Enrichment method of exosome in extracellular matrix, ECM biological material rich in exosome and application

A technology of biomaterials and exosomes, applied to artificial cell constructs, cells modified by introducing foreign genetic material, applications, etc., can solve problems such as high cost and complicated collection process, achieve simple and convenient steps, overcome steps Cumbersome, to achieve the effect of drug delivery

Active Publication Date: 2020-11-20
SUZHOU INST OF NANO TECH & NANO BIONICS CHINESE ACEDEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In experimental research, exosomes are generally extracted by ultracentrifugation, and in order to meet the dosage, cell culture supernatants need to be collected on a large scale, which causes the problem of complicated collection process and high cost

Method used

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  • Enrichment method of exosome in extracellular matrix, ECM biological material rich in exosome and application
  • Enrichment method of exosome in extracellular matrix, ECM biological material rich in exosome and application
  • Enrichment method of exosome in extracellular matrix, ECM biological material rich in exosome and application

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preparation example Construction

[0061] Another aspect of the embodiments of the present invention also provides a method for preparing an exosome-rich ECM biomaterial, which includes:

[0062] The enrichment of exosomes in the extracellular matrix is ​​achieved according to the aforementioned method;

[0063] And, the ECM biomaterial rich in exosomes is prepared by repeated freezing and thawing methods, which can be prepared in liquid or solid form for storage or transportation according to the actual treatment operation requirements.

Embodiment 1

[0068] The present invention provides a technical method of using collagen binding domain polypeptide (Collagen Binding Domain, CBD) to realize the enrichment of exosomes in the extracellular matrix, and then adopts repeated freezing and thawing methods to prepare extracellular matrix rich in a large amount of exosomes biomaterials. The main technical process can be divided into four steps: 1. Transgenic fusion of exosome membrane protein lamp 2b and CBD polypeptide; 2. ECM biological sponge captures exosomes to achieve exosome enrichment; 3. Enrichment Preparation of exosome-containing ECM biomaterials.

[0069] Each step is described in detail below:

[0070] 1. Transgenic fusion of exosomal membrane protein LAMP 2b and CBD polypeptide

[0071] 1. First use trizol to extract the total RNA of HFF cells; then use reverse transcription mix to generate cDNA; use the two primers LAMP2b forward and LAMP 2b reverse to extract the LAMP 2b sequence from the whole genome through PCR...

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Abstract

The invention discloses an enrichment method of exosome in an extracellular matrix, an ECM biological material rich in the exosome and application. The invention further discloses a CBD-LAMP 2b fusiongene. A cell line containing the CBD-LAMP 2b fusion gene can stably express a CBD-LAMP 2b protein and can stably secrete the exosome combined to the extracellular matrix, and the enrichment of the exosome in the extracellular matrix is achieved. The ECM biological material is synthesized by the cell line and secreted to the outside of the cell line. According to the invention, the LAMP 2b fusionprotein carrying the collagen binding region CBD is constructed, so that the exosome has the capability of being combined to collagen I which is one of ECM components. ECM biological sponge realizes grabbing and enrichment of the exosome, and the ECM biological material rich in the exosome is prepared by adopting a repeated freezing and thawing mode and can be used as a drug or miRNA carrier to realize disease drug delivery.

Description

technical field [0001] The present invention relates to a method for enriching exosomes in extracellular matrix, in particular to a technical method for enriching exosomes in extracellular matrix by using collagen binding domain polypeptide (Collagen Binding Domain, CBD), and The preparation and application of extracellular matrix biomaterials rich in a large amount of exosomes belong to the technical field of biomaterials and genetic engineering. Background technique [0002] Exosomes (exosomes) are extracellular vesicles (Extracellular Vesicles, EVs) with a diameter of 50-200 nm formed by the fusion of multivesicular bodies and cell membranes (Gurunathan S, Kang M, Jeyaraj M, et al. Review of the Isolation, Characterization, Biological Function, and Multifarious Therapeutic Approaches of Exosomes.[J].Cells,2019,8(4).), the buoyant density in the sucrose gradient is 1.13-1.18g / ml, and it is cup-shaped with a membrane structure under the electron microscope . In 1983, exos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N15/85C12N15/66C12N5/10C12N5/0797C12N5/0775C12N5/071C12N5/073A61K48/00A61K35/12A61K47/46A61K31/7105
CPCC07K14/70596C12N15/85C12N15/86C12N15/66C12N5/0662C12N5/0686C12N5/0625C12N5/0603C12N5/0623C12N5/0667A61K48/005A61K48/0008A61K35/12A61K47/46A61K31/7105C07K2319/00C12N2740/15043C12N2740/10043C12N2510/00
Inventor 索广力于延珍乔勇
Owner SUZHOU INST OF NANO TECH & NANO BIONICS CHINESE ACEDEMY OF SCI
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