 10 6 Each MSCs can produce 0.76×10 after induction to 1h, 4h, 8h, 16h and 24h respectively 8 pcs, 1.29×10 8 pcs, 1.95×10 8 pcs, 2.48×10 8 pcs, 3.14×10 8 IEVs, it can be seen that after induction to 24h, a single MSC can produce 300 IEVs ( image 3 ).

[0141] In addition, flow cytometry found that the particle diameter distribution of IEVs was concentrated below 1 μm, accounting for 94.97% ( Figure 4A ), the results of side scattered light (SSC) analysis also showed that the scattered light intensity of IEVs was concentrated in the range below 1 μm ( Figure 4B ). Further, the scattered light intensity of IEVs was analyzed by standardized small particle microsph...

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Abstract

The invention belongs to the field of biological medicine, and relates to a vesicle and application thereof. The source of the vesicle comprises stem cells or somatic cells, the marker of the vesicle comprises Syntaxin 4, and the vesicle is an induced vesicle. Compared with exosomes in MSCs, the vesicle provided by the invention can specifically express Syntaxin 4, and can be used for distinguishing MSCs-derived vesicles from characteristic markers of Exosomes. The method for preparing the vesicle has the advantages that the yield is high, 300-2000 vesicles can be produced by single MSCs, the preparation process is simple, the consumed time is short, the requirements on reagents and equipment are low, and the treatment effect is good. The vesicle can play a significant role in coagulation promotion in vitro, can significantly improve the bleeding tendency of hemophilia mice after being injected in vivo, and can be used for treatment for improving the bleeding tendency of hemophilia. Moreover, the vesicle can be discharged through skin and hair and is safe in vivo, so that the vesicle has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a vesicle and an application thereof. Background technique [0002] Extracellular vesicles (EVs) are nanoscale carriers secreted by cells that contain proteins, nucleic acids and various cytokines. Extracellular vesicles can act on target cells in an endocrine or paracrine manner, and play an important role in the process of intercellular material transfer and information exchange. Studies have found that the information exchange mediated by extracellular vesicles plays an important regulatory role in the physiological or pathological processes of the body, involving immune regulation, tumor growth, angiogenesis, and damage repair. Current research in this field is mainly focused on exosomes. Exosomes are extracellular vesicles with a diameter of about 30-150 nm, which contain components such as RNA, lipids, and proteins. Exosomes are widely involved in various physiological / pathologic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/077C12N5/0775G01N33/68C12Q1/6893
CPCC12N5/0696C12N5/0662C12N5/0654G01N33/6893C12Q1/6893G01N2800/085G01N2800/102G01N2800/28G01N2800/285G01N2800/22G01N2800/044
Inventor 张晓寇晓星施松涛
Owner EV CELL BIOTECH GUANGZHOU CO LTD
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