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A method for inducing neural stem cells and its application

A technology of neural stem cells and neural precursor cells, applied in the fields of neural stem cells and biomedicine, can solve the problems of large trauma of fibroblasts, difficulty in culturing fibroblasts, and easy pollution of cell sources.

Active Publication Date: 2021-06-15
WISEHEART MEDICAL VALLEY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

1. The acquisition of fibroblasts is relatively traumatic, and it is difficult to culture fibroblasts in vitro
2. The source of cells is easily polluted. For example, cells obtained from urine are easily polluted during the operation, especially urine obtained from wards is more likely to be polluted
3. Integrative viral vectors may cause gene mutations in host cells
[0005] The cell therapy of Parkinson's disease has attracted the attention of many medical workers and scholars in the field of biology in the past 20 years, but there is no report on the transplantation of dopaminergic neural precursor cells derived from the differentiation of induced neural stem cells

Method used

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  • A method for inducing neural stem cells and its application
  • A method for inducing neural stem cells and its application
  • A method for inducing neural stem cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Inducing Peripheral Blood Mononuclear Cells to Differentiate into Neural Stem Cells

[0079] Step 1: Separation of mononuclear cells in blood.

[0080] (1) At room temperature, 6ml of adult peripheral venous blood was collected, stored in a heparin anticoagulant tube, and mixed upside down 5 times.

[0081] (2) Mononuclear cells were collected by density gradient centrifugation: the peripheral blood was diluted 1:2 with PBS, and stored in a 50ml centrifuge tube at room temperature. If the diluted blood was not enough for 35ml, it was replenished with PBS.

[0082] (3) Take another 50ml centrifuge tube to hold 15ml of Ficoll-Paque Premium, and then tilt it at 45 degrees so that the diluted blood slowly flows into the tube of Ficoll-Paque Premium.

[0083] (4) Centrifuge at 25 degrees for 30 minutes at a speed of 750g, and turn off the centrifuge brake during centrifugation.

[0084] (5) After centrifugation, the upper layer of the centrifuge tube is the plasm...

experiment example 2

[0104] Experimental Example 2 Inducing Neural Stem Cells to Express Neural Stem Cell Marker Proteins

[0105] 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:

[0106] (1) Aspirate the medium, wash twice with PBS, and then add 4% paraformaldehyde to fix for 10 minutes.

[0107] (2) Absorb paraformaldehyde, add 0.3% PBST 1ml, repeat 3 times, each interval is 5 minutes.

[0108] (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) to 1% donkey serum in proportion, and then incubate the cells at 4 degrees overnight.

[0109] (4) Remove the primary antibody and add the corresponding secondary antibody. Donkey anti-mouse FITC corresponds to Nestin at a ratio ...

experiment example 3

[0112] Experimental Example 3 Neural stem cells were differentiated into mature neurons.

[0113] 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 (1:200Mouse Sigma), Neun (1:400RabbitMillpore), expressing mature neuronal protein Map2Neun, such as Figure 9 .

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Abstract

The invention discloses a method and composition for inducing neural stem cells, which can induce peripheral blood mononuclear cells to form neural stem cells. The neural stem cells can express genes related to neural stem cells, and can differentiate into neurons, astrocytes and oligodendrocytes. The dopaminergic neural precursor cells formed by the in vitro induction and differentiation of the above-mentioned neural stem cells can be transplanted into the striatum of the PD mouse model without tumor formation, which can improve the behavior of the mouse Parkinson's disease model and delay the progression of Parkinson's disease. The neural stem cell induction and differentiation method provided by the present invention is simple and quick to operate, less invasive, and has good safety, and is expected to be used in the treatment of Parkinson's disease.

Description

technical field [0001] The present invention relates to the field of biomedicine and the technical field of neural stem cells, in particular to a method for inducing neural stem cells using a non-integrated, temperature-inactivating Sendai virus vector and the use of dopaminergic neural precursor cells of directed differentiation in Parkinson's disease cell transplantation therapy the use of. Background technique [0002] Stem cell transplantation brings hope for the treatment of many neurological diseases, such as Parkinson's disease, amyotrophic lateral sclerosis, brain and spinal cord trauma, etc. Studies have shown that the use of neural stem cells derived from embryonic stem cells has certain effects in the treatment of the above diseases, but the tumorigenicity of embryonic stem cells, immune rejection of allogeneic transplantation, and ethical disputes limit its application. Induced pluripotent stem cells (iPSCs) were obtained from human fibroblasts and differentiate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079C12N5/0793C12N5/0797A61K35/30A61P25/00A61P9/10A61P25/16
CPCC12N5/0623C12N5/0619C12N5/0618A61K35/30A61P25/00A61P9/10A61P25/16C12N2506/11A61P25/28C12N15/86C12N2760/18843C12N2501/60C12N2506/115A61K35/15C12N5/0606C12N2501/01C12N5/0696C12N2501/13C12N2501/15C12N5/0018
Inventor 陈志国袁艳鹏唐玺和张愚
Owner WISEHEART MEDICAL VALLEY CO LTD
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