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Glioma cell exosomes containing mir-124 and preparation method and application thereof

A mir-124, glioma cell technology, applied in the field of glioma cell exosomes and their preparation, can solve the problems of not easy to expand in large quantities, unstable exosome yield and composition, and high cost of cell culture.

Active Publication Date: 2022-07-01
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing research results have confirmed that exosomes carrying miR-124 promote nerve regeneration [7] , inhibit inflammation [8] , improve cognition [9] , control autophagy [10] etc. have significant and efficient effects, but the specific impact on astrocytes has not been reported
And researchers often choose microglia and mesenchymal stem cells as the exosome-secreting parent cells. Although these cells are relatively mature in technology, they are difficult to obtain, not easy to expand in large quantities, and have different cell phenotypes in different culture generations. Instability, instability of exosome production and composition, and high cost of cell culture

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  • Glioma cell exosomes containing mir-124 and preparation method and application thereof

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preparation example Construction

[0046] The present invention provides a preparation method of glioma cell exosomes containing miR-124, comprising the following steps:

[0047] 1) The supernatant obtained by culturing the glioma cell line is separated by solid-liquid to obtain exosomes;

[0048] 2) The exosomes and miR-124 are mixed and incubated together to obtain glioma cell exosomes containing miR-124.

[0049] In the present invention, the supernatant obtained by culturing the glioma cell line is subjected to solid-liquid separation to obtain exosomes.

[0050] The present invention does not specifically limit the type and source of the glioma cell line, and a well-known glioma cell line in the art may be used, such as the U-87 cell line. The present invention does not specifically limit the method for culturing the glioma cells, and the method for culturing glioma cells well known in the art may be used.

[0051] In the present invention, the solid-liquid separation preferably includes centrifugation a...

Embodiment 1

[0062] Isolation and characterization of astrocyte exosomes

[0063] U-87 cells (ATCC Cell Resource Center, USA) were cultured with exosome-free serum medium for 24 h, and the supernatant of U-87 cells was collected. Centrifuge the supernatant at 3000g, 4°C for 20min, and collect the supernatant after centrifugation; centrifuge at 10000g, 4°C for 60min, collect the supernatant after centrifugation, and filter the supernatant with a 0.22 μm filter; Transfer to an ultracentrifuge tube, centrifuge at 120,000g, 4°C for 70min, discard the supernatant after centrifugation, the obtained pellet is exosomes, resuspend the pellet with 100μl PBS buffer and transfer the pellet to a 1.5ml EP tube to obtain Exosome suspension, long-term storage at -80 ℃.

[0064] Add 5 μl of the exosome suspension to the Formvar-carbon sample-loaded copper grid, allow it to adsorb for 4-5 min, then carry out standard uranyl acetate staining, and take electron microscope pictures at 80kV. Electron microsco...

Embodiment 2

[0069] Primary culture and purification of RAs:

[0070] The 1-3 day old rat suckling mice were taken out and immersed in 75% alcohol for 15 minutes and then transferred to a sterile operating table. The surface of the rat suckling mice was washed with PBS and the alcohol was used, and the brains were removed with micro scissors and micro tweezers. , placed in sterile PBS, peeled off the cerebral cortex under the microscope, put it into a non-adherent cell dish containing 10ml DME / F12 (HyClone, SH30023.01), and crushed the cerebral cortex with a 5ml pipette tip particle suspension. The tissue fluid was collected in a centrifuge tube, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded to collect the pellet. Tissues were digested with 0.05% trypsin and DNase (Deoxyribonuclease I, Worthington) for 25 minutes at 37°C with shaking. After the digestion was terminated, a single cell suspension was obtained by filtration through a sterile filter 40 μm-sterileEASYst...

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Abstract

The invention provides miR-124-containing glioma cell exosomes, a preparation method and application thereof, and belongs to the technical field of central nervous system damage repairing drugs. A method for preparing glioma cell exosomes containing miR-124, the supernatant obtained by culturing a glioma cell line is subjected to solid-liquid separation; the separated exosomes and miR-124 are mixed and incubated together to obtain a Glioma cell exosomes of miR‑124. The exosomes prepared above can effectively bind to cholesterol-modified miR-124 and be absorbed by reactive astrocytes, significantly increase the expression of miR-124 in reactive astrocytes, and inhibit astrocytes. Activation and scarring. Therefore, the present invention also provides the application of miR-124-containing glioma cell exosomes in the preparation of a drug for inhibiting astrocyte activation and / or glial scar formation, thereby treating central nervous system injury diseases.

Description

technical field [0001] The invention belongs to the technical field of drugs for repairing damage to the central nervous system, in particular to miR-124-containing glioma cell exosomes and a preparation method and application thereof. Background technique [0002] Astrocytes (As) are the main components of glial cells in the vertebrate central nervous system. When the central nervous system is injured, As is activated into reactive astrocytes (RAs), which synthesize and secrete extracellular matrix mainly composed of chondroitin and keratan sulfate proteoglycan, and interact with microglia, Macrophages, etc. form dense glial scars, affecting the regeneration, elongation and fusion of axons, and compressing microvessels, affecting local blood supply [1] . The glial scars formed by RAs can form a physical barrier and hinder the migration of transplanted cells, so that the use of neural stem cells, mesenchymal stem cells, embryonic stem cells and other cells to treat central...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/46A61K31/7105A61P25/00C12N5/09
CPCA61K47/46A61K31/7105A61P25/00C12N5/0693C12N2509/10
Inventor 池光范葛鹏飞于逸飞
Owner JILIN UNIV
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