Method of inducing differentiation of neural stem cell into dopaminergic neuron by using recombinant slow virus

Abstract

The invention relates to bioengineering technologies, and particularly relates a method of inducing differentiation of neural stem cells into dopaminergic neurons by using recombinant slow virus. The method comprises the following steps: constructing a recombinant slow virus vector; separating, culturing and identifying the neural stem cells; transfecting the recombinant slow virus into NSCs; identifying the NSCs transfected by the recombinant slow virus, wherein the process of transfecting the recombinant slow virus into the NSCs comprises the following steps: digesting NSCs after being subjected to three times of passages to prepare a single-cell suspension, and inoculating into a culture plate; transfecting the TH+Brn4 gene recombinant slow virus; adding the slow virus and ploybrene, uniformly shaking, putting the culture plate into an incubator to culture for 1-2 hours, and replacing fresh differential culture medium; meanwhile, screening by using puromycin to obtain stable transfected cells. The method has the advantages as follows: by constructing the slow virus vectors of target genes TH and Brn4, exogenous genes after transfecting the NSCs can be stably expressed in cells; high proportion of TH positive dopaminergic neuron is generated after differentiation; the Brn4 can promote high expression of neurotrophic factors and has the function of inhibiting apoptosis.

Description

technical field [0001] The invention relates to a method for using recombinant lentivirus to induce neural stem cells to differentiate into dopaminergic neurons. Background technique [0002] Parkinson's disease (Parkinson's Disease, PD) is a common degenerative disease of the central nervous system. , resulting in a decrease in the release of dopamine neurotransmitters in the striatum, resulting in a series of movement disorder symptoms such as tremors and muscle rigidity. [0003] Traditional drug therapy and surgical treatment are limited to improving symptoms, but they cannot prevent the progression of the disease and cannot fundamentally solve the problem. Since this lesion mainly invades the dopaminergic neurons in the substantia nigra of the midbrain, and the striatum, the target area projected by the dopaminergic neurons in the substantia nigra, is clearly positioned, cells that can secrete dopamine are transplanted in the substantia nigra-striatum system to restore...

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Problems solved by technology

However, the molecular mechanism by which Brn-4 promotes the differentiation of NSCs into mature neurons and maintains the survival of transplanted cells has not been studied at home and abroad.

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