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Method for making exosome carry exogenous microRNA

An exosome and exogenous technology, applied in the field of making exosomes carry exogenous microRNA, can solve the problems of low import efficiency, high cost, and cumbersome procedures, and achieve the effects of high import efficiency, simple operation, and simple operation method

Pending Publication Date: 2019-05-14
LANZHOU UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantage: low import efficiency
Disadvantages: The cells after transfection are damaged, a large number of cells die, the number of living cells is significantly reduced, and the amount of exosomes produced is greatly reduced
In addition, for clinical use in the future, the procedures are cumbersome, the cost is high, and the potency is low

Method used

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  • Method for making exosome carry exogenous microRNA
  • Method for making exosome carry exogenous microRNA
  • Method for making exosome carry exogenous microRNA

Examples

Experimental program
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Effect test

Embodiment 1

[0030] The method of the present invention is described below by taking the introduction of exogenous miR-335-5p into exosomes derived from mesenchymal stem cells as an example:

[0031] 1. Extraction of exosomes in cell culture medium:

[0032]Collect the cell culture medium and centrifuge at 300×g (10min), 2000×g (10min), and 10000×g (30min) in order to remove large cells and cell death debris, so keep the supernatant in these steps. Discard the precipitate. Then centrifuge at 100,000×g (70 min) in an ultracentrifuge, discard the supernatant, keep the precipitate, and store at -80°C, which are Exosomes. It can be used in the next experiment.

[0033] 2. Quantification of Exosomes

[0034] The extracted exosomes were quantified using the BCA kit according to the routine protein quantification method.

[0035] 3. Transfection of micro RNA in exosomes

[0036] The micro RNA mimic or micro RNA control NSM used are: hsa-miR-335-5p, miRNA mimic (Life Technologies Corporatio...

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Abstract

The invention provides a method for making an exosome carry exogenous microRNA. The method includes the following steps of transfecting the exogenous microRNA into the exosome, and conducting centrifuging through vivaspin 50,000D. The method has the advantages that existing transfection methods are only used for cell transfection, and the method is used for exosome transfection. In the method, vivaspin is used for concentrating the transfected exosome and removing free microRNA not transfected into the exosome and a transfection agent. The exogenous microRNA cannot be conveyed to a specific position until entering the exosome to be wrapped by the exosome without being degraded in advance. The operation method is simple, various types of microRNA can be introduced, the introducing efficiency is high, and the method can be used on clinic.

Description

technical field [0001] The invention relates to a method for making exosomes carry exogenous microRNA. Background technique [0002] Extracellular vesicles (Extracellular Vesicles, EVs) are a type of vesicles with a membrane-like structure that can be secreted by a variety of cells. The vesicle contains the cytoplasm of the source cell and many proteins of the cell membrane, cytokines, and genetic materials such as DNA and RNA. In recent years, studies have shown that extracellular vesicles are a new tool and method of intercellular communication. Many secreted proteins, cytokines, and genetic materials such as DNA and RNA in cells can be secreted through extracellular vesicles. Transmitted to nearby or distant cells, thereby participating in the regulation of many biological processes. Therefore, cell-to-cell communication mediated by extracellular vesicles plays a very important role in many aspects of the organism, such as in the development of tumors such as tissue inv...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N5/10
Inventor 王芳田宏伟
Owner LANZHOU UNIVERSITY
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