Lamellar extracellular matrix (ECM) material enriched with vascular endothelial growth factor a (VEGFa) as well as preparation method and application thereof

A growth factor and vascular endothelial technology, applied in the field of extracellular matrix preparation, can solve the problems of insufficient application of treatment, limited quantity, high cost, etc.

Pending Publication Date: 2020-05-01
SUZHOU INST OF NANO TECH & NANO BIONICS CHINESE ACEDEMY OF SCI
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Problems solved by technology

The main problems are: (1) Endothelial cell differentiation medium is the most commonly used method to induce mesenchymal stem cells to differentiate into endothelial cells, although the chemical composition of the medium is clear and contains various growth factors that promote the differentiation of mesenchymal stem cells into endothelial cells
B

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  • Lamellar extracellular matrix (ECM) material enriched with vascular endothelial growth factor a (VEGFa) as well as preparation method and application thereof
  • Lamellar extracellular matrix (ECM) material enriched with vascular endothelial growth factor a (VEGFa) as well as preparation method and application thereof
  • Lamellar extracellular matrix (ECM) material enriched with vascular endothelial growth factor a (VEGFa) as well as preparation method and application thereof

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preparation example Construction

[0035] One aspect of the embodiments of the present invention provides a method for preparing a sheet-like extracellular matrix (hereinafter referred to as "ECM") material rich in vascular endothelial growth factor a (hereinafter referred to as "VEGFa"), which includes:

[0036] Constructing a recombinant vector containing a CBD-VEGFa fusion gene, and then using the recombinant vector to introduce the CBD-VEGFa fusion gene into a host cell, and then constructing a recombinant cell capable of expressing the vascular endothelial growth factor a;

[0037] Inoculating the recombinant cells on a culture dish for culturing;

[0038] The recombinant cells attached to the culture dish were freeze-dried to obtain a sheet-like extracellular matrix rich in vascular endothelial growth factor a.

[0039] In some preferred embodiments, the preparation method specifically includes: connecting the CBD-VEGFa fusion gene to the multiple cloning site of the pLVX-IRES-Puro vector, thereby constru...

Embodiment 1

[0056] The preparation method of a kind of extracellular matrix rich in vascular endothelial growth factor-a provided by this embodiment, the main technical process can be divided into four steps (see figure 1 Shown): 1. Vector construction; 2. Cell construction; 3. ECM preparation; 4. ECM performance testing.

[0057] Each step is described in detail below:

[0058] 1. Carrier Construction

[0059] (1) Select the appropriate carrier skeleton. The present invention selects the lentiviral vector pLVX-IRES-Puro (hereinafter referred to as PLVX) as the basic skeleton of the constructed recombinant vector.

[0060] (2) The National Center of Biotechnology Information (NCBI) website (https: / / www.ncbi.nlm.nih.gov / ) found the coding sequence (codingsequence, CDS) sequence of VEGFa and CBD.

[0061] (3) Use the Primer5 primer design software to design the primers required for VEGFa and CBD, and then submit them to the company to synthesize the primers. The list of primers is shown ...

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Abstract

The invention discloses a lamellar extracellular matrix (ECM) material enriched with a vascular endothelial growth factor a (VEGFa) as well as a preparation method and application thereof. The preparation method of the lamellar ECM material enriched with the VEGFa comprises the following steps: constructing a recombinant vector containing a collagenbinding domain-VEGFa (CBD-VEGFa) fusion gene; introducing the CBD-VEGFa fusion gene into host cells by using the recombinant vector so as to construct recombinant cells capable of expressing the VEGFa; inoculating a Petri dish with the recombinant cells so as to carry out cultivation; and then, freeze-drying the recombinant cells attached on the Petri dish so as to obtain the lamellar ECM material enriched with the VEGFa. According to the preparation method of the lamellar ECM material enriched with the VEGFa, VEGFa carrying the CBD is constructed; and then, the VEGFa protein is bound to collagen I of ECM so as to achieve the purpose of enriching the VEGFa protein on the ECM. The lamellar ECM material enriched with the VEGFa prepared by the preparation method can induce mesenchymal stem cells to differentiate into endothelial cells.

Description

technical field [0001] The present invention relates to a preparation method of extracellular matrix, in particular to a VEGFa fusion gene containing CBD (CollagenBinding Domain, short for collagen binding domain, a peptide segment that can bind to collagen), a recombinant expression vector, and a vascular endothelial-rich The sheet-like extracellular matrix material of growth factor a, its preparation method and its application belong to the technical field of biomaterials and tissue engineering. Background technique [0002] Vascular disease is one of the main diseases that threaten human health. Among them, ischemic diseases accounted for nearly 25.6%, especially myocardial infarction, cerebral ischemia and limb ischemia. Under ischemic conditions, tissues cannot effectively exchange gases, nutrients and metabolic wastes, eventually leading to functional impairment and even necrosis. In recent years, more and more experiments have proved that mesenchymal stem cells can ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/66A61K35/12
CPCC12N15/85C12N5/0656C12N15/66A61K35/12C12N2510/00
Inventor 索广力曹俊俊乔勇
Owner SUZHOU INST OF NANO TECH & NANO BIONICS CHINESE ACEDEMY OF SCI
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