Method for co-culturing with nerve stem cell to inducing rASCs to be DA Neuron by Lmx1a mediation

A technology of neural stem cells and neurons, which is applied in the field of treating Parkinson's disease and inducing stem cells to replace defective neuron cells, achieving the effects of low immunogenicity, simple in vitro culture operation, and convenient material collection

Inactive Publication Date: 2013-07-24
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

However, the differentiation of dopaminergic neurons is a complex process induced by multi-factors and multi-steps, which requires the coordinated action of multiple signaling pathways inside and outside the cell at different times to activate a variety of transcription factors and cause changes in the broad gene expression profile of cells

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  • Method for co-culturing with nerve stem cell to inducing rASCs to be DA Neuron by Lmx1a mediation
  • Method for co-culturing with nerve stem cell to inducing rASCs to be DA Neuron by Lmx1a mediation
  • Method for co-culturing with nerve stem cell to inducing rASCs to be DA Neuron by Lmx1a mediation

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Embodiment Construction

[0061] 1. Lmx1a-mediated co-culture with neural stem cells to induce rASCs into DA neurons

[0062] (1) Cloning of Lmx1a gene and construction of recombinant plasmid

[0063] Using Lmx1a whole transcript cDNA (NM_138396) as a template, the primers are:

[0064] P1 up: 5′-GCGTCGACGTACCATGCTGGACGGCCTAAAG-3′,

[0065] P1 down: 5′-CGGGATCCCGCTTATCAAGATGTGAAGTAAG-3′,

[0066] The DNA fragments with SalI and EcoR V restriction sites were amplified by PCR, detected and analyzed by 1% agarose gel electrophoresis, and the amplified bands were consistent with the theoretical molecular weight. The results are shown in figure 1 a. The amplified fragment was inserted into pShuttle-CMV, identified by Sal I and EcoR V double enzyme digestion, and the recombinant shuttle plasmid pShuttleCMV-Lmx1a was obtained, the results are shown in figure 1 b. The recombinant clones were sequenced, and the sequencing results were consistent with the theoretical results.

[0067] The above...

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Abstract

A method for co-culturing with nerve stem cells to inducing rASCs to be DA Neurons by Lmx1a mediation, belongs to a method for treating Parkinson's disease by inducing stem cells to replacing defective Neuron cells. The method of the invention comprises steps of: building Lmx1a gene clone, recombining plasmid, building, packaging and recombining adenovirus Ad-Lmx1a, and co-culturing rASCs and NSCs which are infected by Ad-Lmx1a to differentiating them to be DA Neurons; and culturing rASCs and NSCs passage cells. Protein marker for expressing dopaminergic neuron cells can be differentiated from inducted neuron-like cells in the invention, and the inducted cells can transmit electric signal or secrete neurotransmitters, to prove that the inducted cells are the Neuron cells.

Description

technical field [0001] The invention belongs to molecular cell biology, in particular to a method for inducing stem cells to replace defective neuron cells to treat Parkinson's disease. Background technique [0002] Parkinson's disease (Parkinson's disease, PD) is a common chronic neurodegenerative disease in middle-aged and elderly people. The main pathological feature is the selective degeneration of dopamine (Dopamine, DA) neurons in the substantia nigra The formation of Lewy bodies containing a large amount of α-synuclein (α-synuclein), synphilin-1 and Parkin is very important for Parkinson's disease. The clinical treatment is mainly based on drug therapy, which cannot fundamentally remove the cause of the disease, nor can it delay the progress of the disease, and with the development of long-term medication and disease, the effect is progressively reduced, and the side effects are becoming more and more serious. Under the influence of multiple factors such as the incr...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0793C12N5/0775C12N15/11C12N15/861C12N7/01
Inventor 周艳李鸿钧孙茂盛严敏吴晋元
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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