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Method for inducing differentiation of neural stem cells (NSCs) into dopaminergic neurons

A neural stem cell and dopaminergic technology, applied in the field of stem cell therapy, can solve the problem of low differentiation rate of dopamine neurons

Inactive Publication Date: 2016-02-03
SHANDONG QILU STEM CELL ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the problem that the differentiation rate of NSCs derived from the forebrain into functional mature dopaminergic neurons is not high in the above prior art, the present invention provides a method for inducing neural stem cells to differentiate into dopaminergic neurons with a differentiation rate as high as 30%. method

Method used

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  • Method for inducing differentiation of neural stem cells (NSCs) into dopaminergic neurons
  • Method for inducing differentiation of neural stem cells (NSCs) into dopaminergic neurons
  • Method for inducing differentiation of neural stem cells (NSCs) into dopaminergic neurons

Examples

Experimental program
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Embodiment 1

[0026] Implementation Example 1: Extraction of Forebrain Neural Stem Cells

[0027] This example uses the forebrain of aborted embryos authorized with donor consent as a source of neural stem cells.

[0028] Take a 12-week-old complete aborted embryo, wash it with HBSS (Hyclone) for several times, fix the head of the embryo, use small scissors to cut the entire cranial cavity at the foramen magnum to the occipital side, and gently remove the fetal brain with tweezers. The cranial cavity was peeled off, placed in a disposable sterile petri dish, and washed with a small amount of PBS (Hyclone) buffer. Peel off the meninges with fine forceps and remove the cerebellum. The midbrain and forebrain were separated at the peduncle of the brain, and after the forebrain was ground, the two were resuspended in a 50 mL centrifuge tube with a small amount of DMEM medium. After blowing evenly, filter the suspension with a 100um sieve, and then filter it with a 40um sieve. Centrifuge the f...

Embodiment 2

[0029] Implementation Example 2: Preparation of medium before and after NSC cell induction

[0030] Ordinary medium: NeuralBasal medium (Invitrogen), B27 (1:50, Invitrogen), bFGF (20ng / mL, R&D), EGF (20ng / mL, R&D), heparin sodium (5μg / ml, Changzhou Qianhong Biochemical) , L-Glutamine (2mM, Invitrogen)

[0031]Induction medium: NeuralBasal medium (Invitrogen), B27 (1:50, Invitrogen), fetal bovine serum (1%, Gibco), heparin sodium (5 μg / ml, Changzhou Qianhong Biochemical), L-glutamine (2mM, Invitrogen), TPA (phorbol12-myristate13-acetate, 100nM, sigma), Forskolin (25uM, sigma), acidicFGF (100ng / mL, R&D), BMP-7 (50ng / ml, sigma), pramipexole (10uM, sigma )

Embodiment 3

[0032] Implementation Example 3: Induced Differentiation of NSC Cells by Mixed Inducing Agents

[0033] NSC cells were cultured in the common medium mentioned in Example 2. When neurospheres with a diameter of 0.5 mm were formed, polylysine-coated coverslips were placed in 24-well plates, and 3-5 neurospheres and 1mL of the induction medium mentioned in Example 2, the neurospheres were added dropwise to the central area of ​​the coverslip and cultured until the neurospheres adhered to the wall. The induction medium was changed every 2 days, and the dopaminergic neurons were detected after 6 days of culture.

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Abstract

The invention relates to stem cell differentiation technology in the technical field of stem cell treatment and in particular relates to a method for inducing differentiation of neural stem cells (NSCs) into dopaminergic neurons. The method is characterized by culturing 1-2 generation NSCs from the forebrain with a NSC basal culture medium until the diameters of neural spheres are 0.5-1mm, then changing the culture medium to an induction culture medium to enable the cells to grow adhering to the wall and obtaining dopaminergic neurons after culture for 3-10 days, wherein the induction culture medium contains inducers aFGF, TPA, forskolin, pramipexole and BMP-7. The method has the beneficial effects that after the NSCs from the forebrain are cultured by utilizing the induction culture medium, the proportion of the dopaminergic neurons obtained through differentiation of the NSCs is 30%, thus greatly increasing the differentiation rates of the dopaminergic neurons; the dopamine secretion rates of the neurons are increased by more than 20 times; the NSCs from the forebrain are effectively induced into the dopaminergic neurons.

Description

technical field [0001] The invention relates to stem cell differentiation technology in the technical field of stem cell therapy, in particular to a method for inducing neural stem cells to differentiate into dopaminergic neurons. Background technique [0002] Parkinson's disease (PD) is a neurodegenerative disease that often occurs in middle-aged and elderly people, and its main pathological change is degeneration and necrosis of dopamine neurons in the substantia nigra of the midbrain. The main clinical symptoms are resting tremor, bradykinesia, muscle rigidity, and abnormal posture and gait. Although infusion of levodopa can significantly improve patients' symptoms, Parkinson's symptoms are considered incurable and reversible [Suzuki, NeurosciLett.2015]. Replacement therapy targeting diseased cells, especially the replacement of degenerated dopamine cells with dopamine (Dopamine, DA) neurons to restore dopamine transmission in the nigrostriatum of the brain is consid...

Claims

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Application Information

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IPC IPC(8): C12N5/0793
Inventor 杨洪娜陈恒刘小盾曲廷瑜
Owner SHANDONG QILU STEM CELL ENG
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