Method for inducing neural stem cells in vitro to directionally differentiate into neurons

A neural stem cell and directional differentiation technology, applied in the field of in vitro induction of directional differentiation of neural stem cells into neurons, can solve the problems of lack of uniform standards, low differentiation ratio of neural stem cells, and low purity of neurons, so as to promote pure differentiation and high purity , good repeatability

Active Publication Date: 2020-01-17
XINYANG NORMAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the method of inducing neural stem cells to differentiate into neurons in vitro is still in the exploratory stage, and no unified standard has been formed. The differentiation ratio of neural stem cells into neurons is relatively low, and the purity of neurons obtained is low.

Method used

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  • Method for inducing neural stem cells in vitro to directionally differentiate into neurons
  • Method for inducing neural stem cells in vitro to directionally differentiate into neurons
  • Method for inducing neural stem cells in vitro to directionally differentiate into neurons

Examples

Experimental program
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Embodiment 1

[0028] A method for inducing directional differentiation of neural stem cells into neurons in vitro, comprising the steps of:

[0029] (1) Primary culture of neural stem cells

[0030] Under sterile conditions, 14.5-day-pregnant ICR mice were sacrificed by dislocation of the cervical spine, soaked in 75% ethanol for 5-8 minutes, then transferred to an ultra-clean table for dissection, the fetal mice were taken out, and the brain cortex was separated, and then passed through 1×PBS After rinsing with DMEM / F12 solution, transfer to the growth medium containing 1% N2, 1% B27, 20ng / mL bFGF, 20ng / mL EGF, 97% DMEM / F12, and cut the brain tissue with dissecting scissors, Use a 1mL sterile pipette tip to blow repeatedly into a single-cell suspension. After the single-cell suspension is filtered through a 140-mesh nylon screen, transfer it to a 50ML centrifuge tube. The solution formula is: 1% N2, 1% B27, 20ng / mL bFGF, 20ng / mL EGF, 97% DMEM / F12) to adjust the cell density to 1×10 6 cel...

Embodiment 2

[0038] In step (3), the cell density of plating is 5×10 4 cells / mL, the rest are the same as in Example 1.

Embodiment 3

[0040] In step (3), the cell density of plating is 5×10 6 cells / mL, the rest are the same as in Example 1.

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Abstract

The invention relates to a method for inducing neural stem cells in vitro to directionally differentiate into neurons. The method includes: using fetal rat brain tissue of an ICR (institute of cancerresearch) pregnant rat as a raw material to carry out primary culture on neural stem cells so as to obtain neurospheres, centrifuging to remove supernate, washing precipitate with PBS (phosphate buffered solution), allowing digestion to occur, adding a culture solution to terminate the digestion, centrifuging to remove supernate, adding a growth culture solution into the precipitate, carrying outsecondary culture on the neural stem cells, digesting the fourth-generation or fifth-generation neurospheres to prepare a single-cell suspension, spreading the single-cell suspension to a 12-orifice cell culture plate coated with poly-l-ornithine and laminin under the cell density of 5*104 to 5*106 cells per mL, culturing overnight, adding a differentiation culture solution on the second day, andcarrying out differentiation culture to obtain mature neurons. The method is simple and well repeatable, allows the differentiation ratio of neural stem cells to neurons to be evidently increased, canpromote purity differentiation of neurons and helps attain the neurons with good growth state and high purity.

Description

technical field [0001] The invention relates to a method for inducing directional differentiation of neural stem cells into neurons in vitro, belonging to the technical field of biomedicine. Background technique [0002] Neural stem cells are pluripotent cells with self-renewal and differentiation capabilities in the central nervous system, which can differentiate into neurons, astrocytes and oligodendrocytes. The discovery of neural stem cells is of great significance, providing new research directions for central nervous system development, nerve injury repair, and treatment of neurodegenerative diseases, and has broad prospects for the repair and treatment of nervous system diseases or injuries. Studies have shown that the number of neural stem cells in the adult animal brain is small and most of them are in a resting state. Other studies have shown that most of the exogenous neural stem cells transplanted into the host differentiate into glial cells, and only a small num...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793C12N5/0797
CPCC12N5/0619C12N2501/11C12N2501/115C12N2506/08C12N2533/32C12N2533/52
Inventor 周棋赢韩月华周玉玲刘磊杨宁宁王仪佳王艳丽梁萍
Owner XINYANG NORMAL UNIVERSITY
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