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Method for promoting differentiation of mesenchymal stem cells into neurons

A technology of mesenchymal stem cells and neurons, applied in the field of promoting the differentiation of mesenchymal stem cells into neurons, can solve the problems of high molecular weight of neurotrophic substances, increase the proportion of positive cells, and promote the repair of spinal cord injury.

Active Publication Date: 2019-01-25
NINGBO JINWEI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the discovery of nerve growth factor has brought hope to the clinical drug treatment of central nervous system injury and achieved certain effects, the molecular weight of neurotrophic substances is too large to penetrate the blood-brain barrier and enter the central nervous tissue to exert its activity.

Method used

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  • Method for promoting differentiation of mesenchymal stem cells into neurons
  • Method for promoting differentiation of mesenchymal stem cells into neurons
  • Method for promoting differentiation of mesenchymal stem cells into neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of Human Mesenchymal Stem Cells

[0040] The umbilical cords of newborns were obtained, placed in Hanks' Balanced Salt Solution (HBSS, Gibco, USA), and stored at 4°C. The umbilical cord was disinfected with 75% alcohol for 30 seconds. In HBSS, the umbilical vessels are cleared. Mesenchymal tissue present in Wharton's jelly was cut into approximately 0.5 cm 3, and centrifuged at 1,200 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the precipitated portion of the mesenchymal tissue was washed with serum-free DMEM / F12 medium (Gibco, USA), followed by centrifugation at 1,200 rpm for 5 minutes. The pelleted fraction was enzymatically dissociated using 0.075% collagenase type II (Sigma) at 37°C for 18 hours, and then further digested using 0.125% trypsin / EDTA (Gibco) at 37°C for 30 minutes. The suspension was neutralized with DMEM / F12 containing 10% (v / v) fetal bovine serum, and the cells in the suspension were counted unde...

Embodiment 2

[0042] Example 2 Human mesenchymal stem cells form human mesenchymal stem cells-neurospheres

[0043] Use 0.125% trypsin / 0.02% EDTA to separate the human mesenchymal stem cells of the fourth generation, and the separated human mesenchymal stem cells are divided into 2×10 5 piece / cm 2 The density is seeded into tissue culture low-adhesion plastic flasks containing differentiation medium. The differentiation medium was NB medium (Invitrogen) containing 20 ng / ml epidermal growth factor (EGF), 20 ng / mL basic cytoblastic growth factor (bFGF), and B27 (Gibco) diluted 1:50.

[0044] Cells at 37°C, 5% CO 2 under cultivation. Add fresh growth factors every 3-4 days and change the medium once a week. Spheroid formation can be observed 4-5 days after initiation of differentiation. Cells were maintained in differentiation medium until passaged.

[0045] image 3 It shows the cell morphology of human mesenchymal stem cells reseeded in the neurosphere differentiation medium for 1-2 d...

Embodiment 3

[0048] Example 3 Further differentiation of human mesenchymal stem cells-neurospheres (BDNF induction)

[0049] Collect the human mesenchymal stem cells-neurospheres cultured in Example 2, break them up, and re-inoculate into a six-well chamber containing induction medium double-coated with poly-L-lysine and laminin In slides (chamber slides, NUNC). Cells at 37°C, 5% CO 2 Culture and differentiate for 7-10 days.

[0050] Among them, the induction medium was supplemented with 0.5umol / L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, 1% N2 supplement, 1% penicillin / streptomycin (both purchased from Gibco) and NB medium with 10 ng / mL recombinant human BDNF (rhBDNF, R&D Systems).

[0051] In the control group, the experimental conditions were the same as above, except that the induction medium was replaced with a control induction medium without BDNF, that is, 0.5umol / L all-trans retinoic acid (Sigma), 1% FBS, 5% horse serum, NB medium with 1% N2 supplement and 1% pen...

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Abstract

The invention discloses a method for promoting mesenchymal stem cells to differentiate into neurons, comprising the following steps: 1) preparing human mesenchymal stem cells and subculturing; 2) differentiating the passaged human mesenchymal stem cells into human mesenchymal stem cells in a differentiation medium-neurosphere; 3) differentiating the human mesenchymal stem cell-neurosphere into neurons in an induction culture medium. Neurotrophic factors are added to the induction culture medium to promote the differentiation into neurons, and the results show that expressing mature nerve marker (MAP2ab) and immature nerve marker (beta-tubulin III) were significantly improved relative to the blank control. The method of the invention provides a certain direction for a new treatment method for promoting the repair of spinal cord injury.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for promoting the differentiation of mesenchymal stem cells into neurons. Background technique [0002] Spinal cord injury (spinal cord injury, SCI) is a serious central system disorder, and its most serious consequence is to cause serious dysfunction of the limbs below the injured segment. The patient has brought serious physical and psychological harm, and also caused a heavy burden to the family. [0003] At present, in the treatment of spinal cord injury patients, the use of some drugs and physical therapy are usually used to slow down the degree of paralysis. However, after such treatment, the patient's quality of life still cannot be improved very well. [0004] How to make the repair and functional reconstruction of the central nervous system after spinal cord injury is still a difficult problem. Although the discovery of nerve growth factor has brought ho...

Claims

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Application Information

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IPC IPC(8): C12N5/0793C12N5/0775A61K35/30A61P25/00
CPCA61K35/30A61P25/00C12N5/0619C12N2500/38C12N2501/11C12N2501/115C12N2501/13C12N2501/15C12N2506/1369C12N2533/32C12N2533/52
Inventor 陈金虎
Owner NINGBO JINWEI BIOTECH CO LTD
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