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A method for inducing neural stem cells using a non-integrating plasmid vector and its use

A technology of neural stem cells and plasmid vectors, applied in the field of neural stem cell induction using non-integrated plasmid vectors, can solve the problems of low transdifferentiation efficiency, long somatic cell transdifferentiation cycle, lack of effective treatment of cranial nerve injury and spinal cord injury, etc., and achieve transformation High differentiation efficiency, short induction differentiation cycle, and convenient collection

Active Publication Date: 2018-09-25
WISEHEART MEDICAL VALLEY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the shortcomings of the prior art that the induced neural stem cells cannot be rapidly expanded in vitro, the cycle of somatic cell transdifferentiation is long, and the transdifferentiation efficiency is low, thereby providing a kind of neural stem cells that can be rapidly expanded in vitro, Induction method of neural stem cells with short period of somatic cell transdifferentiation and high transdifferentiation efficiency
[0006] Another technical problem to be solved by the present invention is to overcome the deficiency in the prior art that there is no method for effectively treating cranial nerve injury and spinal cord injury, which is not just a disease of a single nerve cell, so as to provide a method to induce Use of neural stem cells in the treatment of neurocytopathy

Method used

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  • A method for inducing neural stem cells using a non-integrating plasmid vector and its use
  • A method for inducing neural stem cells using a non-integrating plasmid vector and its use
  • A method for inducing neural stem cells using a non-integrating plasmid vector and its use

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Step 1: Separation of mononuclear cells in blood.

[0038] 1. At room temperature, 5-10ml of adult peripheral blood is collected, stored in an anticoagulant tube containing heparin, and mixed up and down 5 times.

[0039] 2. Collect mononuclear cells by density gradient centrifugation. The specific operation is: dilute the peripheral blood with PBS at 1:2, store it in a 50ml centrifuge tube at room temperature, if the diluted blood is not enough for 35ml, fill it up with PBS, The final diluted blood volume was 35ml.

[0040] 3. Take another 50ml centrifuge tube to hold 15ml Ficoll-Paque Premium, then tilt it at 45 degrees, and slowly flow the diluted blood into the Ficoll-Paque Premium tube.

[0041] 4. Centrifuge at 25 degrees for 30 minutes at a speed of 750g. During centrifugation, the brake of the centrifuge should be turned off.

[0042] 5. After centrifugation, the upper layer of the centrifuge tube is the plasma sample, and the cloudy cells in the middle are th...

experiment example 1

[0065] Experimental Example 1 Inducing Neural Stem Cells to Express Neural Stem Cell Marker Proteins

[0066] The induced neural stem cells were divided into 5×10 4 Inoculated on polylysine and laminin-coated 12mm glass slides, cultured in neural stem cell culture medium for 48 hours and then stained. The specific steps are: 1. Absorb the culture medium, wash twice with PBS, and then add 4% more POM fixation for 10 min. 2. Suck off the paraformaldehyde, add 1ml of 0.3% PBST, repeat 3 times, each interval is 5 minutes. 3. Add 3% donkey serum to block for 1 hour at room temperature. 4. After 1 hour, add the primary antibody, respectively add antibodies such as Nestin (1:500Mouse BD bioscience), Sox1 (1:200Goat BD bioscience), Sox2 (1:1000GoatBioscience) to 1% donkey serum in proportion, and then incubate the cells, 4 degrees, overnight. 4. Remove the primary antibody and add the corresponding secondary antibody. Donkey anti-mouse FITC corresponds to Nestin at a ratio of 1:2...

experiment example 2

[0067] Experimental Example 2 Neural stem cells were differentiated into mature neurons.

[0068] Neural stem cells were divided into 2×10 4 Inoculated on polylysine and laminin-coated 12mm glass slides, cultured in neural stem cell medium for 24 hours, then replaced the medium with neuron differentiation medium, the composition of which was DMEM:F12, 1% N2, 1 %B27, 1% Glutamine, 1% non-essential amino acid NEAA (Life Technologies), the medium was changed every other day, and the cells were fixed after 6 weeks for immunocytochemical staining. Specifically, MAP2(1:200 Mouse Sigma), Neun(1:400Rabbit Millpore), GFAP(1:500 Rabbit Dako), such as Figure 10 , Figure 11 shown. The induced neural stem cells can differentiate into mature neurons and express the mature neuronal protein Map2Neun, and the induced neural stem cells can differentiate into astrocytes and express GFAP.

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Abstract

The invention provides a method for inducing neural stem cells by use of non-integrated plasmid vectors. The method comprises the steps of extraction of mononuclear cells from blood, expansion of the mononuclear cells in blood, electrotransfection of non-integrated plasmids and culture of neural stem cells. Mononuclear cells in human peripheral blood are reprogrammed into the neural stem cells which can be expanded by 60 generations or above in vitro to express related genes of the neural stem cells, nerve cells, astrocytes and oigodendrocytes can be differentiated, and the differentiated nerve cells have the electrophysiological characteristic. The method is simple to operate and extremely less in trauma.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to the technical field of neural stem cells, in particular to a method for inducing neural stem cells using a non-integrated plasmid vector and its application. Background technique [0002] Stem cells bring hope for the treatment of many neurological diseases, such as Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease and spinal cord trauma. Studies have shown that the use of embryonic stem cells has a certain effect on the treatment of Parkinson's disease, spinal cord injury and diabetes, but the tumorigenicity of embryonic stem cells, immune rejection of allogeneic transplantation and ethical disputes limit its application. stem cells (iPSCs), and successfully established cell models and animal models for the treatment of related diseases. However, studies have found that the use of iPSCs in clinical treatment still faces many problems, such as tumorigenic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0797A61K35/30A61P25/00A61P25/16A61P25/28A61P3/10A61P37/02A61P9/10
Inventor 陈志国张愚唐玺和
Owner WISEHEART MEDICAL VALLEY CO LTD
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