Method for differentiation of dopaminergic neuron by SHH/Lmx1a signal pathway regulation and control NTN modified rASCs

A dopaminergic and signaling pathway technology, applied in the field of biomedicine, can solve the problems of rising incidence of PD, failing to fundamentally restore the normal function of the lesion, and limiting the long-term treatment of Parkinson's disease

Active Publication Date: 2013-09-04
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

Studies have found that mental workers are susceptible to Parkinson's disease, and the incidence of PD is increasing year by year. The side effects of drug treatment limit

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  • Method for differentiation of dopaminergic neuron by SHH/Lmx1a signal pathway regulation and control NTN modified rASCs
  • Method for differentiation of dopaminergic neuron by SHH/Lmx1a signal pathway regulation and control NTN modified rASCs
  • Method for differentiation of dopaminergic neuron by SHH/Lmx1a signal pathway regulation and control NTN modified rASCs

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Experimental program
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Embodiment Construction

[0049] 1. Cloning of Lmx1a gene and chimeric NTN gene and construction of recombinant plasmid

[0050] Using Lmx1a whole transcript cDNA (NM_138396) as a template, the primers are:

[0051] P1 up: 5'-GCGTCGACGTACCATGCTGGACGGCCTAAAG-3',

[0052] P1 down: 5'-CGGGATCCCGCTTATCAAGATGTGAAGTAAG-3',

[0053] The DNA fragments with SalI and EcoRV 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, and the recombinant clone was identified by double digestion with SalI and EcoRV. The results are shown in figure 1 b. The recombinant clones were sequenced, and the sequencing results were consistent with the theoretical results.

[0054] The above Lmx1a whole transcript cDNA (NM_138396) was produced by GeneCopoeiaTM.

[0055] Connect the NTN mature peptide gene to the art...

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Abstract

The invention provides a method for differentiation of dopaminergic neuron by SHH/Lmx1a signal pathway regulation and control NTN modified rASCs, and belongs to the biomedical method. The coding sequence of Lmx1a gene and chimeric NTN gene are obtained by PCR according to the invention, recombinant adenovirus Ad-JLmx1a and Ad-NTN are respectively packed in the HEK-293 cell after homologous recombination. After rASCs is infected by Ad-Lmx1a, the SHH/FGF-8 and other cytokines induce the rASCs to a standby state, and ad-NTN is infected, and B27, N2supplement and bFGF and other derivants are added. The invention can substantially improve the cell differentiation potential of rASCs to Neuron, and related transcription factor can be applied to the future cell substitution treatment of Parkinsons disease.

Description

technical field [0001] The invention belongs to biomedicine, in particular to a method for inducing stem cells to replace defective neuron cells to treat Parkinson's disease. Background technique [0002] Neurodegenerative diseases (Neurodegenerative Diseases, ND) are a class of diseases characterized by neuron degeneration or neuron cell apoptosis, leading to neurological diseases in the body. Parkinson's disease (Parkinson's Disease, PD) is one of the common diseases of neurodegenerative diseases. Progressive neurodegenerative disease, clinically manifested as resting tremor, muscle rigidity, slowness of movement, and postural instability. PD mainly occurs in the elderly, and its prevalence is second only to diabetes, coronary heart disease, and cerebrovascular disease. Studies have found that mental workers are susceptible to Parkinson's disease, and the incidence of PD is increasing year by year. The side effects of drug treatment limit the long-term treatment of P...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0793C12N5/0775C12N15/11C12N15/861C12N7/01
Inventor 李鸿钧周艳孙茂盛严敏吴晋元
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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