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Application of vesicle in preparation of hemophilia medicine

A technology for hemophilia and vesicles, applied in the field of biomedicine, can solve the problems of easy production of autoantibodies, high treatment cost, and inability to become a treatment method.

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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  • Application of vesicle in preparation of hemophilia medicine
  • Application of vesicle in preparation of hemophilia medicine
  • Application of vesicle in preparation of hemophilia medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The isolation culture of embodiment 1 MSCs

[0069] 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 tissues 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, and centrifuged at 500 g for 5 min. After removing the supernatant, the cell pellet at the bottom was collected, resuspended in PBS, and centrifuged again at 500 g for 5 min to collect the final cell pellet. Then the cells were sorted by flow cytometry, and BMMSCs were sorted out using CD34- and CD90+ as sorting criteria. Finally, resuspend the cells in Dex(-) medium and inoculate them in a 10cm diameter cell culture dish at 37°C, 5% CO 2 nou...

Embodiment 2

[0076] Example 2 Obtaining of inducible vesicles

[0077] The MSCs (bone marrow-derived MSCs) cultured to the second generation in Example 1 were continued to be cultured with the medium (Dex(+) medium) in Example 1 until the cells were confluent to 80%-90%, then rinsed with PBS 2 times, adding serum-free medium (α-MEM medium) containing 500nM STS to induce apoptosis, incubating at 37°C for 24h, collecting the cell supernatant for isolation and extraction of IEVs.

[0078] Separation and extraction of IEVs from the collected medium supernatant, the operation process is as follows figure 2 As shown, the specific steps include: centrifuge at 800g for 10 minutes—collect supernatant—centrifuge at 2000g for 10 minutes—collect supernatant—centrifuge at 16,000g for 30 minutes—remove supernatant, resuspend IEVs in sterile PBS—centrifuge at 16,000g for 30 minutes— Remove the supernatant and resuspend IEVs in 300-500ul sterile PBS.

Embodiment 3

[0082] The analysis of embodiment 3 IEVs

[0083] (1) Quantification of IEVs and analysis of membrane proteins

[0084] 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 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 ).

[0085] In addition, flow cytometry found that the particle diameter distribution of IEVs was concentrated below 1 μm, accounting for 94.97% ( Figure 4A ).

[0086] 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 ).

[0087] Further, the scattered light intensity of IEVs was analyzed by the standardized sma...

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Abstract

The invention belongs to the field of biological medicines, and relates to application of a vesicle in preparation of medicines for treating hemorrhagic diseases. The invention provides an application of a vesicle in preparation of a medicine for treating hemorrhagic diseases. The vesicle is an induced vesicle. The induced vesicle provided by the invention can exert a remarkable coagulation promoting effect in vitro, can remarkably improve the bleeding tendency after being injected in vivo, can be used for improving bleeding caused by blood coagulation factor deficiency, platelet number reduction and / or functional defects, for example, can be used for improving bleeding tendency of hemophilia, lupus erythematosus and treating Chediak-Higashi syndrome, can be discharged through skin and hair, has safety in vivo, and has good application prospects.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to the application of vesicles in the preparation of hemophilia drugs. 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 va...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61K35/545A61P7/04
CPCA61K35/28A61K35/545A61P7/04C12N5/0696C12N5/0663C12N2510/02C12N2502/1323C12N2501/115G01N2333/705G01N2333/4718G01N2333/70546G01N33/6893C12N5/0662
Inventor 张晓寇晓星施松涛
Owner EV CELL BIOTECH GUANGZHOU CO LTD
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