Method for inducing neural stem cells by use of non-integrated plasmid vectors and application of neural stem cells

A neural stem cell and plasmid vector technology is applied in the field of using non-integrating plasmid vector to induce neural stem cells, which can solve the problems of lack of effective treatment for brain nerve injury and spinal cord injury, long somatic cell transdifferentiation cycle, low transdifferentiation efficiency, etc. The effect of short differentiation cycle, convenient collection and high transdifferentiation efficiency

Active Publication Date: 2016-01-06
WISEHEART MEDICAL VALLEY CO LTD
View PDF7 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for inducing neural stem cells by use of non-integrated plasmid vectors and application of neural stem cells
  • Method for inducing neural stem cells by use of non-integrated plasmid vectors and application of neural stem cells
  • Method for inducing neural stem cells by use of non-integrated plasmid vectors and application of neural stem cells

Examples

Experimental program
Comparison scheme
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-PaquePremium, then tilt it at 45 degrees, and slowly flow the diluted blood into the Ficoll-PaquePremium tube.

[0041] Centrifuge at 4.25 degrees for 30 minutes at a speed of 750g. During centrifugation, the centrifuge brake must 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 the mononuclea...

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 paraformaldehyde, add 0.3% PBST1ml, repeat 3 times, each interval is 5 minutes. 3. Add 3% donkey serum to block for 1 hour at room temperature. After 4.1 hours, add the primary antibody, respectively add antibodies such as Nestin (1:500MouseBDbioscience), Sox1 (1:200GoatBDbioscience), Sox2 (1:1000GoatBDbioscience) to 1% donkey serum in proportion, and then incubate the cells at 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 1:200, and ...

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(1:200MouseSigma), Neun(1:400RabbitMillpore), GFAP(1:500RabbitDako), 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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap