Method for directionally differentiating neural stem cells in human embryo midbrain into dopaminergic neuron in vitro

A neural stem cell and dopaminergic technology applied in the biological field to achieve the effect of increasing the success rate of induction

Inactive Publication Date: 2013-04-17
吴卫江
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Problems solved by technology

However, the research on neural stem cells is still in its infancy, and many problems have not been resolved, such as how to differentiate neural stem cells into the specific neural c

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  • Method for directionally differentiating neural stem cells in human embryo midbrain into dopaminergic neuron in vitro

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[0015] Main experimental instruments: ultra-clean workbench (Suzhou Sujing Antai Co.), inverted microscope (Olympus CK40), fluorescence microscope (Olympus BX51), CO 2 Incubator (REVCO), 75cm 2 Petri dishes (Corning, NY). Preparation of cell culture reagents and culture medium: DMEM / F12, B27 (Gibco), EGF (Serotec) and bFGF (Chemicon International), fetal bovine serum (Gibco). Serum-free medium DMEM / F12(1:1)+5%Fcs; serum-free medium DMEM / F12(1:1)+B27(1:50)+EGF(20ng / mL)+bFGF(20ng / mL ). Immunohistochemical antibodies: the primary antibody was rabbit anti-human NF antibody (Sigma), rabbit anti-human GFAP antibody (Sigma), rabbit anti-human TH antibody (Sigma), and the secondary antibody was FITC goat anti-rabbit fluorescent secondary antibody (Chemicon).

[0016] The method for in vitro directed differentiation of neural stem cells derived from human embryo midbrain into dopaminergic neurons comprises the following steps:

[0017] S1: Isolation and culture of primary cells: ta...

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Abstract

The invention provides a method for directionally differentiating neural stem cells in human embryo midbrain into dopaminergic neuron in vitro, and the method comprises the following steps of: isolated culture of primary cells; continuous cell culture of neural stem cells; induced differentiation culture of a nutrient solution containing ascorbic acid; differentiation culture of normal adult cerebrospinal fluid; detection of immumohistochemical staining; and cell counting. According to the method for forming dopaminergic neuron through in vitro induction, ascorbic acid of certain concentration is firstly adopted for induced differentiation, then normal adult cerebrospinal fluid is adopted for further differentiating stem cells in embryo midbrain into dopaminergic nerve cells, so that dopaminergic neuron of the highest proportion can be obtained, and the method provides a new way for generating DA (Dopi Amine) neuron effectively in vitro so as to be applied to curing Parkinson disease through clinical transplantation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for directional differentiation of neural stem cells into dopaminergic neurons in vitro. Background technique [0002] Neural stem cells have broad clinical application prospects. Cell replacement therapy can treat neurodegenerative diseases such as Parkinson's disease, Huntington's disease, and Alzheimer's disease, as well as neurological deficits caused by stroke and traumatic brain injury. As a carrier of gene therapy, stem cells can treat Parkinson's disease, mucopolysaccharidosis and intracranial tumors. However, the research on neural stem cells is still in its infancy, and many problems have not been resolved, such as how to differentiate neural stem cells into the specific neural cells we need in vivo and in vitro, and the relationship between neural stem cells isolated in vitro and those in vivo. Whether there are differences, etc., these need further research. ...

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Application Information

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IPC IPC(8): C12N5/0793C12N5/0797
Inventor 徐杰房文峰朱爱华
Owner 吴卫江
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