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Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts

A neuron cell and fibroblast technology, which is applied in the fields of biotechnology and neurodevelopment, can solve the problem of inability to efficiently obtain inhibitory neurons, and achieve the effect of improving memory and efficient acquisition.

Active Publication Date: 2016-02-17
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Therefore, the object of the present invention is to provide a method for inducing the transdifferentiation of fibroblasts into neuron cells in view of the current inability to efficiently obtain inhibitory neurons

Method used

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  • Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts
  • Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts
  • Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Inducing fibroblasts to transdifferentiate into PV neurons

[0054] Specific experimental process:

[0055] 1) Adenovirus vector construction: the Ascl1 gene was constructed into the adenovirus pAD vector by BamHI+EcoRI double enzyme digestion, and the virus packaging and virus titer were measured respectively;

[0056] 2) Preparation of mouse fibroblast cells: select 14.5-day mouse embryos, remove the head, tail, spine, viscera and gonads, take skin tissue, digest with 0.5% trypsin for 5 minutes, and take the digested skin tissue Spread the fibroblasts into a 10 cm culture dish with DMEM plus 10% fetal bovine serum, culture them, and passage them once after 2 days to finally obtain fibroblasts;

[0057] 3) Virus infection: Spread 60,000 fibroblasts into one well of a 24-well plate, infect the fibroblasts with packaged Ascl1 adenovirus particles, and replace with neuron medium (DMEM, 1×B -27, 10 μg / ml BDNF), add the small compound FSK (10 μM) to the neuron...

Embodiment 2

[0062] Example 2 Transplantation of PV nerve cells to treat epilepsy

[0063] Specific experimental process:

[0064] 1) Establishment of epilepsy mouse model. By intraperitoneally injecting pilocarpine (280 mg / kg) into 6-week-old adult C57 mice, 2 weeks after the drug injection, the epilepsy mouse model was induced. The mice in this experiment were divided into 3 groups, including a group of 10 normal mice, a group of 10 epileptic mice, and a group of 10 transplanted nerve cells to epileptic mice.

[0065] 2) Transplantation of nerve cells. Transplant 50,000 nerve cells into the hippocampus of the mouse brain through stereotaxic and microinjection systems.

[0066] Specific location of mouse hippocampus: Use a positioning needle to locate a point 2mm behind bregma and 2.5mm beside the sagittal suture, which is the plane position of the hippocampus, and then use a drill needle to drill a small round hole on the skull at this point .

[0067] Transplanted cells: the hipp...

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Abstract

The invention provides a composition used for transforming fibroblasts into PV (parvalbumin) neurons, also provides a method for transforming the fibroblasts into neuronal cells and further provides the neuronal cells prepared according to the method. The composition contains one or more of Ascl1 gene and FSK (forskolin). The invention further provides application of the composition and the neuronal cells to preparation of medicines for treating nervous system diseases. According to experiments, the PV neuronal cells can be efficiently obtained by means of transdifferentiation to treat epilepsy after transplantation, memory improvement can be realized by reducing frequency of epileptic seizures, and accordingly a new approach is created for treatment of epilepsy and related diseases.

Description

technical field [0001] The invention relates to the fields of biotechnology and neurodevelopment. The invention specifically relates to a method for inducing fibroblasts to transdifferentiate into neuron cells and its application. Background technique [0002] Regenerative medicine based on stem cell transplantation technology has brought great hope for human beings to overcome intractable degenerative diseases. Induced pluripotent stem cells were once considered a new hope for regenerative medicine because they broke through the ethical barriers of embryonic stem cells. However, its low induction efficiency, batch-to-batch instability and high tumorigenicity make it difficult to be directly used for disease treatment. [0003] Since 2010, some scientists at home and abroad have transformed one type of adult cell into another type of adult cell or adult stem cell by expressing specific transcription factors. This approach is called "transdifferentiation". This technique ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N5/10C12N5/0793A61K35/30A61P25/00A61P25/08
Inventor 焦建伟史子啸
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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