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In-vitro inducing method for high-expression Nurrl gene of human mesenchymal stem cell

A quality stem cell, high expression technology, applied in the field of stem cell induction, to achieve the effect of convenient material acquisition and easy in vitro expansion

Active Publication Date: 2013-01-30
UNION STEMCELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for future clinical applications, genetic modification methods also have certain risks.

Method used

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  • In-vitro inducing method for high-expression Nurrl gene of human mesenchymal stem cell
  • In-vitro inducing method for high-expression Nurrl gene of human mesenchymal stem cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Induction

[0031] (1) Take the P4 generation umbilical cord mesenchymal stem cells, when the cells adhere to the wall and grow to 80%-90% confluence, remove the original culture medium in the culture bottle, and take 10 mL 0.01M phosphate buffered saline (phosphate buffered saline, PBS) Gently add T 75 Wash in the culture bottle and discard the washing solution;

[0032] (2) Add 1.0 mL of 0.25% trypsin-0.01% ethylenediaminetetraacetic acid (ethylenediaminetetraacetates, EDTA) digestion solution, soak to cover the bottom of the bottle, observe under an inverted microscope that the cells are round and floating, add 1.0 mL of fetal bovine Serum suspended trypsinization;

[0033] (3) Add 10 mL of 0.01 M PBS to repeatedly blow and wash, and centrifuge at 1000 rpm for 5 minutes at room temperature; after discarding the supernatant, add 10 mL of DMEM / F-12 to resuspend the cells, 25 Inoculate 5×10 5 Personal umbilical cord mesenchymal stem cells;

[0034] (4) The cultu...

Embodiment 2

[0039] 1. Induction

[0040] (1) Take the P4 generation umbilical cord mesenchymal stem cells, when the cells adhere to the wall and grow to 80%-90% confluence, remove the original culture medium in the culture bottle, and take 10 mL 0.01M phosphate buffered saline (phosphate buffered saline, PBS) Gently add T 75 Wash in the culture bottle and discard the washing solution;

[0041] (2) Add 1.0 mL of 0.25% trypsin-0.01% ethylenediaminetetraacetic acid (ethylenediaminetetraacetates, EDTA) digestion solution, soak to cover the bottom of the bottle, observe under an inverted microscope that the cells are round and floating, add 1.0 mL of fetal bovine Serum suspended trypsinization;

[0042] (3) Add 10 mL of 0.01 M PBS to repeatedly blow and wash, and centrifuge at 1000 rpm for 5 minutes at room temperature; after discarding the supernatant, add 10 mL of DMEM / F-12 to resuspend the cells, 25 Inoculate 5×10 5 Personal umbilical cord mesenchymal stem cells;

[0043] (4) The cultu...

Embodiment 3

[0048] 1. Induction

[0049] (1) Take the P4 generation umbilical cord mesenchymal stem cells, when the cells adhere to the wall and grow to 80%-90% confluence, remove the original culture medium in the culture bottle, and take 10 mL 0.01M phosphate buffered saline (phosphate buffered saline, PBS) Gently add T 75 Wash in the culture bottle and discard the washing solution;

[0050] (2) Add 1.0 mL of 0.25% trypsin-0.01% ethylenediaminetetraacetic acid (ethylenediaminetetraacetates, EDTA) digestion solution, soak to cover the bottom of the bottle, observe under an inverted microscope that the cells are round and floating, add 1.0 mL of fetal bovine Serum suspended trypsinization;

[0051] (3) Add 10 mL of 0.01 M PBS to repeatedly blow and wash, and centrifuge at 1000 rpm for 5 minutes at room temperature; after discarding the supernatant, add 10 mL of DMEM / F-12 to resuspend the cells, 25 Inoculate 5×10 5 Personal umbilical cord mesenchymal stem cells;

[0052] (4) The cultu...

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Abstract

The invention discloses an in-vitro inducing method for a high-expression Nurrl gene of a human mesenchymal stem cell, comprising the following step of inducing the high-expression Nurrl gene of the human mesenchymal stem cell in a DMEM (Dulbecco's Modified Eagle's Medium) / F-12 cultivation system in which RA (Retinoic Acid), CAMP (Cyclic Adenosine Monophosphate) and SHH (Sonic Hedgehog Homolog) are added. By using the in-vitro inducing method instead of a gene modification method, the expression of the Nurrl gene of the mesenchymal stem cell can be remarkably improved (about 23 times that before induction), the Nurrl gene of the mesenchymal stem cell is enabled to differentiate towards the dopaminergic neuron more easily, risks brought by gene modification is avoided, and a more ideal seed cell is provided for the clinical. Compared with an embryonic stem cell and a nerve stem cell, the mesenchymal stem cell is convenient to obtain materials, easy for in-vitro amplification, low in immunogenicity and capable of meeting the requirement of ethics.

Description

technical field [0001] The invention relates to a stem cell induction method, in particular to a method for inducing human umbilical cord mesenchymal stem cells to highly express Nurr1 gene necessary for dopaminergic neuron maturation in vitro. Background technique [0002] Nurr1 gene (Nuclear related factor 1), also known as NOT / TINUR / RNR-1 / HZF-3, as an immediate early gene product, belongs to the orphan nuclear receptor superfamily. Nurr1 protein is almost completely concentrated in the central nervous system, and is closely related to the differentiation of neural stem cells into dopaminergic neurons and the survival and development of dopaminergic neurons. Studies have shown that Nurr1 protein can activate the tyrosine hydroxylase (Tyrosine Hydroxylase, TH) promoter through various ways to promote the expression of TH. In addition, it also affects the metabolism of dopaminergic neurons, promotes the differentiation of dopaminergic neuron precursor cells into dopaminergi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 谢姜刘拥军朱德琳赵雅宁
Owner UNION STEMCELL & GENE ENG
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