A method for in vitro derivativing nerve stem cell directional differentiation to cholinergic neuron

A technology for cholinergic neurons and neural stem cells, applied in the fields of bioengineering and developmental biology, can solve the problems of less research on neurotrophic factors and complex research, and achieve the effect of simple operation and good repeatability

Inactive Publication Date: 2005-09-28
SHANGHAI UNIV
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Problems solved by technology

At present, the research on the differentiation of cholinergic neurons is mainly studied from the aspect of gene regulation. Since the differentiation of cholinergic neurons may be regulated by multiple genes, it is more complicated to use this method to study.
Moreover, most of the studies on the differentiation of t

Method used

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Examples

Experimental program
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Embodiment Construction

[0019] 1. Isolation and culture of neural stem cells

[0020] Take out the striatum of the 14-day rat embryo under sterile conditions, put it into the F12-DMEM (1:1) medium containing 1% B27, 20ng / mL EGF, and 10ng / mL bFGF, and blow it to make a cell suspension , at 37°C, 50mL / L CO 2 Routine cultivation under saturated humidity.

[0021] 2. Identification of Neural Stem Cells

[0022] Cells were grown attached to glass slides and removed after 2 days. Aspirate the culture medium, wash off the residual culture medium with PBS, and fix with 4% (m / m) paraformaldehyde for 20 minutes. Wash twice with PBS (once every 10 minutes); block with 10% normal goat serum for 1 hour; wash three times with PBS; anti-nestin antibody (v / v 1:100, diluted with antibody buffer) overnight at 4°C, wash with PBS for 3 times; secondary antibody (v / v 1:50, diluted with antibody buffer) was incubated at 32°C for 1 hour, washed 3 times with PBS; mounted in 50% (v / v) glycerol, and observed for fluoresce...

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Abstract

The process of extracorporeal inducing nerve stem cell to directionally differentiate into cholinergic neuron includes the following steps: separating, culturing and identifying nerve stem cell; adding bFGF, heparin and laminin into F12-DMEM(1:1) culture medium with B27 in 1 % and EGF in 20 ng/mL; l inducing nerve stem cell to differentiate at 37 deg.c, 50 mL/L CO2 and saturated humidity for 7 days; and the immunogical identification of cholinergic neuron. The said process is simple and good in repeatability, and has differentiating rate over 30 %. The present invention provides new way of obtaining cholinergic neuron.

Description

technical field [0001] The invention relates to bioengineering and developmental biology, in particular to research on the differentiation of neural stem cells, and more specifically to a method for inducing the directional differentiation of neural stem cells into cholinergic neurons in vitro. Background technique [0002] The discovery of neural stem cells is a major breakthrough in the field of neuroscience in recent years, and has become a recent research hotspot, not only because of its importance in studying the development of the nervous system, but also because of its potential therapeutic value [1] . Mckay [2] Neural stem cells refer to cells with self-renewal ability and the ability to differentiate into neurons, astrocytes and oligodendrocytes. Self-renewal and multi-differentiation ability are two basic properties of neural stem cells. [0003] The multi-differentiation potential of neural stem cells has become one of the current research hotspots. When trans...

Claims

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Application Information

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IPC IPC(8): C12N5/06G01N33/533
Inventor 陈付学任雯雯杨洋过七根宋红生文铁桥
Owner SHANGHAI UNIV
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